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STUDIES ON THE TOXICITY OF CADMIUM by Frank W. Bonner A thesis presented for the Degree of Doctor of Philosophy at the University of Surrey. March, 1980 Department of Biochemistry, University of Surrey, Guildford, Surrey.

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Page 1: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

STUDIES ON THE TOXICITY OF CADMIUM

by Frank W. Bonner

A th e s i s p re sen te d fo r th e Degree o f

D octor o f Ph ilosophy a t th e U n iv e rs ity

o f S u rrey .

March, 1980 Department o f B io ch em is try ,

U n iv e rs ity o f S u rre y ,

G u ild fo rd ,

S u rrey .

Page 2: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

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Page 3: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

ABSTRACT

The e f f e c t s o f a cu te p a r e n te r a l , re p e a te d p a re n te r a l and d ie ta r y

a d m in is tra t io n o f cadmium c h lo r id e have been in v e s t ig a te d in th e male

r a t .

A r e le a s e o f k idney enzymes in to th e u r in e occu rred d u rin g th e

developm ent o f cadmium-induced r e n a l damage, and prov ided a u se fu l

c r i t e r i o n o f t o x i c i t y in f u r th e r s tu d ie s .

Cadmium accum ulation in l i v e r and k idney was a s s o c ia te d w ith

in c re a s e s in th e c o n c e n tra tio n s o f z in c and copper in th e se t i s s u e s .

D ie ta ry cadmium exposure d e p le te d th e r e n a l and h e p a tic c o n ten t o f

i r o n . R epeated p a re n te r a l a d m in is tra t io n o f cadmium had a s im ila r

e f f e c t bu t on ly in th e k id n ey , and produced an ex ce ss iv e lo s s o f t r a c e

m eta ls in to th e u r in e , which may have ex ace rb a ted th e d is tu rb a n c e s in

t h e i r m etabolism .

Changes in th e plasm a c o n c e n tra tio n o f t r a c e m eta ls depended upon

th e ro u te o f a d m in is tra t io n and dosage o f cadmium. A s in g le in je c t io n

o f cadmium c h lo r id e in c re a se d th e c o n c e n tra tio n o f copper in plasm a

b u t d ecreased th a t o f z in c and i r o n , w h ile re p e a te d in je c t io n s in c re a se d

th e c o n c e n tra tio n s o f bo th z in c and copper, and decreased i r o n . Long

term d ie ta r y a d m in is tra t io n o f cadmium c h lo r id e r a is e d plasm a copper

b u t decreased bo th z in c and i ro n .

The a d m in is tra t io n o f h igh doses o f z in c su lp h a te a l t e r e d th e

t i s s u e d is p o s i t io n o f cadmium. The p o s s i b i l i t y th a t z in c may p rev en t

some o f th e to x ic e f f e c t s o f cadmium was in v e s t ig a te d , b u t z in c i t s e l f

induced changes in th e hom eostasis o f e s s e n t i a l m e ta ls . •

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C adm ium -treated r a t s showed changes in calcium hom eostasis which

were su g g es tiv e o f an im paired a b so rp tio n , and th e accum ulation o f

t r a c e m eta ls in th e femur was in h ib i te d . These changes were independent

o f th e developm ent o f k idney damage. The d ie ta r y a d m in is tra t io n o f

cadmium a lso d ecreased th e a c t i v i t y o f femur a lk a l in e p h o sp h atase .

This e f f e c t was p rev en ted by d ie ta r y z in c su p p lem en ta tio n .

The e f fe c t iv e n e s s o f a v a r ie ty o f c h e la t in g ag en ts to m o b ilis e

and promote th e e x c re tio n o f cadmium was compared. Some l i p o p h i l i c

d e r iv a t iv e s o f d ie th y le n e tr ia m in e p e n ta a c e tic a c id were v e ry e f f e c t iv e

when ad m in is te red a f t e r a c u te cadmium exp o su re , b u t no compounds

s ig n i f i c a n t ly d ecreased th e body burden o f cadmium in anim als which

had accum ulated th e m eta l in t i s s u e s .

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To my w ife G u itty and

my p a re n ts

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ACKNOWLEDGEMENTS

I am most g r a te f u l to Dr. L .J . King fo r h is in v a lu a b le gu idance ,

and w ith o u t whose h e lp , t h i s p ro je c t would n o t have been p o s s ib le ;

a ls o to P ro fe sso r D.V. Parke fo r h is co n tinued i n t e r e s t and most h e lp ­

f u l a d v ic e , and fo r making th e f a c i l i t i e s o f h is departm ent a v a i la b le

to me.

The f in a n c ia l su p p o rt fo r t h i s work by Rio T in to Zinc L td . i s

g r a te f u l ly acknowledged and th e h e lp and su g g es tio n s o f Dr. A.K. B arbour

and D r. I . O’N e il was v e ry much a p p re c ia te d .

I would a lso l i k e to thank Mr. A. T aylor o f th e Heavy M etals

R eference C en tre , U n iv e rs ity o f S u rre y , fo r h is adv ice on m ethodology

and fo r making equipment a v a i la b le to me; a lso Miss S . Ashby f o r most

u s e fu l d is c u s s io n s th ro u g h o u t th e p r o je c t .

The c o o p e ra tio n o f th e N a tio n a l R a d io lo g ic a l P ro te c t io n B oard,

H arw ell, in su p p ly ing c h e la t in g ag en ts i s acknowledged and p a r t i c u l a r ly

D r. R.A. Bulman fo r h is i n t e r e s t .

I owe a g re a t d e a l to my w ife who has been a c o n s ta n t su p p o r t,

and to my p a re n ts who have always been concerned fo r my e d u ca tio n .

I thank them fo r t h e i r con tinued encouragem ent.

F in a l ly I would l i k e to th an k Mrs. B. Podd fo r h e r m eticu lo u s

ty p in g o f t h i s t h e s i s .

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Knowledge i s as wings to m an's l i f e , and a la d d e r fo r

h is a s c e n t , j I t s ja c q u is i t io n i s incumbent upon everyone.

The knowledge o f such s c ie n c e s , however, should be

acq u ired as can p r o f i t th e peop les o f th e e a r th , and

n o t th o se which b eg in w ith words and end w ith w ords.

B a h a 'u 'l l a h , 1817 - 1892.

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CONTENTS

CHAPTER 1

CHAPTER 2

CHAPTER 3

CHAPTER 4

CHAPTER 5

CHAPTER 6

REFERENCES

APPENDICES

Page

In tro d u c tio n 8

The Value o f U rin ary Enzymes in th e 44

D eterm in a tio n o f Kidney Damage

An I n v e s t ig a t io n o f th e In te r - R e la t io n s h ip s 67

o f Cadmium, Z in c , Copper and Iro n in

th e Rat

The E f fe c t o f Cadmium Upon Calcium 129

H om eostasis and Bone T race M etals in

th e Rat

The Use o f C h e la tin g Agents in th e 156

Therapy o f Cadmium T o x ic ity

G eneral D iscu ss io n 183

197

220

PUBLICATIONS 226

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CHAPTER 1

INTRODUCTION

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CONTENTS

P reface

Sources o f Exposure to Cadmium

O ccurrence and uses o f cadmium; le v e ls

o f cadmium in th e environm ent; ro u te s o f

exposure, to cadmium; h ig h r i s k groups in

th e human p o p u la tio n .

The M etabolism o f Cadmium

A b sorp tion ; t r a n s p o r t ; p ro p e r t ie s o f

m e ta l lo th io n e in ; d i s t r i b u t io n ; e x c re tio n ;

m e ta llo th io n e in s in r e l a t i o n to cadmium

m etabolism .

The Toxicology o f Cadmium

M e ta llo th io n e in in r e l a t i o n to cadmium

to x ic i t y ; s i t e o f exposure; l i v e r ; k idney ;

bone; t e s t i s ; c a rd io v a s c u la r system ;

haem ato p o ie tic system ; m isce llan eo u s e f f e c t s

h e a l th e f f e c t s in humans; h e a l th s tu d ie s

in Japan .

The Im portance o f S tu d ie s on Cadmium T o x ic ity

The r i s k to human h e a l th ; th e d ia g n o sis o f

cadmium t o x ic i t y ; mechanisms o f cadmium

to x ic i t y ; th e tre a tm e n t o f cadmium to x ic i t y ;

c o n tr ib u tio n o f t h i s t h e s i s .

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PREFACE

In re c e n t y e a r s , p roducers of cadmium and re s e a rc h o rg a n is a tio n s

have become in c re a s in g ly aware o f th e p o te n t ia l hazard to human h e a l th

and th e environm ent caused by excess le v e ls o f th e m e ta l. C onsequently

th e re has been p ro g ress in th e e s ta b lish m e n t o f h e a l th c r i t e r i a f o r

cadmium, bo th f o r w orkers exposed in d u s t r i a l l y to th e m e ta l, and f o r

members o f th e g e n e ra l p o p u la tio n . This concern has been promoted by

th e ex p erien ces o f th e o u tb reak o f I t a i - i t a i d is e a se in Japan around

1945-1950, and a g a in has become p e r t in e n t w ith th e re c e n t d isco v e ry

o f h ig h le v e ls o f env ironm enta l cadmium in th e E n g lish v i l l a g e o f Shipham.

1 .1 SOURCES OF EXPOSURE TO CADMIUM

O ccurrence and Uses o f Cadmium

Cadmium belongs to group IIB o f th e p e r io d ic t a b l e , w ith atom ic

number 48, and an atom ic w eight o f 112 .40 . The m eta l' occurs n a tu r a l ly

in th e l i th o s p h e re , no t in a pure s t a t e b u t always in th e p resen ce o f

z in c , and i s p r in c ip a l ly c o n c e n tra te d in su lp h id e d e p o s i t s . The n a tu r a l

c o n c e n tra tio n o f cadmium in rocks i s approx im ate ly 0 .1 5 - 0 .20 pg /g bu t

th e c o n ten t in z in c o res may be c o n s id e ra b ly h ig h e r , v a ry in g from 0 .1

to 5%. The p ro d u c tio n of cadmium as a b y -p roduct o f z in c r e f in in g

began tow ards th e end o f th e n in e te e n th c e n tu ry , bu t in c re a se d in d u s ­

t r i a l demand fo r th e m etal has r a p id ly promoted i t s p ro d u c tio n to th e

e x te n t th a t about 70% o f th e t o t a l w orld p ro d u c tio n has occu rred w ith in

th e l a s t 20 y e a rs . Between 1960 and 1969, an e s tim ated 124,207 m e tr ic

tons o f cadmium were produced w orldw ide. This re p re s e n ts more th a n a

tw e lv e fo ld in c re a se s in c e th e p e rio d 1910 - 1919. The p ro p e r t ie s o f

cadmium make i t s u i ta b le fo r a range o f uses in c lu d in g p igm ents,

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b a t t e r i e s , e l e c t r o - p la t in g , p l a s t i c s s t a b i l i s e r s , so ld e rs and a l lo y s .

The s t a t i s t i c s o f th e p ro d u c tio n and usage o f cadmium have been

th o ro u g h ly review ed (F r ib e rg e t a l . , 1974; C .E .C ., 1978).

L evels o f Cadmium in th e Environm ent

The co n tam in a tio n o f th e environm ent w ith cadmium by n a tu r a l p ro ­

cesses i s in s ig n i f ic a n t compared to th e d i s s ip a t io n o f th e elem ent

b rough t about by th e advancement in i n d u s t r i a l i s a t i o n . However, a n a ly s is

o f th e cadmium c o n c e n tra tio n o f a i r sam ples shows th a t l e v e l s o f th e m eta l

a re g e n e ra l ly low, even in i n d u s t r i a l i s e d a re a s but in c re a s e s ig n i f i c a n t ly

in th e v i c i n i t y o f cadm ium -em itting so u rc e s . Table 1 .1 summarises th e

c o n c e n tra tio n o f cadmium found in v a r io u s env ironm ental com partm ents.

Table 1 .1 The C o n ce n tra tio n o f Cadmium in th e Environm ent

Environm ental Compartment C o n cen tra tio n o f Cadmium

p^n3

AIR - r u r a l 0.0001 - 0.043

urban 0.002 - 0 .7

in d u s t r i a l 0 .01 - 5 .0

y g / i

WATER - m arine 0 .05 - 9 .0

f r e s h 1 .0 - 10.0

d r in k in g <5.0

yg/g

EARTH - e a r th s c ru s t 0 .15 - 0.20

igneous ro ck 0.001 - 0 .6

s o i l <1.0

Data from F r ib e rg e t a l . , (1974) and C.E.C. (1978)

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S im ila r ly in a re as n o t known to be p o llu te d by cadmium, th e con­

c e n t r a t io n o f th e m eta l in w a ter i s low , v a lu es o f l e s s th a n 1 .0 yg/1

b e in g commonly found. In p o llu te d a r e a s , le v e ls a re h ig h e r though a

la rg e p ro p o r tio n o f th e cadmium may be lo c a te d as p a r t i c u la te m a tte r in

sed im en ts .

The c o n c e n tra tio n o f cadmium in s o i l s w i l l be in f lu e n c e d by w a ter

supp ly and a i r d e p o s it io n . Many p la n ts in c lu d in g fo o d s tu f f s a re cap ab le

o f abso rb ing and accum ulating c o n s id e ra b le q u a n t i t ie s o f cadmium from

th e s o i l . T ra n s fe r and c o n c e n tra tio n o f th e m eta l a long food ch a in s may

th u s be im p o rtan t. A wide range o f cadmium c o n c e n tra tio n s have been

re p o r te d in foods and some o f th e se a re d e sc rib e d in Table 1 .2 . A gain,

th e le v e ls d e te c te d in con tam inated a re a s a re g r e a t ly in excess o f th o se

found u s u a lly .\

Table 1 .2 The C o n ce n tra tio n o f Cadmium in Food

F o o d s tu ff C o n ce n tra tio n o f Cadmium

yg/g wet w eight

v e g e ta b le s 0 .04

c e r e a l 0 .03

f r u i t 0 .01

m ilk 0 .04

meat 0 .02 - 0 .10

k idney 0 .04 - 40 .0

f i s h 0.02

o y s te r 0 .1 - 34 .5

D ata from F r ib e rg e t a l . (1974) and C .E.C. (1978)

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There appears to be a tendency tow ards in c re a s in g co n tam in a tio n

o f th e environm ent by cadmium. This tre n d i s r e f l e c te d in a s tu d y

by K je lls trb m e t a l . (1 9 7 5 ), who re p o r te d a s ig n i f i c a n t in c re a s e in th e

cadmium c o n ten t o f c e re a ls h a rv e s te d betw een th e y ea rs 1916 and 1972 in

Sweden.

Routes o f Exposure to Cadmium

Exposure to cadmium occu rs m ain ly th rough th e lu n g s o r th e g a s tro ­

i n t e s t i n a l t r a c t . For in d iv id u a ls i n th e g en e ra l environm ent, n o t

exposed to cadmium o c c u p a tio n a lly , in ta k e o f th e m eta l by in h a la t io n i s

n o t v e ry s ig n i f i c a n t . For persons l iv in g c lo se to a cadm ium -em itting

in d u s try , however, up take by in h a la t io n may become an im portan t so u rce

o f exposure . Tobacco has a ls o been shown to c o n ta in s ig n i f i c a n t amounts

o f cadmium and c o n c e n tra tio n s o f 1 - 2 yg p e r c ig a r e t t e have been

re p o r te d (Nandi e t a l . , 1969 ), o f which about 10% may be in h a le d .

C onsequen tly , smoking may c o n tr ib u te a p p re c ia b ly to d a i ly in ta k e .

T able 1 .3 p ro v id es an e s tim a te o f th e c o n tr ib u t io n o f v a r io u s

so u rces to th e d a i ly in ta k e o f cadmium f o r an in d iv id u a l in th e g e n e ra l

p o p u la tio n . The d a i ly in ta k e r e f l e c t s g eo g rap h ica l v a r ia t io n s in th e

n a tu r a l le v e l o f cadmium, and th e e x te n t o f env ironm enta l c o n tam in a tio n ,

and i s in flu en c ed by c u l tu r e , d ie ta r y h a b i t s , e t c . , b u t i t i s e s tim a te d

th a t th e average d a i ly in ta k e o f cadmium i s around 50 y g , w ith some

c o u n tr ie s h ig h e r ( e .g . Canada - 80 y g ) and o th e rs low er

( e .g . U.K. - 15 - 30 y g ) . This d a i ly in ta k e produces an e s tim a te d

body burden o f about 30 mg o f cadmium by th e age o f 50 y e a rs .

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T ab le 1 .3 C o n tr ib u t io n t o D a i ly Cadmium

In ta k e from V arious Sources

Source C o n tr ib u tio n

yg

AIR - r u r a l 0.0005 - 0 .215

urban 0 .01 - 3 .5

in d u s t r i a l 0 .0 5 - 25.0

WATER 2 - 4

FOOD \ 10 - 80

SMOKING (20 c ig s . /d a y ) 2 - 4

Average d a i ly in ta k e 50

D ata from F r ib e rg e t a l . (1974) and C.E.C. (1978)

High R isk Groups in th e Human P o p u la tio n

Persons exposed to cadmium in in d u s try v ia a i r may be re g a rd ed as

a h ig h r i s k group. C o n s id e ra tio n should a lso be g iven to groups in th e

g e n e ra l p o p u la tio n . The up take and subsequen t t o x i c i t y o f cadmium i s

in f lu e n c e d by a v a r i e ty o f n u t r i t i o n a l f a c to r s (L evander, 1977) and may

be p o te n t ia te d by d e f ic ie n c ie s o f e s s e n t i a l n u t r i e n t s . D ie ta ry d e f ic ie n c ie s

o f ca lc iu m , p ro te in and v ita m in D were undoubtedly s ig n i f i c a n t in th e

a e tio lo g y o f I t a i - i t a i d is e a s e . The in ta k e o f t r a c e e lem ents may be

d e f i c i e n t in some human p o p u la tio n s , e .g . growth r e t a r d a t io n due to

in s u f f i c i e n t d ie ta r y z in c has been re p o r te d (W aslien , 1976). V arious

d is e a s e s t a t e s may a lso d is tu r b e s s e n t i a l m in era l hom eostasis

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(F is h e r , 1975). I f such c o n d itio n s a re combined w ith a h igh exposure

to cadmium, th e n in d iv id u a ls must be reg a rd ed as a h igh r i s k .

1 .2 THE METABOLISM OF CADMIUM

A bso rp tion

There a re im p o rtan t d if f e r e n c e s in th e r a t e o f a b so rp tio n o f

cadmium depending upon th e ro u te o f exposure . E xperim enta l s tu d ie s in

r a t s have shown th a t l e s s th a n 5% o f a s in g le o r a l dose o f th e m eta l

i s absorbed (D ecker e t a l . , 1957; Moore e t a l . , 1973a), w h ile in mice

a b so rp tio n v a r ie s betw een 0 .5 - 8% (C o tz ia s e t al_., 1961). This i s in

agreem ent w ith b a lan ce s tu d ie s perform ed in humans where a b so rp tio n

r a t e s o f around 5 - 6 % have been re p o r te d a f t e r a s in g le o r a l dose o f

cadmium (F r ib e rg _et a l . , 1974). As in d ic a te d in s e c t io n 1 .1 , many

f a c to r s can in f lu e n c e th e up take o f o r a l ly ad m in is te red cadmium.

Washko and Cousins (1976) showed g r e a te r up take o f o r a l ly a d m in is te red

109 . . . .Cd m c a lc iu m -d e f ic ie n t r a t s . The dose o f th e m eta l does n o t seem

to have any e f f e c t upon th e amount absorbed (Moore e t a l . , 1973b), and

cadmium c h lo r id e and th io n e in -b o u n d cadmium a lso seem to have s im i la r

a b so rp tio n r a t e s (C h erian e t a l . , 1978).

A b so rp tio n o f cadmium th rough th e lungs however can be c o n s id e ra b ly

h ig h e r , and v a lu es ran g in g from 10 - 40% have been re p o r te d in an im als

(F r ib e rg e t a l . , 1974; Moore e t a l . , 1973a). Not a l l o f th e cadmium

d e p o s ited in th e lungs i s absorbed and p a r t i c l e s iz e appears to be

im p o rtan t. Some p a r t i c l e s may be t r a n s f e r r e d to th e g a s t r o - i n t e s t i n a l .

t r a c t by m u c o -c ilia ry c le a ra n c e where a b so rp tio n may ta k e p la c e .

D if f e re n t compounds o f cadmium may have v a ry in g a b so rp tio n r a t e s by

in h a la t io n ( P r in c i and G eever, 1950).

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A bsorp tion o f cadmium th rough sk in i s th o u g h t to be

i n s ig n i f i c a n t .

T ran sp o rt

S e v e ra l s tu d ie s have re p o r te d th e b eh av iou r o f cadmium in th e

b lood a f t e r a s in g le p a re n te r a l in j e c t io n to r a t s . Im m ediately fo llo w in g

a d m in is t r a t io n , th e m eta l i s lo c a te d predom inan tly in th e plasm a in

a s s o c ia t io n w ith a -g lo b u lin s (P e r ry e t a l . , 1970; Shaikh and L u c is , 1972).

However, th e m etal r a p id ly d isa p p e a rs from th e b lood w ith in th e f i r s t

few hours (B e r l in and U llb e rg , 1963; L ucis e t a l . , . 1969). H orner and

Sm ith (1975) found more th a n 14% o f th e t o t a l absorbed cadmium in th e

blood o f r a t s 5 min a f t e r in trav en o u s a d m in is tra t io n , b u t by 30 min t h i s

had d ecreased to l e s s th a n 2%. F o llow ing t h i s i n i t i a l r a p id c le a ra n c e

from th e plasm a, th e re i s a slow er phase d u rin g which th e cadmium con­

c e n t r a t io n in c re a s e s in th e e ry th ro c y te s .

M e ta llo th io n e in has been d e te c te d in th e e ry th ro c y te s o f mice a f t e r

s in g le and re p e a te d in je c t io n s o f cadmium, though some cadmium i s a ls o

bound to haem oglobin (N ordberg e t a l . , 1971). I t has been su g g ested

th e re fo re t h a t m e ta llo th io n e in may fu n c tio n in th e t r a n s p o r t o f cadmium

(N ordberg , 1978). Any d is c u s s io n o f th e m etabolism and k in e t ic s o f th e

m eta l n e c e s s i ta te s a c o n s id e ra t io n o f th e s y n th e s is , p ro p e r t ie s and

fu n c tio n o f m e ta l lo th io n e in .

P ro p e r t ie s o f M e ta llo th io n e in

The m e ta llo th io n e in s a re a group o f low m o lecu lar w e ig h t, in d u c ib le

cy top lasm ic p ro te in s w ith a h ig h b in d in g a f f i n i t y fo r c e r t a in m e ta ls ,

p a r t i c u l a r ly cadmium. A cadmium and z in c c o n ta in in g p ro te in was f i r s t

i s o la te d from equine r e n a l c o r te x by K&gi and V a llee (1 9 6 0 ), and sub­

sequen t c h a r a c te r i s a t io n showed th e m e ta l lo p ro te in to have a h ig h c y s te in e

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co n ten t (30%), an absence o f a ro m atic amino a c id s and a m o lecu lar w eigh t

o f around 10,000 (K agi and V a lle e , 1961). P u r i f i c a t io n o f th e p ro te in

re v e a le d a m eta l c o n ten t o f 5.9% cadmium, 2.2% z in c and 0.1% copper.

A more d e ta i le d c h a r a c te r i s a t io n o f r a b b i t l i v e r m e ta llo th io n e in showed

th a t th e p ro te in cou ld be se p a ra te d in to two components c o n ta in in g d i f ­

f e r e n t amounts o f th e v a rio u s c a t io n s (N ordberg e t a l . , 1972).

M e ta llo th io n e in s have s in c e been found in n e a r ly a l l t i s s u e s , and cadmium-

c o n ta in in g th io n e in s have been id e n t i f i e d in th e l i v e r and k idney o f

v a rio u s anim al sp e c ie s (F r ib e rg e t a l . , 1974), in c lu d in g man

(B uh ler and K agi, 1974). C adm ium -thionein may a lso complex w ith z in c in

th e l i v e r and copper in th e k idney (Webb, 1975), though b o th z in c and

copper a re them selves cap ab le o f in duc ing th e sy n th e s is o f th io n e in s

(Bremner and M arsh a ll, 1974; Bremner and D av ies, 1975).

The mechanisms by which cadmium s t im u la te s th e s y n th e s is o f th io n e in

have been in v e s t ig a te d and in r a t l i v e r th e sy n th e s is has been shown to

be c o n tro l le d a t th e t r a n s l a t i o n a l le v e l (Webb, 1972). However, o th e r

w orkers su g g est th a t cadmium re g u la te s th e b io s y n th e s is o f m e ta llo ­

th io n e in in th e l i v e r a t th e t r a n s c r ip t io n a l l e v e l , and in th e k idney

a t th e t r a n s l a t i o n a l le v e l (S haikh and Sm ith , 1977). The d e g ra d a tio n

o f m e ta llo th io n e in s has a ls o been in v e s t ig a te d s in c e t h i s may have a

r o le in th e r e g u la t io n o f th e i n t r a c e l l u l a r c o n c e n tra tio n o f th e p ro te in .

H epatic and re n a l cadm ium -thioneins were shown to have h a l f - l i v e s o f

3 .5 and 3 .7 days r e s p e c t iv e ly , though th e re i s v i r t u a l l y no tu rn o v e r o f

th e m eta l c o n ten t o f th e se t i s s u e s . This in d ic a te s a r e g u la r m etabolism

o f th e p ro te in m oiety and a c o n tin u a l re -b in d in g o f th e cadm ium -cation

by new p ro te in (Feldman e t a l . , 1978a).

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D is t r ib u t io n

D uring c le a ra n c e from th e b lo o d , cadmium shows d i s t i n c t t i s s u e

s p e c i f i c i t y in i t s up take which may be p a r t ly r e la te d to th e a b i l i t y o f

th e organ to s y n th e s is e m e ta l lo th io n e in . The t i s s u e d i s t r i b u t i o n o f a

s in g le o ra l dose o f cadmium has been s tu d ie d in th e r a t (D ecker e t a l . ,

1957), and goat (M ille r e t a l . , 1969). In bo th cases th e l a r g e s t amount

o f cadmium accum ulated in th e l i v e r , w h ile th e h ig h e s t c o n c e n tra tio n was

reached in th e k idney . A pproxim ately 50% o f th e absorbed dose was

r e ta in e d in th e se two o rg an s. T issu e le v e ls o f th e m eta l q u ic k ly reached

a maximum and th e n s low ly d ecreased w ith tim e . A fte r th e in je c t io n o f

cadmium, th e d i s t r i b u t io n fo llo w s a s im i la r p a t te r n though i n i t i a l l y th e

m eta l may p r e f e r e n t i a l l y accum ulate in th e l i v e r (Webb, 1975b).

S u b seq u en tly , l i v e r le v e l s o f th e m eta l may d ec rea se slow ly w ith tim e

w h ile th e c o n c e n tra tio n in th e k idney in c re a se s (H orner and Sm ith , 1975),

i . e . th e p e rcen tag e o f th e body burden in c re a se s in th e k idney .

W ith re p e a te d exposure to cadmium, th e m eta l ag a in accum ulates

m ainly in l i v e r and k idney . In r a t s th e accum ulation has been shown to

occur in a d o s e - re la te d manner a f t e r o ra l a d m in is tra t io n , b u t w ith

h ig h e r doses o f cadmium, th e r a t i o o f l iv e r :k id n e y cadmium in c re a se s

(D ecker jet a l . , 1958; Sugawara and Sugawara, 1974a). In o th e r s tu d ie s

th e l i v e r c o n c e n tra tio n tended to p la te a u w h ile th e c o n c e n tra tio n in th e

k idney co n tinued to in c re a s e (S to n ard and Webb, 1976). S ig n i f ic a n t

amounts o f th e m etal have been d e te c te d in many o th e r t i s s u e s in c lu d in g

p an c rea s , sp le e n , bone, lu n g , h e a r t , stom ach, e tc .

Cadmium d is p o s i t io n may be a f fe c te d by s e v e ra l f a c to r s in c lu d in g

s ex , age and dose. Female mice r e ta in e d more m etal in s o f t t i s s u e s a f t e r

■ sequential in je c t io n s o f ^°^Cd, b u t r e ta in e d le s s o f th e m eta l in th e

s k e le to n compared to m ales (M atsubara-K han and M achida, 1975).

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K ello and K o s t ia l (1977) have shown th a t young r a t s r e t a i n more o f a

s in g le in je c te d dose o f ^ ^ C d in th e body even though l i v e r , k idney

and blood c o n c e n tra tio n s o f cadmium were low er. F in a l ly , th e r a t e o f

accum ulation o f cadmium in th e l i v e r and k idney o f r a t s ad m in is te red

d ie ta r y cadmium was in f lu e n c e d by th e degree o f exposure (B ran cato e t a l . ,

1976).

The l im ite d d a ta fo r human s u b je c ts a ls o in d ic a te s th a t cadmium

^accum ulates p redom inantly in th e l i v e r and k idneys (S y v ersen , 1975;

Gross e t a l . , 1976).

E x c re tio n

Cadmium i s e x c re te d m ainly v ia u r in e and th e a lim e n ta ry t r a c t .

In ex p erim en ta l an im a ls , a f t e r a s in g le dose o f th e m e ta l, th e fa e ce s

seem to be th e predom inant ro u te o f e l im in a tio n . There i s an i n i t i a l

more ra p id phase o f e x c re t io n fo llow ed by a d e c rea s in g r a t e o f e l im in a tio n

w ith . tim e . Horner and Sm ith (1975) showed th a t in r a t s , more th a n 25% o f

109an in tra v e n o u s ly in je c te d dose o f Cd was e x c re te d w ith in 60 days v ia

faeces compared to 0.15% in th e u r in e over th e same tim e p e rio d .

S im ila r ly , Decker e t a l . (1957) measured a 25% e lim in a tio n o f an i n t r a ­

venously in je c te d dose o f cadmium in r a t s by 72 h o u rs , w ith l i t t l e o r no

d e te c ta b le cadmium in th e u r in e . L ucis e t a l . (1 9 6 9 ), a lso u s in g r a t s ,

found sm all amounts o f cadmium in th e u r in e a f t e r p a re n te r a l dosing b u t

p r im a r i ly th e m eta l was e x c re ted in th e f a e c e s . In g o a ts , M il le r e t a l .

(1968) found more th a n 5% o f an in je c te d dose o f cadmium e x c re te d w ith in

5 days v ia th e fa e c e s . The ro u te o f a d m in is tra t io n does n o t appear to

in f lu e n c e s ig n i f i c a n t ly th e r a t e o f e lim in a tio n o f th e m eta l

(Moore e t a l . , 1973a). The m agnitude o f exposu re , however, does have

an e f f e c t s in c e th e re i s ev idence th a t f a e c a l e x c re tio n o f cadmium i s

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d o s e - re la te d (F r ib e rg e t a l . , 1974). U rin ary cadmium e x c re t io n , though

rem ain ing low th roughou t re p e a te d exposu re , may in c re a se w ith th e d u ra ­

t i o n o f exposure and may be d o s e - re la te d (C .E .C ., 1978). The u r in a ry

c o n c e n tra tio n o f cadmium i s s ig n i f i c a n t ly a f fe c te d by th e fu n c tio n a l

s ta tu s o f th e k idney . P a th o lo g ic a l change in r e n a l t i s s u e caused by

cadmium produces a d ram atic in c re a s e in th e u r in a ry e x c re t io n o f th e m eta l

(N ordberg and P is c a to r , 1972), though t h i s cou ld perhaps be reg a rd ed as

a lo s s o f m eta l r a th e r th a n a means o f e l im in a tio n .

In humans, cadmium has a lso been shown to be e lim in a te d v ia fa e ce s

and u r in e , though some re g a rd t h a t u r in a ry e x c re tio n i s more im p o rtan t

th a n th e f a e c a l ro u te (C .E .C ., 1978). C e r ta in ly u r in a ry cadmium le v e l s

appear h ig h e r in people exposed to th e m eta l bu t t h i s w i l l be d isc u sse d

in a l a t e r s e c t io n in r e l a t i o n to th e d ia g n o s is o f cadmium p o iso n in g .

There i s a d i f f i c u l t y in human s tu d ie s in d is t in g u is h in g betw een unabsorbed

cadmium and e x c re te d m eta l in th e fa e c e s .

G a s t r o - in te s t i n a l e x c re t io n o f cadmium could in v o lv e a number o f

ro u te s in c lu d in g th e b i l e , mucosa o f th e gut and g la n d u la r s e c re t io n s such

as s a l iv a and p a n c re a tic ju ic e . The c e l l s o f th e in t e s t i n e have d i f f e r e n t

e x c re to ry c a p a c i t ie s depending upon th e lo c a t io n . Cadmium r a p id ly accu­

m ulates in th e w a ll o f th e stomach o f r a t s ad m in is te red th e m eta l

p a r e n te r a l ly (L ucis e t a l . , 1969) though on ly t r a c e s were d e te c te d in th e

stomach c o n te n ts in d ic a t in g a l im ite d c a p a c ity fo r e l im in a tio n . The m eta l

r a p id ly accum ulates in th e sm all i n t e s t i n e and i t s c o n te n ts , and a ls o to

a c o n s id e ra b le e x te n t in th e la rg e i n t e s t i n e . The appearance o f cadmium

in th e lumen o f th e in t e s t i n e may be th e r e s u l t o f v a rio u s g a s tr o ­

i n t e s t i n a l s e c r e t io n s .

A fte r in trav en o u s a d m in is tra t io n o f cadmium to r a t s , C ik rt and T ichy

(1974) found more th a n 5% o f th e dose was e x c re te d in th e g a s t r o - i n t e s t i n a l

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t r a c t . , though le s s was e x c re te d in th e b i l e th a n v ia th e w a ll o f th e g u t.

The h ig h e s t b i l i a r y e x c re t io n r a t e o f cadmium o ccu rred w ith in 30 min o f

a d m in is tra t io n . K laassen and K otsonis (1977) found marked sp e c ie s v a r i a ­

t i o n in th e a b i l i t y to e x c re te th e m eta l in th e b i l e and th e r a t e o f

e l im in a tio n appeared to -b e d o s e - re la te d . Cadmium ex c re ted in to th e b i l e

has been shown to be a tta c h e d to a low m olecu lar w eight compound, p a r­

t i a l l y c h a ra c te r is e d as g lu ta th io n e (C h erian and V o s ta l , 1977). No cadmium

was found to be bound to h ig h e r m o lecu lar w eight p ro te in s o r m e ta llo ­

th io n e in . F u r th e r work by Nordberg et_ a l. (1977) has a lso dem onstrated

th a t th e b i l i a r y e x c re t io n o f cadmium re p re s e n ts a minor p ro p o r tio n o f th e

f a e c a l c o n ten t o f cadmium, and a lso a r e l a t i v e l y in s ig n i f ic a n t r o le fo r

p a n c re a tic s e c r e t io n s . I t ap p ea rs , th e r e f o r e , th a t tu rn o v e r , shedding and

s e c re t io n s o f th e e p ith e liu m o f th e i n t e s t i n a l mucosa i s la r g e ly re sp o n ­

s ib l e fo r th e cadmium c o n te n t o f fa e c e s .

V arious o th e r ro u te s may c o n tr ib u te to cadmium e x c re t io n , though by

in s ig n i f ic a n t amounts. M easurable q u a n t i t ie s o f th e m etal have been

d e te c te d in s a l iv a (D re izen e t a l . , 1970), and m ilk (M ille r e t a l . , 1967).

H igher c o n c e n tra tio n s o f cadmium have been found in h a i r , b o th in e x p e r i­

m ental anim als (N ordberg and N ishiyam a, 1972) and humans (S ch ro ed e r and

Nason, 1969).

The m etabolism o f cadmium is summarised in F ig u re 1 .1 .

M e ta llo th io n e in s in R e la t io n to Cadmium M etabolism

The b io lo g ic a l r o le o f th e m e ta llo th io n e in s i s u n c le a r . I t has been

e s ta b l is h e d th a t th e p r o te in p lays an im portan t r e g u la t in g r o le in th e

m etabolism o f z in c , p a r t i c u la r ly in th e c o n tro l o f i n t e s t i n a l a b so rp tio n

and h e p a tic uptake o f th e m eta l (Bremner and D av ies, 1975; R ichards and

C ousins, 1975a, 1976a), and s im i la r r o le s have been proposed in th e

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Figu

re

1.1

THE

MET

ABO

LISM

OF

C

AD

MIU

M

Dis

trib

utio

n

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m etabolism o f copper (E vans, 1973; Bremner and M arsh a ll, 1974). The

p resence o f cadm ium -thionein in i n t e s t i n a l mucosa and th e organs where

th e m etal i s s to re d would a ls o in d ic a te a r o le fo r th e p ro te in in th e

up take and s to ra g e o f cadmium. The k in e t ic s of th e d is p o s i t io n o f cadmium

may a ls o be in flu en c ed by i t s a b i l i t y to b ind to o th e r p e p tid e s in c lu d in g

g lu ta th io n e ( P e r r in and W att, 1971) and h igh m o lecu lar w eight macro­

m olecules ( F r a z ie r and P u g le se , 1978).

The p ro cess o f cadmium uptake and accum ulation has been in v e s t ig a te d

in more d e t a i l . The i n i t i a l up take o f th e m eta l in to male r a t h ep a to cy te s

i s com plete w ith in one hour fo llo w in g a b so rp tio n and b in d in g i s predom i­

n a n tly to th e h ig h m o lecu lar w eight p ro te in s a t t h i s s ta g e , as d e sc r ib e d

by F r a z ie r and P uglese (1 9 7 8 ), w h ile th e re i s a la g phase o f 3 - 4 hours

b e fo re th e o n se t o f m e ta l lo th io n e in s y n th e s is in th e l i v e r (Cempel and

Webb, 1976). Once induced , th io n e in sy n th e s is co n tin u es and th e cadmium

c a t io n i s t r a n s f e r r e d from th e h ig h m o lecu lar w eight p ro te in s to th e

a p o p ro te in . The h e p a tic c o n ten t o f b o th m eta l and th io n e in th e r e f o r e

in c re a s e s . Cadmium a lso r a p id ly accum ulates in th e k idney , a lth o u g h th e

sy n th e s is o f r e n a l - th io n e in i s not induced w ith in 48 h o u rs .

I t has been su g g ested th a t cadm ium -th ionein may be tr a n s p o r te d in

th e blood from th e l i v e r to th e k idney ( P is c a to r , 1964) where th e p r o te in -

bound m etal i s f i l t e r e d th rough th e glom erulus and alm ost co m p le te ly

reab so rb ed th ro u g h th e proxim al tu b u le s (C h erian and S ha ikh , 1975;

Nomiyama and F o u lk es , 1977). Thus u r in a ry e x c re t io n o f th e m eta l i s

ex trem ely low. W ith in th e k idney , cadmium i s d e p o s ited m ain ly in th e

c o r te x and th e c o n c e n tra tio n o f th e m eta l h e re i s about 50% h ig h e r th a n

whole k idney (F r ib e rg e t a l . , 1974).

The tim e -c o u rse o f s y n th e s is o f m e ta l lo th io n e in i s a lso im p o rtan t

fo r th e b i l i a r y e x c re tio n o f cadmium. The i n i t i a l h igh r a t e o f b i l i a r y

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cadmium e x c re t io n occurs b e fo re th io n e in sy n th e s is i s m axim ally induced .

Once adequate amounts o f th e p ro te in a re produced, th e c o n c e n tra tio n o f

cadmium in th e b i l e d e c re a se s . P re - tre a tm e n t w ith cadmium, which induces

th io n e in s y n th e s is , causes a d ec rea se in th e e lim in a tio n o f cadmium v ia

b i l e when th e m eta l i s su b seq u en tly ad m in is te red (C h erian , 1977a).

E stim a tes o f th e b io lo g ic a l h a l f - l i f e o f cadmium range from 200 days

in r a t s to 2 y ears in monkeys (Webb, 1975c), w h ile in man i t may be as

lo n g as 20 - 40 y ears (F r ib e rg a t a l . , 1974).

1 .3 THE TOXICOLOGY OF CADMIUM

M e ta llo th io n e in in R e la tio n to Cadmium T o x ic ity

In a d d it io n to i t s in f lu e n c e on th e k in e t ic s o f th e d i s t r i b u t i o n o f

th e m e ta l, th e b in d in g o f cadmium by m e ta llo th io n e in can m odify th e

developm ent o f th e to x ic re sp o n se . M e ta llo th io n e in may be in v o lv ed in th e

p ro te c t io n a g a in s t cadmium to x ic i t y b u t has a lso been im p lic a te d as th e!

to x ic ag en t, j I t s r o le th e re fo re appears ob scu re .

C e r ta in ly , m e ta llo th io n e in can be co n sid e red as f u l f i l l i n g a p ro te c ­

t i v e r o le in th e acu te t o x i c i t y o f cadmium, s in c e in th e p re sen ce o f t h i o ­

n e in , th e c a t io n i s e f f e c t iv e ly se q u e s te re d and th e re fo re n o t a v a i la b le to

i n t e r a c t w ith c e l l u l a r c o n s t i tu e n ts . The p re - tre a tm e n t o f an im als w ith a

low dose o f cadmium p ro te c ts a g a in s t a subsequent norm ally to x ic dose o f

th e m e ta l, because once induced , th e sy n th e s is o f th io n e in may proceed

w ith o u t a la g phase upon f u r th e r exposure to cadmium (T erh aa r e t a l . , 1965;

Leber and Miya, 1976; Webb and V ersch o y le , 1976). The a cu te to x ic r e a c t io n

to cadmium, e .g . in th e t e s t i s , may th e re fo re develop due to in te r f e r e n c e

o f th e m etal w ith s e n s i t iv e c e l l u l a r t a r g e t s in th e absence o f a s u i ta b le

s e q u e s te r in g a g en t.

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A fte r re p e a te d exposure to cadmium however, th e r o le o f m e ta llo ­

th io n e in may be co n sid e red d i f f e r e n t l y , and can be r e la te d to th e concept

o f c r i t i c a l c o n c e n tra tio n s d iscu ssed by Nordberg (1976). The developm ent

o f a c r i t i c a l e f f e c t in a c r i t i c a l o rgan re q u ire s th e accum ulation o f

c r i t i c a l c o n c e n tra t io n s ' o f th e m etal w ith in th a t t i s s u e ( th e c r i t i c a l

o rgan being d e fin e d as th e organ t h a t f i r s t a t t a in s th e c r i t i c a l concen­

t r a t i o n o f m e ta l, i . e . th e c o n c e n tra tio n a t which u n d e s ira b le fu n c tio n a l

change o c c u rs ) . The b in d in g o f cadmium by th io n e in allow s s to ra g e o f th e

m eta l in a n o n -to x ic form b u t a t th e same tim e f a c i l i t a t e s r e t e n t io n o f

th e m etal and th e accum ulation o f c r i t i c a l c o n c e n tra tio n s , i . e . a s ta g e

i s reached when a l l a v a i la b le cadm ium -binding s i t e s on m e ta l lo th io n e in a re

s a tu r a te d and th io n e in s y n th e s is i s in s u f f i c i e n t to a d eq u a te ly s e q u e s te r

th e c a t io n upon f u r th e r exposure .

The up take o f cadmium in th e l i v e r by th e h ig h m o lecu lar w eigh t p ro ­

t e in s and th e subsequent in c o rp o ra tio n in to m e ta llo th io n e in have d o se-

dependent k in e t i c s , and th e s e two d i s t i n c t p ro cesses may be s a tu r a te d

( F r a z ie r and P u g le se , 1978). S im ila r ly , C o lucci e t a l . (1975) and

K otson is and K laassen (1977) have r e l a t e d th e developm ent o f t o x i c i t y to ,

th e b in d in g c a p a c ity o f m e ta l lo th io n e in and have found th a t t h i s i s th e

c r i t i c a l d e te rm in in g f a c to r in p roducing c e l l u l a r damage. The m an ifes­

t a t i o n o f t o x i c i t y fo llo w in g s a tu r a t io n o f th e b in d in g c a p a c ity o f m e ta llo ­

th io n e in would su g g est t h a t th e c a t io n i s th e c a u s a tiv e agen t o f t o x i c i t y ,

bu t th e re i s ev idence th a t th e cadm ium -th ionein complex i t s e l f has th e

p o te n t ia l to produce c e l l damage. The movement o f cadm ium -th ionein from

mucosal c e l l s in to th e lumen o f th e i n t e s t i n e has been shown to produce

s ig n i f i c a n t u l t r a s t r u c t u r a l change in mice (V alberg e t a l . , 197 7 ), and

s im i la r ly th e uptake o f th e cadm ium -thionein complex by th e proxim al tu b u le s

o f th e k idney can cause sev e re n e p h ro to x ic ity (C h e rian e t a l . , 1976).

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Webb and E tien n e (1977) have dem onstrated t h a t cadmium bound to th io n e in

was 7 to 8 tim es more to x ic th a n io n ic cadmium. However, re c e n t work by

Suzuki e t a l . (1979) has su g g ested th a t th e n e p h ro to x ic ity o f m e ta llo ­

th io n e in was dependent upon th e c o n te n t o f cadmium in th e p r o te in , i . e . th e

e x te n t o f n e c ro s is in c re a se d w ith in c re a s in g c a t io n c o n te n t.

D esp ite th e se a p p a re n tly c o n tra d ic to ry v iew s, i t i s e v id e n t th a t th e

s y n th e s is o f m e ta l lo th io n e in p lays an im p o rtan t r o le in th e developm ent o f

th e cadmium-induced to x ic re sp o n se .

S i t e o f Exposure

The developm ent o f sy stem ic t o x i c i t y to cadmium i s dependent to an

e x te n t upon th e ro u te o f exposure and subsequent up take in to o rg a n s , b u t

to x ic changes may a lso occur a t th e s i t e s o f exposu re , p a r t i c u l a r ly a f t e r

a cu te exposure.

M orphological and b iochem ica l changes a re ap p aren t in th e lungs o f

r a t s exposed to a s in g le tre a tm e n t o f cadmium a e ro s o l . Palm er e t a l . (1975)

and Hayes e t a l . (1976) observed c e l l u l a r p r o l i f e r a t i o n and an a s s o c ia te d

in c re a se d c o n ten t o f l i p i d and DNA. V arious enzymes such as l a c t a t e

dehydrogenase and g lu co se -6 -p h o sp h a te dehydrogenase showed e le v a te d

a c t i v i t i e s . H is to lo g ic a l changes in c lu d ed a lv e o la r sw e llin g and d is te n s io n

of m ito ch o n d ria . S im ila r r e s u l t s were re p o r te d by Bus e t a l . (1978) who

d e sc rib e d decreased lung fu n c tio n , pulmonary oedema and i n t e r s t i t i a l

pneum onitis . A fte r re p e a te d in h a la t iv e exposure in r a t s , S n id e r e t a l .

(1973) observed emphysema l ik e changes and a cu te v a s c u la r c o n g e s tio n .

P rigge (1978) a lso re p o r te d h y p e rp la s ia and im paired lung fu n c tio n (d e c re a se d

gas exchange).

A fte r re p e a te d o r a l a d m in is tra t io n o f cadmium to r a t s (100 p .p .m . fo r

30 d a y s ) , Sugawara and Sugawara (1977) re p o r te d a s ig n i f ic a n t in c re a s e in

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th e m ucosal w eight o f th e duodenum and a lso observed th e lo c a l i s a t i o n o f

cadm ium -thionein in t h i s t i s s u e . There was a cadmium-induced a l t e r a t i o n

o f v a r io u s enzymes in th e duodenum. The in g e s t io n o f cadmium has a lso been

a s s o c ia te d w ith m orpho log ica l changes o f th e i n t e s t i n e in r a t s , in d ic a t iv e

o f an in c re a se d c e l l tu rn o v e r (Sugawara and Sugawara, 1974b). P a th o lo g ic a l

changes in th e i n t e s t i n e have a lso been d e sc rib e d in Japanese Q uail

(R ichardson e t a l . , 1974) and damage to mouse i n t e s t i n e was induced by th e

m eta l in v i t r o (V alberg e t a l . , 1977).

L iv e r

The l i v e r accum ulates la rg e amounts o f cadmium a f t e r bo th a cu te and

long term exposure to cadmium. H epatic c e l l u l a r d e g e n e ra tio n , and d i l a t i o n

and d e g ra n u la tio n o f smooth endoplasm ic re tic u lu m have been observed a f t e r

th e re p e a te d p a re n te r a l a d m in is tr a t io n o f v a r io u s doses o f cadmium to r a t s

(C o lu cc i e t a l . , 1975j F aeder elt a l . , 1977). S im ila r f in d in g s have b een '

re p o r te d a f t e r con tinuous o r a l a d m in is tra t io n o f th e m eta l to r a b b i t s

(160 p .p .m . f o r 200 days) (Stowe e t a l . , 1972). Faeder e t a l . (1977) demon­

s t r a t e d th a t th e se m orpho log ica l changes were accompanied by an e le v a t io n

o f c e r t a in plasm a enzymes which a re n o rm ally in d ic a t iv e o f l i v e r damage.

An enhancement o f g luconeogenesis in r a t s r e p e a te d ly in je c te d w ith cadmium

(1 mg/kg f o r 45 days) has a lso been shown, s in c e th e re were s ig n i f i c a n t

in c re a s e s in th e enzyme a c t i v i t i e s a s s o c ia te d w ith th e p ro c e ss , and a d e p le ­

t i o n o f h e p a tic g lycogen (S in g h a l e t a l . , 1974).

P o te n t ia t io n o f drug re sp o n se due to an in h ib i t i o n o f h e p a t ic drug

m e ta b o lis in g a c t i v i t y by th e a cu te a d m in is tra t io n o f cadmium has been

re p o r te d in mice and r a t s . A decreased m icrosom al c o n ten t o f cytochrom es

P-450 and b5 , to g e th e r w ith s ig n i f i c a n t ly decreased a c t i v i t i e s o f am ino-

p y rin e dem ethy lase , a n i l in e h y d ro x y lase , n i t r o re d u c ta s e and 0 -dem ethy lase

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have been observed (Unger and C lausen , 1973; Hadley e t a l . , 1974;

Jo h n sto n e t a l . , 1975; K rasny and H olbrook, 1977; T eare e t a l . , 1977).

A s e x - r e la te d d if f e r e n c e seems to e x i s t in th e a b i l i t y o f cadmium to a l t e r

drug a c t io n in th e r a t s in c e fem ales a re l e s s s u s c e p t ib le to th e e f f e c t

(S c h n e ll e t a l . , 1976; Pence e t a l . , 1977).

However, t h i s in h ib i to r y e f f e c t has n o t been no ted a f t e r o r a l t r e a t ­

ment w ith cadmium. Using r a t s , W agstaff (1973) and H ie tanen (1978) bo th

no ted a tendency tow ards s t im u la t io n o f h e p a tic m icrosom al enzyme a c t i v i t i e s

b u t th e le v e ls o f d ie ta r y cadmium were h ig h (up to 10,000 p .p .m ., and

250 p .p .m . r e s p e c t iv e ly ) .

Kidney

The k idney i s reg a rd ed as th e c r i t i c a l organ a f t e r long te rm exposure

to cadmium a lth o u g h c o n s id e ra b le c o n c e n tra tio n s o f th e m eta l accum ulate

a f t e r acu te tre a tm e n t a ls o . There i s l i t t l e ev idence th a t a cu te adm in is­

t r a t i o n o f cadmium s a l t s causes s ig n i f i c a n t r e n a l damage. When th e m etal

i s complexed w ith th io l-com pounds, how ever, r e t e n t io n in the- k idney i s p ro ­

moted and t o x i c i t y may be induced . A s in g le dose o f a m ix tu re o f cadmium

c h lo r id e (10 ymol/kg) and m ercap to e th an o l caused in te r f e re n c e w ith bo th

g lo m eru lar and proxim al tu b u le fu n c tio n in r a b b i t s (G ieske and F o u lk e s ,

1974). A d m in is tra tio n o f a cadm ium -th ionein complex a ls o produces morpho­

lo g ic a l changes in th e proxim al tu b u le s (C h e rian e t a l . , 1976).

A fte r re p e a te d p a re n te r a l a d m in is tra t io n o f cadmium to r a b b i t s

(0 .2 5 m g/kg/day f o r v a rio u s t im e s ) , h i s to lo g ic a l and b iochem ical changes

have been produced in th e k idney (A xelsson and P is c a to r , 1966). Cadmium

was d e p o s ited m ain ly in th e r e n a l c o r te x and d e g e n e ra tiv e changes were con­

f in e d to th e proxim al tu b u le s , b u t w ith in c re a se d exposure tim e , p a th o ­

lo g ic a l changes were ev id en t in o th e r r e g io n s . Renal d y s fu n c tio n was

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c h a r a c te r is e d by a p r o te in u r ia , p redom inan tly o f a and g g lo b u l in s , and

was a s s o c ia te d w ith an in c re a se d u r in a ry e x c re tio n o f cadmium, g lucose

and amino a c id s .

Stowe e t a l . (1972) have a lso in v e s t ig a te d th e r e n a l e f f e c t s o f

cadmium in r a b b i t s b u t a f t e r long te rm , o ra l tre a tm e n t (160 p .p .m . fo r

200 d a y s) , th e k idneys o f cadmium-exposed anim als e x h ib ite d p a th o lo g ic a l

a l t e r a t io n s in c lu d in g i n t e r s t i t i a l f i b r o s i s and n e c r o s is , and th e re was

a lso ev idence o f c o lla g e n d e p o s it io n in th e g lom eru lus. Cadmium-induced

b iochem ical changes in th e k idney have a lso been observed . In swine

r e c e iv in g d ie ta r y cadmium (0 - 1350 p .p .m . f o r 42 d a y s ) , a d ecreased

a c t i v i t y o f r e n a l le u c in e am inopeptidase was observed (C ousins e t a l . ,

1973), and in r a t s , a f t e r p a re n te r a l tre a tm e n t (1 mg/kg fo r 45 d a y s) ,

enhanced a c t i v i t i e s o f th e r e n a l g luconeogenic enzymes have been re p o r te d

and were accompanied by p ro te in u r ia and g lu c o su r ia (S in g h a l e t a l . , 1974).

Such changes may p e r s i s t even a f t e r exposure to cadmium has ceased and

th e re fo re su p p o rts th e view th a t cadmium-induced changes in th e k idney

a re i r r e v e r s i b l e .

Nomiyama e t a l . (1973,1975) have in v e s t ig a te d re n a l fu n c tio n a f t e r ,

b o th p a re n te r a l (1 .5 - 15 mg/kg d a i ly f o r v a rio u s tim es) and o r a l

(300 p .p .m . f o r 54 weeks) tre a tm e n t w ith cadmium. In bo th c a s e s , cadmium

induced in c re a s e s in u r in a ry enzymes, amino a c id s , p ro te in s and g lu c o se ,

in d ic a t in g c e l l u l a r damage. Changes in r e n a l fu n c tio n a f t e r cadmium

in je c t io n s ( e .g . decreased in u l in c le a ra n c e , and maximum tu b u la r c a p a c ity

f o r s e c re t in g p -am inoh ippura te ) su g g ested d is tu rb a n c e s o f b o th g lo m eru la r

and tu b u la r fu n c tio n . O ther s tu d ie s have re p o r te d s im ila r f in d in g s in

r a t s a f t e r o ra l cadmium a d m in is tra t io n (K otson is and K laassen , 1978) and

a f t e r p a re n te r a l a d m in is tra t io n a mixed type p r o te in u r ia was confirm ed

(B ernard e t a l . , 1978).

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I t a p p ea rs , th e r e f o r e , th a t cadmium-induced r e n a l damage i s mainly-

co n fin ed to th e proxim al tu b u le s b u t a f t e r p a re n te r a l tre a tm e n t, th e re

i s ev idence o f g lom eru lar damage a ls o .

Bone

Cadmium does no t accum ulate to any c o n s id e ra b le e x te n t in osseous

t i s s u e so an e f f e c t o f th e m eta l on th e bone i s g e n e ra lly co n sid e re d to

be secondary to d is tu rb a n c e s o f calc ium and phosphate m etabolism caused

by th e cadmium-induced r e n a l damage, and such e f f e c t s p robab ly th e re fo re

r e q u ire a long tim e to develop .

An a cu te and su b acu te e f f e c t o f cadmium on th e bone h a s , however,

been re p o r te d in r a t s . F u ru ta (1978) ad m in is te red cadmium c h lo r id e by

subcutaneous in je c t io n a t a s in g le dose o f 11 .2 mg/kg, o r re p e a te d ly a t

1 .7 m g/kg/day f o r 21 d ay s, and observed th in n in g o f th e ep ip h y sea l

c a r t i l a g e , th e e f f e c t b e in g more sev e re a f t e r re p e a te d d o s in g . In c re a se d

numbers o f o s te o c la s t s , and o s te o p o ro tic changes were n o ted .

Itokaw a e t a l . (1973) a d m in is te red d ie ta r y cadmium to r a t s (200 p .p .m

f o r r e l a t i v e l y s h o r t p e rio d s (30 days) and observed some changes in th e

m eta l c o n ten t o f bone, such as a d ecreased c o n c e n tra tio n o f z in c and

magnesium. There a re a ls o r e p o r ts o f h i s to lo g ic a l changes in th e bone,

e s p e c ia l ly th in n in g o f th e ep ip h y sea l c a r t i l a g e and c o r t i c a l t i s s u e ,

a f t e r th e a d m in is tra t io n o f d ie ta r y cadmium (Itokaw a e t a l . , 1974, 1978;

Takashima e t a l . , 1978). These changes were accompanied by p a th o lo g ic a l

changes in th e k id n ey s. Y oshik i e t a l . (1975) have dem onstrated th e

p resence o f o s te o p o ro tic changes in th e bones b e fo re th e developm ent o f

r e n a l damage, in r a t s fed d ie ta r y cadmium (10 - 300 p .p .m . f o r 12 w eeks).

O ther w orkers have shown an e f f e c t o f cadmium upon v a r io u s a sp e c ts

o f calc ium m etabolism , e .g . Kawamura e t a l . (1978). Ando e t a l . (1977 ,

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1978) u sin g r a t s dosed o r a l l y w ith cadmium (10 mg/kg d a i ly f o r up to

24 weeks) observed a d ecreased i n t e s t i n a l a b so rp tio n and bone up take o f

ca lc iu m , w h ile th e h a l f - l i f e o f calc ium d ecreased and i t s e x c re t io n was

enhanced. The decreased calcium a b so rp tio n in r a t s has been r e la t e d to

a d ecreased b in d in g a c t i v i t y o f th e ca lc iu m -b in d in g p ro te in in th e duo­

denum and k idney (Sugaw ara, 1977). In v i t r o s tu d ie s have p rov ided s im i la r

ev idence f o r cad m iu m -in te rfe ren ce in th e up take and m etabolism o f calc ium

(G ruden, 1977; H am ilton and Sm ith , 1978; Yuhas e t a l . , 1978). I t i s w e ll

documented th a t c a lc iu m -d e fic ie n c y s ig n i f i c a n t ly enhances th e cadmium-

induced changes in bone and calcium hom eostasis ( e .g . L arsson and P is c a to r ,

1971; Itokaw a e t a l . , 1978).

Feldman and Cousins (1973) have dem onstrated an in h ib i t i o n by cadmium

of th e r e n a l h y d ro x y la tio n o f 2 5 -h y d ro x y c h o le c a lc ife ro l in c h ic k s , in v i t r o ,

and s im i la r f in d in g s have been re p o r te d in v iv o in r a t s exposed to cadmium

in d r in k in g w a te r (L o ren tzo n and L a rsso n , 1977). A cadmium-induced d i s ­

tu rb an ce in v ita m in D m etabolism may th e re fo re be im p o rtan t.

T e s tis

The in d u c tio n o f t e s t i c u l a r damage in r a t s and mice by th e a cu te p a ren ­

t e r a l a d m in is tra t io n o f cadmium was d e sc rib e d by P a riz e k (1957)!. The e f f e c t . . _ i

i s r e s t r i c t e d m ainly to th o se sp e c ie s p o sse ss in g s c r o ta l t e s t e s . This

s e le c t iv e re sp o n se i s produced by cadmium a t doses o f ap p ro x im ate ly

0.012 mmol/kg (1 .3 5 mg/kg) (Gunn e t a l . , 1968a), and c o n s is ts o f haem orr-

h ag ic necrosis w ith in 48 h o u rs , fo llow ed by a tro p h y and perm anent s t e r i l i t y ,

B iochem ical changes such as a d ec rea se in th e t e s t i c u l a r c o n te n t o f DNA,

RNA and z in c -c o n ta in in g enzymes such as carb o n ic anhydrase have been

observed (Johnson and W alker, 1970; Webb, 1972). An a c tio n o f cadmium on

th e u t i l i s a t i o n o f z in c by sperm iogenic c e l l s has a lso been in d ic a te d as

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a p o s s ib le mechanism f o r th e to x ic e f f e c t (Lee and Dixon, 1973), w h ile

Chen e t a l . (1974) have su g g ested th a t th e most p la u s ib le t a r g e t o f cad­

mium i s an u n id e n t i f ie d cadm ium -binding p ro te in o f m olecu lar w eigh t

30 ,000 . There does no t appear to be a long term e f f e c t o f cadmium upon

th e t e s t i s .

C a rd io v asc u la r System

An a cu te e f f e c t o f cadmium upon th e c a rd io v a s c u la r system i s depen­

den t upon th e d o se , ro u te o f a d m in s tra tio n and s p e c ie s . Both in c re a s e s

and d ecrea se s in b lood p re s su re have been observed (P e rry and Y unice, 1965

P e rry e t a l . , 1970). The developm ent o f h y p e rte n s io n has been s tu d ie d

a f t e r long term exposure to cadmium. S chroeder and V inton (1962) induced

h y p e rte n s io n in r a t s w ith ch ro n ic a d m in is tra t io n o f low le v e l s o f d ie ta r y

cadmium (<5 p .p .m .) , b u t a t h ig h e r le v e l s o f cadmium (50 p .p .m .) , blood

p re s su re may be reduced (P e r ry and E r la n g e r , 1974). A b ip h a s ic a c t io n o f

cadmium i s th e re fo re p o s s ib le . O ther w orkers have produced h y p e r te n s io n

in r a b b i ts a f t e r re p e a te d p a re n te r a l a d m in is tra t io n o f cadmium (2 mg/kg)

and dem onstrated d ecreased v a s c u la r r e a c t i v i t y to a n g io te n s in in v i t r o

(F is h e r and T hind, 1971; T h ind , 1972). D is tu rb an ces in th e r e n in -

a ld o s te ro n e system have a lso been in d ic a te d (P e r ry and E r la n g e r , 1973),

and in h ib i t i o n o f th e enzymes re s p o n s ib le f o r th e d e a c t iv a t io n o f c a te c h o l

amines in th e a o r ta has a ls o been d e sc r ib e d (R e v is , 1978).

H aem atopoietic System

Anaemia i s a common f in d in g a f t e r cadm ium -treatm ent. R epeated

p a re n te r a l a d m in is tra t io n o f th e m eta l to r a b b i t s (1 mg/kg) produced an

in c re a se d d e s t r u c t io n o f e ry th ro c y te s , p a th o lo g ic a l changes in th e bone

marrow, and low ered in c o rp o ra tio n o f i ro n , which caused a m ic ro c y tic ,

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hypochromic ty p e o f anaemia (B e r l in and F r ib e rg , 1960; B e r l in e t a l . ,

1961). D ecreased h a em a to c rit and haem oglobin le v e ls have been commonly

re p o r te d a f t e r bo th a cu te o ra l dosing (K o tson is and K laassen, 1977) and

re p e a te d d ie ta r y a d m in is tra t io n o f cadmium ( e .g . Jacobs e t a l . , 1969;

Doyle e t a l . , 1974; P rig g e e t a l , , 1977). M orphological changes in th e

s p le e n o f r a t s have been re p o r te d a f t e r re p e a te d p a re n te r a l dosing

(P e r e t a l . , 1977).

M isce llaneous E f fe c ts

V arious o th e r to x ic m a n ife s ta tio n s o f cadmium tre a tm e n t have been

re p o r te d in ex p erim en ta l an im a ls . A p p reciab le amounts o f cadmium a lso

accum ulate in th e pancreas and p a re n te r a l a d m in is tra t io n o f th e m eta l to

mice (2 .0 - 6 .0 mg/kg) has been shown to produce hyperg lycaem ia and g lu ­

cose in to le ra n c e (G hafghazi and M ennear, 1973), a s s o c ia te d w ith a s ig ­

n i f i c a n t d e c rea se in c i r c u la t in g serum in s u l in . O ther s tu d ie s in r a t s

u s in g low er doses o f cadmium (0 .2 5 - 0 .5 mg/kg) f a i l e d to show s ig n i f i c a n t

changes in b lood g lucose and in s u l in , though g lucose m etabolism was

a f f e c te d , e .g . g ly c o ly s is and t r i c a r b o x y l ic a c id c y c le ( I th a k i s s io s e t a l . ,

1974).

Haem orrhagic le s io n s in sen so ry g a n g lia o f th e nervous system have

been re p o r te d a f t e r p a re n te r a l a d m in is tra t io n o f h ig h doses o f cadmium

to r a t s (2 .5 - 28 mg/kg) (G ab b ian i, 1966 ), and b eh av io u ra l e f f e c t s such

as dep ressed spontaneous locom otor a c t i v i t y have been observed in r a t s

fo llo w in g p re n a ta l exposure to th e m eta l (H astin g s e t a l . , 1978).

O ther e f f e c t s in c lu d e d is tu rb a n c e s in p ro te in and n u c le ic a c id

s y n th e s is , and in h ib i t io n o f RNA polym erase a c t i v i t y in r a t s a f t e r in je c te d

cadmium (H idalgo e t a l . , 1976; S to l l e t a l . , 1976). Cadmium exposure may

a ls o in f lu e n c e an tibody p ro d u c tio n and th e immune re sp o n se (Cook e t a l . ,

1975; R o lle r e t a l . , 1975).

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S ev era l in v e s t ig a to r s have dem onstrated th e developm ent o f sarcomas

a t th e in je c t io n s i t e a f t e r doses o f b o th cadmium m eta l (H eath e t a l . ,

1963) and m etal s a l t s (Roe e t a l . , 1964). Cadmium has a lso been im p li­

c a te d in m utagenic and te r a to g e n ic p ro cesses ( S h i r a is h i e t a l . , 1972;

B a rr , 1973).

H ealth E f fe c ts in Humans

In human b e in g s , th e main t a r g e t s o f cadm ium -tox ic ity a f t e r a cu te

exposure to th e m eta l a re th e g a s t r o - in t e s t i n a l t r a c t fo llo w in g in g e s t io n ,

and th e lungs fo llo w in g in h a la t io n . A f te r long term exposu re , th e lungs

and th e kidneys a re prim ary t a r g e t o rgans.

Acute o ra l t o x i c i t y has u s u a lly r e s u l te d from th e a c c id e n ta l in g e s ­

t io n o f contam inated f o o d s tu f f s , and produces symptoms such as nausea and

o th e r d is tu rb a n c e s o f g a s t r o - in t e s t i n a l fu n c tio n . L iv e r and k idney

damage may fo llo w and in sev e re in to x ic a t io n , d ea th may r e s u l t (C .E .C .,

1978).

Acute p o iso n in g due to in h a la t io n i s u s u a lly an o c cu p a tio n a l h a za rd ,

and produces b ro n c h ia l and pulmonary e f f e c t s . O ther symptoms in c lu d e

n au sea , headache, d iz z in e s s , e t c . (C .E .C ., 1978). The p o s s i b i l i t y o f

long term e f f e c t s develop ing a f t e r a cu te in h a la t iv e exposure has n o t been

p ro p e r ly in v e s t ig a te d .

The most common form of ch ro n ic cadmium po ison ing in man r e s u l t s

from th e long term in h a la t io n o f cadmium d u s t or fumes. Pulmonary oedema,

p neum onitis , emphysema and o th e r changes in lung morphology have been

r e p o r te d , and produce an im paired r e s p i r a to r y fu n c tio n (K azan tz is e t a l . ,

1963; G i l l , 1978; Lauwerys e t a l . , 1978; Sm ith , 1978).

Exposure to cadmium v ia in h a la t io n in e v i ta b ly le a d s to an accumu­

l a t i o n o f th e m eta l in o th e r t i s s u e s , e s p e c ia l ly l i v e r and k idney .

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R enal tu b u la r m a lfu n c tio n , u s u a lly c h a ra c te r iz e d by p ro te in u r ia has

been commonly re p o r te d , u s u a l ly in i n d u s t r i a l w orkers (K azan tz is e t a l . ,

1963; Adams e t a l . , 1969; F r ib e rg et^ a l . , 1974; C .E .C ., 1978; K a z a n tz is ,

1978). O ccas io n a lly th e se e f f e c t s have been a s s o c ia te d w ith o s teo m alac ia

l i k e changes. K je lls tro m e t a l . (1977a) have dem onstrated an in c re a se d

p rev a len ce o f 02”m c ro Slot>ul i n in th e u r in e of w orkers exposed to cad­

mium i n d u s t r i a l l y .

O ther h e a l th e f f e c t s o f cadmium in humans have no t been reco rd ed

in d e t a i l , a lth o u g h th e p o te n t ia l o f th e m eta l to be c a rc in o g e n ic , and

to produce h y p e rte n s io n have been co n s id e re d . Some re s e a rc h e rs have

in v e s t ig a te d th e in c id en ce o f m alignancies in cadmium w orkers ( e .g . C.E.C

1978), and th e developm ent o f h igh b lood p re s su re has been r e l a t e d to

blood and t i s s u e cadmium l e v e l s , and c o r r e la te d th e e x te n t o f p o l lu t io n

by cadmium to m o r ta l i ty r a t e s caused by c a rd io v a s c u la r d is e a s e s . H ealth

s tu d ie s o f o c c u p a tio n a lly exposed p e rso n n e l have no t shown a tendency

tow ards h y p e rte n s io n ( e .g . C .E .C ., 1978).

H ea lth S tu d ie s in Japan ( in c lu d in g I t a i - I t a i D isea se )

M ention was made p re v io u s ly o f th e occu rrence o f cadmium p o iso n in g

in Japan due to env ironm enta l exposure - th e s o -c a lle d i t a i - i t a i d is e a s e .

I t i s beyond th e realm s o f t h i s th e s i s to rev iew th e l i t e r a t u r e and

op in ions on t h i s s u b je c t ( re a d e rs shou ld r e f e r t o , e .g . F r ib e rg e t a l . ,

1974; E d ited P roceedings F i r s t I n te r n a t io n a l Cadmium C onference , 1978).

However, some o f th e im portan t f e a tu re s w i l l be in d ic a te d .

V arious re g io n s o f Japan have been found to c o n ta in h igh concen­

t r a t i o n s o f cadmium in th e environm ent, p a r t i c u la r ly th e Toyama re g io n .

A h ig h in c id en ce o f an unusual d is e a se c h a ra c te r iz e d by sev e re p a in in

th e jo in t s and bones, and o th e r s k e le t a l m alfo rm atio n s , was no ted around

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1948. Subsequen tly in v e s t ig a t io n s have in d ic a te d a p o s s ib le r o le fo r

cadmium in th e a e tio lo g y o f th e c o n d itio n , which occu rred m ain ly in

post-m enopausal, m u lti-p a ro u s women. The s k e le t a l c o n d itio n was a sso ­

c ia te d w ith r e n a l damage and th e symptoms o f th e d is e a s e were e x ac e r­

b a ted by d ie ta r y f a c to r s such as d e f ic ie n c ie s o f ca lc ium , p ro te in and

v ita m in D. Some w orkers have doubted th e im portance o f th e r o l e p layed

by cadmium in th e p a th o g en es is of th e d is e a s e . F u r th e r in v e s t ig a t io n s

o f th e r e n a l e f f e c t s o f cadmium in th e a re a where I t a i - i t a i d is e a s e was

endem ic, and in o th e r cadm ium -polluted re g io n s o f Japan have been

re p o r te d (Nomiyama e t a l . , 1973a; Nogawa e t a l . , 1975; K je lls tro m e t a l . ,

1977b; Kojima e t a l . , 1977; S h ir o is h i e t a l . , 1977; Nogawa e t a l . , 1978).

1 .4 THE IMPORTANCE OF STUDIES ON CADMIUM TOXICITY '

The R isk to Human H ealth

The occu rren ce o f cadmium in th e environm ent has been rev iew ed in

e a r l i e r s e c t io n s . S im ila r ly , th e m etabolism and t o x i c i t y o f th e m eta l

a f t e r v a rio u s ro u te s o f exposure has been d is c u s se d , m ain ly in r e l a t i o n

to anim al s tu d ie s . The im portance o f th e se f in d in g s to p o s s ib le h e a l th

e f f e c t s in humans i s a complex q u e s tio n . In an a ttem p t to r e l a t e expo­

su re and accum ulation o f th e m e ta l, an e la b o ra te e igh t-com partm ent k in e t ic

model has been fo rm u la ted by K je lls tro m and Nordberg (1978). A ccep ting

th e l im i t a t io n s o f such a m athem atica l m odel, i t may be u s e fu l f o r

d e f in in g exposure s ta n d a rd s , and p re d ic t in g r i s k s .

The p re se n t view o f r i s k i s based on th e assum ption th a t a c r i t i c a l

c o n c e n tra tio n o f cadmium needs to be a t ta in e d in th e c r i t i c a l o rgan .

This has been e s tim a te d to be about 200 yg/g in th e r e n a l c o r te x . We can

th e re fo re a ttem p t to e s tim a te th e exposure re q u ire d to reach t h i s

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c r i t i c a l c o n c e n tra tio n , and th u s p rov ide some measure o f a s a f e ty

m argin . However, th e e x is te n c e o f a c r i t i c a l c o n c e n tra tio n o f cadmium

in human re n a l c o r te x has been q u estio n ed (Nomiyama, 1977).

A fte r making assum ptions f o r th e r e t e n t io n and e x c re tio n r a t e s ,

and f a c to r s such as th e p ro p o r tio n o f th e dose d e p o s ite d in th e k id n ey ,

i t has been e s tim a te d th a t a d a i ly o r a l in ta k e o f about 250 yg o f cad­

mium fo r a 50 y e a r p e rio d i s s u f f i c i e n t to re a ch th e c r i t i c a l concen­

t r a t i o n in th e r e n a l c o r te x (F r ib e rg e t a l . , 1974). When we c o n s id e r

t h a t th e average d a i ly in ta k e o f cadmium i s around 50 yg and th a t

smoking may s ig n i f i c a n t ly c o n tr ib u te to th e body bu rden , th e n th e s a f e ty

m argin fo r cadmium i s low. I t shou ld a ls o be in d ic a te d th a t no e f f e c t

le v e l s fo r long term exposure to cadmium in humans a re no t a d eq u a te ly

d e f in e d .

The in c re a s e in th e body burden o f cadmium from a n e g l ig ib le amount

in a newborn human (< 1 yg) to about 30 mg in a 50 y e a r o ld a d u l t ,

i l l u s t r a t e s th a t exposure to and accum ulation o f th e m etal i s u n av o id ab le ,

and p ro g re s s iv e . The c o n tin u in g demand f o r th e m eta l would su g g es t t h a t

exposure w i l l in c re a s e due to g re a te r env ironm enta l co n tam in a tio n .

S o c ie ty however, demands th e b e n e f i ts o f th e uses o f cadmium, b u t we

must pay th e c o s t o f an in c re a se d h e a l th h aza rd . A g re a te r u n d e rs tan d in g

o f th e to x ic o lo g y and m etabolism o f cadmium would th e re fo re seem e sse n ­

t i a l to th e w e llb e in g o f our s o c ie ty . The stu d y d e sc rib e d in t h i s th e s i s

was designed to in v e s t ig a te what th e au th o r co n sid ered to be some o f

th e most im p o rtan t problem s, namely:

1. The d ia g n o s is o f cadmium to x i c i t y ;

2. Mechanisms o f cadmium t o x ic i t y ;

3. The tre a tm e n t o f cadmium t o x i c i t y .

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The D ia g n o s is o f Cadmium T o x ic i t y

There i s a need to be a b le to a sse ss th e m agnitude o f exposure to

cadmium, and th e re fo re r i s k o f develop ing to x i c i t y , b o th fo r th e g en e ra l

p o p u la tio n exposed to low c o n c e n tra tio n s o f th e m e ta l, and f o r in d u s­

t r i a l w orkers exposed to much h ig h e r c o n c e n tra tio n s in th e w orkp lace.

T r a d i t io n a l ly , th e h e a lth h azard has been e s tim a te d from d e te rm in a tio n s

o f th e c o n c e n tra tio n o f cadmium in blood and u r in e , b u t th e se may n o t

be r e l i a b l e in d ic a to r s . I t i s no t understood in man w hether b lood and

u r in e cadmium c o n c e n tra tio n s r e f l e c t c u r re n t exposure o r body burden .

The c o n c e n tra tio n o f th e m eta l in blood may be c o n s id e ra b ly h ig h e r in

exposed w orkers th a n in peop le w ith o u t known exposure b u t i t does n o t

r e l a t e to th e d is p o s i t io n o f th e m eta l among o rg an s , o r to th e whole

body c o n c e n tra tio n o f cadmium. I t seem s, th e r e f o r e , to r e f l e c t c u r re n t

exposure (Lauwerys e t a l . , 1976). C o n verse ly , a good c o r r e la t io n has

been dem onstrated between b lood cadmium and u rin e cadmium a f t e r long

te rm , low le v e l exposu re , o r a f t e r c e s s a t io n o f exp o su re , in d ic a t in g

th a t blood cadmium may, in some c ircu m stan ces r e f l e c t th e body burden

( P is c a to r , 1977).

An in te r p r e ta t io n o f th e v a lu e o f u r in a ry cadmium i s a lso complex

and ag a in c o n tra d ic to ry r e s u l t s have been re p o r te d (Lauwerys ^ t a l . ,

1976; C .E .C ., 1978). There i s ev idence th a t u r in a ry cadmium r e f l e c t s

th e body burden , s in c e , f o r exam ple, a p ro g re s s iv e in c re a s e o f th e con­

c e n t r a t io n o f m eta l in th e u r in e w ith age has been observed . However,

th e re i s a lso ev idence th a t cadmium in th e u r in e may r e f l e c t c u r r e n t

ex p o su re , s in c e o th e r in v e s t ig a to r s have no t found a c o r r e la t io n betw een

le n g th o f exposure and th e u r in e cadmium c o n c e n tra tio n . Lauwerys e t a l .

(1976) have su g gested th a t a t low le v e l s o f exposure , i . e . when th e re

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i s s u f f i c i e n t m e ta l lo th io n e in sy n th e s is e d to b ind cadmium, u r in a ry

cadmium i s in e q u ilib r iu m w ith th e body bu rden , b u t a t h ig h e r l e v e l s o f

ex p o su re , i . e . when a l l cadmium b in d in g s i t e s a re s a tu r a te d , th e u r in a ry

c o n c e n tra tio n o f cadmium may be a r e f l e c t i o n o f i n t e n s i t y o f exposu re .

I t has a ls o been su g g ested th a t th e c o n c e n tra tio n o f cadmium in

h a i r may se rv e as a u s e fu l in d ic a to r o f exposure and p o s s ib ly accumu­

l a t i o n o f th e m e ta l, s in c e m easurable c o n c e n tra tio n s accum ulate in t h i s

t i s s u e p o s s ib ly by b in d in g to c y s te in e re s id u e s which a re abundant in

h a i r (Hammer e t a l . , 1971). However, i t i s no t th ough t th a t cadmium

in th e h a i r r e f l e c t s t i s s u e s to r e s (S ch ro ed e r and Nason, 1969).

B rancato e t a l . (1976) have shown th a t h a i r le v e ls o f cadmium d id n o t

c o r r e la te w ith t i s s u e accum ulation o f th e m etal a f t e r con tinuous o r a l

exposure in r a t s , and in f a c t th e c o n c e n tra tio n was observed to d ec rea se

w ith in c re a s in g organ r e te n t io n . S im ila r f in d in g s have been re p o r te d

fo r humans (G ross e t a l . , 1976). The p resen ce o f cadmium in h a i r may,

th e r e f o r e , on ly in d ic a te some exposure to th e m e ta l, and does n o t p ro ­

v id e an index o f r e t e n t io n o f cadmium.

S ince th e c o n c e n tra tio n o f cadmium in blood and o th e r b io lo g ic a l

sam ples does n o t always q u a n t i t a t iv e ly c o r r e la te w ith th e e x te n t o f

exposure o r accum ulation o f th e m e ta l, th e n t h e i r v a lu e in th e d ia g n o s is

o f cadmium p o iso n in g i s l im i te d . P aram eters r e f l e c t i n g th e fu n c t io n a l

s ta tu s o f c r i t i c a l organs may be o f g r e a te r d ia g n o s tic v a lu e in th e

assessm ent o f t o x i c i t y . As p re v io u s ly d is c u s s e d , th e developm ent o f

r e n a l d y sfu n c tio n may be reg a rd ed as a c r i t i c a l consequence o f lo n g term

cadmium exposure . Cadmium-induced nephropathy i s a s s o c ia te d w ith a

v a r ie ty o f r e a b s o rp t io n d e fe c ts o f te n le a d in g to p r o te in u r ia . T his

tu b u la r type o f p ro te in u r ia i s c h a ra c te r iz e d by a p reponderance o f

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$2“nri.croglobulin and th e q u a n t i ta t iv e a n a ly s is o f t h i s low m o lecu lar

w eigh t p ro te in has re c e iv e d p a r t i c u la r a t t e n t io n . The e x c re tio n o f

32"mic ro g lo b u l in in to th e u r in e has been in v e s t ig a te d in humans a f t e r

bo th in d u s t r i a l exposure and env ironm enta l exposure to cadmium

(Nomiyama e t a l . , 1973a; K je lls tro m e t a l . , 1977a ,b ; Kojima e t a l . , 1977

S h ir o is h i e t a l . , 1977). A dose re sp o n se r e la t io n s h ip e x is t s betw een

d u ra t io n o f exposure and occu rren ce o f 3 2~m c ro S lo t> u linu ria and s in c e

p r o te in u r ia i s th o u g h t to p recede o th e r fu n c tio n a l l e s io n s , i t appears

th a t a measure o f th e e x c re t io n o f 32“mi c ro S lobul i n th e uri.ne i s

p robab ly th e most s e n s i t iv e param eter c u r r e n t ly a v a i la b le f o r th e a s s e s s

ment o f th e fu n c tio n a l s t a t e o f th e k idney .

The r e le a s e o f k idney enzymes in to th e u r in e may a lso g ive an

e a r ly in d ic a t io n o f cadmium to x i c i t y , p reced in g fu n c tio n a l d is tu rb a n c e s

and p o s s ib ly p reced in g p r o te in u r ia (Nomiyama e t a l . , 1973bj Nomiyama

et_ a l . , 1975). This p o s s i b i l i t y should be in v e s t ig a te d , p a r t i c u l a r l y

in r e l a t i o n to th e accum ulation o f cadmium in th e k idney .

Mechanisms o f Cadmium T o x ic ity

At p re se n t th e re i s no adequate e x p la n a tio n f o r a mechanism o f

t o x i c i t y o f cadmium, and i t i s even d i f f i c u l t to e s ta b l i s h th e a c tu a l

to x ic ag en t, because as d isc u sse d in s e c t io n 1 .3 , b o th cadm ium -th ionein

and th e cadm ium -cation have been proposed . A f u r th e r problem l i e s in

th e f a c t th a t cadmium does n o t produce a s p e c i f ic e f f e c t in b io lo g ic a l

sy stem s, bu t th e t o x i c i t y may be m an ife s t in s e v e ra l organ system s a t

v a r io u s m olecu lar s i t e s . The m u l t i p l i c i t y o f m etab o lic t a r g e t s o f

to x ic m eta ls has been in d ic a te d by Webb (1 9 7 7 ), and th e v a rio u s m acro-

m o lecu lar components of th e c e l l which could p rov ide r e a c t iv e s i t e s

have been d isc u sse d .

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The in te r a c t io n s o f cadmium w ith e s s e n t i a l m eta ls in b iochem ical

system s may a ls o be im p o rtan t. Such in te r - r e l a t io n s h ip s depend upon

th e physico -chem ical p ro p e r t ie s o f th e m eta l ions and i t has been

p o s tu la te d t h a t because o f th e s im i l a r i t y o f th e cadmium io n w ith , fo r

exam ple, th e z in c and cuprous io n s , t h a t p a r t o f th e t o x i c i t y o f cadmium

may be due to i t s a c t in g as an a n tim e ta b o l i te o f th e se e s s e n t i a l m eta ls

( H i l l and M atrone, 1970). There i s c o n s id e ra b le ev idence th a t cadmium

causes changes in th e d is p o s i t io n and hom eostasis o f v a rio u s m eta ls

in c lu d in g z in c , copper and iro n a f t e r bo th p a re n te r a l and o ra l adm inis­

t r a t i o n ( e .g . Bremner, 1974). I n te r a c t io n s betw een th e se m e ta ls may

in v o lv e s e v e ra l mechanisms though u s u a lly th e y in v o lv e c o m p e titio n fo r

b in d in g and t r a n s p o r t s i t e s on m e ta llo p ro te in s and o th e r fu n c t io n a l •

components o f th e c e l l . For in s ta n c e , th e exchange o f cadmium f o r z in c

in enzymes n e ce ssa ry f o r th e re a b s o rp t io n and c a tab o lism o f p ro te in s

in th e k idney has been su g g ested as a p o s s ib le mechanism f o r th e cadmium-

induced r e n a l damage (F r ib e rg e t a l . , 1974).

F u r th e r in fo rm a tio n on th e i n te r - r e l a t io n s h ip s o f cadmium w ith

e s s e n t i a l m eta ls would be u s e f u l , p a r t i c u l a r ly th e im portance o f such

r e la t io n s h ip s in m ed ia tin g to x ic i ty .

The Treatm ent o f Cadmium T o x ic ity

A tre a tm e n t fo r cadmium p o iso n in g could be made p o s s ib le by

s e v e ra l m eans:

1 . P rev en tio n o f th e cadmium-induced to x ic re sp o n se ;

2. Removal o f cadmium from th e body;

3 . S to rag e o f cadmium in a non to x ic form w ith in th e body.

The s to ra g e o f th e m eta l in a non to x ic form i s a fu n c tio n p re s c r ib e d

to m e ta l lo th io n e in , and t h i s has been d is c u s se d . However, as we have

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n o te d , th e s to ra g e c a p a c ity may be s a tu r a te d w ith subsequent t o x i c i t y

r e s u l t in g . U n til th e u n d e rs tan d in g o f th e mechanisms of t o x i c i t y o f

cadmium is more tho rough , a ttem p ts to p rev en t th e to x ic resp o n se

develop ing a re u n lik e ly to be s u c c e s s fu l . However, th e re i s some e v i­

dence th a t z in c may a l l e v i a t e some o f th e t o x i c i t y o f cadmium, because

o f th e in t e r - r e l a t io n s h ip s d iscu ssed in th e p rev io u s s e c t io n . The p re - /

v e n tio n o f t o x i c i t y by m a in ta in in g th e hom eostasis o f th e e s s e n t i a l

t r a c e m eta ls may prove to be u s e fu l and should be in v e s t ig a te d f u r t h e r /

The f i n a l means o f t r e a t i n g th e to x ic re sp o n se i s by rem oving th e

cadmium from th e body, p re fe ra b ly b e fo re c r i t i c a l c o n c e n tra tio n s have

accum ulated. The use o f c h e la t in g ag en ts f o r th e rem oval o f o th e r heavy

m e ta ls , p a r t i c u l a r ly le a d and m ercury , i s f a i r l y s u c c e ss fu l and q u ite

w e ll documented. However, a ttem p ts to remove cadmium by s im i la r means

have no t been s u c c e s s fu l , p robab ly because o f th e g re a te r a f f i n i t y of

m e ta l lo th io n e in fo r cadmium. The developm ent o f novel compounds w ith

c h e la t in g c a p a c ity would be i n t e r e s t i n g , p a r t i c u la r ly i f th o se compounds

p o ssessed su lp h y d ry l groups.

C o n tr ib u tio n o f t h i s T hesis

The work p re sen te d in t h i s th e s i s i s a s tudy o f v a r io u s a sp e c ts

o f th e to x ic o lo g y and k in e t ic s of cadmium, p a r t i c u l a r ly r e le v a n t to th e

d ia g n o s is and tre a tm e n t o f cadmium t o x i c i t y and th e mechanism o f th e

to x ic e f f e c t . The stu d y may be d iv id e d in to v a rio u s s e c t io n s :

1 . An in v e s t ig a t io n o f th e v a lu e o f enzym uria in th e d e te c t io n o f

cadmium-induced r e n a l damage in th e r a t , a f t e r bo th s h o r t term

and long term exposure to th e m e ta l. In th e absence o f a method

f o r th e measurement o f 32~m -c ro S l° k u l in in th e r a t , c o n s id e ra t io n

is g iven to th e use o f enzym uria in e s ta b l is h in g a model f o r

fo llo w in g th e cou rse o f cadmium t o x ic i t y .

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The e f f e c t o f cadmium upon th e m etabolism and e x c re tio n o f z in c ,

copper and iro n i s s tu d ie d a f t e r v a r io u s le n g th s o f cadmium

tre a tm e n t, and th e im portance o f th e se in te r a c t io n s in th e develop­

ment o f t o x i c i t y i s c o n s id e re d . The e f f e c t o f changes in th e

normal hom eostasis o f th e se e s s e n t i a l m e ta ls on th e m etabolism

o f cadmium i t s e l f i s a lso in v e s t ig a te d .

In view o f th e s im i la r p h y sico -chem ical p ro p e r t ie s o f cadmium and

z in c , a ttem p ts to a l l e v i a t e o r p rev en t th e t o x i c i t y o f cadmium by

supplem ents o f z in c a re d is c u s se d , a f t e r bo th o ra l and p a re n te r a l

a d m in is tra t io n o f cadmium.

The p o s s i b i l i t y t h a t cadmium-induced changes in calc ium hom eostasis

and th e bone may occur b e fo re o r in d ep en d en tly o f k idney damage i s

in v e s t ig a te d .

The p o te n t ia l o f v a r io u s novel c h e la t in g ag en ts to remove cadmium .

from th e body a f t e r bo th acu te and ch ro n ic cadmium tre a tm e n t i s

s tu d ie d , and compared to th e e f f ic a c y o f s tan d a rd c h e la t in g com­

pounds c u r r e n t ly a v a i la b le fo r th e tre a tm e n t o f o th e r heavy m eta l

p o iso n in g .

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CHAPTER 2

THE VALUE OF URINARY ENZYMES IN THE

DETERMINATION OF KIDNEY DAMAGE

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CONTENTS

Page

In tro d u c tio n 46

E xperim en ta l 48

M a te r ia ls ; exp erim en ta l d e s ig n ;

a n a ly t ic a l m ethods. -

R e su lts 51

Repeated p a re n te r a l a d m in is tra t io n o f

cadmium; d o se-resp o n se s tu d y ; d ie ta r y

a d m in is tra t io n o f cadmium.

D iscu ssio n 62

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2 .1 INTRODUCTION

The purpose o f t h i s p a r t o f th e s tu d y was to in v e s t ig a te th e

r e le a s e o f enzymes in to th e u r in e d u ring th e course o f cadmium t r e a t ­

ment.

The presence o f enzyme a c t i v i t y in th e u r in e has been known fo r

a c o n s id e ra b le tim e bu t more r e c e n t ly a t t e n t io n has been fo cussed on

th e d ia g n o s t ic p o te n t ia l o f u r in a ry enzyme d e te rm in a tio n s , p a r t i c u l a r ly

when i t became e v id en t t h a t th e a c t i v i t i e s o f c e r ta in enzymes in u r in e

in c re a se d in d is e a s e s o f th e k idney and u r in o g e n i ta l t r a c t . Under

normal c o n d itio n s , s e v e ra l so u rces c o n tr ib u te to th e enzyme a c t i v i t y

of u r in e . Plasma p ro te in s w ith a m o lecu lar w eight o f le s s th a n about

69,000 a re f i l t e r e d a t th e glom erulus and may pass in to th e u r in e , and

g la n d u la r s e c re t io n s and c e l l u l a r tu rn o v e r o f th e u r in o g e n i ta l t r a c t

c o n tr ib u te to th e enzyme c o n te n t . Renal t i s s u e must be reg a rd ed as

th e m ajor source o f enzymes in th e u r in e however. Kidney t i s s u e i s

p a r t i c u l a r ly r i c h in enzymes such as l a c t a t e dehydrogenase, y -g lu tam y l

t r a n s p e p t id a s e , a m in o tra n s fe ra se s , am in o p ep tid ases, e t c . , and th e

p h y s io lo g ic a l tu rn o v e r o f th e b ru sh b o rd e r e p ith e liu m o f th e nephrons

is p robab ly s u f f i c i e n t to account fo r most o f th e enzymic a c t i v i t y

p re se n t (M attenheim er, 1971; Raab, 1972).

Under p a th o lo g ic a l c o n d itio n s marked changes may occur in th e

enzyme co n ten t o f th e u r in e , th e e x te n t o f which w i l l depend m ain ly on

th e s e v e r i ty o f th e damage, and th e l o c a l i s a t i o n o f th e enzyme in th e

k idney . These changes may ta k e th e form of in c re ase d o r d ecreased

a c t i v i t i e s o f th e enzymes norm ally p re s e n t , o r new enzymes may ap p ea r.

The most p robab le cause o f enzym uria i s r e le a s e o f th e enzyme c o n te n t

o f d is ru p te d r e n a l c e l l s bu t changes in g lom eru lar and tu b u la r fu n c t io n

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may a lso occu r. The measurement o f enzyme a c t i v i t y in human u r in e

has been used c l i n i c a l l y in an a ttem p t to d e te c t r e n a l cancer and o th e r

k idney d is e a se s (Wacker and Dorfman, 1962; Amador e t a l . , 1965;

Dance e t a l . , 1970), to examine th e co u rse o f damage fo llo w in g su rg e ry

to th e kidney (P r ic e e t a l . , 1970), and as an e a r ly in d ic a t io n o f

r e j e c t i o n a f t e r r e n a l t r a n s p la n ts (Wellwood e t a l . , 1973). U rin a ry

enzyme e x c re t io n has been in v e s t ig a te d in anim als t r e a te d w ith a v a r ie ty

o f to x ic chem icals which produce a range o f k idney d is o rd e r s ,

e .g . e th y len e im in e-in d u ced p a p i l la r y n e c ro s is ( E l l i s and P r ic e , 1975),

am inonucleoside-induced g lom eru lar n ep h ro s is (Sack and W ilhelm , 1975)

and m ercury-induced tu b u la r damage (S tro o and Hook, 1977).

U rinary enzyme e x c re t io n may th e r e f o r e prove to be a s e n s i t i v e ,

n o n - in v a s iv e , n o n -d e s tru c tiv e t e s t o f r e n a l t o x i c i t y p ro v id in g

( i ) th e e a r ly d e te c tio n o f cadmium-induced k idney damage, and

( i i ) a means o f t r a c in g th e course o f n e p h ro to x ic ity over a p e rio d o f

tim e o f cadmium tre a tm e n t. The te ch n iq u e may be p a r t i c u l a r ly s u i ta b le

s in c e Sack and W ilhelm (1975) have concluded th a t th e main d ia g n o s t ic

v a lu e o f u r in a ry enzymes was in th e d e te c t io n o f chem ica l-in d u ced tu b u la r 1

l e s io n s . For ex p erim en ta l in v e s t ig a t io n s , th e r a t i s reg a rd ed as th e

most a p p ro p r ia te anim al because th e enzyme d i s t r i b u t io n in th e k idney

o f th i s sp ec ie s i s s im ila r to th a t in th e v a rio u s p a r ts o f th e human

k idney (Raab, 1972). The v a lu e o f th e measurement o f 8^ -m ic ro g lo b u lin

e x c re tio n f o r th e e a r ly d e te c t io n o f p a th o lo g ic a l change in human k idney

has been in d ic a te d in a p rev io u s s e c t io n . However, no q u a n t i t a t iv e

method a t p re s e n t e x is t s a llo w in g t h i s to be used in o th e r s p e c ie s .

Enzymuria in th e r a t cou ld th e re fo re p rov ide a u s e fu l model f o r in v e s ­

t i g a t in g cadmium to x ic i t y a f t e r a v a r i e ty o f exposures to th e m e ta l.

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2 .2 EXPERIMENTAL

M a te ria ls

Enzyme s u b s t r a te s were o b ta in e d from Sigma Chemical Co. L td . ,

P oo le , D o rse t, excep t fo r 4 -n itro p h en y lp h o sp h a te which was su p p lie d

by B.D.H. Chem icals L td . , P o o le , D o rse t.

O ther chem icals in c lu d in g cadmium c h lo r id e were o b ta in ed th ro u g h

B.D.H. Chem icals L td . A ll chem icals were o f A nalar o r e q u iv a le n t

g rade . N e u t r a l i t T es t S t r ip s were produced by Merck, D arm stadt.

E xperim ental D esign

In a p re lim in a ry experim en t, te n W ista r a lb in o r a t s (U n iv e rs i ty o f

S u rrey Animal House S to c k ) , i n i t i a l body w eight 100 - 120g, were housed

in d iv id u a l ly in g la ss m etabolism cages which allow ed s e p a ra te c o l le c ­

t io n o f e x c re ta . 24 hour u r in e sam ples were c o l le c te d a t 4°C in g la s s

c o n ta in e r s , s im ila r to th e manner d e sc rib e d by Leathwood and Plummer

(1 9 6 9 ), d a i ly fo r f iv e days. A f te r t h i s i n i t i a l a c c l im a t is a t io n p e r io d ,

anim als were r e s te d b e fo re dosing commenced. Animals were th e n d iv id e d

in to two equal g roups. C o n tro ls were g iven 0.9% s a l in e (0 .2 ml) as a

subcutaneous in je c t io n , d a i ly fo r 21 d ay s, and t e s t anim als w ere g iven

2+ . cadmium as CdCl^ a t a dose o f 1 .5 mg Cd /k g as a subcutaneous i n j e c t i o n

(0 .2 m l) , d a i ly , fo r 21 days. P e r io d ic a l ly anim als were p laced in

m etabolism cages f o r u r in e c o l le c t io n , o th e rw ise th e y were housed c o l ­

l e c t i v e ly in t h e i r g roups. At c e r t a in tim es th roughou t th e ex p erim en ta l

p e r io d , r e n a l fu n c tio n t e s t s were perform ed. At a l l tim es an im als were

p rovided w ith s ta n d a rd , ro d en t d ie t and w a te r ad l ib i tu m . The com posi­

t i o n o f th e d ie t i s d e ta i le d in Appendix I . U rine samples were c e n t r i ­

fuged to remove c e l l d e b r is and were an a ly sed im m ediately fo r enzyme

c o n te n t .

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In a second experim en t, male W ista r a lb in o r a t s , 130 - 150g body

w eig h t, in groups o f fo u r , were g iven 0.9% s a l in e (0 .2 ml) o r cadmium

2+as CdCl^ a t doses o f 0 ,7 5 , 1 .5 and 3 .0 mg Cd /k g , d a i ly in 0 .2 ml

s a l in e by subcutaneous in je c t io n s f o r up to 42 days. The h ig h dose

group were k i l l e d a f t e r 18 day s. S tandard ro d e n t d i e t and w a ter were

prov ided ad l ib i tu m . Animals were housed c o l le c t iv e ly in t h e i r groups

bu t a t fre q u e n t in te r v a l s th e y were p laced in m etabolism cages f o r

u rin e c o l le c t io n as d e sc rib e d above.

A long term s tu d y was a lso perform ed u s in g d ie ta r y a d m in is tra t io n

of cadmium. Male W ista r a lb in o r a t s , i n i t i a l body w eigh t 60 - 80g,

were random ly d iv id e d in to g roups. One group re c e iv e d powdered d i e t

(o f id e n t ic a l com position to t h a t d e sc rib e d p re v io u s ly ) , and w a te r

ad l ib i tu m . The o th e r group re c e iv e d powdered d ie t and a supplem ent o f2+

cadmium as CdC^ (75 p .p .m . Cd ) . At in te r v a l s anim als were removed

and p laced in m etabolism cages f o r u r in e c o l le c t io n as b e fo re .

A n a ly tic a l Methods

U rin a ry c r e a t in in e was m easured by th e method o f B a r te ls and

Bohmer (1971) and in o rg a n ic phosphate was m easured by th e ammonium

vanadate /m olybdate method (B ioch im ica T e s t, B o eh rin g er).

The fo llo w in g enzyme a c t i v i t i e s were determ ined : a s p a r ta te amino­

t r a n s f e r a s e (EC 2 .6 .1 .1 ) u s in g 0.1M a s p a r ta te , a la n in e a m in o tra n s fe ra se

(EC 2 .6 .1 .2 ) u s in g 0.2M a la n in e , a lk a l in e phosphatase (EC 3 .1 .3 .1 ) u s in g

lOmM or 15mM* 4 -n itro p h e n y lp h o sp h a te , y -g lu tam yl tra n s p e p tid a s e

(EC 2 .3 .2 .2 ) u s in g 4.2mM L -y -g lu ta m y l-p -n i t ro a n i l id e , and le u c in e

am inopeptidase (EC 3 .4 .1 1 .2 ) u s in g 1.2mM L - le u c in e - p - n i t r o a n i l id e .

A ll enzyme a c t i v i t i e s were determ ined u sin g op tim ised methods d e sc r ib e d

*15mM s u b s t r a te was used f o r th e long term d ie ta r y s tu d y which gave

co rre sp o n d in g ly h ig h e r enzyme a c t i v i t y .

Page 51: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

by Bergmeyer (1974) and m easurements were perform ed u sin g a CECIL 303

sp ec tro p h o to m ete r.

Renal fu n c tio n was e v a lu a te d by p h en o lsu lp h o n th a le in e x c re t io n ,

c o n c e n tra tio n and d i lu t i o n t e s t s , and u r in e pH.

P h e n o lsu lp h o n th a le in e x c r e t io n . Each r a t was g iven an i n i t i a l w a ter

lo ad o f 2 .0 ml by g a s t r i c in tu b a t io n . PSP in s a l in e was in je c te d

in tra m u sc u la r ly a t a dose o f 0 .1 mg/kg. The t o t a l u r in e produced

in 60 min. was c o l le c te d and th e c o n c e n tra tio n o f PSP was e s tim a te d

by spec tro p h o to m etry a t a lk a l in e pH. An a l iq u o t o f u r in e was made

up to 3 .0 ml w ith 25% (w /v) NaOH and th e e x t in c t io n read a t 520 nm.

C o n cen tra tio n t e s t . This and th e d i l u t i o n t e s t were c a r r ie d o u t in

a s im i la r manner to th a t d e sc rib e d by S h a r r a t t and F ra z e r (1 9 6 3 ).

Each r a t was d ep riv ed o f w a te r fo r 16 h o u rs . The s p e c i f ic g ra v i ty

o f th e u r in e produced d u rin g t h i s tim e was m easured u sin g a r e f r a c t o -

m ete r.

D ilu t io n t e s t . Each r a t was g iven 2 .0 ml o f w a te r by g a s t r i c in tu b a ­

t i o n and th e u r in e c o l le c te d f o r 60 m in. The s p e c i f ic g ra v i ty o f

th e sample was de term ined .

U rine pH was measured u s in g N e u t r a l i t T es t S t r ip s (pH 5 -1 0 ).

S t a t i s t i c a l com parison o f groups was made u s in g S tu d en ts

t - t e s t .

Page 52: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

2 .3 RESULTS

( i ) REPEATED PARENTERAL ADMINISTRATION OF CADMIUM: P re lim in a ry Study

The u r in a ry e x c re t io n o f a lk a l in e phosphatase and y -g lu tam y l

tr a n s p e p t id a s e was m easured over a p e rio d o f 5 d ays, d u rin g which tim e

anim als were housed in m etabolism cages bu t n o t s u b je c t to any t r e a t ­

ment (T ab le 2 .1 ) . Enzyme a c t i v i t y in th e u r in e i s c a lc u la te d as

U/mmole c r e a t in in e . The u r in a ry e x c re tio n o f th e se two enzymes was

no t s ig n i f i c a n t ly a f f e c te d by th e le n g th o f tim e th e anim als were k ep t

in th e m etabolism c ag e s , a lth o u g h th e r e s u l t s do in d ic a te a s l i g h t

tendency tow ards be in g h ig h e r i n i t i a l l y and a t th e end.

Table 2 .1 D a ily V a r ia t io n in th e U rin ary

E x c re tio n o f A lk a lin e P hosphatase and

-G lutam yl T ran sp e p tid ase by Rats

U/mmol c r e a t in in e

Day A lk a lin ephosphatase

y-G lutam yl tra n s p e p tid a s e

1 3 .5 ± 0 .2 57 ± 2

2 3 .3 ± 0 .2 52 ± 2

3 3 .0 ± 0 .2 54 ± 1

5 3 .5 ± 0 .1 56 t 2

V alues a re ex p ressed as mean £ S.E.M. o f 5 anim als

(d u p l ic a te o b s e rv a tio n s ) .

Page 53: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

The u r in a ry e x c re t io n o f v a r io u s enzymes a f t e r re p e a te d paren­

t e r a l a d m in is tra t io n o f cadmium i s d e sc rib e d in Tables 2 .2 and 2 .3 .

Cadmium tre a tm e n t caused an i n i t i a l in c re a s e in e x c re tio n fo r a l l

enzymes and v e ry s ig n i f i c a n t ly e le v a te d le v e l s were found around day 2.

This in c re a se d enzym uria was t r a n s i e n t and le v e l s re tu rn e d to normal

around day 4. A second phase o f enzym uria began around days 18 - 21

which p e r s is te d fo r s e v e ra l days a f t e r cadm ium -treatm ent had ceased

on day 21 bu t by day 28 th e u r in a ry e x c re t io n o f a l l enzymes was n o t

s ig n i f i c a n t ly d i f f e r e n t from c o n tro l an im als. Cadm ium -treatm ent d id

no t have any s ig n i f i c a n t e f f e c t upon c r e a t in in e e x c re tio n (T ab le 2 .4 ) .

The r e s u l t s o f th e r e n a l fu n c tio n t e s t s perform ed on th e se

anim als a re g iven in Table 2 .5 . C o n tro l r a t s produced an amber

co lou red u r in e o f app rox im ate ly n e u tr a l o r s l i g h t l y a c id pH w h ile

cadm ium -treated r a t s produced a d a rk e r co lo u red u rin e a f t e r day 14.

The a lk a l i n i t y o f th e u r in e o f th e s e anim als in c re a se d g ra d u a lly th ro u g h

ou t th e ex p erim en ta l p e r io d , and t h i s change con tinued even a f t e r

cadm ium -treatm ent had ceased . The a b i l i t y o f th e r a t s to remove p h en o l-

su lp h o n th a le in was n o t in flu en c ed by cadmium, and a l l an im als e x c re te d

about 50% o f th e in je c te d dose w ith in th e f i r s t hour. S im ila r ly , th e

a b i l i t y o f th e kidneys o f cadm ium -treated anim als to c o n c e n tra te and

d i lu te th e u r in e was n o t im paired .

( i i ) REPEATED PARENTERAL ADMINISTRATION OF CADMIUM: Dose-Response Study

E f fe c ts o f Cadmium on U rinary E x c re tio n o f Enzymes

The u r in a ry e x c re t io n o f a s p a r ta te a m in o tra n s fe ra se , a la n in e

a m in o tra n s fe ra se , a lk a l in e p h o sp h atase , y -g lu tam y l tr a n s p e p t id a s e and

le u c in e am inopeptidase a l l showed s ig n i f i c a n t in c re a se s on th e second

day o f dosage which su b seq u en tly re tu rn e d to normal (T ab les 2 .6 and 2 .7 )

Page 54: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 2.

2 Th

e E

ffec

t of

Rep

eate

d P

aren

tera

l A

dmin

istr

atio

n of

Cadm

ium

on th

e U

rinar

y E

xcre

tion

of V

ario

us

Enzy

mes

<U3•H3

•rlPcdcuPo

mo

COoQ

CUtocO

• 3cu •rH CO CM CM3 P • • •

•p i c x o O oa (U3 Du + 1 + 1 •HCU o

• P 3 0 0 ON 0 0•H • • •

§CO CO CO

cuCO

pH cd>N 3£ • r lCO P

P o . c o < r CM3 <U

rH c u + 1 + i + 1O CO

1 3 < r VO? - cd in m < r

PP

cu $CU CO3 cd pH CM CM

•p i P • . • •pH cd o o Ocd , 3

X D . + 1 + 1 + 1pH CO< O I " - CM

• • 9

CU CO < r CO

CUCOcdP CM

CU cu o o o3 4 h • • •

•H CO o o o3 3cO cd + 1 + i + 1

pH P< P pH CM o

O • • •3 pH rH rH

•rH

CUCOcd

CU PP cu pH CM rHcO m . • 9

P CO O O ap 3cO cd + 1 + 1 + 1CU PCO P CM CO pH

< O • m •3 CM CM CM

• H

0 0h inM *■._ •

3 bD o pH OO S

3O

•rHP

4 h cdO p

p pHt o COcd • r l

Q 3•pi£

3< !

*CO

o

+1CO

in

%tin+1coCO

i0

+ «< r

vo

1<ro

o

+1COrH

1pH

O+1<fCO

inrH

CM

CM

o+iCM

co

CM

-HOm

CM

o

+1< r

CO

<ro

o+i

pHo

+ iCO

CM

o

+iCOCO

CO

+1ONin

tCO

o + iVO

9

<r

**CMo

o + i

' pH

1pH

o

+1

CO

in

co

pH

o

+1<rCO

pH

o

+1pH

CO

COOo+iON

o

o

+1mCO

CM CM pH CM CM CM

+ 1 +J +1 + 1 + 1 -H

in < r ON CM pH o< r < r < r in m in

o

+1voCO

VO O - •

o

+1o

9

rH

rH rH• •

o o

-H +1rH <T

• •CM CM

inpH

pH

o+ivO

CO

CM

o+iCOCO

o

+ 1

CO

COo

o+1

o

+1CO

CO

<ro

o+ i

CM

pH

o + 1

CO

pH

o+1CO

inpH

in

coo

+lCM

CO

CM

o

+ 1

< r

CO

o

o+iCM

rH

o

+1pH

CM

CM

O+1ON

<N

r-*

o

-HCM

CO

COo

d-H

O + 1

ON

in

Val

ues

are

expr

esse

d as

mea

ns

i S.

E.M

. of

6 an

imal

s (d

upli

cate

ob

serv

atio

ns).

S

tati

stic

ally

si

gn

ific

ant

diff

eren

ces

betw

een

test

gr

oup

and

cont

rols

ar

e sh

own:

**

*p

<0.

001,

*

*p

<0

.01

.

Page 55: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 2.

3 Th

e E

ffec

t of

Rep

eate

d P

aren

tera

l A

dmin

istr

atio

n of

Cadm

ium

on

the

Urin

ary

Exc

retio

n of

Var

ious

Enzy

mes

(c

onti

nued

)

CUCO iscd 1c rTO * i t

CU •rl rH rH H rH CN CN CN CN rH rH rH rHPi p • • . • • • a • a • a •

•H Cu O O O O . O O o O o O o Oo

, p0Cu +1 +1 +1 + 1 + 1 -H +1 +1 +1 +1 +1 +1

cup

oPS in cn in <r <r O CO C\ rH CN CN o

•H • • • • • • a • a • • aCO CO CO CO CO CO CO CO CO <r CO CO

cuCO

rH cdrH V *

•rl t *Cu P i t ■jc i tP cx CN rH CO CO CN CO CN vo CN CN rH CN3 curH Cu -H -H +1 +1 +1 +1 +« +1 +1 +1 +1 -H01

COPi CT\ 00 <3- CO l"> r- CO in o CO rH o

J- ccJ <r m <r . <T in vo in inPi4->

0PS CU * r

•rl CU CO ■kPS PS 03 CN H CN rH CN CN CN CO rH rH CN rH

•H •H p • • a • a • • • • • • a

P rH cd O O o O o O O o O O o ocdCU

cdPU + 1 +1 +1 +1 +1 +1 + 1 + 1 +1 +1 +1 -H

£-1Oi-H CO

o CO CO <r C\ CN LO CO CO rH <r CN rH,c • • m • a a • • a • • •

i—I P CO CO CO CO CO CO CO vo co CO CO COo

.J CU *p CO iscd 4«

Pi in <r CT\ <r lO ID in r . in o inCU cu o o o o o O o o o o rH os 4h . • • • a a • • a • • •

•H CO o o o o o o o o o o O oPScd S + 1 -H +1 +1 +1 -Pi +1 +1 +1 + i +1 +1H<

piXI CN CN CO rH CN CN CO CO o rH CN rHo • • • • • a • • • • • • •PS rH rH rH rH rH rH rH rH rH rH rH rH

•H

0’

CO iscd iSCU Pi ■5< ■ic 1'p 0 rH rH CN rH CM rH rH CN rH rH rH rHcd 4-i • • • • • a • • • • • •P CO o o O o o o o O o O o OS £

cd -pi -H +1 -H +1 +1 -H +1 +1 +1 + 1 -HCXCO Pi

P CN O rH O rH in rH vO o VO CN Oo • •' • • • a • a • • • •PS CN CN CN CN CM CN CN CO CN CN CN CN

•H

4HO

+QJJ

m in in in in incu CN . a a a a •CO TJ "bB O rH O rH o rH O rH o rH o rHo O sQ V-X

PSo•Hp4h cdo pp CN in CD rH CO CO

to CO rH H rH CN CN CMCd •rlQ PS

s< V

alue

s ar

e ex

pres

sed

as m

eans

i

S.E.

M.

of 6

anim

als

(dup

lica

te

obse

rvat

ions

).

Sta

tist

ical

ly

sig

nif

ican

t di

ffer

ence

s be

twee

n te

st

grou

p an

d co

ntro

ls

are

show

n:

***p

< 0.

001,

**

p<0.

01,

*p

<0

.05

.

Page 56: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

T ab le 2 .4 The E f f e c t o f Cadmium on th e U rin ary

E x c re tio n o f C re a tin in e in th e Rat

u r in a ry c r e a t in in e

Day o f ym ol/24 h

. . . 2+A d m in is tra tio n c o n tro ls Cd - t r e a te d

1 80 + 3 o 1+ 4

2 70 + 4

+io 6

3 80 + 4

+lo00 6

■ 4 80 + 6

+1o00 4

5 90 ± 5 90 ± 3

10 100 + 5 100 ± 9

12 100 + 5 100 ± 5

15 100 + 6 110 ± 6

18 * 90 + 3 90 ± 3

21 100 + 5 vo o 1+ 3

23 90 + 6 110 ± 6

28 110 + 2 100 t 3

V alues a re exp ressed as means £ S.E.M. o f 6 an im als.

Page 57: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 2.

5 Th

e E

ffec

t of

Rep

eate

d P

aren

tera

l A

dmin

istr

atio

n of

Cadm

ium

on Re

nal

Func

tion

in

pccSpiCl)AP

•( . £CuO

u•H O o o O O o in inp CN CN CO CN CO CN CN CN

• £ o O o O o O o o(X r-4 • • • • • • • •CO •H 1—4. i—1 r-4 r-4 r-4 r-4 r-4 r-4

XSN—/

/"N£O

•H• pp CCS o o o o o o O oGjD p in in in in in in in in

p o o o o Q o o o• £ • • • • • • • •

(X cu i—i i—4 r-4 r—1 r-4 r-4 r-4 r-4CO o£oo

V—'

£oPh

•Hp /- \ cr» o o\ VO OV VO Ov

CO as 5-S <r in <r <r <r <r <rfp P V -/o

Xas

cu£ cc o o pH r-4 CO♦H Ph • • • • • • • •p r> VO r^H CO CO

D

4-4 /—\O + 60

CN in in in mas •d • • • •CO o 'ao o r-4 O r-4 o r—4 o r-4o gQ V_y

£O

•HP4-4 CCS0 pp r ^ <r r-4 COCO r—1 CN CN

CCS •HQ £

•Hg<

CO1—4dg•H£

VO4-4OCO£ccJOSgCOCCS'dcucococupaXcucu8COcu£

r-4ccS>

Page 58: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 2.6

Th

e E

ffec

ts

of R

epea

ted

Par

ente

ral

Adm

inis

trat

ion

of Ca

dmium

on

the

Urin

ary

Exc

retio

n of

Var

ious

En

zym

es;

Init

ial

Phas

e of

Enzy

me

Los

s

- 57 -

• r ic

rtaPci

rHo

tHociCOoQ

01tor t

•o f t01 *H CM CM r-lC P • • • •

•H O . O © OCl 0)5 C. + 1 1 +1 +101 0PJ C vO Of VO

•H • • •B cn cn mxt t

01co

rH Cj>N *3B *Hr t P

p a vo vo3 01

rH C . -H » + t io co1 3 CM rH>- i n m

pP

0101 10 f tc rt cn cm cn

•H P • • •rH rt © o ©rt 40a +! t +1 +1

rH CO< o 00 o> r*.r“ • • •& CO cn < r

01cor tp

05 01 rH rHC IH • •

•H CO o Oc cr t r t + 1 1 + 1 1

rH p< H) rH CMo • •

c fH fH•H

01COrt f t

01 P f tP 01 rH rH rHr t vh • • •

P CO © O ©P Cr t r t + i . i ' + 1 -Ha p10 P o © vo< o • • •c CM CM CM

•H

m•/-N rH ©

to • in•o •£ « cn n*© in •

00 o r>. o og • cV-/ © rt

Go•HP

4-i r to p

p rH CNrP COr t »h

© C•HE•o<

ftr>.

ftftc

o o+1 +1

CM CM • •

in vo

ftVO

f t CM I—i-Hmx m

ov

f t

*in*

o-HMX

in©■Hmvo

•ft*CM

O+!CM

cn

inr-i

o■Hvo|H

ftfeftCM

O•HC•a-

ocn

cno

+iOv

cn

cm in

<r co in vo

MXo

+!O<r

trH

•O+ 1CO

rH

o+1

CM

rH

o+iCO

m

o o

cn

*ftftvo

ftcno o

+1 +1rH CM

• ' •in n .

ftcn

ft<uVcn

+1 +1 +1 +1oOv

cn m • •

o o+1 +1co m

• •in vo

ftftrHO+1CM

CM

O-HinCM

V > ,vVCM VO

• •o o

+« +1vo <r

• •CM CM

in ocn

CM.

o+1

cn

ftrH

ftCM

o o

I +1 +1«H

• •'C1 *9“

CM CM CM CN

+1 +1 +1 +1CM C \in in

CM

o+1

ocn

CM

o+1MXrH

CM

©+1r>.rH

o o

CMVO vo

I I

m m ©cn

cn©+«COcn

cn•Hcnin

cn©

©

|H

©■H<■rH

i i

ftrH

©I +1

CO•

fH

rH

©•H I ©CM

inrH

m© f"*ocn

ino

•oVa

ft

0» •CO ©o Vp a '

*crt •»

rHCO ©G1 o3 •rH or t> V

r*.fH fto ftp ftpr* »•o cCl •>

o>N r“

rH *wG

© ciprt•

n COCO rHc Oo P•H Pp crt o> ClP01 BCO o& po tp01 pp Grt 01Cl P•H arH VHa vp3 •H

•a

CO rHrH rHrt r tB o•H •Hc Prt CO

•rH• «a- P

rtIH po CO. G1s prtw

45-CO Cl

•H+1 43

SCOc COr t 01ai 3B rHrt0) >Prt rH

r tCO p01 G3 01

, rH Br t •H> P01rH arH X< a

2

Page 59: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 2.7

Th

e E

ffec

ts

of R

epea

ted

Pare

nter

al

Adm

inis

trat

ion

of Ca

dmium

on

the

Urin

ary

Exc

retio

n of

Var

ious

En

zym

es;

Seco

nd

Phas

e of

Enzy

me

Loss

- 58 -

a)e•Hc•r-44-Jrt0)rtOr—I O

4-4

rtCOrtX}rt -H r-4

fi 4-> ••H ft ort rtrt ft +1rt oi-3 rt <r•r-4 •

E cnrt

rtCOr-4 rt>? X}E *Hrt 4-J4-> ft cnrt rtr-4 ft -HO COi rt in^ u

in4-J

rtrt COrt rt r-4•H 4-J *r-4 rt oCCi ,rtX ft -Hr-4 CO< o•fi •

ft cn

r tCOr tr t

r t r t r-4c 4-4 •

•r-4 CO or t r tr t r t -H

t—4 S-4< 4-J CM

O •r t r—4

•H

0)4-Jrt4-JrtrtCuco<

CUCOrtrtrt

4-1CO

§J-44-Jort•H

4-4O

D

6

fio•rH4-JrtCi4-JCO•Hc•He*0<

r—l

O-H iOCM

cno+ioLO

r-4

O + 1

r-4

LT)+1cn

•Jc■Jc•5cCM

O+1< r

LO

1r-4

O-HOcn

$CM

o+ir-4

cn

O 00 LOTO X in o

r t O •— • • •CO 00 o o 1—4 cn

> r—4

o+1r-4

cn

t—4

+1ft

CM

o+1

cn

cn

r-4

O-H< r

i—i

CM

o+1CM

CM

00r-4

O-Hin

in

cn

I +lvoft

<r o

i +ir-4

VO

r-4

O+1in

CM

r-4 CM rH• • •

o o o+ 1 + ! -H

oo VO f t• • •

CM <r r—4

m *r- in o• • •

o r-4 cn

o+1n-cn

CM

+1om

cn

o+tocn

r-4

o-Hvo

r-4

inin

-}C-JC< r r—4

• •o o

+ i -H

cn r-4

vo O ’

■jc

tcn CM

+ l +1

82 50

•JC•Jc■5cCM CM

o O

-H •H

f t r -• •

in cn

r-4 r—4

O O

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00 00 00 00r ft

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• • • • r-4 COo O o o r t

o r t+1 •H -H +i

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• • • • covo cn VO vo CO t—i

r t oo r t

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• • • • CO o •r4O o o o X r t CO

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• • • r tr-4 o r—4 t—i CO E

C CO •r-4r t r t cr t rt r tE r—4rt f trt > rtr t or t r—4 r t

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The in te n s i ty o f t h i s in c re a s e was d o s e - re la te d and was most marked

w ith a s p a r ta te a m in o tra n s fe ra se and a lk a l in e p h o sp h ata se , which showed

in c re a s e s a t a l l th r e e d o ses .

A second phase o f enzym uria began around day 15 o f a d m in is tra t io n

and p e r s is te d to th e end o f th e experim en t. Animals re c e iv in g th e h igh 2+

dose (3 .0 mg Cd /k g /d ay ) e x h ib ite d s ig n i f i c a n t in c re a s e s in a l l f iv e2+

enzymes by th e 15 th day, anim als on th e median dose (1 .5 mg Cd /k g /d a y )

showed s ig n i f i c a n t in c re a se s in a l l enzymes by day 21 , and th e anim als2+

on th e low dose (0 .7 5 mg Cd /k g /d a y ) d id n o t e x h ib i t an in c re a s e in

a l l enzymes u n t i l day 38 o f a d m in is tra t io n (F ig u re 2 .1 ) .

E f fe c ts o f Cadmium upon U rin a ry In o rg an ic Phosphate E x c re tio n

The u r in a ry e x c re tio n o f in o rg a n ic phosphate was in c re a se d a t

48 hours in th e h ig h dose group o n ly , to 10 mmol/mmol c r e a t in in e , and

su b seq u en tly re tu rn e d to th e c o n tro l l e v e l (app rox . 5 mmol/mmol c r e a t in in e )

th ro u g h o u t th e rem ain ing p e rio d (38 days) o f cadmium a d m in is tra t io n .

( i i i ) DIETARY ADMINISTRATION OF CADMIUM

The u r in a ry e x c re t io n o f enzymes by r a t s t r e a te d w ith d ie t a r y cad­

mium was m easured r e g u la r ly over a 72 week p e rio d and i s d e sc rib e d in

T able 2 .8 . Enzyme a c t i v i t y in th e u r in e rem ained w ith in c o n tro l l im i t s

u n t i l around 36 weeks. At 48 weeks, cadm ium -treated anim als showed a

s ig n i f i c a n t ly r a is e d le v e l o f a lk a l in e phosphatase (p < 0 . 01 ) and

y-glutam yl t r a n s p e p tid a s e (p < 0 .0 0 1 ). At 72 weeks, a lk a l in e p h o sp h a ta se ,

y -g lu tam yl tra n s p e p tid a s e and le u c in e am inopeptidase a l l showed s ig n i ­

f i c a n t l y in c re a se d le v e ls in th e u r in e . However, c o n tro l an im als a lso

showed a tendency tow ards in c re a se d enzyme e x c re tio n in th e u r in e by

th i s s ta g e and so th e d if f e r e n c e between t e s t and c o n tro l groups i s

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Figu

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Page 62: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

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•H a g o vo vo <r in in <r in m in in vo CUDPCO

a) CU03 . prt ■Jc

•d cH CM CM rH «H rH rH pH pH CM |H CM £a; •H • • • • • • • • • • • • cu£ P o O O o o o O o o o o o <u

•H a &O CUp, + i -H +1 +1 -H -H +1 -H •H +1 +1 +1 p

cud) O in vo CO oo vo <r OO 00 CM 00 00 CO pP £ ■ • • • • • • • • • • •

•pi 00 00 oo oo CO CO oo oo <r oo COg curt o

£cup

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i—l rt 4h>> X3 * •rHg •pi ■JC •Jc *drt p CM 00 CM 00 CM oo CM CM pH CM CO CMP a p3 0) + 1 +1 +1 +1 -H -H -H -H +1 + 1 -H +1 £rH a rtO 03 VO in VO oo o\ o VO pH VO CM rH o1 £ vo vo in in <r in uo in <r in in VO •rH

rt 4hP •HP £

bD•rH

CU CO£ d) •Jc ■Jc

•H CU 03 •Jc •Jc »>>£ £ rt 00 r - oo 00 in CM in in CM CO 00 pH

•H •rl P • • • • • m • • • • • • pHP pH rt o o o o o o o o o o o o rtrt rt jC oCU X a +1 •H +1 +1 + i -H +» -H •H + 1 -H -H •rH£ pH 03 Po < o G\ in CM pH vo CM pH CM O CM in pH CO

,£ • • • • « • • • • • • • •rH1—I a G\ CM OV a \ 00 o\ 00 00 o OO o OO P0 pH pH pH pH pH rtg P£ CO

<13P 03

rt <r oo < t CM CM in vo o \ l". in CM •P o O o o O O o o o o o pH CO

<D cu m • • ■ • • • • • • • • ££ 4h o O o o o o o o o o o o . O

•H 03 •rH£cd

£cd +1 +1 +1 +1 -H -H -H +1 -H +1 -H -H P

cdfH p pH o CM pH pH O CM CM CO pH oo <r >< p • • ■ * • * • ■ • ■ • • p

o fH fH pH pH rH pH rH rH rH pH fH |H cu£ CO

•H6 ort

vo4h

“ d) O03rt •

<y P fH |H pH pH fH pH pH pH fH CM rH rH SP cu • • ■ • • • • • • • • • •rt 4-1 o o o o o o O O o O o o wP 03 •a £ -H -H +1 -H + i -H +1 +1 -H +1 +1 -H cna p O O o pH o O o pH CM <r <r m + i03 P m • • ■ • • • ■ • • • •< o CM CM CM CM CM CM CM CM CM CM OM CM CO

£ £•pi rtg curt S

COrt

P£ CO CO CO CO CO CO •dCU pH pH pH pH pH pH cua O + O + O + o + O + O + COp P CM P CM P CM P CM P CM P CM COrt P •d P *d P ■d P ■d P *d P rd cud) £ o £ CJ £ o £ o £ o £ o pP O o O O O O aH O u o V o O Xcu

d)4h P pO £ ✓*>. rt

d) 03•a 6 X 00 VO <r VO 00 CM COo P CU pH CM 00 <r cu

•H rt cu dp cu £ rH0) P w rtPH H >

moova*

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£§.C03

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£rt

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le s s s ig n i f i c a n t . The e x c re tio n o f in o rg a n ic phosphate in to th e u r in e

was u n a ffe c te d by cadmium tre a tm e n t and by 72 weeks, anim als showed

la rg e in d iv id u a l v a r ia t io n s .

2 .4 DISCUSSION

The v a rio u s methods which have been co n sidered fo r t h e i r p o te n t ia l

to f a c i l i t a t e th e e a r ly d ia g n o s is o f cadmium po ison ing have been d i s ­

cussed in s e c t io n 1 .4 , and th e v a lu e o f v a rio u s p a ram ete rs , p a r t i c u l a r ly

$2~ m icrog lobu lin , fo r r e f l e c t i n g th e fu n c tio n a l s ta tu s o f th e k id n ey ,

has been in d ic a te d . This p re se n t in v e s t ig a t io n has examined th e u r in a ry

e x c re tio n o f enzymes in th e r a t , a t v a rio u s le v e ls o f cadmium exp o su re ,

in r e l a t i o n to th e developm ent o f r e n a l to x i c i t y .

An i n i t i a l s tu d y dem onstrated th a t p la c in g th e anim als in m eta­

bo lism cages fo r p e rio d s o f up to 5 days d id no t have any s ig n i f i c a n t

e f f e c t upon th e u r in a ry e x c re t io n o f th e enzymes m easured. In subsequen t

exp erim en ts , th e r e f o r e , i t was n o t thought n ecessa ry to allow th e an im als

to a c c lim a tis e to th e u rin e c o l le c t io n procedure and so random 24 hour

u r in e c o l le c t io n s were perform ed when re q u ire d .

Although le s s r e le v a n t th a n prolonged in g e s t io n to th e problem

o f human cadmium p o iso n in g , th e p a re n te r a l a d m in is tra tio n o f cadmium

was used to produce a range o f body and organ burdens o f th e m eta l w ith in

r e l a t i v e l y sh o r t p e rio d s o f tim e . The s ig n i f i c a n t changes in th e u r in a ry

e x c re t io n o f enzymes was co n sid e red to be ev idence o f r e n a l damage.

C onventional r e n a l fu n c tio n t e s t s , however, d id n o t r e v e a l any in d ic a ­

t i o n o f k idney d y s fu n c tio n a t t h i s s ta g e . A fte r cadmium tre a tm e n t had

ceased , enzyme e x c re tio n g ra d u a lly re tu rn e d to normal even though th e

cadmium-induced r e n a l damage i s though t to be i r r e v e r s ib le

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(F r ib e rg et_ a l . , 1974). This f in d in g was s u rp r is in g s in ce th e concen­

t r a t i o n o f cadmium in r e n a l t i s s u e w i l l co n tin u e to in c re a se even when

exposure has ceased due to r e - d i s t r i b u t io n from o th e r o rg an s .

The co u rse o f k idney damage, and th e re fo re th e r e le a s e o f enzymes

in to th e u r in e , i s p robab ly r e la te d to th e le v e l o f cadmium in th e

k idney . Thus, a dose-resp o n se s tu d y was designed to in v e s t ig a te th i s

f u r th e r . A s im ila r p a t te r n o f enzym uria developed in t h i s experim ent

and th e e x te n t o f th e i n i t i a l damage and tim e o f on set o f th e second

phase o f enzym uria were d o s e - r e la te d . M e ta llo th io n e in sy n th e s is in th e

k idney fo llo w s a la g phase o f about 48 hours a f t e r exposure to cadmium

(Cempel and Webb, 1976). I t i s su ggested th a t in th e absence o f adequate

amounts o f t h i s b in d in g p ro te in , cadmium may cause t i s s u e damage and

th e r e le a s e o f enzymes from re n a l c e l l s in to th e u r in e . T h is could

account fo r th e i n i t i a l peak o f enzym uria, where th e degree o f damage

was r e la te d to th e amount o f m etal a d m in is te red . W ith c o n tin u in g

exposure to cadmium, th e m etal accum ulated u n t i l th e b in d in g c a p a c ity

o f th io n e in was s a tu r a te d , i . e . th e c r i t i c a l c o n c e n tra tio n was reach ed .

F u r th e r a d m in is tra t io n o f th e m eta l may th e n r e s u l t in c e l l u l a r damage

and u l t im a te ly th e developm ent o f r e n a l d y s fu n c tio n . The h ig h e r th e

d ose , th e sooner th e c r i t i c a l c o n c e n tra tio n i s exceeded as in d ic a te d

by th e tim e o f on set o f th e second p h a se .o f enzym uria.

Nomiyama e t a l . (1973b, 1975) have a lso d e sc rib e d enzym uria in th e

r a b b i t fo llo w in g bo th re p e a te d p a re n te r a l and d ie ta r y a d m in is tra t io n o f

cadmium, and found th a t th e r e n a l d y s fu n c tio n p re sen ted as a p ro g re s s iv e

leakage o f c e l l u l a r components in to th e u r in e in th e fo llo w in g o rd e r :

enzymes, amino a c id s , p ro te in s and g lu co se . I t was concluded , th e r e f o r e ,

t h a t th e r e le a s e o f k idney enzymes in to th e u r in e may g ive an e a r ly

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in d ic a t io n o f cadmium in to x ic a t io n , p reced in g fu n c tio n a l d is tu rb a n c e s

and p o s s ib ly a ls o p reced ing p r o te in u r ia . The r e s u l t s p re sen ted h e re

su p p o rt t h i s co n c lu s io n . I t i s g e n e ra lly co n sidered th a t in c re a se d

u r in a ry $2”ra^c ro 6! ° b u^ n th e f i r s t d e te c ta b le m a n ife s ta tio n o f

cadmium-induced r e n a l t o x i c i t y (s e e s e c t io n 1 .4 ) , so i t i s u n fo r tu n a te

th e re fo re th a t in th e absence o f a q u a n t i ta t iv e method fo r th e m easure­

ment o f t h i s p ro te in in th e r a t , a com parison between g2”ini c r 08^0^ui i n

and enzym uria a f t e r cadmium tre a tm e n t cannot a t p re se n t be made. I t i s

ap p aren t from th e r e s u l t s o f th e d o se-resp o n se s tu d y th a t enzym uria

o ccu rred a t an e a r l i e r s ta g e th an some o th e r s ig n s o f d y s fu n c tio n ,

e .g . p h o sp h a tu r ia , which i s reg ard ed as an o th er m a n ife s ta tio n and

c h a r a c te r i s t i c o f cadmium-induced r e n a l damage.

The d a ta o f Nomiyama e t a l . (1973b, 1975) allow com parison o f th e

u r in a ry e x c re tio n o f enzymes w ith fu n c tio n a l d is tu rb a n c e s , b u t n o t w ith

th e cadmium c o n c e n tra tio n in b io lo g ic a l f lu id s o r t a r g e t t i s s u e s . This

p re se n t in v e s t ig a t io n has examined th e r e le a s e o f enzymes in to th e

u r in e a t low er le v e ls o f cadmium exposure th a n th o se used by Nomiyama

and cow orkers, and has a lso r e l a t e d th e developm ent o f n e p h ro to x ic ity

to changes in th e u r in a ry e x c re tio n o f cadmium and th e d is p o s i t io n o f

o th e r e s s e n t ia l t r a c e m e ta ls . T his i s d iscu ssed in a l a t e r s e c t io n ( 3 .4 ) .

The p a re n te r a l a d m in is tra tio n o f cadmium has produced some

in te r e s t in g d a ta , bu t more u s e fu l to th e problem o f human h e a l th e f f e c t s

i s th e a d m in is tra tio n o f low er le v e ls o f th e m eta l in th e d i e t fo r

lo n g e r p e rio d s o f tim e. Thus cadmium was g iven to r a t s as a d ie ta r y

supplem ent fo r p e rio d s which re p re se n te d a c o n s id e ra b le p a r t o f t h e i r

l i f e s p a n . The e lev a te d le v e l s o f a lk a l in e phosphatase and y -g lu tam y l

tra n s p e p tid a s e a re again reg ard ed as ev idence o f some re n a l damage

a f t e r 48 weeks o f cadmium exposure. At 72 weeks, however, c o n tro l

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anim als a lso showed a h ig h e r e x c re tio n o f u r in a ry enzymes compared to

c o n tro l anim als a t an e a r l i e r age, and h i s to lo g ic a l exam ination o f th e

k idneys re v e a le d th a t th e se r a t s d isp lay ed ev idence o f ch ro n ic n e p h r i t i s .

This a g e - re la te d change in th e c o n tro l anim als obscures any cadmium-

r e la te d change in t e s t anim als o f a s im ila r age. I t a p p ea rs , th e r e f o r e ,

th a t th e measurement o f u r in a ry enzymes may no t be very s e n s i t iv e a t

t h i s s ta g e .

The k idney has a la rg e fu n c tio n a l re s e rv e c a p a c ity and s ig n i f ic a n t

h i s to lo g ic a l damage i s n ecessa ry b e fo re changes a re apparen t in r e n a l

fu n c tio n ( E l l i s e t a l . , 1973). Thus enzyme e x c re tio n may prove to be

a more s e n s i t iv e in d ic a to r o f nephropathy . The kidney i s d iv id e d in to

d i s t i n c t m orpho log ica l and fu n c tio n a l re g io n s , each having a c h a r a c te r i s ­

t i c complement o f enzymes (M attenheim er, 1974), so enzymes o f g e n e ra lis e d

in t r a c e l l u l a r o r ig in such as th e a m in o tran sfe ra se s may be expected to be

re le a s e d in to th e u r in e on ly a f t e r c o n s id e ra b le and perhaps sev e re c e l l

l y s i s . Enzymes such as a lk a l in e phosphatase and le u c in e am inopep tidase ,

however, a re lo c a l is e d in th e b rush b o rder o f th e proxim al tu b u le o f

th e r a t , and as d e g en e ra tio n o f th e b rush bo rd er occurs e a r ly in

t o x i c i t y (S tro o and Hook, 1977), lo s s o f th e se enzymes may p ro v id e a

more s e n s i t iv e index o f cadmium-induced in ju r y , p a r t i c u la r ly s in c e such

d eg en e ra tio n may occur w ith o u t o b lig a to ry , i r r e v e r s ib le c e l l damage.

The r e s u l t s o f th e experim ents d e sc rib e d h e re do in f a c t in d ic a te th a t

a lk a l in e phosphatase was a f fe c te d b e fo re changes in o th e r enzymes were

a p p a re n t.

I t i s im portan t to in d ic a te th a t th e u r in a ry e x c re tio n o f enzymes

can v a ry g r e a t ly between in d iv id u a ls , and w ith tim e . The r e s u l t s o f

t h i s s tu d y a re only s ig n i f ic a n t on a group b a s is and enzym uria may n o t

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be u se fu l th e re fo re fo r id e n t i fy in g in d iv id u a ls a t r i s k . There i s a

tendency f o r r e n a l fu n c tio n t e s t s to r e tu r n tow ards normal when le s io n s

become ch ro n ic (S h a r r a t t and F ra z e r , 1963) and th e apparen t la c k of

s e n s i t i v i t y to change a f t e r th e long term d ie ta r y a d m in is tra tio n o f

cadmium may in d ic a te th a t u r in a ry enzyme e x c re tio n e x h ib its a s im ila r

p a t te rn .

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CHAPTER 3

AN INVESTIGATION OF THE INTER-RELATIONSHIPS

OF CADMIUM, ZINC, COPPER AND IRON IN THE RAT

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CONTENTS

In tro d u c tio n

E xperim ental

M a te r ia ls ; experim en ta l d e s ig n ;

a n a ly t ic a l m ethods.

R esu lts

Repeated p a re n te r a l a d m in is tra tio n o f

cadmium (d o se -re sp o n se s tu d y ) ; acu te

p a re n te r a l a d m in is tra tio n o f cadmium and

z in c ; re p e a te d p a re n te ra l a d m in is tra tio n

o f cadmium to r a t s given a d ie ta r y

supplem ent o f z in c ; d ie ta r y a d m in is tra tio n

o f cadmium and z in c .

D iscu ssio n

Page

69

70

75

114

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3 .1 INTRODUCTION

The p re se n t u n d e rs tan d in g o f th e m etabolism and to x ic o lo g y o f

cadmium inv o lv es th e c r i t i c a l o rgan concept d esc rib ed in s e c tio n 1 .3 .

Fundam ental to t h i s concept i s th e " th re sh o ld dose" f o r th e occu rren ce

o f ad verse e f f e c t s w ith in an organism , and th e b a s is f o r such a t h r e s ­

ho ld dose l i e s in th e in te r f e r e n c e o f cadmium w ith c e r ta in b iochem ical

p ro cesses o r s t r u c tu r a l components (N ordberg , 1976). The mechanism o f

cadmium to x ic i t y i s as y e t u n re so lv ed , and in view o f th e v a r ie ty o f

to x ic e f f e c t s produced, th e re may be s e v e ra l p rocesses in v o lv ed . The

developm ent o f such to x ic e f f e c t s , however, w i l l be in flu en c ed by th e

a b so rp tio n , d i s t r i b u t io n and e x c re tio n o f cadmium, and in tu rn th e

k in e t ic s o f th e m eta l may be a l t e r e d by in te r a c t io n s w ith o th e r m eta ls

in th e body.

The in te r a c t io n o f m e ta ls in b io lo g ic a l systems depends upon th e

p hysico -chem ical p ro p e r t ie s o f th e m etal io n s , and i t i s p o s tu la te d

th a t ions w ith s im i la r v a len cy s h e l l e le c t r o n ic s t r u c tu r e s a re l i k e l y

to be b io lo g ic a l ly a n ta g o n is t ic ( H i l l and M atrone, 1970). Zinc and

2+ 2+copper a re bo th e s s e n t ia l m eta ls and th e Zn and Cu ions have th e2+ .same e le c tro n ic s t r u c tu r e o f th e v a len ce s h e l l as th e Cd io n . Cadmium

may th u s in te r f e r e w ith th e involvem ent o f z in c and copper in v i t a l

fu n c tio n s . C onverse ly , th e t o x i c i t y o f cadmium may be reduced by sup­

plem ents o f z in c and copper. Cadmium can a lso in t e r a c t w ith i r o n , and

th e in te r - r e la t io n s h ip s o f a l l th e se m eta ls have been review ed

(Brem ner, 1974; Spivey-Fox, 1974; S an d stead , 1976),

S ev e ra l mechanisms u n d e r lie th e in te r - r e la t io n s h ip s o f t r a c e

e lem en ts , and th e y may be m ediated a t s e v e ra l s ta g e s , e .g . a b s o rp tio n ,

d i s t r i b u t io n and e x c re tio n . I n te ra c t io n s o f cadmium w ith z in c and

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copper may be a consequence o f t h e i r isomorphous rep lacem ent in b io ­

chem ical p ro c e sse s , b u t a com plete u n d ers tan d in g o f th e se i n t e r ­

r e la t io n s h ip s must in v o lv e a r o le fo r th e m e ta llo th io n e in s , s in c e

th e se p ro te in s can b ind no t on ly cadmium but a lso z in c and copper

( s e e s e c t io n 1 .2 ) . Changes in th e d is p o s i t io n and b io a v a i l a b i l i ty o f

th e se t r a c e m eta ls may s ig n i f i c a n t ly modify th e to x ic resp o n se to

cadmium ( e i t h e r in c re a se o r d ecrease i t ) .

Experim ents were perform ed to in v e s t ig a te th e e f f e c t o f b o th

p a re n te ra l and d ie ta r y a d m in is tra t io n o f cadmium on th e hom eostasis o f

z in c , copper and iro n in th e r a t . The in flu en c e o f a z in c supplem ent

on th e developm ent o f t o x i c i t y and k in e t ic s o f cadmium was a ls o s tu d ie d .

3 .2 EXPERIMENTAL

M a te r ia ls

M etal s a l t s were o b ta in ed from BDH Chemicals L td . , P o o le , D o rse t.

Acids fo r atom ic a b so rp tio n sp ec tro sco p y were su p p lied by F isons

S c ie n t i f i c A pparatus, Loughborough, L e ic e s te r s h i r e . S u b s tra te s fo r

th e enzyme d e te rm in a tio n s were o b ta in ed from Sigma Chemical Company L td . ,

P oo le , D o rse t, except fo r 4 -n itro p h en y lp h o sp h a te which was from

BDH Chem icals L td . A ll chem ical compounds were o f A nalar o r e q u iv a le n t

g rade . Sodium p en to b a rb ito n e was produced by May and B aker, Dagenham,

E ssex .

E xperim ental D esign

( i ) The e f f e c t o f re p e a te d p a re n te r a l a d m in is tra tio n o f cadmium:

a dose-resp o n se s tu d y

Young male W istar a lb in o r a t s , i n i t i a l body w eight 130 - 150g,

were d iv id ed in to groups o f fo u r . Animals were g iven cadmium (0 .7 5 ,

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2+1 .5 and 3 .0 mg Cd /k g /d a y , as CdCl^ m 0 .2 ml s a l in e ) by subcutaneous

in je c t io n fo r 42 days, excep t fo r th e h igh dose group which was k i l l e d

a f t e r 18 days. C on tro l anim als were g iven d a i ly subcutaneous in je c ­

t io n s o f s a l i n e . S tandard ro d en t d ie t and w ater were provided ad l ib i tu m .

Animals were housed in d iv id u a l ly in g la ss m etabolism cages fo r

s e p a ra te c o l le c t io n o f u r in e and fa e c e s . 24 hour u r in e samples were

c o l le c te d on days 1 , 2 , 3 , 4 , 10, 15, 16, 18, 19, 21, 23, 24, 25, 28,

30, 31, 36, 37, 38, 39 and 42. At th e te rm in a tio n o f th e experim en t,

anim als were a n a e s th e tis e d w ith sodium p e n to b a rb ito n e . L iv er and k id ­

neys were removed and w eighed, plasma was sep a ra te d from venous b lood ,

u r in e s were c e n tr ifu g e d to remove c e l l d e b r is , and samples o f each o f

th e se were s to re d a t -20°C f o r subsequent a n a ly s is o f m etal c o n te n t .

( i i ) The e f f e c t o f acu te p a re n te r a l a d m in is tra tio n o f cadmium and z in c

Young male W istar a lb in o r a t s , body w eight 180 - 200g were d iv id ed

in to groups o f f iv e an im als. One group was in je c te d in t r a p e r i to n e a l ly2+

w ith a s o lu t io n o f z inc su lp h a te (5 .0 mg Zn /k g as ZnS0^.7H20 ) .

24 hours l a t e r , anim als were t r e a te d as fo llo w s : -

C o n tro ls - s in g le subcutaneous in je c t io n o f is o to n ic s a l i n e .

2+s in g le subcutaneous in je c t io n o f 1 .5 mg Cd /k g as

CdCl2 . 2%H20 .2+

s in g le subcutaneous in je c t io n o f 1 .5 mg Cd /k g and

2+s in g le in t r a p e r i to n e a l in je c t io n o f 5 .0 mg Zn /k g .

s in g le subcutaneous in je c t io n o f i s o to n ic s a l in e and

2+s in g le in t r a p e r i to n e a l in je c t io n o f 5 .0 mg Zn /k g .

(z in c p re tre a te d ) - s in g le subcutaneous in je c t io n o f

1 .5 mg Cd2+/ k g . *

Group Is

Group 2:

Group 3 :

Group 4:

Group 5:

t2+In the results group 5 is designa ted : Zn p retrea ted

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W ater and s ta n d a rd ro d en t d i e t were p rovided ad l ib i tu m . Animals were

housed in d iv id u a l ly in ac id washed g la ss m etabolism cages fo r a 24 hour

c o l le c t io n o f u r in e . At th e te rm in a tio n o f th e experim ent, anim als

were a n a e s th e tis e d w ith sodium p e n to b a rb ito n e , and a venous blood sample

was c o l le c te d . L iv er and kidneys were removed and t i s s u e s , whole blood

and plasma were s to re d a t -20°C fo r measurement o f th e cadmium, z in c ,

copper and i ro n co n ten t by atom ic a b so rp tio n sp ec tro m etry .

( i i i ) The e f f e c t o f re p e a te d p a re n te r a l a d m in is tra tio n o f cadmium in

anim als g iven a d ie ta r y supplem ent o f z in c

Young male W istar a lb in o r a t s , i n i t i a l body w eight 150g, were

d iv id ed in to groups o f f iv e an im als. The anim als were prov ided w ith

w ater and s tan d a rd ro d en t d i e t ad l ib i tu m except fo r one group which2+

was a lso g iven a d ie ta ry z in c supplem ent (2 ,0 0 0 p .p .m . 2h as ZnSO^).

A fte r 7 day s, anim als were t r e a te d as fo llow s

d a i ly subcutaneous in je c t io n o f i s o to n ic s a l in e .

2+d a i ly subcutaneous in je c t io n o f 1 .5 mg Cd /k g as

CdCl2 .2%H20.2+

d ie ta r y z in c supplem ent (2 ,0 0 0 p.p .m . Zn ) and2+d a i ly subcutaneous in je c t io n o f 1 .5 mg Cd /k g .

2+d ie ta r y z in c supplem ent (2 ,0 0 0 p.p .m . Zn ) and

d a i ly subcutaneous in je c t io n o f is o to n ic s a l in e .

( z in c p re tr e a te d ) d a i ly subcutaneous in je c t io n o f

1 .5 mg Cd2+/k g . *

The tre a tm e n t con tinued f o r 28 d ay s, d u rin g which tim e body w eigh ts

and food in ta k e were m easured. At r e g u la r i n t e r v a l s , anim als were

p laced in m etabolism cages (days 1 , 2 , 4 , 10, 15, 20, 24 and 28) fo r

24 hour c o l le c t io n o f u r in e . At th e te rm in a tio n o f th e experim en t,

Group Is

Group 2:

Group 3:

Group 4:

Group 5:

t2 +In the results group 5 is designated : Zn pretreated

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anim als were a n a e s th e tis e d w ith sodium p en to b arb ito n e and venous blood

c o l le c te d . L iv e r and k idneys were removed and weighed. T is s u e s , whole

b lood and plasm a were s to re d a t -20°C f o r m etal d e te rm in a tio n s . U rinary

enzymes were measured on f r e s h , c e n tr ifu g e d u rin e samples and th e

rem ainder s to re d a t -20°C f o r m etal d e te rm in a tio n s .

( i v ) The e f f e c t o f d ie ta r y a d m in is tra tio n o f cadmium

Young male W ista r a lb in o r a t s , i n i t i a l body w eight 60g, in groups

o f 18, were fed s tan d a rd ro d e n t d ie t (pow dered), ad l ib i tu m . One group

2+o f anim als re c e iv e d a d ie ta r y supplem ent o f 75 p .p .m . Cd as CdCl2 ,2+

a n o th er group a combined supplem ent o f 75 p.p .m . Cd as CdCl2 and

2+300 p .p .m . Zn as ZnSO^, and a f u r th e r group re c e iv e d on ly a supplem ent 2+of 300 p .p.m . Zn as ZnSO^. Body w eights were r e g u la r ly re c o rd ed . At

in te r v a ls o f 8 , 16, 24, 36, 48 and 72 weeks, 24 hour c o l le c t io n s o f

u r in e were ta k e n u sin g g la s s m etabolism cages. Animals were an aes th e ­

t i s e d using sodium p e n to b a rb ito n e and venous blood c o l le c te d . L iv e r and

kidneys were removed and w eighed. F resh , c e n tr ifu g e d u rin e sam ples were

used fo r enzyme d e te rm in a tio n s and sam ples o f u r in e , whole b lo o d , plasma

and t i s s u e s were s to re d a t -20°C fo r m eta l d e te rm in a tio n s .

A n a ly tic a l Methods

U rinary c r e a t in in e , a lk a l in e ph o sp h atase , y -g lu ta t iy l t r a n s p e p tid a s e

and le u c in e am inopeptidase were measured as d e sc rib e d in s e c t io n 2 . 2 .

M etals were measured by atom ic a b so rp tio n sp ec tro m etry u s in g an

IL 353 sp ec tropho tom eter ( In s tru m e n ta tio n L ab ora to ry I n c . , L ex in g to n ,

M ass .) , w ith m icrosam pling accesso ry and background c o r re c t io n .

M etals in T issu es

The c o n c e n tra tio n o f cadmium, z in c , copper and iro n in l i v e r and

k idney was m easured a f t e r d ig e s t io n o f 0 .5 g t i s s u e w ith a m ix tu re o f

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n i t r i c /p e r c h lo r i c a c id s , by th e method o f Delves e t a l . (1971).

This method g ives re c o v e r ie s of >98% fo r cadmium, z in c , copper and

i ro n a t a c o n c e n tra tio n o f 40 yg/100 m l, and a c o e f f ic ie n t o f v a r ia ­

t i o n o f < - 8% a t a c o n c e n tra tio n o f 10 yg/100 ml. P r a c t ic a l d e ta i l s

a re provided in Appendix I I .

M etals in Blood and Plasma

The c o n c e n tra tio n o f cadmium in whole blood and plasma was d e te r ­

mined using th e m icro-sam pling method o f Delves (1 9 7 0 ), which gave a

reco v ery o f >93% - 6% a t th e range o f c o n c e n tra tio n s m easured. Plasma

z in c and copper were measured fo llo w in g d i lu t io n w ith w ater to remove

in te r fe re n c e by plasma p ro te in s . Plasma iro n was determ ined a f t e r

removal o f haem oglobin by p ro te in p r e c ip i ta t io n to e lim in a te i n t e r ­

fe ren ce from h aem olysis , and e r ro rs due to enhancement o f l i g h t absorp

t io n o f iro n by sodium and potassium ions were avoided by a d d it io n o f

th e se c a tio n s to b lank and s ta n d a rd s . These methods gave re c o v e r ie s

o f >93 i 4%. F u r th e r p r a c t ic a l d e t a i l s a re provided in Appendix I I .

M etals in U rine

Cadmium in th e u r in e was measured by th e m icro-sam pling method

d esc rib ed above. U rinary z in c , copper and iro n c o n c e n tra tio n s were

measured by th e method o f s tan d a rd a d d itio n s (C h r is t ia n and Feldman,

1970) which gave re c o v e r ie s o f >94 £ 6% a t th e range o f c o n c e n tra tio n s

measured (Appendix I I ) .

S t a t i s t i c a l com parisons o f groups were made u s in g S tu d en ts t - t e s t

a n a ly s is o f v a ria n c e and m u ltip le range t e s t s .

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3 .3 RESULTS

( i ) REPEATED PARENTERAL ADMINISTRATION OF CADMIUM: Dose-Response Study

F ig u re 3 .1 shows th e in c re a se in body, w eight o f th e d i f f e r e n t groups

2+o f r a t s . A fte r day 18, th e h igh dose group (3 .0 mg Cd /k g /d ay ) were

k i l l e d because o f abscess fo rm ation a t th e s i t e s o f in j e c t io n , bu t a

d o s e -e f fe c t r e la t io n s h ip may s t i l l be seen . C on tro l r a t s gained s ig ­

n i f i c a n t ly more w eight over th e experim en tal pe rio d th a n any o f th e th re e

groups o f cadm ium -treated an im als. At th e te rm in a tio n o f th e experim ent

(6 w eeks), low and medium dose groups o f th e cadm ium -treated r a t s had

body w eights o f 78% and 67% o f th e c o n tro l an im als. Cadmium tre a tm e n t

a lso r e s u l te d in in c re a se s in l i v e r and kidney w eights r e l a t i v e to body

w eights (T ab le 3 .1 ) .

E f fe c ts o f Cadmium on L iv e r and Kidney C o n cen tra tions o f T race M etals

From Table 3 .1 i t may be seen th a t cadmium accum ulated in th e l i v e r

and k idney , in r e l a t i o n to dosage. S im ila r ly , th e re was an accum ulation

o f z in c in th e l i v e r and to a l e s s e r e x ten t in th e k idney , and accumu­

l a t i o n o f copper in b o th t i s s u e s . The a d m in is tra tio n o f cadmium had no

s ig n i f ic a n t e f f e c t on th e c o n c e n tra tio n o f i ro n in th e l i v e r , bu t

decreased th e c o n c e n tra tio n in th e k idney in low and medium dose g roups.

E f fe c ts o f Cadmium on Blood C o n cen tra tio n s o f Trace M etals

The p o st mortem blood c o n c e n tra tio n s o f t r a c e m eta ls fo llo w in g

tre a tm e n t w ith cadmium a re shown in Table 3 .2 . The c o n c e n tra tio n o f

cadmium in whole blood was h ig h e r in th e m iddle dose group ( 1 .5 mg/kg)

compared to th e low dose group (0 .7 5 m g/kg), and as th e c o n c e n tra tio n

fo r th e h igh dose group was o b ta ined a f t e r 18 days tre a tm e n t, th e

b lood c o n c e n tra tio n o f cadmium probab ly shows a d i r e c t d o se -re sp o n se .

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Page 80: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

A d m in is tra tio n o f cadmium a t a l l dose le v e ls in c reased th e plasm a

c o n c e n tra tio n s o f z in c (40 - 50%) and copper (60 - 80%), bu t th e con­

c e n tr a t io n o f i ro n was m arkedly d ecreased (60%).

E f fe c ts o f Cadmium on th e U rin ary E x c re tio n o f Trace M etals

The u r in a ry e x c re tio n o f cadmium, z in c , copper and iro n was

measured a t in te r v a l s th roughou t th e experim en t, and th e notew orthy

r e s u l t s a re summarised in Table 3 .3 . R e su lts a re expressed as y mol/mmol

c r e a t in in e , to avoid e r ro r s due to incom plete c o l le c t io n o f 24 hour u r in e

sam ples. E x c re tio n o f cadmium in u r in e in c reased w ith th e le v e l o f

dosage and th e d u ra tio n o f a d m in is tra tio n . The lo s s o f cadmium in to th e

u r in e in c re a se d s ig n i f i c a n t ly a t days 40 (low d o se ) , 28 (medium dose)

and 15 (h ig h d o se ) . In th e h igh dose group o n ly , th e re was an i n i t i a l

peak o f e x c re tio n a t 48 hours a f t e r beg inn ing cadmium a d m in is tra tio n .

U rin a ry e x c re tio n o f z in c , copper and i ro n in c reased in a l l cadmium-

t r e a te d groups a t tim es th a t p robab ly r e f l e c t th e development o f r e n a l

damage. T reatm ent w ith cadmium a t 3 .0 mg/kg caused an in c re ase d e x c re ­

t io n o f copper w ith in 48 h o u rs .

Enzymuria R e la ted to U rinary Cadmium E x c re tio n

The developm ent o f enzym uria in r a t s t r e a te d w ith cadmium has been

d e sc rib e d p re v io u s ly ( s e c t io n 2 .3 ) . F ig u re 3 .2 shows th e e x c re t io n o f

a s p a r ta te a m in o tra n s fe ra se , a lk a l in e phosphatase andy-glutam yl t r a n s ­

p e p tid a se in r e l a t i o n to th e lo s s o f cadmium in to th e u r in e f o r th e

medium dose group. U rinary cadmium c o n c e n tra tio n s d id not p a r a l l e l th e

i n i t i a l phase o f enzymuria a t 48 h o u rs , b u t th e d ram atic in c re a s e o f

cadmium e x c re tio n a t l a t e r s tag e s was accompanied by s ig n i f ic a n t in c re a se s

in th e e x c re tio n o f a l l th e enzymes m easured.

Page 81: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 3.

3 Th

e E

ffec

ts

of R

epea

ted

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ente

ral

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inis

trat

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of Ca

dmium

on

the

Urin

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£ • • • • . • • . • • • . • •O O o o o o o o O o o o O o o£hw +1 +1 +1 + i +1 + i +1 +1 +1 -H -H +1 +i -H

1 . o !>» <r 00 CM CO 00 o co CM 00 COi". 1 00 vo 00 pH r>. o VO Os in

• • • • • • • • • • • • • •o o O o o o rH rH o o rH O O rH

a * •it * * •J* a■Jc ■}c * V •it a •5c i t■*C * * * i t i t ■it i t

rH o os in in <r pH Os rH o CO 00 VO COU rH CM pH CM o pH CO CM rH <r pH rH CM0) • • • • • • • • • • • • • •(X o o O o o o o O O o o o O ocuo +1 +1 +1 +1 +1 + 1 -H +1 +1 -H +1 +1 +1 -Ho rH in pH vo O o in CO in o

CO CO on o CO CO rH Os CM Os 00 vo• ' • . . • • • • • . • • • •

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• • • . • • • • • • • • • •|—1 H rH CM rH pH rH CM rH rH rH rH CM

pH Os CO OV Os Ov o <r <rO pH CM O o CO O <r CM pHO O o o o O O rH O CM

rH • • • • • • • • • •3 o o O o o o o o O o o

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Page 82: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Figu

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Page 83: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

( i i ) ACUTE PARENTERAL ADMINISTRATION OF CADMIUM AND ZINC

The acu te a d m in is tra tio n o f cadmium (1 .5 m g/kg), o r z in c (5 .0 mg/kg)

d id no t have any s ig n i f ic a n t e f f e c t upon th e body o r organ w eights o f

th e r a t s 24 hours l a t e r ( r e s u l t s n o t shown).

E f fe c ts o f Cadmium and Zinc on L iv e r and Kidney C o n cen tra tio n s o f

Trace M etals

In Table 3 .4 i t may be seen th a t a s in g le in je c t io n o f cadmium

caused an accum ulation o f th e m eta l in l i v e r and k id n ey s. Animals g iven

cadmium and z in c s im u ltan eo u sly showed an in c re a se d uptake o f cadmium

in bo th o f th e se o rg an s , w h ile anim als p re tre a te d w ith z in c showed an

in c re ase d uptake o f cadmium on ly in th e l i v e r . The accum ulation o f

cadmium was a s s o c ia te d w ith in c re a se d c o n c e n tra tio n s o f z in c in bo th

l i v e r and k id n ey s. Zinc tre a tm e n t alone s im i la r ly caused in c re a se d con­

c e n tra tio n s o f z in c in l i v e r and k idneys. Only r a t s t r e a te d w ith cad­

mium alone showed an in c re a se d c o n c e n tra tio n o f copper in th e l i v e r ,

and th e re were no s ig n i f ic a n t changes in th e le v e l o f copper in th e

k id n ey s. The c o n c e n tra tio n s o f h e p a tic i ro n were u n a ffe c te d by any

tre a tm e n t, bu t z in c t r e a te d and cadmium (z in c p re tre a te d ) an im als showed

a decreased accum ulation o f i ro n in th e k idney compared to c o n tro ls and

cadmium and z in c t r e a te d r a t s .

E f fe c ts o f Cadmium and Zinc on Blood C on cen tra tio n s o f Trace M etals

The c o n c e n tra tio n s o f cadmium in whole b lood , and z in c , copper and

iro n in plasma a t th e te rm in a tio n o f th e experim ent a re shown in

Table 3 .5 . The sim ultaneous a d m in is tra tio n o f cadmium and z in c caused

in c re a se d c o n c e n tra tio n s o f cadmium in th e blood compared w ith o th e r

cadm ium -treated g roups. Animals g iven cadmium and cadmium (z in c p r e t r e a te d )

Page 84: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

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Page 85: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

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5 Th

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ffec

ts

of A

cute

P

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tera

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dmin

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Cadm

ium

and

Zinc

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d. CM ON ON in <r• • • • •LO CO CO o <r<r CO CM <r co

•dPcd cd cd

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3. H ON 00 CM 00• • • • •

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4h p ••pH o CD o■—pH • 4hO a •H •dg • ' TO CD3 w p• P cdCO CO £ CDcd cd P

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3 •H PCO pH P Pp cd CO £pH > •H O3 P oCO pH cdCD pH P ••P5 < CO cd

Page 86: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

had plasma c o n c e n tra tio n s o f z in c decreased by 25% and 20% re s p e c t iv e ly

compared to c o n tr o ls . Zinc a d m in is tra tio n alone caused a 25% in c re a se

in th e plasma c o n c e n tra tio n o f t h i s m e ta l, and th e sim ultaneous adm inis­

t r a t i o n o f z in c w ith cadmium preven ted th e cadmium-induced d ecrea se s

seen in o th e r groups. A ll cadm ium -treated groups showed an in c re a se d

c o n c e n tra tio n o f copper in th e plasma (14 - 40%) and a decreased con­

c e n tr a t io n o f i ro n (25 - 35%) compared to c o n tro ls . These changes were

most marked in anim als g iven cadmium and z in c to g e th e r . Zinc a lo n e d id

no t s ig n i f i c a n t ly in f lu e n c e plasma copper o r iro n compared to c o n tro ls .

E f fe c ts o f Cadmium and Zinc on th e U rinary E x c re tio n o f Trace M etals

24 hours a f t e r t re a tm e n t, anim als in je c te d w ith cadmium e x c re te d

sm all amounts o f th e m etal in th e u r in e , bu t th e re were no d if f e r e n c e s

betw een groups (T ab le 3 .6 ) . U rinary z in c e x c re tio n was s ig n i f i c a n t ly

in c re a se d in a l l z in c t r e a te d an im als, p a r t i c u la r ly th o se g iven z in c

a lo n e which showed alm ost a th re e fo ld in c re a se compared to c o n tro ls and

cadm ium -treated an im als. The a d m in is tra tio n o f cadmium and z in c in

i s o l a t i o n bo th caused a decreased e x c re tio n o f copper bu t no tre a tm e n t

had any e f f e c t upon th e u r in a ry e x c re tio n o f iro n .

( i i i ) REPEATED PARENTERAL ADMINISTRATION OF CADMIUM TO RATS GIVEN A

DIETARY SUPPLEMENT OF ZINC

F igure 3 .3 shows th e in c re a se in body w eight o f anim als over th e

ex p erim en ta l p e r io d . From Table 3 .7 i t can be seen th a t a t th e te rm in a ­

t i o n o f th e experim en t, c o n tro l and z in c - t r e a te d anim als had gained

s ig n i f i c a n t ly more w eight th a n any o f th e cadm ium -treated an im a ls . R ats

in je c te d w ith cadmium showed a decreased food consum ption, w h ile th e

d ie ta r y z in c supplem ent p robab ly in c re a se d consum ption. A ll cadmium-

exposed anim als had a decreased w eight g a in to food in ta k e r a t i o compared

Page 87: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 3.

6 Th

e E

ffec

ts

of A

cute

P

aren

tera

l A

dmin

istr

atio

n of

Cadm

ium

and

Zinc

on

the

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o o o o o S «• +CO CNcu ts £P o tort r-I

r-I ••CO O CU0) 4-1d ••»

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p • • • • • CU rtcu O o o O o r—1 P CUp , r-I rt Pf t +1 +1 +1 -H +1 < po COCJ vo r-I CN f t +CN <r r-I m • d CN• • • • • CU o £

r—I r-I r-I H rH £ P„ to•rl 40£•H £ •dP CUrt CU

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00 00 vo vo ov P rtO CN 00 ON r-I >> CU

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Page 88: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

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Page 89: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

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foll

ows:

Page 90: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

to c o n tro ls and z in c supplem ented an im als, a lthough th e z in c supplem ent

a lso decreased th e r a t i o compared to c o n tro l an im als. Cadmium caused

an in c re a se d organ to body w eight r a t i o fo r both l i v e r and k idney , and

anim als g iven on ly z in c showed a s im ila r in c re a se on ly fo r th e k idney

(T ab le 3 .8 ) . The in c re a se d k idney to body w eight r a t i o was more marked

in z in c-su p p lem en ted , cadm ium -treated an im als.

E f fe c ts o f Cadmium and Zinc on L iv e r and Kidney C o n cen tra tio n s o f

Trace M etals

Table 3 .9 shows th e d is p o s i t io n o f cadmium, z in c , copper and i ro n

in th e l i v e r and k idneys. Z in c -p re tre a tm e n t, and th e sim ultaneous

a d m in is tra tio n o f z in c to cadmium-exposed r a t s caused an in c re a se d r e te n ­

t io n o f cadmium in th e l i v e r b u t no t th e k idney . Both cadmium and z in c

tre a tm e n t produced in c re a se d c o n c e n tra tio n s o f h e p a tic and r e n a l z in c ,

and th e com bination o f cadmium and z in c made th i s e f f e c t more e v id e n t .

The cadm ium -treated group a lso had in c re a se d c o n ce n tra tio n s o f copper in

l i v e r and k idney , and in c re a se d h e p a tic i r o n , but d ecreased k idney con­

c e n tr a t io n s o f i ro n . Zinc a lone d id no t in f lu e n c e th e d i s p o s i t io n o f

copper o r i ro n , n e i th e r d id z in c supplem ents in f lu e n c e th e cadmium-induced

changes.

E f fe c ts o f Cadmium and Zinc on Blood C o n cen tra tio n s o f Trace M etals

Zinc d id no t have any e f f e c t upon th e c o n c e n tra tio n s o f cadmium

measured in whole b lood a t th e te rm in a tio n o f th e experim ent (T ab le 3 .1 0 ) .

Cadmium-treatment a lone s ig n i f i c a n t ly in c re a se d th e plasm a c o n c e n tra tio n

o f z in c by 30%, and copper by 130%, bu t th e c o n c e n tra tio n o f i r o n was

d ecreased by 40%. Z inc-supplem ented , cadm ium -treated anim als showed

an even g re a te r in c re a se in plasma z in c (85%), though th e e f f e c t on

plasm a copper was le s s marked (60%). P re trea tm en t w ith z in c d id n o t

Page 91: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

T able 3 .8 The E f f e c t s o f R epeated P a r e n te r a l A d m in is tr a tio n

of Cadmium on L iv e r and Kidney W eights o f R ats

Given a High D ie ta ry Supplement o f Zinc

T issue Treatm ent Organ Weight

(g )

Organ/Body Wt,

R a tio

L iv er C on tro ls

Cd2+

Cd2+ & Zn2+

2+Zn

2+Zn p re tr e a te d

13.45 i o .64

13.08 ± 0.41

12.43 ± 0 .36

13.46 ± 0.27

13.64 ± 0.32

3.94 i 0 .07

5.00 ± 0 .0 7 ad

5.00 ± 0 .1 2 ad

3 .98 i 0 .05

5.46 ± 0 .16ad

Kidney C o n tro ls 1 .87 + 0.05 0.55 + 0.01

Cd2+ 1.69 + 0.05 0 .65 + o .o i 1

Cd2+ & Zn2+ 1.87 + 0.09 0.76 + 0 .0 4 12+Zn 2.07 + 0 .07be 0.61 + 0 .01

2+Zn p re tr e a te d 1 .79 + 0.04 0.71 + 0 .01

A ll v a lu es a re means - S.E.M. o f 5 an im als.

S t a t i s t i c a l l y s ig n i f ic a n t d if f e r e n c e s (p< 0 .0 1 ) between groups

a re as fo llo w s :

2+ 2+ 2+ a: c o n tro ls ; b: Cd t r e a te d ; c: Cd & Zn t r e a te d ;

2+ 2+ d: Zn t r e a te d ; e : Zn p re tre a te d

Page 92: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 3.

9 Th

e E

ffec

ts

of R

epea

ted

Par

ente

ral

Adm

inis

trat

ion

of Ca

dmium

on

the

Con

cent

ratio

n of

Met

als

in th

e Li

ver

and

Kid

ney

of Ra

ts gi

ven

a H

igh

Die

tary

Su

pple

men

t of

Zin

crt

CM o\ inr-I o o o

HD • • 9 •P — O o o oO r-IP O + 1 +1 +1 + iw s3. ON r-I <r CM

as <r rH as• • • 9

f—1 CM CM rH

•d •drt rt

CM 00 ino O o oo O o o

P ClD ■ • * ■CD o o o oa t—1p ,/■n oc3 +1 +1 +1 + iwo

C3P- 00 CO CM 00o r-I H o

• • • 9

o O O o

rt•d

•d jart rt rt

r-I CO a \ r-IHD O O o O

o • • ■ 9

c r-I o o o a•H oCM e +1 +1 +1 + i

2. on CM <r<r CM in in• • • •o rH rH o

tOin ino t—i

fi HD ■ •3 o o

•H r-I X} 'de O p + i + i pert 3- as vo

O CM 1• •CO CO

+P CMP CO Pd H CMs O + +p p CM CMrt p •d Prt p o + CMP O CMH o •d

o

rt3 f-lCO rtCO >•H •HH

X) XS •dr t r t rt

r-I VO in VO < r inr—1 O o o o o• • • • • •O o o o o o

+1 +1 •H +1 +i +ivo co as VO CM voCO co 00 00 co n-

• • • • • •CM r-I o o rH o

*d 'd *d •drt rt rt rt

r-I < r . in CM Or-I o CM CM rH HO o o O O o

• ' • 9 • • •O o o o o o

+1 +1 +1 +1 + ! +1<r ON VO o\ voi—i r-I CO CO rH CO

• • • • • •o O o o o o

•drt r t r t rt rt<r < r in CO i—i r-lo o O o o O• 9 • 9 9 •o o o o a o

+i +1 -H +1 +i +1<r CM CM ON i—i oCM <r VO vo in VO• • • • • •rH o o o o o

jaCO 00 H 00i—i o r-l o

• • • •o o o o

•d XS+i P +1 + 1 p +1in VO m oVO o o o

• • • •CO CM CM CM

•d 'dr t . r tp + pr t CM r tr t CO P r tP r-i CM PP o + + Pr t p CM CM rtP p X3 P Pa p O + CM D,o CM

+ o •d +CM o CMP PCM CM

rtp•d•rlUi

•*drtp• rt

CO rtH Prt PE rt

•rl PP ar t

•• +i n CO CM

& PIH o CMo r-l

r-l ••9 O r t

E 4-1 • 9K,W CO X3

r t CDCO P

r t rt+t P r t

r t PCO Pp COr t P . +rt 3 CME O P

P CMrt HDp ••r t P *d

r tCO r tCL) ts •dd P CD

1—1 rt prt .O rt> rt

Pr-l r—1 Pr-l O< • +

o CMV P

• CMP a& v ^

JS CO +CO rt CMrt o -dp P o

4-1 r tP ••

HD rt o4-1

r-l 4-1O • r l •dE • d CDd- p

p r tCO P CDr t r t P

a PX5 •Hr t 4-1 +CO 9 •rl CMCO rt P •dr t r—1 HD oP .Q •Ha rt CO ••X P • a<d o r*N

rt l—laj p r-l COp CL) r t r-lr t •d O O

•rl PCO p P PP o CO Pr-l p •H O3 P oCO •• r tr t *d p ••P5 P CO rt

Page 93: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 3.

10

The

Eff

ects

‘of

Rep

eate

d P

aren

tera

l A

dmin

istr

atio

n of

Cadm

ium

on th

e

apCU

•H

x:bO•Hsc

sicu>

•HbOCOprtP5mo73OOrHPQ

CU. 3P

Pi•H03rHrtPCUS44oPio

•HPrtPPPicuoPioo

copM oB3.

00

r-1+iinCN<r

pcuCUao

CJ

rHOB■PL

VO

o+100

Ov|H

Op ;

•HCMr4OB3.

ino

+io\crvr4

OPi

•HCN

44O

£5cuScur~4cua3CO

•HS73rt

CJ

ofi3-

73Pi

PPi<USPrtcuPH

coi—•op

£oCJ

73rtO<N

+iCN

•VOCN

73art<rCN

+1ovin<r

rtoo•

r-i

+iCT\mCN

inoo

+1inin

+CN

73CJ

73rtinrH

-Hoo00CN

73rtonCN

-Hr-i

•r*4on

CU£xrto•

CN

■HC\

•voon

r4o

+!onin

ft

+CN

73O

r-4

CN

+1

CN<r

vo . •o

+i•

r-iCN

<uXXrtoo

o+1o<ron

73Pi

PiCN +cd CN

piCN

r4 •rt 73

73 P CUrt cu P

O S rt• cu

r-4 P Pcu p

-H •fl cup po o Cu

00 P • • +CN o 03 CN

44 is 3o CN

rt fHB r4 ••to O CUrt 44

73 t-4O D, 03 73rt rt CU

r4 H • P• cu CU rt

CN r-4 r-4 p cuQ . a rt p

+1 fi rt p3 . p CO

o o a +• 73 cu 3 CNcn Pi p O 3<r r t cu P CN

73 bO* ••

g p 3 733 O cu

•r4 3 cu • Kfi is 73

73 •• p CUrt 73 cu P

rt o 3 . .o rtcn cu

• p P|H o rH p

44 O•H • +

73 • o ONvo O 03 V/ 3, • o «-4 V CNvo i-4 rt CuCN XX fi v y cd

•HCU 3 03 +t-4 rt CU CNo O 73

,3 in 3 CJis CU

C\ 44 p ••O r4 o cu O

• 44o p4 • 44 • *\

o JS •H 73-H B 73 CU

3L w P00 • P rtm to co 3 CU

• rt rt pr-4 +1 a p

73 •H(U 03 44 +03 3 •r4 CN03 rt 3 73CU cu bD CJP B •H

73 iX 03 ••CU X cu XXP a) P tnr t r t |H ••kcu cu t-j top P CQ rt «Hp r t CU o O<u 3 •H Pp 03 r4 P PP . P rt CO 3

H > •H O+ 3 P aCN 03 r4 rt

Pi CU rH P ••CN P3 < CO rt

Page 94: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

have any e f f e c t upon th e cadm ium -induced changes in plasm a m e ta ls .

Animals g iven on ly th e z in c supplem ent showed an e lev a te d plasm a z inc

c o n c e n tra tio n (70%) b u t th e copper and iro n le v e l s in th e plasm a o f

t h i s group d id no t d i f f e r s ig n i f i c a n t ly from c o n tro ls .

E f fe c ts o f Cadmium and Z inc on th e U rinary E x c re tio n o f Trace M etals

The u r in a ry e x c re tio n o f cadmium, z in c , copper and iro n was measured

th roughou t th e exp erim en ta l p e r io d . The r e s u l t s a re summarised in

Tables 3 .11 and 3 .1 2 , and f u r th e r d e t a i l s a re shown in F ig u res 3 .A - 3 .6 .

Cadmium e x c re t io n was i n i t i a l l y low bu t in c re a se d w ith tim e in a l l cadmium

t r e a te d groups. Zinc d id no t have any s ig n i f ic a n t e f f e c t upon th e p a t­

t e r n o f e x c re tio n . A fte r about day 15, th e re was a marked in c re a s e in

th e u r in a ry e l im in a tio n o f cadmium, which p e r s is te d u n t i l th e te rm in a­

t io n o f th e experim en t. Cadmium tre a tm e n t decreased u r in a ry z in c ex cre ­

t io n i n i t i a l l y , b u t a t l a t e r s ta g e s ( a f t e r day 1 5 ) , th e re was an in c re a se d

lo s s o f th e m eta l in to th e u r in e . As cadmium tre a tm e n t c o n tin u ed , t h i s

e f f e c t became more s ig n i f i c a n t and by day 28, u r in a ry z in c e x c re t io n was

in c re a se d by over 100%. The z in c supplem ent d ra m a tic a lly in c re a se d z in c

e x c re tio n and z in c supplem ented cadmium t r e a te d anim als showed h ig h e r

le v e ls th a n th o se anim als re c e iv in g o n ly z in c . Z inc p re tre a tm e n t caused

an i n i t i a l h ig h e r e x c re tio n o f z in c compared to c o n tro ls and cadmium-

tre a tm e n t a lo n e . Cadm ium-treatment a ls o in c re a se d th e lo s s o f copper

and iro n in to th e u r in e in a s im ila r manner. Z inc d id no t have any s ig ­

n i f i c a n t e f f e c t upon th e e x c re tio n o f e i th e r m e ta l, though th e r e was a

c o n s is te n t tendency fo r z in c supplem ented anim als to e x c re te l e s s copper

compared to c o n tr o ls .

Page 95: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 3.

11

The

Eff

ects

of

Rep

eate

d P

aren

tera

l A

dmin

istr

atio

n of

Cadm

ium

on th

e U

rinar

y E

xcre

tion

od•HCN4-4Ortd0ea)HPa3CO>*uarta)•HQx:40•H

dcu>•HocortrtPid•HOrt•HCNTJrtrt

rtO4-4O

0 •*o 0 COrt rt r-4rt rt rt rt rt

00 00 CN CN *3’ o CO i—i Sr-4 i i—1 r-4 i—1 CN CO CN •rt• • • • • • • • dCO O o O O O rH o O rt

CN +1 +i +1 +1 +1 +i ■H +1 in •frtLO co CN o 00 CN o\ 4-4 0I—1 CN CN CO o\ CN in VO O rt• • • • • > • • rtCN CN CN r—4 CN 00 in CN ■ 0S r t

■ rtw 0• r t

0 CO PT ) 0r t r t -H •• +rt r t r t CO CN

O CO o\ O r-l 00 O 00 co £ dCN r-4 r—4 CN <r O d o CN

• • • • • • • • rt r—lLO o o o O O o o o 0 r-4 • •t—4 e O 0+1 +1 +1 +1 +1 +i +i +1 4-1

0 99*CO 00 o\ vo <r r-i 00 00 r t CO •do o o CN vo CN <r o r t r t 0

!>J • • • • • • • • r tr t 1—1 i—4 rH rH iH in CN co 0 rtQ 0 rt 0

d rt rtr—4 rt

0 rt CO0 o > a +r t r t d CN

VO in VO cd r t r t r—1 o do o o O'v CO CN in 00 r—4 rt CNo o o O fH 00 00 o < . 40

• • • • • • • • ••<r o o o o O o o o d *d

• 0+1 +1 +1 +1 +1 +1 +i +1 0r t

0>>99*

in i n i n vo CN o r—4rt

•H►>rt AJ

0o o o 00 rH i n 00 O d 0 r t

■ ■ • • • • • • • rl , o r to o o i—4 rH <r 00 CN r t 0

r t / '-s r t0 r—4 r tr t Oc • +

r t r t r t O CNCO CN t—4 rt r t r t do O O r-l rH 00 CN r t V CNo o O i—1 H CN CO r-4 r t p

• • • • • • • d v >o o o O o O o O •H

t—4 r t CO ++1 +1 +1 +1 +1 +1 +1 +1 d 0 CNo •d

CO CN CN CO CO 00 C \ CO r-4 d CJo o O F^. <r 00 CN O 0

• • 9 • • • • • g rt ••o o o r-4 r—4 CN CO CN g 0 o

4-1r-4 lHO • r l *d6 *d 0

• d •d : p . rt0 0 rt rt

+ rt + r t CO d 04-> CN rt CN rt rt r t rtr t r t 0 10 c 0 o rtcu CN rt i—I CN rt •d •rte + rt O + + r t 0 4-4 +r t CN cS 0 rt CN CN 0 CO •H CNrt T3 rt rt TO d rt CO d *d0 O + a rt o + CN P 0 40 ort CN o CN rt •rlE-4 TO + o • a + P CO ••

O CN o CN Xrt d 0CN CN r-4

0 r-4 COrt rt r-4rt o O

• r l rtg CO rt rt

t—4 3 rt CO drt •H o t—i •r-4 ort e rt d r t o0 ' a •r-4 CO rtS rt CN 0 rt ••

o P i CO rt

Page 96: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 3.

12

The

Eff

ects

of

Rep

eate

d P

aren

tera

l A

dmin

istr

atio

n of

Cadm

ium

on

the

Urin

ary

Exc

reti

on

aG•HIN4-1O

e!cu6CUHrta3C/0

8Gcu•HQ•s•Hsc

GCD>•H40COGCtJ(GG

•rl

GOGM73§

■ 8P.aoomo

00(N

in

&Q

G<U6Grt(UGH

r—IrtGCUS

OSO

00CO

CO r—I •o

+1oo00

inr-l•O

+1Osin

ino•o

+i!■>•vo

70rtCNVO

nrto<r

o\inin

o+l0000

70rto\CN

o+1<rCN

VOOO

+1Os00

CO I-IoG£oo

G<uartoo

+CN70O

COr-

o-Hooin

ONCN

mo

•HOoCN

CNCN•O

+1oON

m

ON

+ON

GCNc3

VOO

+1 +1 -H +1inoo

*73O

O+ 1

CNrH

rH|H

O+ 1<r<r

curtCTirH•O

+1asON

ON

+ON

GCN

73rtooo

+1vo00

73 73 73rt rt rt<r m ovo <r o <r

o-Hooo\

ooON

o-Hoo00

oo

+tr-'-vo

73CUGrtcuGGCUGrt

+ON

GCN

CNO•o-HONin

ino•o

-H<r

voO•o

-Hooo

voo•o

-H00vo

corHOGGGO

CJ

GOGM

73rtOOO•O

-HooOs

oooO

-Hin

aso•o

-Hn-in

inoo

•Hoo

+ON

73a

73rtoo

+1Os

00oo

+1<rin

|H

o+1oVO

CNoo

+1ovo

+ON

GCN

v3+ON

73O

•co

73 |Hrt rt

00 <r Eo o •H

• • Go o rt

+1 -H in 73CU

VO VO 4-1 Gm as o r t

• • CUo o • G

s G• CU

w G• rtcn

+-H •• CN

CO GVO 00 CO £ INO o G O

• • r t rHo o cu rH ••

E O cu+1 -H 4-1

cuCN G CO 73in in rt rt CU

• • Go ' o CO a> rt

cu G cuG rt G

rH Grt CO> rt +G CN

rH O G00 CO i—1 G tNo o <! 40

• •o o G 73• a)

+1 -H CU <u • «vG £ 73

vo oo •H G CUI'- I''. G cu G• • •H G rto o G CU

r t Ga> rH GG Oa ■ +

O CNV G

VO in G CNo o rt rt

• • G Wo o •H

G CO ++1 ■H 3 cu CN

o 73Os <r rH G ovo vo O CU

• ■ g G • •o o g CU o—. 4-1

rH 4-1 • •vO •H 73E 73 CU

73 ■p. GCU G rtG CO G <urt rt rt Gcu a GG 73 •r-l

+ G CU 4-1 +ON CU CO •H ON

G G CO G 73CN rt cu 40 CJ

G •H+ rt CO • •CN KG cu !>>

CN |Hcu rH COG r t rHrt o O

•H GCO G GG CO G|H •H OG G OCO r tcu G ••(G cn rt

Page 97: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

urin

e co

pper

(%

of co

ntro

ls)

urin

e zi

nc

(% o

f^co

ntr

ols

)

u rin ary L n -ieL iu n u i ^ iiiu , uupptjr aim n u n a iu e i ncpcai-eu ra i.cn k ci.a i

A d m in is tra tio n o f Cadmium to R ats Fed a High D ie ta ry Supplement o f Zinc

Zinc

6 0 0

5 0 0

4 0 0

3 0 0

200

100

10 20 30Day o f a d m in is tra tio n

Copper

3 0 0

200

100

10 3020

Day o f a d m in is tra tio n

Page 98: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

urin

e iro

n (%

of co

ntr

ols

)

Iro n

100

5 03010 20

Day o f a d m in is tra tio n

2+Cd - t r e a te d2+ 2+Cd and Zn - t r e a te d

■------■ Zn^+- t r e a te d2+A----- A Zn -p r e t r e a te d

Page 99: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

E f f e c t s o f Cadmium and Z inc on th e U rin ary E x c r e tio n o f Enzymes

The u r in a ry e x c re tio n o f a lk a l in e p h o sphatase , y -g lu tam y l t r a n s ­

p e p tid a se and le u c in e am inopeptidase was s ig n i f i c a n t ly in c re a se d w ith in

48 hours o f cadmium tre a tm e n t commencing. The p re tre a tm en t o r sim ul­

taneous a d m in is tra t io n o f z in c p reven ted o r decreased t h i s e f f e c t

(T ab les 3 .13 - 3 .14 and F ig u re 3 .7 ) . A fte r t h i s i n i t i a l phase o f enzym­

u r i a , v a lu es re tu rn e d to normal u n t i l around day 15 when a l l cadmium-

t r e a te d groups o f anim als showed e le v a te d le v e ls o f enzyme e x c re t io n

in to th e u r in e . This p a t te r n con tinued u n t i l th e te rm in a tio n o f th e

experim ent. Z inc supp lem en ta tion d id no t a l t e r th e developm ent o f th e

second phase o f enzym uria.

( iv ) DIETARY ADMINISTRATION OF CADMIUM AND ZINC

The d ie ta r y a d m in is tra t io n o f cadmium, z in c o r a com bination o f

cadmium and z in c d id no t s ig n i f i c a n t ly a f f e c t th e growth r a t e o f r a t s

over a 72 week ex p erim en ta l p e rio d (F ig u re 3 .8 ) .

E f fe c ts o f Cadmium and Zinc on L iv e r and Kidney C o n cen tra tio n s o f

T race M etals

F ig u re 3 .9 shows th e g radual accum ulation o f cadmium in th e l i v e r

and k idney . The c o n c e n tra tio n o f th e m eta l in r e n a l t i s s u e was ap p ro x i­

m ately tw ice t h a t in th e l i v e r . Both th e l i v e r and kidney showed d ecreased

c o n c e n tra tio n s o f cadmium a f t e r 48 weeks o f cadm ium -treatm ent. A z in c

supplem ent d id no t in f lu e n c e th e d is p o s i t io n o f cadmium in th e s e two

o rgans. Z inc a ls o accum ulated in th e l i v e r and k idney a f t e r cadmium

exposure (T ab le 3 .1 5 ) . The h e p a tic z in c c o n c e n tra tio n was in c re a se d by

30% compared to c o n tro ls a t 8 weeks, and th e r e a f t e r rem ained h ig h e r

u n t i l th e te rm in a tio n o f th e experim ent. The sim ultaneous a d m in is tra t io n

Page 100: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

T able 3 .1 3 The E f f e c t s o f R epeated P a r e n te r a l A d m in is tr a tio n o f Cadmium

on th e U rinary E x c re tio n o f Enzymes in Rats g iven a High D ie ta ry Supplement o f Zinc

TWr A lk a lin e y-G lutam yl LeucineDay Treatm ent , ^ ,phosphatase t r a n s p e p tid a se am m opeptidase

C o n tro ls 10 .5 + 0 .3 52 + 2 A .l + 0 .1

Cd2+ 12.6 ± 0 . 7aCe 76 ± ^acde 5.2 ± 0.A61 Cd2+ & Zn2+ 9.9 + 0 .1 53 + 3 A.2 + 0 .2

Zn2+ 11.3 + 0 .2 60 ± 1 A.6 + 0. A2+Zn p re tre a te d 9 .A + 0 .3 57 ± 1 3 .9 + 0 .2

C o n tro ls 9 .7 + 0 .3 5A ± 3 A .l + 0 .2Cd2+ 1A.2 ± 0.AaCde 81 ± .acdeA 6.8 + 0.Aade

2 Cd2+ & Zn2+ 10.1 + 0 .6 62 ± 3 6 .0 ± 0 . 2ade

Zn2+ 10.7 ± 0 .3 62 ± 1 A .2 + 0 .2Zn2+ p re tre a te d 9.3 + 0 .2 59 + 1 A .l + 0 .1

C o n tro ls 8.6 + 0 .6 56 + 2 A .l + 0 .2Cd2+ 7 .A + 0.Ad 6A + A .8 + 0 .2 d

A Cd2+ & Zn2+ 7.9 + 0.Ad 61 + 2 A.A + 0 .2Zn2+ 10.1 + 0 .3 59 + 2 A. A + 0 .2

Zn2+ p re tre a te d 7.9 + 0 .5 d 56 ± 2 3 .8 + 0 .2

C o n tro ls 9 .A + 0 .3 61 + 1 A. 5 + 0 .1Cd2+ 11.9 + 1 .1 58 + 2 A .6 + 0 .2

10 Cd2+ & Zn2+ 10.6 + 0. A 55 + 2 A.O + 0 .1Zn2+ 10.0 + 0 .5 57 + 2 A .l + 0 .1

2+Zn p re tr e a te d 11.2 + 0 .5 53 + 3 A.3 + 0 .1

R esu lts a re expressed as U/mmol c r e a t in in e . A ll v a lues a re means * S.E.M. o f 5 an im als .

S t a t i s t i c a l l y s ig n i f ic a n t d if f e re n c e s ( p < 0 .0 1 ) between groups a re shown: a : c o n tro ls ; b : Cd2+ t r e a te d ; c : Cd^+ & Zn2+ t r e a te d ;

d: Zn2+ t r e a te d ; e: Zn2+ p r e t r e a te d .

Page 101: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

T ab le 3 .1 4 The E f f e c t s o f R epeated P a r e n te r a l A d m in is tr a tio n o f Cadmium

on th e U rinary E x c re tio n o f Enzymes in Rats given a High

D ie ta ry Supplement o f Zinc

T)pV TTPafmpnt* A lk a lin e y-Glutamyl Leucinephosphatase tra n s p e p tid a s e am inopeptidase

C ontro ls 7 .6 + 0 .2 54 + 1 4 .1 + 0 .1Cd2+ 12.7 + 0 . 5ad 57 + ^ad 5 .4 + 0 .2 ad

15 Cd2+ & Zn2+ 11.9 + 0 . 6ad 55 ± ^ad 6 .0 + 0 .2 abdSZn2+ 8.4 + 0 .2 55 ± 2 4 .2 ± 0 .1

9+Zn^ p r e t r e a t ed 13.2 + 0 . 7ad 52 ± ^ad 5.1 ± o . i ad

C on tro ls 7 .8 + 0 .4 57 + 2 3 .9 + 0 .2Cd2+ 14.0 + 0 . 5ad 84 + 3ad 6 .6 + 0 . 2ad

20 Cd2+ & Zn2+ 13.9 + 1.0** 82 + 4ad 6 .4 + 0 .4 adZn2+ 8.6 + 0 .5 57 + X 4 .0 + 0 .1

9+Zn"1 p re tre a te d 14 .4 + 0 . 6ad 80 + 3ad 6.7 + 0 .2 ad

C on tro ls 8 .6 + 0 .2 53 + 2 4 .0 + 0 .2Cd2+ 18.3 + 1 . 5ad 88 + 4ad 8.1 + 0 . 5ad

24 Cd2+ & Zn2+ 18.1 + 0 . 8ad 92 + 3ad 6 .9 + 0 .3 abd

Zn2+ 8.9 + 0 .2 58 + 1 4 .5 + 0 .1

Zn2+ p re tre a te d 18 .8 ± 1 .2 ad 88 + . ad 4 7.0 ± o . i ad

C on tro ls 9 .2 + 0 .2 60 + 1 4 .2 ± 0 .2

Cd2+ 18 .5 + 0 . 7ad 95 + ,ad6 7 .4 ± 0 .3 ad

28 Cd2+ & Zn2+ 18.2 + 0 .9 ad 93 + ,ad4 7 .2 ± 0 .3 ad

Zn2+ 10.9 + 0 .2 55 + 2 4 .3 ± 0 .1

Zn2+ p re tre a te d 17.9 + 0 . 7ad 85 + 4ad 7 .2 ± 0 .3 ad

R esu lts a re expressed as U/mmol c r e a t in in e . A ll v a lu es a re means - S.E.M.

o f 5 an im als .S t a t i s t i c a l l y s ig n i f ic a n t d if f e r e n c e s (p < 0 .0 1 ) between groups a re shown:

9+ 9+ 9+a: c o n tro ls ; b: Cd t r e a te d ; c: Cd & Zn t r e a te d ;9+ 9+d: Zn t r e a te d ; e: Zn p r e t r e a te d .

Page 102: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Figu

re

3.7

Urin

ary

Exc

retio

n of

Alk

alin

e Ph

osph

atas

e af

ter

Rep

eate

d P

aren

tera

l A

dmin

istr

atio

n

T3dPrtdpp •d1 d

+ pT3 CM •d rt0) £ d dP N p Prt rt pd T3 d dp Pi P Pp1 rt p■ cxI1

+ +•

+1

+CM CM CM CM

•d Pi £o CJ N

1 1 1 !aPi•HCSJ

4-1OPdCDedrHaadco

PrtPd•HQrtPid>•HOCOPrtP3

'H

rto4-tO

( siojljuoo jo %) uoTjaaoxe eui/Czue jCaruxan

Day

of ad

min

istr

atio

n

Page 103: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Figu

re

3.8

Body

W

eigh

t Ga

in

afte

r D

ieta

ry

Adm

inis

trat

ion

of Ca

dmium

an

d Z

inc

TSd)4-1rt(Uu4-1I

+•o CM TJ0) c QJ4-1 CM 4-»rt rt

CO CL) •d CL)pH U S3 ShO 4-> rt 4->u 1 14-1 + + +CM CM CMO T5 Go CJ O CM

1 1 1 1

m o w o ioh- (0 "'t co

(9 ) q.q9xaM jCpoq

Time

(m

onth

s)

Page 104: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Figu

re

3.9

Acc

umul

atio

n of

Cadm

ium

in Li

ver

and

Kid

ney

afte

r D

ieta

ry

Adm

inis

trat

ion

of

T3(UPrtd)UPI+TJ CM

d Pi CMP

rt d U P I+ +CM CM 'd *d o o

d

•H

TJPirt

II

TJd)Prtd)PiPI

•d CMd) CJp CMrtd •du pip1 rt1

+ +CM CMra •do o

Pid>•H

O <

op;•HCM•dPirt

rtO

O

oco

o

o

oco

oCM

|_____________I_________________ L.__ _________ I---------------------- 1------O CO <0 ^ CMr - o o o o

(9/loiur/) uo-prj^uaouoo +^P0

Time

(w

eeks

)

Page 105: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 3.

15

The

Eff

ects

of

Die

tary

A

dmin

istr

atio

n of

Cadm

ium

and

Zinc

on

the

Con

cent

ratio

n of

Zinc

in

the

Liv

er

CUrt

ft

rt

r t• d ■d • d o

r t r t r t r t •Ho - 0 0 H CM O ' O ' CM f to O 1—1 O O o o • o r t

• • • • • • • 9 j>CM o o O o o o o a Hr>* cu

+ 1 -H + 1 -H + 1 + 1 -H •H CO

1—1 CM r>* i n O ' CM i n VO oi n l^» v o O ' CO i n i n CO

• • • • • 9 • • Q)o O o o o O o o ■P

luoTi T3 • d • d • Hr t r t r t r t i—l

CM o CO I—I I—1 CM O ' CM f tO o o o o O o O d

• • • • 9 • • • • d0 0 o o o o o o o o< r

+ 1 + 1 + 1 + 1 + 1 + 1 -H -H COi—l

CM o i n o CO c o O ' v o r ti n v o i n c o o o O ' g

• • 9 • • • • • •Ho o O o o o o o rt

r t

• d • d * d • d COr t r t r t r t

< r r^ . c o CM l—l r - i CM l—l 4-1o o o O o o o o . O

• • • •• • • ■ • •v o o o o o o o o o ■ ••CO s CO

+ 1 + 1 + 1 + 1 + 1 + 1 •H + 1 • gS~\ w oCO 0 0 CO i n o o G \ 1—1 • Hft < r v o v o i n CO i n i n O ' CO l—l •0) m • • • • 9 • • o • dCU o o o o o O o o - H 4-1 cui* p

v _ / CO CO rt• d • d • d • d rt rt cu

CL) r t r t r t r t r t Pg < r i n CM I—I CM CO CO CU cu p

• H o o o O o O O o . 6 pH • • • • • • 9 • r t +

< f o o o o o o O o CL) CMCM P CO r t

+ 1 + 1 + i + 1 + 1 + 1 -H + 1 r t f t•3

EM

VO Ov o O ' 1—1 0 0 i—l CM COf-*o . .

< r i n v o O ' O ' o i n O ' Q) ft„ • d• - • • • • • • 9 r t 4 0

o o o o o o o O i—ir t r t> cu • d

• d d i • d • d cu cur t r t r t r t r - l & p

r - i CM CM r—1 I—I CM i—i r - l i—l p r to O O o o O o o < <u a )

• • • • • • • • f t PVO o o o o o o o o pt—1 • S~s

+ 1 - H + 1 + 1 + 1 + 1 + 1 + 1 f t r - l +O CM

CO a\ O i n C \ o \ CO CO • rt< r i n v o O ' CO o i n O ' ft O CM

• • • • 9 • • • CO yo o o o o o o o CU c dft ft

4-1 v - ' +• d • d • d • d CMrt r t r t r t 4 0 CO • d

< r CO i n I - l r - l i—i CM CU oo O o o o O o o i—l Q9 9 • • 9 ■ • • r t r t • •

0 0 O O o o o O o o P cu ocu P

+ 1 + 1 + 1 + i + 1 + 1 + 1 + 1 g cu4-1

o - 0 0 a\ 0 0 CO o o\ l—l 4-1 • do i n i n CO CO c o CO CO o •H a )

• • • • 9 • • • g • d po o o o o o o o ■ rt, r t

p <uCO rt Prt rt p

a+ + • d •H +

f t CM CM cu 4-1 CMrt CO rt co rt CO •H • d<u r-1 CM I—I CM CO rt og o + + o + + cu 4 0f t f t CM c d CM p CM c d CM P • r l • •r t f t • d c f t • d r t f t CO . acu r t a + CM r t o + CM Kf t o CM o CM cu t>>

H o • d o • d l—lo o <u r - l CO

p r t i—irt O o• r l p

CO P pa ) P CO r t0 p a) H • H oCO a) r t r t P aCO > * d CO rt

•H •H • H cu P • •H ft ft CO rt

Page 106: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

o f z in c d id no t a l t e r th e cadmium-induced changes in z in c d is p o s i t io n . -

The z in c supplem ent a lone d id n o t in f lu e n c e th e accum ulation o f z inc in

th e l i v e r . In th e k id n ey , no s ig n i f ic a n t changes were ap p aren t u n t i l

16 weeks when cadm ium -treated anim als showed a r e n a l z in c c o n c e n tra tio n

about 30% h ig h e r th a n c o n tro ls . This change p e r s is te d th roughou t th e

experim en t. A gain, th e z in c supplem ent had no e f f e c t upon th e r e n a l

c o n c e n tra tio n o f z in c .

Cadm ium-treatment a ls o caused th e accum ulation o f copper, m ainly

in th e k idneys (T ab le 3 .1 6 ) , b u t betw een 16 - 36 weeks anim als g iven

o n ly cadmium a lso showed an in c re a se d l i v e r copper c o n c e n tra tio n . The

h e p a tic c o n c e n tra tio n o f copper was no t s ig n i f i c a n t ly d i f f e r e n t from

c o n tro ls a t 48 o r 72 weeks. Zinc d id no t have any e f f e c t upon th e cad­

mium-induced changes in th e k idney c o n c e n tra tio n o f copper, and adm inis­

t r a t i o n o f z in c a lone d id no t produce any a l t e r a t i o n in th e d is p o s i t io n

o f copper. Cadmium tre a tm e n t caused a s ig n i f ic a n t d ecrease in l i v e r

s to re s o f i ro n a f t e r 8 weeks and k idney s to re s o f iro n a f t e r 16 weeks

(T ab le 3 .1 7 ) . These changes were p e r s i s te n t th roughout th e experim en t,

and a t 72 weeks cadm ium -treated r a t s had h e p a tic and r e n a l c o n ce n tra ­

t io n s o f i ro n o f about 60% and 45% o f c o n tro ls r e s p e c t iv e ly . Zinc d id

no t in f lu e n c e th e d is p o s i t io n o f i r o n , nor a l t e r th e re sp o n se to cadmium.

E f fe c ts o f Cadmium and Zinc on Blood C o n cen tra tio n s o f Trace M etals

Cadmium was d e te c te d in th e blood and plasm a o f cadm ium -treated

anim als a t a l l s ta g e s o f th e experim ent (T ab le 3 ,1 8 ) . The plasm a con­

c e n tr a t io n o f cadmium was 20 - 40% o f th e c o n c e n tra tio n found in whole

b lo o d . Cadmium le v e ls d id no t show any tre n d w ith tim e o f exposure to

th e m e ta l, b u t z in c supplem ented, cadm ium -treated r a t s had a c o n s is te n t ly

low er whole blood cadmium c o n c e n tra tio n though t h i s was on ly s ig n i f i c a n t

a t 8, 36 and 48 weeks.

Page 107: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 3.

16

The

Eff

ects

of

Die

tary

A

dmin

istr

atio

n of

Cadm

ium

and

Zinc

on

the

Con

cent

ratio

n of

Copp

er

in th

e L

iver

cu£-d

•H

•O£rt

•d •d00 CM vo CM rt rt •o O o o <r vo o CM S~so O o o o o r-l o CO

m • • • • • • • £CN o o o o o o O o 0

•r-l+ i -H + 1 +1 +1 +1 +1 +1 P

rt>Ov o\ o 1—1

VO VO vo vo CO o <r <r £O O o O r-l CO CM i—i CU• • • • • • • • ' COo o o o O o O o JO

o

'd •d cu1—1 co CN CM rt rt po o o o i—l CM vo CO rto o o o o O O o o• • 9 • • • 9 • •H00 o o o o o o o o i—l

<r a+1 -H +1 +1 +1 +1 +1 +1 df''. vo 00 in Vm/v-/in vo VO in C\ o \ ino O o o r-l CO CO CM CO• • • • • • • • rHo o o o O o o o rt

•H•d •a £rt rt •d -d rt

CM <r CM CM rt rtO o o O CM Os vo r-l COo o o o O O o O• 9 • • ■ * 9 • 4-ivo o o o o O o o o O00 +1 +1 +1 +1 +1 +1 +1 +1 •00 VO 00 VO

CO vo 00 vo in vo 00 VO HO o o O i—i <r <r r-l •cu • • • • • • • • COcu o o o o o o o O12 +1v-/

COCU rt •d •d £g CM CO CO <r rt rt rt

•rl O o o o i—i <r r- i—1 cuH o o o o o o o o 6• • • • • • • •

o o o o o o o o cuCM £+1 +1 +1 +1 +1 +1 +1 +1 rt

vo <r in in COvo vo m vo CO in vo CUO o o o i—l CO CO i—1 3• • • • • ■ • • i—lo o o o o o o o rt

>•d r-lrt *d •d r-l

CM r-l in CT\ rt rt <O O o o l—l <r CO <ro O o o o o O o• 9 9 • • • • - 9 9

VO o o o o o o o O Pi—1 »2+1 +1 +1 +1 +1 +1 +1 +1 r*

o o 00 vo rHCO1". 00 vo o\ m CM <r cu

o o o o H <r CM £• • • • • • • • 4-1o o o o o o o O 00

•d r-ll—l CO CO CO rt rt rto o o o CO <r CM CM Po o o o o o O O CU• • • • • • • • • go o o o o o o o00 r—1+1 +1 +1 +1 +1 +1 +1 +1 OaCM 00 00 p.vo vo in in vo r-l m oO o o o r-l CO CM CM CO• • • • • • • • rto o o o O O o o

•dcu+ + co

P CM CM CO£ CO £ CO £ cucu r-l CM l—l CM £g O + + O + + aP £ CM CM £ CM CM Xrt P •d £ P *d £ cucu £ a + CM £ CJ + CM£ O CM O CM cuH CJ •d CJ •d £CJ CJ rt

COcu P£ £ cu iHCO CU £ 3CO > "a CO

•H •H •rl cuH *4 Pi

COJ2ol—ll—l •o •d4-1 cu

pCO rtrt cu£cu P£rt +CMCO £a CM3O —£ •d00£ 90sCU •dcu cu12 pp rtcu cu•a £

P/-\l—l +o CM• £o CMVCU

+CMCO •dcu CJo£ ••cu a£cu4-1 99s4-1•H CU•d Prtp cu£ £rt Po

•rl +4-1 CM•rl •d£ CJ00•rl 99CO &r—1i—l COrt r-lo o•H £P PCO £•rl OP artp ••CO rt

Page 108: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 3.

17

The

Eff

ects

of

Die

tary

A

dmin

istr

atio

n of

Cadm

ium

an

d Zi

nc

on th

e C

once

ntra

tion

of Ir

on

in th

e L

iver

CDd

•d•H

drt

•d -d T5 •drt r t rt rto\ ov CN oo m O <r

O tH o CN CN rH co rH• • • • • • • •CN o o o o o O o Or-

+1 +1 +1 +1 +1 +1 +1 + 1vo vo in o 00 00|H CO in <r CN G\ CN tH

• • • • • • • •CN r— —1 CN CN O 1—I CN

XJ TO •d •drt rt rt rto oo in in in m VO1—1 o rH rH o o CN O• • • • • • • •

00 o o o O o o o o<r

+1 +1 +1 +1 +1 +1 +1 +1o in CN OO uo vo o\rH vo rH o o CN CN 00

• • a • • - • . • •CN o tH CN CN rH tH l—l

'd 'd •d 'drt rt rt rtrH <r <r 00 in 00 rH orH o tH o o o O l—l• • • • • • ■ •

vO O o o o o o o o00

+1 +1 +1 +1 +i +1 +1 +1/'"NCO LO in 00 rH CO G\ o\ 00

00 o 00 00 1^ ov 00CD • • • • • • a a

CD rH o rH r—1 i—i o o rH&W

•d ■d TJ •dCD r t r t r t r t

g CO VO in CN CO o CN 00•H O rH O CN l—l tH rH OH • • • • • • a a

<r O o o O o o o oCN

+1 +1 + i +1 +1 +1 +1 +1CN CT\ in n* n* <rI'- o vo in o CN• • • • • • a •rH o I—I 1—1 r—1 o tH l—l

•d •d •d •drt rt rt rt00 CN VO o CO o\ in inO O o i—i i—l o o rH

• • • • • • a a

VO o o o o o o o orH

+1 +1 +1 +1 +1 +1 +1 +1rH <1- ov 00 vo in <r

vo o\ CN o\ CO• • • • • • • •|—1 O o l—l tH o o l—l

•d •drt rtI—1 00 rH vo <r in vo VOo o rH rH • o rH O T—1• • a • • • • •00 o o o O o o o o

+1 +1 +1 +1 +1 +1 +1 +1CN r>. o\ rH 0\ 1—1 i—ivo in n* m rH o CM

a • • • • • • a

tH o o tH rH o T—I rH

+ +P CN CNC CO d co dCD tH CN tH CN6 O + + O + +P p CN iZ CN P CN <Z CNr t P TJ d P •d dCD d O + IN d CJ + CNP o CN o CN

H o •d o *dcj o

CDd P CDCO CD dCO >•H •H •HH

COdo•HPrt>PCDCOPOCDPrtO•HtH(Xd

•d

COrHrt.6•H

drt••

oo COs4-1 o

o tHrH

a Os 4-1•w CO

rtCO g •

X3+1 g CD

o PCO .c rtd CO CDrt PCD CD PS p

rt +CD ONP CO drt ex

dCN

CO o ••CD p •dd 40

tHrt d aav

> CD *dCD CD

t—i S PtH P rtc CD CD

P PP

a /~SP tH +£ o CN

a dA o CNCOCD V *3p (X4-1 w

2+

40 CO •dCD orH O

rt d ••P CD oCD pg CD

4-1 aas

tH 4-1 •dO •H CD1 •d p

rtp CD

CO d Prt r td

P•d •H +CD 4i CNCO •H *dCO d CJCD 40P •H «•P. CO .QXCD >•»rHCD I—1 COP r t |Hr t o O

•H PCO P PP CO dtH •H od P oCO rtCD P ••

cd CO rt

Page 109: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 3.

18

The

Eff

ect

of D

ieta

ry

Adm

inis

trat

ion

of Ca

dmium

an

d Zi

nc

on th

e C

once

ntra

tion

of Ca

dmiu

m

and

Zin

c

T*OorHPQ0)rdP

•H

CMr>»

oo<r

voCO

CO

cucu£0)s•HH <r

CM

VOi-1

00

d<uEprtcupH

rtPcuS

'd T3<r rH rH rt rt

o o O O CM o\ <r• • • • • • • •o o o O o o o o

+1 +1 +1 + 1 +1 + 1 +1 +i COLO CM 00 din <r rH rH r'. in CM <r o .• • • • • • • a •Ho o O O CM 00 tH P

CM rH rH CM rt>P

p 'd *d CUm r—1 rH rH rt rt COo O O O CM o\ VO 00 .o• • • • • a • a oo o O o O o o o Q)

+ i +1 +1 + 1 +1 + 1 +1 +1 pcd

CO 00 Ov o aVO co. i—1 CM <r CO o <r •H• • • • • • • • tHO o o o rH vo 00 «H PX

CM i—1 tH CM d•d

.a 'd •dCO CM rH |H rt rt COo O O o t'''. 00 <fr CO H• • • • a • • • rto o O o o o o o e a.

•H CO-H +1 +1 +1 + i + 1 +1 +1 a

r t go\ 00 00 rHin CO rH rH CO 00 rH o\ CO |H •

• • • • • • • • O *do o O o CM in rH 4-1 4-4 cu

CM rH rH CN O pCO rta d cu

’d *d s PCM CM rH rH rt rt • cu po o O O in 00 VO r-1 H p• • • • • • • • • rt +o o O O o o o O CO CM

CO d-H +1 +1 + 1 + i +1 +1 + 1 -H p.•3 CMI—1 00 vo CO

I—1o ..

<r CO rH tH CM 00 r . rH Pi f t •d• • • • • • • • rt 00o o O O CM iH cu

CM rH rH CN £ pi aavcu -dcu cu cuTO & t2 p

m vo tH rH rt rt Iv P rto o O O CM 00 in CO CO CU cu• • • ■ a • • • Cl) p Po o o o rH o o o 0 p

rH-H +1 +1 +1 +1 +1 •H + ! cd►> rH +CNG\ o iH iH

w• d<T |H r-l C\ 00 CO o rH o CM• • • • a • a • rHO o o O CO in 00 <r < V

CM rH tH CM PXV-/ +

'd a CMp P o *d tH CO •d

<r 1—I i—i iH rt rt -■— CU oo o o O CO r > <r in I—1 o• • • • a • a • rt d ••o o o o . o o o o P cu o

cu p■H +1 +1 +1 +1 + i •H + i £ cu

4-1 ••vin CM 00 H 4-1 •d

in CO CM rH o rH O •H cu• • • • a • • a E •d po o O O rH rH 00 o : p. rt

CM |H rH CM p cuCO d Prt rt p

o+ + + •d •H +CM CM CM cu 4-4 CM

pi Pi CO d CO •H •dCM CM tH CM CO d o

+ + O + + cu 00CM CM p CM CM P •H ••

•rt •a p •d Pi a CO Po + o + pi o + CM KCM CM o CM cu rO

TU •d o •d |H aa>o o cj cu rH CO

p rt rHrt o O/“s •H Prt rt CO P P9 a) r-N 9 E E P CO d

•rl rH TO •H CO CO tH •H o6 O O 6 rt o rt d P u•d & o TO «H Pi rH CO rtrt S |H rt P. •H a cu P a«o '-n & o ^ N cd co rt

Page 110: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tables 3 .1 8 and 3 .19 show th e e f f e c t o f cadmium and z in c on th e

plasm a c o n c e n tra tio n s o f z in c , copper and i ro n . The z in c and i ro n con­

c e n tr a t io n s were s ig n i f i c a n t ly dep ressed in bo th groups o f cadmium-

t r e a te d anim als from 8 weeks onwards compared to c o n tro ls and anim als

supplem ented o n ly w ith z in c . A z in c supplem ent d id no t s ig n i f i c a n t ly

in f lu e n c e th e e f f e c t o f cadmium. N e ith e r z in c nor cadmium a l te r e d th e

plasm a le v e l o f copper u n t i l 72 weeks when bo th cadm ium -treated groups

o f anim als had s ig n i f i c a n t ly e le v a te d le v e l s o f copper (up to 40%

in c re a s e d ) .

E f fe c ts o f Cadmium and Zinc on th e U rin ary E x c re tio n o f Trace M etals

Low c o n c e n tra tio n s o f cadmium were d e te c te d in th e u r in e o f cadmium

exposed r a t s (T ab le 3 .2 0 ) . From 8 - 3 6 weeks, u r in a ry cadmium e x c re tio n

rem ained betw een 0 .03 - 0 .06 pmole/mmole c r e a t in in e , b u t s ig n i f i c a n t ly

in c re a se d a t 48 and 72 weeks. C on tro l anim als e x c re te d r e l a t i v e l y

c o n s ta n t amounts o f z in c th roughou t th e experim ent bu t z in c supplem ented

anim als had a c o n s is te n t ly e le v a te d le v e l o f e x c re tio n fo r th e f i r s t

24 weeks. Cadmium tre a tm e n t d id no t s ig n i f i c a n t ly a f f e c t th e e x c re t io n

o f z in c in th e u r in e . In T able 3 .21 i t can be seen th a t cadmium d id not

in f lu e n c e th e u r in a ry e x c re tio n o f copper u n t i l a t 72 weeks when r a t s

exposed to th e m eta l were e x c re tin g s ig n i f i c a n t ly more copper. Zinc

a lone s ig n i f i c a n t ly dep ressed th e normal u r in a ry e x c re tio n o f copper a t

8 and 16 weeks b u t su b seq u en tly re tu rn e d to norm al. N e ith e r cadmium no r

z in c a f fe c te d th e u r in a ry e x c re tio n o f i ro n .

E f fe c ts o f Cadmium and Zinc on th e U rin a ry E x c re tio n o f Enzymes

Cadmium and z in c tre a tm e n ts d id n o t have any s ig n i f ic a n t e f f e c t

upon th e u r in a ry e x c re tio n o f enzymes u n t i l 48 weeks. At t h i s t im e ,

Page 111: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 3.

19

The

Eff

ect

of D

ieta

ry

Adm

inis

trat

ion

of Ca

dmium

an

d Zi

nc

on th

e C

once

ntra

tion

s of

Copp

er

and

Iro

nCM

oo

vooo

CO

0)a)£0)S•HH <r

CM

VO r—I

00

*dOOrHPQ

0)r£P

£•H

P!CUB

CUPH

rtP0)s

TJ •o TOrt rt rt r t

CO 00 o vo oo CN <r in• • • • • ' • • •

«H iH tH «H rH «H CN o •

■H •H + i +1 + i •H +1 + i corio \ uo CO 00 00 <r CN CN

>—• o• • 9 • • • • 9 •H

oo oo o in rH <r in CN PCN 00 CN CN <r oo CO <r rt

>Hcu

T3 TO wrt rt P

OV vo OO in vo 00 O o• • • • • • • 9

o rH rH tH o O pH O cup

-H + i + i + i +1 ■H + i +1 rto

rH < r oo oo 1—I rH H <r •H• • • • • • • • rH

00. o \ t ''. rH <r vo o aCN CN ON CN co 00 <r d

v-/

•d TO COrt rt rH

<r O rH |H vo in a \ rt• • • 9 ■ • • 9 6o o rH O iH rH |H O •H ••

£ CO■H -H + i +1 +1 -H + i +1 rt gin O o 00 00 rH in VO 00 rH

• • • • • • 9 9 rH •CN rH oo rH o o \ rH <p O X3CN CN CN CN <r CN CN < r O P CU

P9 CO rt

s rt cuTJ TJ Prt r t w <u p

<r o 00 00 tH <r rH P• • • • • • • 9 CO rt +o o rH o o rH CN tH CN

+1 CO £-H ■H +1 +1 +1 •H +1 + i a CN

CO 3in 00 o tH oo in CO £ o ••

• • • • • • • • rt u •dCN CN ON 00 iH o \ 00 CN a) bDCN CN CN CN <r CN CN <r g

£ 99k

cu CU *dP CU cu

Xi xs rt & prt rt p r t

O < r 00 tH o vo CN vo CO cu cu• ■ 9 • • • • • cu p P

|H o O o rH o fH o 3 prH ✓“S

-H •H +1 +1 +1 +1 +i +1 rt rH +i> O ON

CN 00 C\ o O o o o \ • £• • • • • • • 9 tH o CN

CN o a \ rH o rH <r 00 rH VON CN rH CN < r 00 oo oo C ry

+• CNXl 'd rH CO •drt r t <u o

VO 00 in 00 in vo 1^. tH a• • • • • • • • r t £ ••

O o o o o rH |H CN P cu acu P

*H -H + i +1 +1 •H +1 +1 6 cup 99s

oo VO H vo in oo OO vo «H p X5• • • 9 • • 9 • O •H CU

iH |H fH o 00 00 1". tH B •d PCN ON CN CN CN 00 p. r t

p cuto £ prt r t p

o+ + •d •H +CN CN cu P CN

CO £ co £ CO •H xsrH CN tH CN CO £ CJO + + O + + cu 00P CN CN P CN U) CN P •H ••P *d £ P •a £ a CO .3£ o + CN £ CJ + CN XO CN O CN cu r*»O • a CJ •d tH •»

CJ o cu rH COp rt rHrt V O

/~ s •H Prt rt to P P

P g g P CO £CU CO co |H •H Oa , rt £ rt 3 P aCX iH O rH CO rtO a £ a cu P ••

CJ ^ H PS CO rt

Page 112: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 3.

20

The

Eff

ect

of D

ieta

ry

Adm

inis

trat

ion

of Ca

dmiu

m

and

Zinc

on

the

Urin

ary

Exc

retio

n of

Cadm

ium

an

d 1—1 vo 00 CM in ino o 00 00 i—i CM• • • • • •

CM T3 o o •d o o o od d

+1 -H +1 +1 +i +11—1 vo VO CM in o\ CO<r oo <r <r o\ o rH• • • • • • rto o rH i—i rH CM g•H

dJO rtrt rt •l—l l—l VO vo O vo CO CUo o |H 00 CM CM I—l• • • • • • m JO

00 •d o o •d O o O O o rt<r d d P

+ 1 -H +1 +1 +1 +1 ■ • as •• cu

CM r-l rH uo o Os • CO pCM CM CN 00 CM Os w % cu• • • • • • • o •dO O r-| iH CM i—1 00 I-ll—l p•rt +1 O o0 4-1 dvo rt CO

l—l O Os o VO d CO ••o O a 1—1 CM i—1 rt rt •d• • • • • a> dVO •d o o 'd o o O o S CUCO d d P

+1 -H +i +i +! +1 cu rtpco LO vo o in OO os rt /'“N

X o o VO i—i 1—1 Os r—1 •CU • • • • • • CO O •dCU o o i—1 r—1 CM tH cu • cu|2 p O pr—1 v rt

JO rt cuCU LO 00 rt > a pg o o r>. <r CM CM V_y p•H o o i—i i—i oo O i—iH • • • • • • i-i CO +<r TO o o 'd o o o o < cu CMCM d d a d

+1 +1 +i +i +1 +1 d CM• cu

<r < r 00 Os l—l H cu P ••o o oo a OS d cu •d

• • • • • • •H 4-1o o |H rH rH rH d 44•H •H • •v

P •d •d& rt cu00 r- rt cu p po o o |H o\ Os P d rto o CM rH o O o rt cu

• • • • • • o PVO •d o o 'd o o o o •H piH d d p 4-1+1 +1 +1 +1 +1 +1 rt •H +d d CM

<r <r 00 I—l vo oo •H 40 do o CM r*. o P •H CM

• • • • • • p COo o rH i—l i—i CM id|H >>O i—l +

JO E i—l CM1—1 rt E rt *do o o 00 00 ■"_ o CJo o CM o o i—i iH •rl

• • • • • • rt P ••00 •d o o •d ' O o o o P CO o

d d cu •H+1 +1 +1 +1 +1 +1 g Prt00 oo CM oo r - 00 |H P *do o <r in o o CO cu

• • • • • g Po o r-l rH i—i CM a rt

• cuCO Pr t CO

r* P

+ + •d O +P CM CM cu •H CMd CO d CO d CO P •dCU i—i CM i—l CM CO rt og o + + O + + cu >4-) p CM id CM p CM id CM p P ••Cti P 'd d p •d d a, cu .QCU d o + CM d a + CM X COP o CM o CM cu .aH o *d o •d o

CJ CJ cu COp cu l—lrt p ort pg co o p

i—l p p •ri drt •rl i—i i—i oP o P a oCU •g d CO pS rt •H cu •d ••

o CM P5 rt

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Tabl

e 3.

21

The

Eff

ects

of

Die

tary

A

dmin

istr

atio

n of

Cadm

ium

and

Zinc

on

the

Urin

ary

Exc

retio

n of

Cop

per

-ddrt

rdrt

CO CM m vo rH VO voCM r—l i—i rH rH rH rH rH• • • • • • • •

CM O O o O o o o O+1 +1 +i +1 +1 -H +1 +1

<r a \ o 00 O vo <r CO CO00 CM 00 CM o o\ I-l &• • • • • • • • rt o1—1 CM CM i—i o rH I-l o E l—l•H 1—1

d ort 4-1

<r i—l VO <r vo rH vo CO COCO CM O o O CM o rH rt■ • • • • • • 4-i

00 o o o o o O o O O rt<r d

+1 +1 +1 +1 +1 -H +1 +1 *5- rtVO <r CM co o\ CM 00 o a CO

in O 00 I". rH 00 00 w S*• • • • • • • • a dr—i CM CM rH o rH o o CO o

d+1 00

CO d<r VO r>. rH CO rH 00 vo d cui—i i—1 o CO o rH o rH rt cu■ • « • • • • • rt is

VO o o o O o O o O E dco cu

+i +! +1 +1 +1 +1 +1 +1 rt jad

CO rH ov 00 o rt ✓-vX 00 CO o <r CO CO <r r-ld) • • • • • • ■ a CO O •0) 1—1 CM CM rH o o o o rt •d£ d O cui—i \/ d

rt V rtrt > . a rts <r rH <r 00 00 00 vo d•H CO o rH CO o o o O r-l dH • • • • • • • • r-l CO +

<r o o O o o o o o < rt CMCM a d

+1 +1 +! +1 +1 +1 +1 +1 d CMa cu

o CO <r rH CO 00 CO 00 r t d a*<r <r <r in <r CO CM d cu TJ• • • • * • • a •H 4-ti—i i—i rH rH o o o o d 4-1

•H •rl a atd TJ •dr t r t

r t r t d d<r rH CO CO <r d d r t

r-i i—i rH rH 1—1 o o o o rt cu• • • • • • ■ a d dVO o o o o o o o o •rl drH d 4-1+1 +i +1 +i +1 +1 +1 +1 rt •H +d d CMco in l—l o\ in CO 00 uo •H 00 dCO in UO CM <r CO <r uo d •H CM• • • • • • • a d COrH rH rH rH o o o o

rH >>O l—1 +£ rH CMr t § r t •d

uo Q\ rH <r CM rH CO CO a oi—i rH rH o O rH rH l—l rH •H• • • • • • • a rt d •a

00 o o o o o o O o d CO or t •rl

+ i +1 +1 +1 +1 +1 +1 + 1 E dr t

00 CO in vo 00 l"» CO i—i d *di—i rH o UO uo UO o CO cu

a • • • • • • • E d1—1 rH rH rH o o o o d. r t

a r tCO O dr t CO (—1 d

+ + •dVho +

d CM CM CU •H CMd CO d CO d CO d •drt 1—1 CM |H CM CO rt CJs o + + O + + rt >d d CM CM J-l CM u3 CM d d ••rt d *d rt d •d d d. cu .art d o + N a o + CM X COd o CM o CM rt .oH o <j O aa>

O O rt COd rt rHrt d O

rt dCO rt dr-C u d •rl drt rt i—i rH od a d d a rtrt (X o CO ds o d rt •d aa

a M P5 rt

Page 114: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 3.

22

The

Eff

ect

of D

ieta

ry

Adm

inis

trat

ion

of Ca

dmium

an

d Zi

nc

on th

e U

rinar

y E

xcre

tion

of

CDCOccS£03XaCOo

xCL,<Dc

•rt r—Icc5X

•d T3CCS CCS

CO f''. r . in• • • •CM o o o or .

+1 +1 +1 +iin rH CM o

a • • ao CO <r CMrH rH rH rH

•doccS

r>. CM m 00• • • a

00 o o o o<r

+ i +1 + i +io CM in• • • ao CO o 00rH rH rH

in in CM <r• • • a

o o O ovoCO . +1 +1 + 1 +i

CO rH CM CM oX • • acu 00 00 G\ o\cu&v_cu in CM CM rH6 ■ • • ■•H o o o OH <rCM + i +1 + 1 +1

vo CM CM CM• • • •00 o O

rH rH

CO CO rH <r• • • ao o o o

VOrH + l +1 +1 +i

CM o• • • a

o \ o> 00 o \

00 CM C\• • • ao o rH o

00 +1 +! + i + iCJ\ in r-'- rH• • • •o CM rH rH

rH rH rH

+£ CM£ CO £cu rH CMs O£ £ + +CCS £ CM CMcu £ TO + ££ O O CM CMH o TS

o

ccSS•H£ccS

CO

4-cO

5w

co+l

COaccSa)6a)yCOcu3

rHCCS>rHrHc

CU£•H£•H£ccSCUuO>>£rt£

•rH

o4h

COCCS

cu£CCS

COa£O&£cucuI*4->as

x

ov

cocuo£cu£cu

4 h4 h•HT3£Sa•Hm

•Hs•HCO

i—IrtO•H

TSCU£ccS<u££

+CM£

CM

T5(U£ccSCU££

+CM

£CMces

+CM'do

£ £ ••d CO OrH

•H£

O CCSg £ •dg CO cu— £P a)• cuCO £ccS

•dpHo +

cu •H CMCO £ •dCO CCS ocu£ & a#a as XX COcu X

ocu CO£ cu rHa) £ O

CCS £CO a ££ •rH £rH rH O3 a aCO 3cu •d ••£3 CCS

Page 115: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

cadm ium -treated anim als showed an e le v a te d e lim in a tio n o f a lk a l in e

phosphatase and Y -glutam yl tra n s p e p tid a s e compared to o th e r groups

(d e sc r ib e d in s e c t io n 2 .3 ) . This tre n d was a lso seen a t 72 weeks and

was noted in th e z in c-supp lem en ted , cadm ium -treated group. Zinc a lone

had no e f f e c t upon th e normal le v e l o f u r in a ry enzyme e x c re tio n . Only

d a ta fo r a lk a l in e phosphatase a re provided s in c e o th e r enzymes responded

in th e same manner (T ab le 3 .2 2 ) .

3 .4 DISCUSSION

Exposure to cadmium causes d is tu rb a n c e s in th e normal m etab o lic

h an d lin g o f z in c , copper and i ro n . A c o n s id e ra tio n o f th e mechanisms

o f th e se in te r - r e la t io n s h ip s must in c lu d e th e p ro p e r t ie s o f th e m e ta llo -

th io n e in s , th e tim e -co u rse o f sy n th e s is o f th e se b in d in g p r o te in s , and

th e development o f c r i t i c a l c o n c e n tra tio n s o f cadmium in t a r g e t t i s s u e s .

The changes in th e d is p o s i t io n o f z in c and copper a f t e r th e .a c u te

a d m in is tra tio n o f cadmium may be r e la te d to th e sy n th e s is o f cadmium-

th io n e in , s in ce th io n e in can a lso bind z in c and copper (s e e s e c t io n 1 .2 ) .

H epatic cadm ium -thionein sy n th e s is i s induced in r a t s w ith in a few

hours o f exposure to th e m etal (Cempel and Webb, 1976), and t h i s p robab ly

accounts fo r th e in c re a se d accum ulation o f z in c a t 24 hours in th e l i v e r .

Renal cadm ium -thionein however has a much lo n g e r la g phase o f about

48 hours b e fo re sy n th e s is i s m axim ally induced . I t i s no t s u rp r is in g

th e re fo re th a t no changes in th e c o n c e n tra tio n o f z in c o r copper were

ap p aren t in th e k idney 24 hours a f t e r in j e c t io n o f cadmium.

The m ajor m eta l component o f h e p a tic cadm ium -thionein , in a d d it io n

to cadmium, i s z in c (Webb, 1975a), bu t an in c re a se d copper c o n c e n tra tio n

in th e l iv e r s o f r a t s exposed to a v a r ie ty o f doses o f cadmium has been

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a common f in d in g in t h i s la b o ra to ry . I t has been dem onstrated th a t

a cu te p a re n te r a l a d m in is tra tio n o f cadmium a t doses s im ila r to th o se

used in t h i s s tu d y , caused a marked in h ib i t io n o f b i l i a r y copper ex cre ­

t io n , le a d in g to an accum ulation o f th e m etal in th e l i v e r (Ashby e t a l .

1979).

The changes in t i s s u e z in c and copper induced by th e s in g le in je c ­

t i o n o f cadmium a re accompanied by changes in th e plasma c o n c e n tra tio n s

o f th e se m eta ls (T ab le 3 .2 ) . The d ecreased plasma c o n c e n tra tio n o f

z in c could be caused by th e in c re ase d requ irem en t fo r z inc in th e l i v e r ,

and i t i s suggested th a t th e accum ulation o f copper in th e l i v e r s tim u­

la t e s th e sy n th e s is o f cae ru lo p lasm in which consequen tly r a i s e s th e

plasma le v e ls o f copper. F u r th e r ev idence fo r a d is tu rb a n ce in copper

m etabolism is shown by th e decreased e x c re tio n o f t h i s m etal in th e

u r in e , su g g es tin g e i th e r an in c re ase d requ irem en t fo r copper e lsew h ere ,

o r an im paired e x c re to ry p ro cess . Changes in t i s s u e iro n c o n c e n tra tio n

were n o t ap p aren t in acu te -d o sed anim als a t 24 h o u rs , bu t S to n ard and

Webb (1976) have suggested th a t cadmium may in h ib i t th e uptake and

r e le a s e o f i ro n th rough b in d in g to f e r r i t i n , which could be expected to

low er th e plasma c o n c e n tra tio n o f i ro n q u ite q u ic k ly , b u t a lo s s o f i ro n

from h e p a tic s to re s would ta k e lo n g e r . This could account f o r th e

o b se rv a tio n s in th e p re se n t s tu d y .

When cadmium i s ad m in is te red re p e a te d ly , m e ta llo th io n e in s t i l l

p lay s an im p o rtan t r o le in d e te rm in in g th e in te r a c t io n s o f t r a c e m eta ls

bu t th e s a tu r a t io n o f th e b in d in g c a p a c ity o f th e p ro te in s and th e

development o f c r i t i c a l c o n c e n tra tio n s o f cadmium now become im p o rtan t.

The d o s e - re la te d r e ta r d a t io n o f growth which occurred a f t e r th e re p e a te d

p a re n te ra l a d m in is tra tio n o f cadmium i s a g en era l in d ic a t io n o f t o x i c i t y

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and r e f l e c t s th e in h ib i to r y e f f e c t s o f cadmium on many a sp e c ts o f

m etabolism (K otson is and K laassen , 1977). The d o s e - re la te d in c re a se s

in l i v e r and k idney w eigh ts r e l a t i v e to body w eight p robably r e f l e c t

th e accum ulation o f m eta ls w ith in th e se organs and th e a s s o c ia te d t i s s u e

damage (T ab les 3 .1 and 3 .8 ) . The accum ulation o f h igh c o n c e n tra tio n s

o f z in c and copper in th e l i v e r and kidney which accompanied th e r e te n ­

t i o n o f cadmium, was no t d o s e - re la te d . This may in d ic a te t h a t e i th e r

th io n e in has a l im ite d c a p a c ity to bind th e se c a t io n s , o r i t may be a

r e f l e c t i o n o f th e l im ite d d ie ta r y a v a i l a b i l i t y o f th e m e ta ls . A lthough

r e n a l cadm ium -thionein i s though t to b ind m ainly copper in a s s o c ia t io n

w ith cadmium (S to n ard and Webb, 1976), t h i s s tudy confirm s th e observa­

t i o n made in a v a r ie ty o f sp ec ie s t h a t z in c a lso accum ulates in th e

k idney (Pow ell e t a l . , 1964; Cousins e t a l . , 1973; Doyle and P fan d e r,

1975; Seth e t a l . , 1976). I t i s no t known w hether t h i s z inc i s bound to

m e ta llo th io n e in o r to some o th e r p ro te in .

This accum ulation o f z in c in th e l i v e r and kidney may cause s ig n i ­

f i c a n t b io lo g ic a l changes in c lu d in g d e p le t io n o f th e m etal in o th e r

o rg an s , such as m uscle and bone. This p o s s ib i l i t y has been f u r th e r

in v e s t ig a te d in C hapter 4 . A n e g a tiv e z in c b a lance has been shown to

occur in lambs a f t e r cadmium tre a tm e n t (Doyle e t a l . , 1974). In c re a se d

m o b ilis a tio n o f z in c may account fo r th e in c reased plasma le v e l s o f z in c

found a f t e r re p e a te d cadmium in je c t io n s , a lthough th e converse r e s u l t

has o f te n been d e sc rib ed a f t e r o ra l cadmium tre a tm e n t ( e .g . Pow ell e t a l .

1964; Cousins e t a l . , 1977), and was found in t h i s s tu d y w ith a c u te

exposure to cadmium. Our r e s u l t s may th e re fo re r e f l e c t a d is tu rb a n c e

in an a sp ec t o f z in c hom eostasis th a t i s m an ife st on ly a t h ig h e r con­

c e n tr a t io n s o f cadmium.

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The e f f e c t s o f re p e a te d cadmium a d m in is tra tio n on copper m etabolism

were s im ila r to th o se on z in c , and h igh c o n ce n tra tio n s o f copper in

t i s s u e s c o n ta in in g h ig h le v e ls o f cadmium a re ag a in l i k e ly to be due to

b in d in g to th io n e in . The in c re ase d c o n c e n tra tio n o f copper in h e p a tic

t i s s u e and plasm a d e sc rib e d a f t e r acu te exposure to cadmium, was even

more marked a f t e r re p e a te d cadmium tre a tm e n t. Julsham n e t a l . (1977)

showed an i n i t i a l in c re a s e in th e h e p a tic c o n ten t o f copper a f t e r d ie ta r y

a d m in is tra tio n o f cadmium to r a t s , which le v e l le d o f f a t l a t e r s ta g e s ,

bu t u su a lly in c re a se d r e te n t io n o f copper has on ly been no ted in th e

k idney .

The s ig n i f i c a n t in c re a se in th e h e p a tic i ro n c o n c e n tra tio n and

d ecrease in k idney le v e ls o f th e m eta l a f t e r re p e a te d in je c t io n s o f cad­

mium i s an i n t e r e s t in g r e s u l t (T ab le 3 .9 ) . A low ering o f th e iro n con­

t e n t o f th e l i v e r as a r e s u l t o f d ie ta r y cadmium tre a tm e n t i s a common

f in d in g (Whanger, 1973; S tonard and Webb, 1976). In t h i s experim en t,

th e decreased plasma c o n c e n tra tio n o f i ro n f u r th e r in d ic a te s a d is tu rb e d

hom eostasis o f th e m e ta l, bu t i t i s d i f f i c u l t to f in d an adequate

ex p lan a tio n fo r th e changes in th e l i v e r . M o b ilis a tio n o f i ro n in o th e r

t i s s u e s by cadmium could r e s u l t in accum ulation o f iro n in th e l i v e r ,

bu t r a is e d le v e l s o f th e m eta l in plasm a would th e n be ex p ec ted . However,

cae ru lo p lasm in i s n ece ssa ry fo r iro n m o b il is a t io n (F is h e r , 1975). I t i s

though t th a t i r o n , absorbed as F e ( I I ) i s o x id ise d by cae ru lo p lasm in

( fe r ro x id a s e -1 ) to F e ( I I I ) fo r s to ra g e in th e l i v e r as f e r r i t i n

(F r ie d e n , 1973). The h ig h le v e ls o f copper in th e plasma o f th e se

an im als , and presum ably cae ru lo p lasm in (s in c e cae ru lo p lasm in accounts

f o r up to 99% o f plasma copper in th e r a t ) would suggest t h a t re p e a te d

p a re n te r a l a d m in is tra t io n o f cadmium i s producing a s im ila r e f f e c t upon

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copper m etabolism as t h a t d e sc rib e d a f t e r a cu te exposure. An in c re a se d

sy n th e s is o f cae ru lo p lasm in in th e l i v e r which promotes th e s to ra g e o f

iro n w ith in h e p a tic t i s s u e may th e re fo re be a p o s s ib le ex p lan a tio n fo r

th e observed r e s u l t s .

The concept o f a c r i t i c a l c o n c e n tra tio n fo r cadmium i s an im p o rtan t

one when c o n s id e rin g th e developm ent o f r e n a l damage. When cadmium i s

p re se n t w ith in t i s s u e s as th e th io n e in complex, th e re i s l i t t l e b io ­

chem ical o r fu n c tio n a l d is tu rb a n c e , and m e ta llo th io n e in appears to be

f u l f i l l i n g a p ro te c t iv e r o le . However, b e fo re in d u c tio n o f th e sy n th e s is

o f th io n e in , o r when th e c o n c e n tra tio n o f cadmium exceeds th e maximum

a v a ila b le th io n e in , f r e e cadmium i s p re se n t to m an ife st t o x i c i t y . The

mechanisms o f r e n a l damage caused by cadmium a re s t i l l unknown though i t

has been suggested th a t cadmium may exchange fo r z inc in enzymes n e ce ssa ry

fo r i n t r a c e l l u l a r p ro te in ca tab o lism in th e proxim al tu b u le s (F r ib e rg

e t a l . , 1974). There i s a lso d oub t, as d iscu ssed in an e a r l i e r s e c t io n

(1 .3 ) w hether th e cadmium c a tio n o r cadm ium -thionein complex i s th e

to x ic a g en t.

I f a lo s s o f enzymes in to th e u r in e does r e f l e c t r e n a l damage, th e n

th e e a r ly phase o f enzymuria w ith in 48 hours seen in r a t s g iven re p e a te d

p a re n te ra l cadmium, i s p robably caused by th e cadm ium -cation s in c e a t

t h i s s ta g e th io n e in sy n th e s is shou ld no t be maxim ally induced in th e

k idney . An a l t e r n a t iv e e x p lan a tio n im p lic a tin g cadm ium -thionein ds th e

to x ic agen t could be th e t r a n s f e r o f th e m e ta l-p ro te in complex from th e

l i v e r to th e k idney . This i n i t i a l damage does n o t appear to be i r r e v e r ­

s ib l e , o r a t l e a s t i s no t so sev e re as to cause permanent fu n c tio n a l

d is tu rb a n c e . With re p e a te d cadmium a d m in is tra t io n , however, a second

phase o f enzym uria, and by im p lic a t io n , r e n a l damage d ev e lo p s . I f th e

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concept i s v a l id , th e n t h i s would be expected to occur a t th e c r i t i c a l

c o n c e n tra tio n and i s though t to r e s u l t from th e inadequacy o f th io n e in

to be sy n th es ised a t a r a t e s u f f i c i e n t to s e q u e s te r a l l th e cadmium

being re ta in e d in th e k idney . I t would be re a so n a b le to h y p o th e sise

th e re fo re t h a t in th e p resen ce o f excess cadmium, c e l l u l a r damage i s

caused by f r e e c a tio n . I t could a lso be su g g ested , however, th a t i t i s

a t t h i s s ta g e th a t th e cadm ium -thionein complex may become m o b ilised o r

reach such a le v e l as to produce th e damage. F u r th e r work i s needed

to re so lv e t h i s problem.

Changes in th e u r in a ry e x c re tio n o f cadmium a lso su g g est th e

development o f r e n a l damage. E x c re tio n o f th e m eta l in to th e u r in e i s

norm ally low due to re a b so rp t io n o f cadm ium -thionein th rough th e p ro x i­

mal tu b u le s , bu t a s ig n i f ic a n t in c re a se d lo s s o f th e m etal in to th e

u r in e i s known to occur a f t e r r e n a l damage. The in c re a se in e x c re tio n

has been shown to c o in c id e w ith th e appearance o f p a th o lo g ic a l p ro te in ­

u r ia in ex p erim en ta l anim als (F r ib e rg e t a l . , 1974). The developm ent

o f enzymuria accompanied th e d ram atic lo s s o f cadmium in to th e u r in e

o f r a t s g iven re p e a te d in je c t io n s o f th e m etal and th e re was a con­

com itan t r e le a s e o f z in c , copper and iro n in to th e u r in e which was d o se -

r e la te d . These changes appear th e re fo re to c o r r e la te w e ll w ith , th e

development o f cadmium-induced re n a l damage, and th e lo s s o f t r a c e

m eta ls may cause a d ecrea se in th e r e n a l c o n c e n tra tio n o f th e m e ta ls .

This may p a r t ly account fo r th e o b se rv a tio n th a t th e k idney c o n c e n tra tio n

o f cadmium was no t as h ig h as expected in th e medium-dose group.

The re p e a te d p a re n te ra l a d m in is tra tio n o f cadmium allow ed an in v e s ­

t i g a t io n o f th e in te r a c t io n s o f cadmium w ith z in c , copper and i ro n d u rin g

th e developm ent o f c r i t i c a l c o n c e n tra tio n s o f cadmium, over a r e l a t i v e l y

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sh o r t p e rio d o f tim e . I t must be borne in mind, however, th a t i f th e

dose o f cadmium i s to o la r g e , changes in th e normal p a t te r n o f p ro te in

b in d in g may be caused (Webb, 1975b), thus producing e f f e c t s th a t may no t

be ap p aren t a f t e r long term , low le v e l exposure to cadmium. The d ie ta r y

a d m in is tra tio n o f low er le v e ls o f cadmium (75 p .p .m .) over a c o n s id e ra b le

tim e may p rov ide d a ta t h a t a re more a p p lic a b le to th e s i t u a t io n o f human

exposure. The le v e l o f cadmium in th e d ie t o f r a t s was s u f f i c i e n t to

cause b iochem ical changes, bu t th e body w eight changes o f cadmium-

t r e a te d anim als compared to c o n tro ls shows th a t th e le v e l was no t so h igh

as to produce sev e re t o x i c i t y and i l l - h e a l t h .

A fte r prolonged o ra l in g e s t io n o f cadmium, th e m etal accum ulated

to i t s h ig h e s t c o n c e n tra tio n in th e k idney , and to i t s h ig h e s t co n ten t in

th e l i v e r . This i s in accordance w ith p rev ious s tu d ie s , which have a lso

shown th a t s tea d y s t a t e c o n c e n tra tio n s o f cadmium a re u s u a lly a t ta in e d

in th e l i v e r w hile th e k idney ten d s to co n tin u e accum ulating th e m eta l

(S tonard and Webb, 1976). The observed d ecrease in bo th th e h e p a t ic ,

and p a r t i c u la r ly r e n a l c o n c e n tra tio n o f cadmium in th i s s tu d y a f t e r

48 weeks was th e re fo re in te r e s t in g . Such a change could be r e la te d to a

d iv e rs io n o f cadmium to o th e r t i s s u e s du rin g l a t e r s tag e s o f exposure ,

bu t t h i s cannot be confirm ed by a v a i la b le d a ta . A more f e a s ib le exp lana­

t io n i s th a t th e e x cessiv e lo s s o f cadmium in to th e u r in e a t t h i s tim e

i s l i k e ly to cause a low ering o f th e r e n a l c o n c e n tra tio n o f th e m e ta l.

S ince a t 72 weeks th e r a t e o f lo s s o f cadmium in to th e u r in e was e q u i-2+

v a le n t to about 4 pg Cd / day, i t can be assumed th a t th i s was s u f f i c i e n t

to cause a s ig n i f ic a n t decrease in th e r e n a l cadmium c o n c e n tra tio n , d e s­

p i te co n tin u in g exposure to th e m e ta l. There a re s e v e ra l r e p o r ts th a t

th e le v e l o f cadmium in th e k idneys o f humans d ecreases a f t e r about

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50 y e a rs o f age (S chroeder e t a l . , 1967; Gross et_ a l . , 1976), and i t

has been suggested th a t changes in th e environm ental exposure to cadmium

th a t have occurred over th e l a s t fo u r decades ( c o n s is te n t w ith th e f a c t

70% o f th e t o t a l w orld p ro d u c tio n o f th e m eta l has been w ith in th e l a s t

20 y e a rs ) may be th e most p la u s ib le e x p lan a tio n fo r t h i s (C .E .C ., 1978).

I t a i - i t a i p a t ie n ts however a lso showed a much lower c o n c e n tra tio n o f

r e n a l cadmium th a n expec ted , d e s p ite h igh h e p a tic c o n c e n tra tio n s

(F r ib e rg , 1978). The a l t e r n a t iv e e x p la n a tio n fo r th e se o b se rv a tio n s

forw arded by Webb (1975c) i s th a t changes in re n a l fu n c tio n and m etabolism

may o ccu r. This seems more l i k e l y and i s c e r ta in ly supported by th e se

r e s u l t s .

The accum ulation o f cadmium a f t e r o ra l a d m in is tra tio n was ag a in

accompanied by changes in th e d is p o s i t io n o f z inc and copper, which

could be expected to c o r r e la te w ith th e sy n th e s is o f th io n e in , as ex p la in ed

e a r l i e r , and d e sc rib e d more f u l l y by S tonard and Webb (1976). In our

s tu d y th e c o n c e n tra tio n o f z in c in th e l i v e r and kidney d id n o t co n tin u e

to in c re a s e s ig n i f i c a n t ly w ith th e accum ulation o f cadmium a f t e r about

16 weeks. P is c a to r (1975) has re p o r te d th a t in th e h o rse , a t low er con­

c e n tra t io n s o f r e n a l cadmium, th e in c re a se in z in c was equim olar to th e

in c re a s e in cadmium, but w ith h ig h e r c o n ce n tra tio n s o f cadmium, z in c

d id n o t in c re a se to th e same e x te n t ; This may be in te r p r e te d as a d i s ­

tu rb an ce in th e cadmium-zinc r e la t io n s h ip . The cadmium:zinc r a t i o in

b o th th e l i v e r and kidney th e re fo re in c re a se d s ig n i f ic a n t ly w ith tim e ,

and i t has been suggested th a t i t i s th e cadmium:zinc r a t i o r a t h e r th a n

th e cadmium c o n c e n tra tio n p er se th a t i s th e im portan t f a c to r in th e

developm ent o f to x i c i t y ( e .g . S chroeder e t a l . , 1967).

Changes in th e plasm a c o n c e n tra tio n s o f z in c , copper and i ro n

a f t e r d ie ta r y cadmium exposure i s f u r th e r ev idence o f d is tu rb e d hom eostasis

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of th e se e s s e n t ia l m e ta ls . Cadmium i s known to in t e r f e r e w ith th e

g a s t r o - in t e s t i n a l a b so rp tio n o f z in c ( e .g . L ease , 1968), copper

( e .g . Van Campen, 1966) and iro n ( e .g . Hamilton and V alberg , 1974).

T hioneins a re b e lie v ed to be im portan t in th e uptake o f z in c and copper,

p o s s ib ly in th e c o n tro l o f i n t e s t i n a l ab so rp tio n (R ichards and C ousins,

1976a; Bremner e t a l . , 1978a). Thus cadmium may in te r f e r e w ith z in c

and copper a b so rp tio n by com peting fo r b ind ing and t r a n s p o r t s i t e s

(D avies and Campbell, 1977), and co m p etitio n fo r s im ila r up take p ro cesses

may a lso u n d e r lie th e decreased i ro n ab so rp tio n (H am ilton and V alberg ,

1974).

A lthough a d ecreased uptake o f z in c and iro n may p a r t i a l l y account

fo r th e decreased plasma le v e ls o f th e se m e ta ls , copper was n o t a f fe c te d

in th e same way. The cadmium-induced in h ib i t io n o f b i l i a r y copper

e x c re tio n , mentioned a f t e r a cu te exposure to cadmium, has a ls o been

observed a f t e r d ie ta r y cadmium tre a tm e n t (Ashby e t a l . , unpub lished

o b s e rv a tio n s ) , and t h i s may account fo r th e in c reased c o n c e n tra tio n o f

copper in th e plasm a a t 72 weeks. This e f f e c t was much le s s s ig n i f i c a n t

th a n a f t e r p a re n te ra l cadmium tre a tm e n t. The in c reased plasm a copper

re q u ire d a long p e rio d o f cadmium exposure to become a p p a re n t, b u t a t

th e tim e t h i s e f f e c t was s ig n i f i c a n t , th e c o n c e n tra tio n o f copper in th e

l i v e r appeared to have d ecrea sed . This could in d ic a te some m o b il is a t io n

o f h e p a tic copper s to r e s . I t i s in te r e s t in g to no te t h a t o th e r w orkers

have found decreased c o n c e n tra tio n s o f copper and cae ru lo p lasm in in

th e plasm a, and low ered h e p a tic copper a f t e r th e d ie ta r y a d m in is tra t io n

o f low le v e l s o f cadmium to lambs (M ills and D algarno , 1972; Bremner

and Cam pbell, 1978). The d if f e r e n c e in th e se f in d in g s may be r e l a t e d

to th e dosage o f cadmium used .

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D ie ta ry cadmium tre a tm e n t f o r 48 weeks was s u f f ic ie n t to cause2+th e accum ulation o f about 115 yg Cd /g which i s e q u iv a len t to a re n a l

2+c o r te x c o n c e n tra tio n o f Cd o f about 175 y g /g , s in c e th e c o n c e n tra tio n

o f th e m etal in th e c o r te x i s about 50% h ig h e r . The ex cessiv e lo s s o f

cadmium in to th e u r in e a f t e r t h i s tim e , to g e th e r w ith th e s ig n i f ic a n t

in c re a se in some u r in a ry enzymes in d ic a te s th e o n se t o f r e n a l damage in

th e se an im als. The e s tim ated c r i t i c a l c o n c e n tra tio n o f 200 yg/g in th e

r e n a l c o rte x th e re fo re appears to be a rea so n a b le e s tim a te , though because

o f th e lo s s o f m eta l from th e kidney between 48 and 72 weeks, i t i s no t

p o s s ib le to know th e peak c o n c e n tra tio n o f cadmium a t ta in e d in t h i s t i s s u e

There have been s e v e ra l a ttem p ts to a l l e v i a t e o r p reven t th e to x i ­

c i t y o f cadmium in a v a r ie ty o f experim en ta l an im als , by a d m in is te r in g

a supplem ent o f z in c , e i th e r p a re n te r a l ly o r in th e d i e t . The p ro te c t iv e

a c t io n o f z in c when in je c te d b e fo re an acu te dose o f cadmium i s known to

be r e la te d to th e in d u c tio n o f m e ta llo th io n e in sy n th e s is (Webb, 1972c).

P re - tre a tm e n t w ith cadmium i t s e l f w i l l s im i la r ly p ro te c t a g a in s t a sub­

sequen t dose o f th e m etal (T erhaar _et a l . , 1965). However, any p ro te c ­

t iv e e f f e c t o f z in c a f t e r ch ro n ic exposure to cadmium may in v o lv e d i f ­

f e r e n t mechanisms. I f p a r t o f th e t o x i c i t y o f cadmium i s due to an i n t e r ­

a c t io n w ith z in c in e s s e n t ia l components such as enzymes, th e n p ro te c t io n

by z in c may a ls o be produced by i t s co m p etitio n w ith cadmium a t th e

m olecu lar l e v e l , f o r in s ta n c e in t r a n s p o r t and b in d in g s i t e s . Indeed ,

d ie ta ry supplem ents o f z in c s a l t s have been shown to reduce some o f th e

to x ic symptoms o f cadmium, e .g . d ecreased growth r a t e in r a t s (P e te r in g

e t a l . , 1971), ch ick s ( H i l l e t a l , , 1963) and c a lv e s (P ow ell e t a l . , 1964)

and a combined supplem ent o f s e v e ra l t r a c e elem ents was p a r t i c u l a r ly

e f f e c t iv e (Bunn and M atrone, 1966; Jacobs e t a l . , 1978),

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The ex perim en ta l r e s u l t s confirm th a t cadmium exposure causes an

in c re a se d demand f o r z in c . In o rd e r fo r z in c to a f fo rd some p ro te c t io n

a g a in s t cadmium t o x i c i t y , i t i s l i k e ly t h a t excess le v e l s o f z in c a re

r e q u ire d . Very h ig h le v e ls o f d ie ta r y z in c a re re q u ire d in o rd e r to

induce th e sy n th e s is o f z in c - th io n e in in th e r a t (Chen e t a l . , 1977),

bu t z in c bound to z in c - th io n e in can be r e u t i l i s e d in th e body and z in c -

th io n e in may conserve z in c and p rov ide a source of th e m etal d u rin g

d e p r iv a tio n (C h e ria n , 1977b). Most o f th e a ttem p ts to in v e s t ig a te th e

p ro te c t iv e r o le o f z in c have n o t used s u f f i c i e n t ly h igh le v e ls o f z in c

to induce z in c - th io n e in , and th e re fo re an excess o f th e m etal was

p ro b ab ly no t a v a i la b le .

I t has been shown th a t a dose o f 2 mg/kg o f z in c i s s u f f i c i e n t to

induce th e z in c -b in d in g p ro te in in r a t s (Bremner and D avies, 1975).

I t was no t s u rp r is in g th e re fo re th a t anim als given a s in g le in je c t io n

o f 5 mg/kg o f z in c had a r a i s e d plasma c o n c e n tra tio n o f z in c . I t has

been suggested th a t z in c hom eostasis may invo lve a mechanism whereby

z in c - th io n e in sy n th e s is i s c o n tro lle d a t th e t r a n s c r ip t io n a l s ta g e by

the plasm a z in c c o n c e n tra tio n . When plasma z in c le v e ls a re h ig h , l i v e r

z in c - th io n e in sy n th e s is i s s tim u la te d which f a c i l i t a t e s th e up tak e o f

th e m e ta l" in to h ep a to cy te s where i t rem ains p ro te in -b o u n d u n t i l needed

to meet c e l l u l a r req u irem en ts (R ichards and C ousins, 1975b, 1976b).

C onsequently , th e sim ultaneous a d m in is tra tio n o f cadmium and z in c d id

no t have th e ad v erse e f f e c t upon plasma z in c th a t i s norm ally seen a f t e r

th e a cu te a d m in is tra tio n o f cadmium a lo n e . H epatic z in c - th io n e in i s

m axim ally induced a f t e r 18 hours in th e r a t and has a ra p id tu rn o v e r

s in c e z in c b in d in g to th e p ro te in d ecreases a f t e r 24 hours (D avies

e t a l . , 1973). Thus th e p r e - in je c t io n o f z in c 24 hours b e fo re cadmium

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d id no t m a in ta in th e plasm a c o n c e n tra tio n o f z in c in th e same way th a t

th e sim ultaneous a d m in is tra tio n o f z in c w ith cadmium d id . The e x c re tio n

o f h ig h le v e l s o f z in c in th e u r in e o f z in c - t r e a te d r a t s p robab ly r e f l e c t s

f u r th e r th e a ttem p ts to r e s to r e normal hom eosta tic c o n tro l .

The in c re a se d t i s s u e b in d in g o f cadmium in anim als p r e - t r e a te d w ith

z in c i s p robab ly accounted f o r by th e p re - in d u c tio n o f z in c - th io n e in ,

which can b ind cadmium as w e ll (L eber and Miya, 1976) o r a llow cadmium-

th io n e in sy n th e s is to occur w ith o u t th e la g phase seen upon exposure to

cadmium a lone (Webb, 1972b). The d e p le t io n o f k idney iro n a f t e r some

z in c - tre a tm e n ts has been re p o r te d b e fo re . D ie ta ry z in c o v erload has been

shown to have a s im ila r e f f e c t on t i s s u e iro n as cadmium (S e t t le m ire and

M atrone, 1967) which may be due to a lo s s of f e r r i t i n (Coleman and M atrone,

1969). This may account fo r th e f a c t th a t th e sim ultaneous a d m in is tra tio n

o f z in c and cadmium caused a g re a te r d ecrease o f plasma iro n th a n e i th e r

cadmium o r z in c g iven in d iv id u a l ly . Zinc a lso ex acerb ated th e e f f e c t o f

cadmium upon plasm a co p p er> which may be evidence o f a z in c -co p p e r

in te r a c t io n . Z inc (and cadmium) have been shown to d is p la c e copper from

su lp h y d ry l b in d in g s i t e s on m e ta llo th io n e in (Evans e t a l . , 1970).

The le v e l o f d ie ta r y z in c supplem ent used fo r th e re p e a te d p a re n te ra l

a d m in is tra t io n o f cadmium was a lso s u f f i c i e n t to induce z in c - th io n e in

(Chen e t a l . , 1977). R ats fed t h i s h ig h supplem ent f o r 28 days showed

no a l t e r a t i o n o f growth r a t e compared to c o n tro l an im als, b u t th e in c re a se d

food consum ption may in d ic a te some d is tu rb a n c e s in m etabo lic p ro c e sse s .

The z in c supplem ent d id n o t however m a in ta in th e growth o f cadm ium -treated

r a t s and th e decreased r e n a l cadmium:zinc r a t i o compared to non-supplem ented

anim als d id no t appear to be b e n e f ic ia l s in c e th e o n se t o f th e second

p e r s i s te n t phase o f enzym uria was th e same in a l l groups. Z inc p re ­

tre a tm e n t however d id com plete ly a b o lis h th e i n i t i a l phase o f enzym uria

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seen w ith in 48 hours o f commencing cadm ium -treatm ent. This p ro te c t io n

i s p robab ly due to th e p resence o f z in c - th io n e in in th e k idney , so th a t

th e cadmium c a tio n i s seq u e s te red r a th e r th a n f r e e to in t e r a c t w ith

c e l l u l a r c o n s t i tu e n ts and produce damage. The tim e-co u rse o f sy n th e s is

o f z in c - th io n e in in th e k idney i s n o t as w e ll known as th e cadmium-

b in d in g p ro te in , b u t i f i t i s o f th e same o rd e r o f tim e (and h e p a tic

sy n th e s is o f th e two th io n e in s i s s im i la r ) , th e n th e degree o f p ro te c t io n

p rov ided by th e sim ultaneous a d m in is tra tio n o f z in c and cadmium i s

s u rp r is in g . This could be ex p la in ed though by an i n i t i a l in c re a se d

uptake o f cadmium in to th e l i v e r . Zinc supplem ented anim als showed a

h ig h e r cadmium c o n c e n tra tio n in th e l i v e r a t th e te rm in a tio n o f th e

experim en t, presum ably because o f a d d it io n a l b in d in g s i t e s made a v a i la b le

on z in c - th io n e in . Thus, on i n i t i a l exposure to cadmium, a g re a te r p ro ­

p o r tio n o f th e m etal may have been r e ta in e d w ith in h ep a to cy te s r a th e r

th a n be ing tra n s p o r te d to th e k id n ey , and th e re fo re th e e x te n t o f i n i t i a l

damage was l e s s . In th e long te rm , z in c could be envisaged as hav ing a

d e le te r io u s e f f e c t upon cadmium t o x i c i t y i f th e in c reased a v a i l a b i l i t y

o f b in d in g s i t e s promotes th e accum ulation o f cadmium w ith in t a r g e t

o rg an s . The r o le o f synergism which may modify th e outcome o f exposure

to a to x ic m eta l has been co n sid ered to be im portan t (Magos, 1976).

The c o n s id e ra b ly h ig h e r e x c re tio n o f u r in a ry z in c by anim als g iven cad­

mium w ith a z in c supplem ent, compared to th o se anim als g iven cadmium, o r

z in c a lone may in d ic a te t h a t such a p rocess i s o ccu rrin g .

Zinc i s a lso known to in t e r a c t w ith copper in b io lo g ic a l sy stem s,

h ig h le v e l s red u c in g th e i n t e s t i n a l a b so rp tio n o f copper (Van Campen and

S c a ife , 1967), The p a t te r n o f u r in a ry copper e x c re tio n may in d ic a te

some d is tu rb a n c e in normal h o m eo stas is . A fte r an i n i t i a l in c re a se d

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e x c re tio n o f copper a t 24 h o u rs , anim als fed th e z inc supplem ent showed

a p e r s i s t e n t , though no t s t a t i s t i c a l l y s ig n i f ic a n t d ecrease in copper

e x c re t io n , which may r e f l e c t m a lab so rp tio n o f th e m e ta l, i . e . an a ttem p t

to conserve copper by red u c in g e x c re tio n .

The d ie ta r y z in c supplem ent used in th e long term fe e d in g s tu d y

was p robab ly no t s u f f i c i e n t to induce th e sy n th e s is o f z in c - th io n e in , b u t

s in c e th e aim was to a ttem p t to r e s to r e normal z in c hom eostasis in cad­

m ium -treated an im als , i t was decided th a t v e ry high le v e l s o f d ie ta r y

z in c fed over a long p e rio d o f tim e may in f a c t cause f u r th e r d is tu rb a n c e s

in th e m etabolism o f o th e r t r a c e elem ents l i k e copper and i r o n , which

would f u r th e r com plica te th e developm ent o f cadmium to x ic i ty .

Nomiyama e t a l . (1978) have re p o r te d th a t in mice exposed to cad­

mium o r a l ly , th e le v e l o f d ie ta r y z in c in flu en c ed th e accum ulation o f

cadmium in th e l i v e r and k idney . Low doses o f z inc (0 - 100 p .p .m .)

in c re a se d cadmium accum ulation in th e se t i s s u e s , w h ile h igh doses

(100 - 3 ,000 p .p .m .) dep ressed i t . This o b se rv a tio n i s d i f f i c u l t to

ex p la in in view o f th e f a c t t h a t th e p ro cesses o f z in c a b so rp tio n and

i t s r e g u la t io n in v o lv in g m e ta llo th io n e in a re no t com pletely re s o lv e d .

I t i s known th a t z in c a b so rp tio n i s re sp o n siv e to z in c s ta tu s and homeo­

s t a s i s i s ” m ediated v ia f lu c tu a t io n s in th e plasma z in c c o n c e n tra tio n .

When exposure to z in c i s low, a b so rp tio n o f th e m etal i s prom oted.

C onverse ly , when exposure i s h ig h , mucosal c e l l m e ta llo th io n e in i s syn­

th e s i s e d in resp o n se to an in c re a s e in z in c s t a t u s , and may fu n c tio n to

b ind z in c and p rec lu d e i t s up take (R ichards and C ousins, 1975, 1976b).

Thus any cadmium-binding to th e z in c -b in d in g p ro te in may a lso b lo ck

some a b so rp tio n o f th e cadmium.

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However, th e d ie ta r y supplem ent o f 300 p .p .m . o f z in c d id not

cause any changes in th e d is p o s i t io n o f cadmium, nor d id i t in f lu e n c e

z in c accum ulation . Plasma le v e ls o f z in c tended to be h ig h e r in cad­

m ium -trea ted , z in c supplem ented r a t s compared to th o se anim als g iven

only cadmium, b u t t h i s e f f e c t was no t s t a t i s t i c a l l y s ig n i f ic a n t excep t

a t 8 w eeks, in d ic a t in g th a t th e supplem ent d id no t s ig n i f i c a n t ly

in f lu e n c e th e z in c s ta tu s o f th e an im als. The decreased c o n c e n tra tio n

o f cadmium in th e blood o f z inc-supplem ented anim als a t some tim es

th roughou t th e experim ent may in d ic a te a cadmium-zinc in te r a c t io n however.

The plasma cadmium c o n c e n tra tio n , which norm ally r e f l e c t s exposure to

th e m e ta l, was u n a ffe c ted su g g es tin g th a t th e in te r a c t io n may have

occu rred a t a l a t e r s tag e th a n a b so rp tio n , p o s s ib ly d u rin g t r a n s p o r t .

Thus, d e sp ite some minor changes in z in c m etabolism , th e z in c supplem ent

d id no t s ig n i f i c a n t ly a l t e r th e b io lo g ic a l resp o n se to cadmium as

measured by changes in th e d is p o s i t io n and e x c re tio n o f t r a c e m e ta ls ,

and th e u r in a ry e x c re tio n o f r e n a l enzymes.

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CHAPTER 4

THE EFFECT OF CADMIUM UPON CALCIUM HOMEOSTASIS

AND BONE TRACE METALS IN THE RAT

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CONTENTS

4 .1 In tro d u c tio n

4 .2 Experim ental

M a te r ia ls ; experim en ta l d esig n ;

a n a ly t ic a l m ethods.

4 .3 R esu lts

Acute a d m in is tra t io n o f cadmium and

z in c ; re p e a te d p a re n te ra l a d m in is tra tio n

o f cadmium; d ie ta r y a d m in is tra tio n o f

cadmium and z in c .

Page

131

131

134

4 .4 D iscu ssio n 149

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4 .1 INTRODUCTION

The e f f e c t s o f cadmium upon th e hom eostasis o f z in c , copper and

iro n have been d e sc rib e d in C hapter 3 . I n te r a c t io n s between th e se

m eta ls may be im p o rtan t in th e development of th e cadmium-induced bone

m alfo rm ations. D e f ic ie n c ie s o f z in c and copper a re b o th known to

cause s k e le ta l d e fe c ts (A slin g and H urley , 1963) and s k e le ta l changes

have a lso been re p o r te d in cases o f iro n d e f ic ie n c y (A scen z i, 1976).

Most r e p o r ts o f a cadmium-induced e f f e c t upon th e bone em phasise

th e need fo r long term exposure to th e m eta l in o rd e r f o r changes to

develop . However, w ithou t ex p erim en ta l ev id en ce , an a cu te o r subacu te

e f f e c t o f cadmium upon th e bone cannot be excluded.

This p re se n t s tudy has in v e s t ig a te d th e p o s s i b i l i t i e s th a t cadmium

may produce an e f f e c t upon th e bone m ediated by mechanisms o th e r th a n

th e one in v o lv in g re n a l damage, and may be produced in th e s h o r t term

as w e ll as th e ch ro n ic s i tu a t io n . In te r a c t io n s between cadmium and

z in c a re in v e s t ig a te d f u r th e r by examining th e e f f e c t o f z in c su p p le ­

m en ta tio n , p a r t i c u l a r ly in r e l a t i o n to th e z in c -c o n ta in in g enzyme,

a lk a l in e phosphatase which i s known to f u l f i l an im portan t r o le in

bone c a l c i f i c a t i o n .

4 .2 EXPERIMENTAL

M a te r ia ls

Chemical compounds (A n a la r grade) were o b ta in ed from B.D.H.

Chem icals L td . , P oo le , D o rse t, and F isons S c ie n t i f i c A pparatus,

Loughborough, L e ic e s te r s h ir e .

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• E xperim ental Design

( i ) The e f f e c t o f acu te p a re n te r a l a d m in is tra tio n o f cadmium and z in c

Young male W istar a lb in o r a t s , body w eight 180 - 200g in groups

o f f iv e were t r e a te d as fo llo w s :

s in g le subcutaneous in je c t io n o f is o to n ic s a l in e

2+s in g le subcutaneous in je c t io n o f 1 .5 mg Cd /k g

(a s CdCl ,2%H20 ) . ■2+s in g le subcutaneous in je c t io n o f 1 .5 mg Cd /k g and

2+s in g le m t r a p e n to n e a l in je c t io n o f 5 .0 mg Zn /kg

( as ZnSO^. 7H20 ) .

s in g le subcutaneous in je c t io n o f is o to n ic s a l in e and

2+s in g le m t r a p e n to n e a l in je c t io n o f 5 .0 mg Zn /k g .

Animals were housed in d iv id u a l ly in g la ss m etabolism cages fo r

u r in e c o l le c t io n and provided w ith s tan d a rd ro d en t d ie t and w ater

ad l ib i tu m . They were k i l l e d 24 hours a f t e r in je c t io n .

( i i ) The e f f e c t s o f re p e a te d p a re n te r a l a d m in is tra tio n o f cadmium

in anim als g iven a d ie ta r y supplem ent o f z in c

Young male W ista r a lb in o r a t s , body w eight 180 - 200g in groups

o f f iv e were t r e a te d as fo llo w s :

Group 1: d a i ly subcutaneous in je c t io n s o f is o to n ic s a l in e .

. . . 2+Group 2 : d a i ly subcutaneous in je c t io n s o f 1 .5 mg Cd / kg.

2+Group 3 : d a i ly subcutaneous in je c t io n s o f 1 ,5 mg Cd /k g and

2+a d ie ta r y supplem ent o f 2,000 p .p .m . Zn .

Group 4: d a i ly subcutaneous in je c t io n s o f is o to n ic s a l in e and

2+a d ie ta r y supplem ent o f 2,000 p .p .m . Zn .

Animals were prov ided w ith s tan d a rd ro d e n t d ie t (powdered) and

w ater ad l ib i tu m . The tre a tm e n t con tinued fo r 28 d ays, d u rin g which

tim e re g u la r u r in e samples were c o l le c te d . Animals were k i l l e d

24 hours a f t e r th e f i n a l in je c t io n .

Group 1:

Group 2:

Group 3:

Group 4:

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( i i i ) The e f f e c t o f d ie ta r y a d m in is tr a t io n o f cadmium and z in c

Young male W ista r a lb in o r a t s , i n i t i a l body w eight 60g, in groups

o f 18 were p rovided w ith s ta n d a rd ro d e n t d i e t (powdered) and w ater

. . . . . 2+ad l ib i tu m . Animals re c e iv e d d ie ta r y supplem ents o f 75 p.p .m . Cd ,2+ 2+ 2+75 p .p .m . Cd and 300 p .p .m . Zn , o r 300 p .p .m . Zn . At in te r v a ls

u rin e sam ples were c o l le c te d and 3 anim als p e r group were k i l l e d .

At th e te rm in a tio n o f each experim en t, venous blood sam ples were

ta k en f o r s e p a ra tio n o f plasm a. Both femurs were removed from each

an im al. Samples o f u r in e , plasm a and femur were s to re d a t -20°C fo r

m etal d e te rm in a tio n s by atom ic a b so rp tio n sp ec tro sco p y .

A n a ly tic a l Methods

In o rg an ic phosphate was determ ined in f r e s h samples o f u r in e and

p lasm a u sin g th e ammonium vanadate /m olybdate method (B iochim ica T e s t ,

B oehringer Manheim D ia g n o s tic s ) . Plasma a lk a l in e phosphatase was

measured using th e op tim ised method d e sc rib ed p re v io u s ly in s e c t io n 2 .2 .

The c o n c e n tra tio n o f calc ium in plasma and u rin e was m easured by

atom ic a b so rp tio n sp ec tro sco p y a f t e r d i lu t io n o f samples w ith a s o lu ­

t io n o f 0.1% w/v lanthanum c h lo r id e (s e e Appendix I I ) .

The femurs were c a r e f u l ly c leaned o f adhering t i s s u e , and s p l i t

lo n g i tu d in a l ly so th a t th e bone marrow could be removed by w ashing in

co ld s a l in e (Rucker e t a l . , 1969). From each an im al, one femur was

re ta in e d fo r m eta l an a ly ses by atom ic a b so rp tio n sp ec tro sco p y , a f t e r

d ig e s t io n w ith n i t r i c /p e r c h lo r i c a c id s , as d e sc rib ed in Appendix I I .

A lk a lin e phosphatase a c t i v i t y was determ ined in th e o th e r femur a f t e r

e x tr a c t io n by hom ogenisation o f th e bone fragm ents in 1.0M d ie th a n o l-

amine b u f f e r , pH 9 .8 , c o n ta in in g 0 .5 mM-MgC^ fo r 5 m inu tes . The homo-

genate was c e n tr ifu g e d a t 8,000 x g (4°C) fo r 20 m in u tes . Heat

Page 135: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

d e n a tu ra tio n a t 56°C d estro y ed 90% o f th e a lk a l in e phosphatase a c t i ­

v i ty o f th i s e x t r a c t , in d ic a t in g th e p resence o f th e h e a t - l a b i l e bone

isoenzym e. A lk a lin e phosphatase a c t i v i t y in th e su p e rn a ta n t was d e te r ­

mined usin g 15 mM 4 -n itro p h e n y l phosphate as d e sc rib ed in s e c t io n 2 .2 .

S u p ern a tan t p ro te in was measured by th e method o f Lowry e t a l . (1951).

S t a t i s t i c a l com parison o f groups was made u sin g a n a ly s is o f

v a r ia n c e and m u ltip le range t e s t s .

4 .3 RESULTS

The E f fe c ts o f Acute A d m in is tra tio n o f Cadmium and Zinc

Table 4 .1 shows th e e f f e c t o f acu te doses o f cadmium an d /o r z in c

on plasm a and u r in a ry calc ium , phosphate and a lk a l in e phosphatase .

S in g le doses o f cadmium, z in c , and cadmium and z in c to g e th e r caused

a s ig n i f ic a n t d ec rea se in th e plasma c o n c e n tra tio n o f calcium a t 24 h o u rs ,

and a decreased e x c re tio n o f calc ium in th e u r in e . Cadmium a d m in is tra ­

t io n a lone caused a s ig n i f ic a n t e le v a tio n o f u r in a ry a lk a l in e phosphatase b u t

t h i s was p rev en ted by th e sim ultaneous a d m in is tra tio n o f z in c . N e ith e r

cadmium nor z in c tre a tm e n ts had any e f f e c t upon th e plasm a c o n ce n tra ­

t io n o f in o rg a n ic phosphate , th e a c t i v i t y o f a lk a l in e phosphatase o r

th e u r in a ry e x c re tio n o f phosphate .

Low le v e ls o f cadmium accum ulated in th e femur o f r a t s in je c te d

w ith cadmium, and th e sim ultaneous a d m in is tra tio n o f z in c d id n o t

in f lu e n c e t h i s accum ulation (T ab le 4 .2 ) , Cadmium a lo n e produced a s ig ­

n i f i c a n t d ec rea se in th e femur z in c c o n c e n tra tio n , though th e concen­

t r a t i o n s o f copper and iro n were no t d i f f e r e n t from c o n tro ls . There

was a ls o a d ec rea se in th e femur c o n c e n tra tio n o f calc ium in cadmium-

t r e a te d an im als. Zinc a d m in is tra tio n caused an in c re a se d accum ulation

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Tabl

e 4.

1 Th

e E

ffec

ts

of A

cute

P

aren

tera

l A

dmin

istr

atio

n of

Cadm

ium

and

Zinc

on

Plas

ma

and

Page 137: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 4.

2 Th

e E

ffec

ts

of A

cute

P

aren

tera

l A

dmin

istr

atio

n of

Cadm

ium

an

d Zi

nc

on th

e

ftCUftftd•HcuCOcdftcdftftCOoftftCUd•H

PcdftP<!O

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d p vo <r CM CO CO•H cd •Hr-l f t +1 + i +1 +1cd f t <Uf t CO CM VO VO 10H O CM CM <r <r cdC ft i—I i—1 p p p

f t cdftf tCO

cd oVO CO < r < r ftO P p p f t

g to • • • •o o o o cu

•H ft da o + 1 +1 +i + i •Hp g ftcd g rH CO <r CO cdCJ 00 CO in f t• • • • P

CO CO CO CO cd4-1O

CM CO p <ro o o o• • • • P

to o o o o •Hd >o p +1 +1 +1 +1 •Hp o Pft g CM CM o 00 C

ft VO vo vo in cd. • • •o o o o 0)

BCM CM p pO O o o •o O o o p

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g

m <r CO• « cdg to o o3 - f t

•H p ft +i + i x} cug o d d COft g o\ p COcd 3 • . cuCJ o i—i Pp p ft

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P CM pd CO d cdcu p CMg o + + COP p CM CM pcd ft ft d pcu d o + CM 3P o CM COH CJ ft cu

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Page 138: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

o f z in c in th e fem ur, bu t d id no t in f lu e n c e th e femur co n ten t o f

co pper, i ro n o r calc ium . I t a lso p reven ted th e cadmium-induced d ecrease

in femur z in c . Both z in c - t r e a te d groups showed e lev a te d a c t i v i t y o f

bone a lk a l in e phosphatase compared to c o n tro ls and anim als g iven on ly

cadmium.

The E f fe c t o f R epeated P a re n te ra l A d m in is tra tio n o f Cadmium

Repeated doses o f cadmium to anim als g iven normal and z in c sup­

plem ented d ie t s had no e f f e c t upon th e plasma c o n c e n tra tio n o f calc ium ,

bu t th e re was a s ig n i f i c a n t ly e le v a te d plasma c o n c e n tra tio n o f in o rg a n ic

phosphate compared to c o n tro ls and r a t s g iven on ly th e z in c supplem ent

(T ab le 4 .3 ) . Plasma a lk a l in e phosphatase was s ig n i f i c a n t ly in c re a se d

in bo th groups o f anim als re c e iv in g h igh d ie ta r y z in c . The u r in a ry

e x c re tio n o f calc ium was s ig n i f i c a n t ly decreased in cadm ium -treated r a t s

th roughou t th e exp erim en ta l p e rio d (F ig . 4 .1 ) , and a t th e te rm in a tio n

o f th e experim en t, th e c o n c e n tra tio n o f calcium in th e u r in e o f th e

cadmium, and cadmium (z in c supplem ented) anim als was approx im ate ly 50%

and 30% o f c o n tro ls r e s p e c t iv e ly . The u r in a ry e x c re tio n o f in o rg a n ic

phosphate was u n a ffe c te d by e i th e r cadmium o r z in c tre a tm e n t, and rem ained

w ith in c o n tro l l im i t s (ap p ro x im ate ly 6 mmol/mmol c r e a t in in e ) th roughou t

th e experim ent. A lk a lin e phosphatase e x c re tio n in to th e u r in e fo llow ed

th e p a t te r n d e sc rib e d e a r l i e r ( s e c t io n 3 .3 ) , and a t th e te rm in a tio n o f

th e tre a tm e n t p e r io d , bo th cadm ium -injected groups o f r a t s had an

enzyme a c t i v i t y in th e u r in e o f about 200% o f c o n tro ls .

Repeated in je c t io n s o f cadmium produced a s ig n i f ic a n t accum ulation

o f cadmium in th e fem ur, and anim als g iven th e z in c supplem ent in

a d d it io n had s ig n i f i c a n t ly more cadmium in t h i s bone (T able 4 .4 ) . The

s ig n i f ic a n t d ec rea se in th e femur z in c co n ten t o f anim als g iv en cadmium

Page 139: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 4.

3 Th

e E

ffec

ts

of R

epea

ted

Par

ente

ral

Adm

inis

trat

ion

of Ca

dmium

on

Plas

ma

and

Uri

nary

C

alci

um,

Inor

gani

c Ph

osph

ate

and

Alk

alin

e P

hosp

hata

se,

in Ra

ts gi

ven

High

D

ieta

ry

Supp

lem

ent

of Z

inc

Page 140: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

urin

e ca

lciu

m

(mm

ol/m

mol

cr

eati

nin

e)

F ig u r e 4 .1 U rin ary E x c r e t io n o f C alcium a f t e r R epeated P a r e n te r a l

A d m in is tra tio n o f Cadmium

0-9

0-6

0-3

0 20

Day o f a d m in is tra tio n

3 0

0 — 0 c o n tro ls 2+• ----- • Cd - t r e a t e d

(B ars r e p re s e n t S .E .M .)

Page 141: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 4.4

Th

e E

ffec

ts

of R

epea

ted

Par

ente

ral

Adm

inis

trat

ion

of Ca

dmiu

m

on th

e

§Cu<u.c44d•HCUCOrt4-1CCSxzaCO

acud•HHrt5

mo

44•H.a4-1<C!0)CO r—1rt4-1CUs4hoao•H4-1rtdtiCUHoo

CUCO xz xz

cu rt rt rtsc 4-1 CM CO in CM

•H rtrH .d +1 +1 + l + 1rt CUX CO <r r-l <r rHi—i o 00 00 rH O< .c «H r-l

CU

tC •drt rt

<r r>- i -o o o o£ 00 • • • •

3 o o o o•H r-lo O +1 +1 +1 +1rH grt g CM o in

c_> H to vo CT\• • 9 .<r CO CO CO

•O •drt rt

H CM CM CMO O O o• • • •

00 o o o oSC —_o I—1 +1 +1 +1 +1Cl oM E t". o rH

p. vo m in vo• • • •o o o o

rt rt rt1—1 r-l r-l tHo O O Oo O O O• • • •

u 00 o O o ocu —cu r-l +1 -H +1 • +1cu Oo e o CO o COo rt. <r CO CO CO

o o o o• • • •o o o o

TOort rt rt

r—1 CM CO voO O o o

00 • • • •O o o o

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•H — •d +1 + i •de H sc d*o O rH 00rt g • •o 3 CT\ 00

CO <r

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o

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T5tH 2 CUO o

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*rtrto

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•rl rH CUcu a) rH CO 44d 4-1 rt rH CUrt O o O •d

d •H dCO a 4-1 44 444-1 CO d Ol-H 00 •H o dd e 4-1 oCO rt ••cu 44 •• 'dPZ 6 CO rt d

Page 142: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

was p rev en ted by th e d ie ta r y z in c supplem ent, and anim als g iven z in c

a lone showed in c re a se d z in c le v e l s in th e bone compared to c o n tro ls .

Both cadmium and z in c tre a tm e n ts caused a d ecrease in th e c o n c e n tra tio n

o f copper in th e bone, and cadmium a lso produced a d ecrease in th e

bone i ro n c o n te n t . The calcium co n ten t o f th e femurs o f bo th cadmium,

and cadmium (z in c supplem ented anim als was s ig n i f i c a n t ly decreased

compared to c o n tro ls and z in c supplem ented an im als , bu t th e a c t i v i t y

o f a lk a l in e phosphatase in th e femur was u n a ffe c te d by cadmium, and

in c re a se d by z in c .

The E f fe c t o f D ie ta ry A d m in is tra tio n o f Cadmium and Zinc

The long term exposure o f r a t s to d ie ta r y cadmium and z in c d id no t

have any e f f e c t upon th e c o n c e n tra tio n s o f calcium and in o rg a n ic phos­

phate in th e plasm a (T able 4 .5 ) . The u r in a ry e x c re tio n o f calc ium was

co n s ta n t in a l l groups up to 24 weeks, a f t e r which tim e , cadmium and

cadmium (z in c supplem ented) anim als e x c re te d s ig n i f i c a n t ly le s s calc ium .

At th e te rm in a tio n o f th e experim en t, th e r a t e o f u r in a ry calc ium e x c re ­

t io n was approx im ate ly 55% and 40% o f c o n tro ls r e s p e c t iv e ly . The u r in a ry

e x c re tio n o f in o rg a n ic phosphate was n o t in flu en c ed by cadmium o r z in c .

There was a g rad u a l accum ulation o f cadmium in th e femur w ith

tim e (T ab le 4 .7 ) , b u t th e t i s s u e c o n c e n tra tio n was low er a t 72 weeks

th an 48 weeks. The sim ultaneous a d m in is tra t io n o f z in c w ith cadmium

d id no t in f lu e n c e th e accum ulation o f cadmium in th e fem ur. The femur

z in c c o n c e n tra tio n o f c o n tro l anim als in c re a se d s ig n i f i c a n t ly w ith age.

Zinc a lso accum ulated in th e femur o f cadm ium -treated anim als b u t to a

low er l e v e l . A f te r 8 weeks o f cadmium a d m in is tra t io n , anim als had a

z in c c o n c e n tra tio n in th e femur o f about 65% o f c o n tro ls . This

decreased accum ulation p e r s is te d th roughou t th e p e rio d o f cadmium-

Page 143: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 4.

5 Th

e E

ffec

t of

Die

tary

A

dmin

istr

atio

n of

Cadm

ium

and

Zinc

on

the

Plas

ma

Con

cent

ratio

n of

Calc

ium

an

d

Q)4->rt45CmCOo45PmO•HartbDPo(5

pH <r COO O o o pH pH pH CM• • • • • • • •o o o o O O O oCM

+1 +1 +1 +1 + 1 +1 + 1 +1r>. a* in o\CM pH CM pH in 00 00• • • • • • • •CM CM CM CM rH rH rH rH

00 in h* inO o O o rH rH rH rH

• • • • • • • •o o o o o o o o00

<r +1 +1 +1 +i +1 +1 +1 +1r . in vo COCO CM CO 00 o o o\• • • • • • • •CM CM CM CM pH CM CM rH

VO CO <fro O O o rH CM rH rH• • • • • • • -•o o o o o o o ovo

CO +1 •H +1 +1 +i -H +i +1/~ \CO C \ CT\ CO in

CO CM CO CO o pH o pHcu • • • • • • • •cu CM CM CM CM CM CM CM CM£

CUc2G•rH CM pH CO VOH o O o O CM CM rH pH• • • • • • • •

o O o o O O o OCM +1 -H +1 +1 +1 + 1 +1

CO O 00 n-CO CO CO CO rH pH ov o• • • • • • • •CM CM CM CM CM CM rH CM

CO VO VO COO O O O pH pH rH rH• • • • • • • •o o o o O O o o

VOi—1 +1 •H +1 +1 -H -H +! +1

<r vo VO <r<r CM CO CO O C\ CM CM• • • . • • • • •CM CM CM CM CM rH CM CM

<r VO CO oo O o pH pH CM pH CM• • • • • • • •o O o O O O O O00

+1 +1 +1 +1 -H •H +1 + 1CM o COuo in <r <r o\ CO CO pH• • • • • • • •CM CM CM CM pH CM CM CM

+ ++4 CM CM£ CO (5 CO C!CU 1—1 CM pH CM& o + + O + +P p CM CM P CM CMrt +4 •d £ P X3 £cu £ o + CM (5 O + CMP O CM O CMH o a X)

o o

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o M cm

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Page 144: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 4.

6 Th

e E

ffec

t of

Die

tary

A

dmin

istr

atio

n of

Cadm

ium

and

Zinc

on

the

Urin

ary

Exc

retio

n of

Calc

ium

an

d

Q)PrtAaCOoAPHo•HS3rt00ShOS3M

'drt rt

rH rH CM <rO O O o C\ CO vo CM• • • • • • • •O O o o O H o oC\l

+1 + 1 +1 +1 + 1 +i +i +1o CM CMCO rH rH CO 00 CO rH• • • • • • • •o O O o vo in 00

X3 *drt rtCM CM CM rHO O O O m rH CO CO• • • • • • • •o o O O o O o o

00<r +1 +1 + 1 +1 +1 +1 +i +1

1". <fr in CM<r CM CM <r rH m CO o• • • • ' • » • •o O o o in in vo

T5 •drt rt

CO rH CM rHo O O O CO rH rH rH• • • • • • • •in o O o O o O O o00 +1 +1 +1 +1 +i +1 +1 +1

CO CO <r oX <r CO CO <r 00 co 1 .cu m • • • • • • •CU►» o o o o m in m <r►s

(Uc!c•H rH CM <r CMH O O o O CM CM rH rH• • • • • • • •

O o o o o o o oCM +1 +1 +1 +! +1 +1 +1 +1

rH rH o CM<r <r <r <r rH CO CM• • • • • • • •o o o o m m in

CM CO rH HO o O o CM in <r CO• • • • • • • •o o O o o o o oVO

rH +1 +1 +1 +! +1 +i +i +1o in CM<r <r <r 00 co o o• • • • • • • •o o o o <r in vo in

CM CM CM <rO o O o CO rH CO CM• • • ♦ • • • •O o o o o O o o00

+1 +1 -H +1 +i +1 +1 +1VO CO CM CO •<r •a- <r rH 1 CO• • • • • • • •o o o o VO VO VO VO

+ +P CM CMS3 CO S3 CO S3CU rH CM rH CM£ O + + O + +p U CM CM M CM CMrt P •d S3 P T3 S3cu S3 o + CM S3 O + CMSh O CM O CMH O •d CJ 'd

cj CJ

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Tabl

e 4.

7 Th

e E

ffec

ts

of D

ieta

ry

Adm

inis

trat

ion

of Ca

dmiu

m

and

Zinc

on

the

Con

cent

rati

ons

of Ca

dmium

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d Zi

nc

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C\ <r vo inCO vo O o o o■ • • • • •o o o o o o

+1 +1 ? +1 +! +1 + io\ vo in in

CO 00 CO CM 00 CM •• • • 9 • • CO

OV 00 CM rH iH CM iHrtS’d •H

o £rt rt rt rtCO <r in

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<r CO in CM £ 4-1 rtCM CM o o o O 4-1 <U cu• • 9 • • • XI *4o o o o o o rt) 4-1

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CJ a rH a

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exposure . The z in c supplem ented, cadm ium -treated anim als had a h ig h e r

femur z in c c o n c e n tra tio n th a n c o n tro ls up to 36 weeks, bu t a t 72 weeks

th e se anim als a lso showed a s ig n i f ic a n t d e c rea se . The z in c supplem ent

a lone caused an in c re a se d z in c accum ulation on ly up to 48 weeks.

Table 4 .8 a lso shows th e e f f e c t o f cadmium and z in c upon th e con­

c e n tr a t io n s o f copper and iro n in th e fem ur. At 8 weeks o f tre a tm e n t,

bo th cadmium, and cadmium (z in c supplem ented) anim als showed a s ig n i ­

f i c a n t ly decreased femur co n ten t o f i ro n , though th e copper c o n c e n tra tio n

was u n a ffe c te d . T his d ec rea se p e r s is te d th roughou t th e experim ent and

a f t e r 48 weeks th e copper c o n c e n tra tio n was a lso low ered. Over th e

f i r s t 36 w eeks, copper and iro n con tinued to accum ulate in th e fem ur,

b u t a t a slow er r a t e th an c o n tr o ls . At th e te rm in a tio n o f th e e x p e r i­

m ent, cadm ium -treated anim als had femur c o n ce n tra tio n s o f copper and

i ro n o f le s s th a n 80% and 50% o f c o n tro ls r e s p e c t iv e ly . The adm inis­

t r a t i o n o f z in c a lone d id no t s ig n i f i c a n t ly in f lu e n c e th e accum ulation

o f iro n in th e femur b u t a f t e r 48 weeks th e se anim als showed a tendency

tow ards decreased c o n c e n tra tio n s o f copper which was s t a t i s t i c a l l y

s ig n i f ic a n t .

The c o n c e n tra tio n o f calc ium in th e femurs o f c o n tro l anim als

in c reased g ra d u a lly up to 48 w eeks, a f t e r which i t d e c lin e d . Cadmium-

tre a tm e n t had no e f f e c t u n t i l 48 weeks when th e re was a s ig n i f i c a n t ly

decreased calcium c o n c e n tra tio n . The a d m in is tra tio n o f a z in c su p p le ­

ment to cadm ium -treated anim als p reven ted t h i s e f f e c t . i (T able 4 .9 ) j

Plasma a lk a l in e phosphatase a c t i v i t y was no t a f fe c te d by e i t h e r

cadmium o r z in c tre a tm e n ts , b u t showed an a g e - re la te d d e c re a se .

S im ila r ly , femur a lk a l in e phosphatase a c t i v i t y was h ig h e r i n i t i a l l y

bu t decreased g ra d u a lly up to 36 weeks. A fte r t h i s tim e i t rem ained

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Tabl

e 4.

8 Th

e E

ffec

ts

of D

ieta

ry

Adm

inis

trat

ion

of Ca

dmium

an

d Zi

nc

on th

e C

once

ntra

tion

s of

Copp

er

and

Iron

in

Page 148: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 4.

9 Th

e E

ffec

ts

of D

ieta

ry

Adm

inis

trat

ion

of Ca

dmium

an

d Zi

nc

on th

e C

once

ntra

tion

of

Calc

ium

in

1a)Ua

cuJSP

TJOCj

C\ hN in CMO rH rH rH9 • • •O O O OCM

r>s +1 +1 +1 + 1rH CM o OSCO VO CM CO• • • •<r • CO <r <r

•doCj

vo VOrH o o o• • • •O o o o

00<r +1 +! +1 +1

CO h- VO o .00 rH in os co• • • • «H<r <r <r <r Cjc:H•H

rtcjo\ co m VOo o o O CO• • • •o o o o m

VO o ••co +1 +1 + i +1 CO• Is

Ov VO CO CM 2 oco CM Ov |H CO • rHX • • • • H rH •CU <r CO <r <r • O *dCU CO m cuIs p

■H CO Cjcj cu

Q) VO CO o 00 co Pe rH rH rH o c cu p•H • • • • cj PH O o O o Q) Cj +

<r e CMCM +1 +1 +1 +1 CO . rt

cu ex CMrH o\ CO 00 p rtOs vo in 00 Cj o • •

• 9 • • P„ *dCO CO CO CO co 00

curt rtrH CUCj cu CU

00 o 1^ CM > is Po CM |H rH p Cj• • • • rH cu CU

VO o o o O «H .Q pi—l < p

+1 +1 +1 +1 /-srH +rH OS <r vo 9 O CM

CO vo 00 P ■ rt• • • 9 .rt O CMco CO CO CO CUD

•H VCU exIS +CM

CO Os c \ CT\ X CO •do o o O CO cu cj• • ■ • cu o

o o o o P r t ••00 Mh <u a

+1 + 1 +1 +1 P00 (Uvo co 00 00 ---- VH00 00 r>. rH m •d• • • ■ O •H cuCO CO CO CO B •d p

s . pCUcu

CO rt pCj cj p

+ op CM 'd •H +r t CO r t cu IP CMa) 1—1 CM CO •H *dB o + + CO rtn op p CM CM cu 00cj p •a rt P •H • •

CU rt CJ + CM a CO AP o CM XH o •d cu rO

* CJ rH 99kcu rH COp Cj rHcj O O

•H Ps CO P PP CO rt

r*1 •H |H •H oCj o rt P oP iH CO CjCU Cj <u p ••s O P5 CO Cj

Page 149: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

Tabl

e 4.

10

The

Eff

ects

of

Die

tary

A

dmin

istr

atio

n of

Cadm

ium

an

d Zi

nc

on th

e A

ctiv

ity

of

gI

CL)PP*d§rtscort

r—IPM

rt•Ha)wrtprtPaCOoPPm

CUrt•Hf—IrtP

■d•

<ua rtrt o

CM iH CO CO CM CM CO uo pCM +1 -H +1 + l + 1 -H + l •H PACM vo 00 pH VO vo CO CM U4hin in in VO <r CM in VO

M•Hcuprt rt o

rH CO VO CO CO CM CO CM p ••00 Cu CO<r +1 •H +1 +1 +1 -H + 1 •H rt00 •

VO 00 00 o CM o\ 00 a rt •duo in UO uo vo CO VO a cup" O pa P rt

CO cu'd P

rt rt rt cu p<r CM <r CM CM CM CM CO rt PVO rt +~\ CO +i +l +1 +1 +1 -H + 1 -H rt CM

CO 6 CO rtP CM rH pH CM uo CM 0'» rH co a CM<u VO VO VO VO uo UO VO rt rt(U rH o ••£ cu p„oo

rHcu rt rt rt •*s i—1 CM CM CO CM CM CO CO P cu *d

•h <r cu cuH CM +1 +1 +! +1 +1 -H + 1 -H CO t2 p

rt p rtpH O vo uo VO vo o cu cuvo VO vo vo uo in VO 'd p Pcu Pco /"N

CO rH +cu O CMrt P • rt

pH CM CM vo co CM in CM cu • o CMVO X wrH +1 +1 +1 +1 +1 -H + l +1 cu iH

nJVrv

CO 0 \ o rH CO vo CO B ••—H

+r>. r^ 1. 00 00 •H •rH CMrt CO •d

CU rt CU CJCO ort CO rt ••

rt P cu oCM in <r CM vo uo <r rt 4n P

P O cuCO +1 +1 +1 + i +1 +1 +1 +i Cu IH

CO ■ m •dCO 00 o CM CO |H. O 33 •rH cu

o rH rH CM pH rH rH CM P •d prH rH rH rH i—1 rH rH rH Cu W rt• P cu

CU CO rt Prt rt p•H +1 o

+ + rH •H +P CM CM rt 10 4h CMrt CO rt CO rt P rt •H xscu rH CM rH CM rH rt rt CJS O + + O + + rt cu 00p P CM CM tH CM CM a •H ••rt P TO rt P •d rt 4h CO pcu rt O + CM rt o + CM o cuP o CM o CM pH o •d o •d {*> rt |H

o CJ p pH CO•rH CO rt rH> cu o O

•rH rt •H PP rH P Prt O rt CO rta p rt > •H o

CO rt P ort 6 cu rH rtrH cu p rH P ••Pm Pm H <s CO rt

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c o n s ta n t in c o n tro l an im als . A fte r 48 weeks o f cadm ium -treatm ent,

cadmium s ig n i f i c a n t ly decreased th e a c t i v i t y o f femur a lk a l in e phos­

ph a tase (T ab le 4 .1 0 ) , b u t when cadmium was ad m in is te red w ith a z inc

supplem ent, t h i s d e c rea se was p rev en ted . The a d m in is tra tio n o f z in c

a lone produced a s ig n i f i c a n t ly e le v a te d bone a lk a l in e phosphatase

a c t i v i t y th roughou t th e experim en t, excep t a t 72 weeks.

4 .4 DISCUSSION

The p re se n t experim ents have i l l u s t r a t e d th a t cadmium may a f f e c t

th e bone a f t e r a c u te , subacu te and long term d ie ta r y a d m in is tra tio n o f

th e m e ta l, though th e e f f e c t s may invo lve d i f f e r e n t mechanisms depending

upon th e dosage regim en o f cadmium.

D istu rb an ces in calc ium hom eostasis a f t e r th e s h o r t term adm inis­

t r a t i o n o f cadmium a re in d ic a te d in th i s s tu d y , s in c e th e calc ium con­

c e n tr a t io n o f th e femur was decreased a f t e r bo th acu te and re p e a te d

p a re n te ra l a d m in is tra t io n . In th e p rocess o f calcium h o m eo stas is , th e re

i s a tendency f o r th e organism to m a in ta in th e plasma c o n c e n tra tio n o f

calcium a t th e expense o f th e bone. The bone dom inates th e c o n tro l o f

plasma calcium in th e s h o r t term (R obertson , 1976). T his may account

fo r th e decreased c o n c e n tra tio n o f calcium in th e femur a f t e r th e

a d m in is tra t io n o f a s in g le dose o f cadmium. W ith re p e a te d cadmium

d o sin g , however, plasm a calcium was m ain ta ined b u t th e decreased u r in a ry

e x c re tio n o f calc ium was ev idence o f a d is tu rb e d m etabolism . H y p o calc i-

u r ia was a s u rp r is in g f in d in g , s in c e , as m entioned in s e c t io n 1 .3 , th e

converse o b se rv a tio n has o f te n been re p o r te d , and i f a cadmium-induced

nephropathy was p re s e n t , an ex ce ss iv e lo s s o f calcium in to th e u r in e

could be expec ted . The re p e a te d dosage regim en o f cadmium in th e

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p re se n t s tudy was s u f f i c i e n t to produce some kidney damage, s in c e

an ex cessiv e lo s s o f a lk a l in e p h o sp h atase , and o th e r enzymes ( s e e 3 .3 )

in to th e u r in e was observed . The h ig h c o n c e n tra tio n o f in o rg a n ic

phosphate in th e plasma o f th e se anim als a lso in d ic a te s some k idney

d y s fu n c tio n .

A p o s s ib le e x p la n a tio n fo r th e se e f f e c t s i s th a t th e accum ulation

o f cadmium in th e k idney caused some d is tu rb a n c e in v itam in D m etabolism .

This would r e s u l t in a reduced c a p a c ity to absorb s u f f ic ie n t calc ium

from th e d ie t (Wasserman and T a y lo r, 1968; Wasserman e t a l . , 1974).

H y p o ca lc iu ria in f a c t u s u a lly deno tes calc ium m alab so rp tio n . A re sp o n se

to th e decreased a b so rp tio n could be an in c reased p ro d u c tio n o f p a ra ­

th y ro id hormone, which would reduce th e re n a l lo s s o f calcium by

in c re a s in g tu b u la r re a b so rp t io n (d e s p i te th e p resence o f some k idney

dam age), and could cause th e m o b ilis a tio n o f calcium from th e bone.

In a d d it io n , a h igh c o n c e n tra tio n o f in o rg a n ic phosphate in th e plasma

i s known to b lock th e sy n th e s is o f 1,25-D ihydroxy c h o le c a lc i f e r o l

(W ilk inson , 1976).

The plasm a calcium c o n c e n tra tio n was s im i la r ly m ain ta ined in r a t s

exposed to long term d ie ta r y a d m in is tra t io n o f cadmium, bu t a g a in th e

s ig n i f ic a n t d ecrease in th e femur calc ium co n ten t a f t e r 48 w eeks, and

h y p o c a lc iu r ia a f t e r cadmium exposure were in d ic a t iv e o f a d is tu rb e d

calcium h o m eostasis . H y p o ca lc iu ria occurred ag a in d e sp ite th e p resence

o f some kidney d y sfu n c tio n as in d ic a te d by e le v a te d u r in a ry enzymes

( s e e 3 .3 ) , bu t was ev id en t b e fo re th e o n se t o f enzym uria.

In a d d it io n to th e mechanism in v o lv in g v ita m in D th a t has been

proposed a f t e r re p ea ted p a re n te r a l a d m in is tra tio n o f cadmium, d ie ta r y

a d m in is tra t io n o f th e m etal may in f lu e n c e calcium m etabolism a t th e

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a b so rp tio n s ta g e . Cadmium in th e gut may compete w ith calcium fo r

b in d in g to th e ca lc iu m -b in d in g p ro te in as cadmium and calcium have

s im ila r b in d in g a f f i n i t i e s f o r t h i s p ro te in (Bredderman and Wasserman,

1974). T his in te r a c t io n can le a d to an enhanced uptake o f cadmium,

p a r t i c u la r ly in c o n d itio n s o f d ie ta r y calcium d e fic ie n c y (Washko and

C ousins, 1976), and co n sequen tly calcium a b so rp tio n may be f u r th e r

im paired . V itam in a lso s t im u la te s th e a c t i v i t y o f a lk a l in e phos­

p h a tase which i s lo c a l i s e d e x c lu s iv e ly in th e b rush b o rd e r o f th e

i n t e s t i n a l mucosa o f some s p e c ie s . A r o le fo r t h i s enzyme in calc ium

t r a n s p o r t has been suggested (Norman e t a l . , 1970). A f u r th e r p o s s ib le

in te r fe re n c e o f calcium m etabolism by cadmium could th e re fo re be

envisaged s in c e d ie ta r y cadmium tre a tm e n t can produce p a th o lo g ic a l

changes in th e i n te s t in e (Sugawara and Sugawara, 1977; V alberg e t a l . ,

1977).

In a d d itio n to th e in te r f e r e n c e w ith calcium h o m eo stas is , t h i s

s tu d y in d ic a te s t h a t cadmium may induce changes w ith in th e bone i t s e l f .

These changes d id no t need long te rm exposure to cadmium s in c e s im ila r

e f f e c t s were noted in anim als g iven s in g le and re p e a te d in je c t io n s o f

th e m e ta l. The d ecreased c o n c e n tra tio n s o f z in c and copper in th e

femur p robab ly r e f l e c t th e in c re a se d requ irem en t fo r th e se m e ta ls

caused by th e sy n th e s is o f th io n e in , e s p e c ia l ly in th e l i v e r and k id n ey .

The r e s u l t s th e re fo re su p p o rt th e su g g es tio n o f Sugawara e t a l . (1978)

t h a t th e accum ulation o f th e se m e ta ls w ith cadm ium -thionein may le a d

to a t r a n s f e r from o th e r o rgans. The d ecrease in femur i ro n may

r e f l e c t d is tu rb a n c e s in iro n m etabolism as d e sc rib e d in s e c t io n 3 .3 ,

and d iscu ssed by S tonard and Webb (1976). The developm ent o f k idney

damage need no t th e re fo re be a p r e r e q u is i te f o r d is tu rb a n c e s in th e

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bone, b u t th e d ecreased a v a i l a b i l i t y o f z in c , copper and iro n may be

ex acerb a ted by an in c re a se d lo s s o f th e se m eta ls in to th e u r in e i f

nephropathy i s caused .

O ther w orkers have a lso observed an e f f e c t o f cadmium on bone65m e ta ls . Lease (1968) showed th a t th e uptake o f Zn in to bone t i s s u e

was in h ib i te d by cadmium when ad m in is te red o r a l ly to c h ic k s , and

Itokaw a e t a l . (1973, 1974) have d e sc rib ed d is tu rb a n c e s in magnesium

and z in c le v e ls in th e bones o f r a t s a f t e r d ie ta r y cadmium exposu re ,

which was a s s o c ia te d w ith a decreased calc ium co n ten t and h i s to lo g ic a l

changes c h a r a c t e r i s t i c o f o s teo m alac ia . R e ta rd a tio n o f growth and

s k e le ta l fo rm ation have been re p o r te d in r a t s and th e o f f s p r in g o f

sheep fed very low le v e ls o f cadmium (Campbell and M il ls , 1974;

Campbell and M il ls , c i te d in Bremner and Cam pbell, 1978), and th e se

e f f e c t s were r e l a t e d to cadmium-induced d is tu rb a n c e s in copper m etabolism .

However, th e copper co n ten t o f th e bone i t s e l f was no t in v e s t ig a te d .

Changes in th e c o n c e n tra tio n o f z in c , copper and iro n in th e

bone caused by cadmium tre a tm e n t a re p a r t i c u la r ly in te r e s t in g because

i t has been known f o r some tim e th a t th e se m eta ls may f u l f i l im p o rtan t

fu n c tio n s in bone fo rm ation (A s lin g and H urley , 1963; Calhoun e t a l . ,

1974), and s k e le ta l d e fe c ts have been re p o r te d a f t e r d e f ic ie n c ie s o f

z in c , copper and iro n (A slin g and H u rley , 1963; F is h e r , 1975;

A scenzi, 1976), W hile th e ex ac t fu n c tio n o f th e se m eta ls rem ains

o b scu re , i t i s th ough t th a t z in c as a component o f a lk a l in e phosphatase

i s invo lved in th e fo rm atio n o f th e m in e ra l m a trix o f osseous t i s s u e

( U r i s t , 1976), and copper, as a component o f ly s y l ox idase i s in v o lv ed

in c o lla g e n c ro s s - l in k in g in th e o rg an ic m a trix o f bone (O’D e ll , 1976).

The decreased a c t i v i t y o f femur a lk a l in e phosphatase observed in

th i s s tu d y may th u s have im portan t consequences fo r bone c a l c i f i c a t i o n .

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D istu rb an ces in th e a c t i v i t i e s o f z in c and copper c o n ta in in g enzymes

in th e bone have been re p o r te d in anim als s u f fe r in g from d e f ic ie n c ie s

o f th e se two m eta ls (P ra sad e t a l . , 1967; Rucker e t a l . , 1969). I t

rem ains u n c le a r why th e cadmium-induced d ecrease in femur a lk a l in e

phosphatase was only ev id en t a f t e r 48 weeks o f d ie ta r y cadmium t r e a t ­

m ent, d e s p ite s ig n i f i c a n t ly decreased c o n c e n tra tio n s o f bone z in c a t

a much e a r l i e r s ta g e . The e f f e c t may be r e la te d to th e d i s t r i b u t io n

o f th e enzyme in th e bone, s in c e i t has been re p o r te d th a t th e e f f e c t

o f z in c d e f ic ie n c y upon a lk a l in e phosphatase depends upon th e s i t e

o f th e enzyme w ith in th e t i s s u e (W estmoreland and H oekstra , 1969).

The la c k o f e f f e c t o f cadmium on bone a lk a l in e phosphatase a f t e r

re p e a te d p a re n te r a l a d m in is tra tio n was perhaps s u rp r is in g s in c e th e

bone c o n c e n tra tio n o f z in c was s ig n i f i c a n t ly d ecreased .

The e f f e c t on bone a lk a l in e phosphatase seen in th e d ie ta r y s tu d y

o ccu rred even though th e c o n c e n tra tio n o f cadmium in osseous t i s s u e

was low. However, i t has been re p o r te d t h a t , in c o n tra s t to l i v e r

and k idney , on ly 20% o f th e cadmium p re s e n t in th e bone i s bound to

m e ta llo th io n e in (Kimura e t a l . , 1974). Thus, most o f th e cadmium in

bone may be a v a i la b le to i n t e r a c t , p o s s ib ly by d is p la c in g z in c from

a lk a l in e pho sp h atase . An a l t e r n a t iv e mechanism fo r th e e f f e c t i s

th a t th e p ro d u c tio n o f th e enzyme r a th e r th a n i t s a c t i v i t y i s d is tu rb e d

by cadmium, s in c e i t has been suggested th a t z in c may be n e c e ssa ry fo r

th e sy n th e s is o f a lk a l in e phosphatase (W estmoreland and H o ek stra ,

1969).

The most n o ta b le e f f e c t o f z in c su pp lem en ta tion in t h i s s tu d y was

i t s in c re a se d accum ulation in bone. This len d s f u r th e r su p p o rt to th e

id e a th a t th e bone may a c t as a s to r e f o r z in c which may be m o b ilised

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i f re q u ire d bu t accum ulated i f in ex cess. An in c reased accum ulation

o f z in c in th e bone to g e th e r w ith in c re ase d a c t i v i t y o f a lk a l in e

phosphatase has been d e sc rib e d a f t e r d ie ta r y su p p lem en ta tion (O berleas

and P rasad , 1969; Calhoun e t a l . , 1974). In th e p re se n t ex p erim en ts,

anim als exposed to cadmium bu t supplem ented w ith z in c d id n o t show th e

decreased c o n c e n tra tio n o f z in c in th e bone th a t was e v id en t in anim als

g iven on ly cadmium, and i t i s in te r e s t in g th a t th e cadmium-induced

d ecrease in bone a lk a l in e phosphatase a c t i v i t y a f t e r long term d ie ta r y

a d m in is tra tio n o f cadmium was com plete ly p rev en ted . This may have con­

t r ib u te d to th e f in d in g th a t th e se anim als had a bone calc ium co n ten t

w ith in c o n tro l l im i t s .

O ther changes in calc ium hom eostasis brought about by z in c sup­

p lem en ta tio n may n o t be advantageous, bu t were on ly e v id e n t a f t e r th e

a d m in is tra tio n o f z in c a t le v e ls s u f f i c i e n t to induce z in c - th io n e in .

The d ecreased plasma calcium c o n c e n tra tio n a f t e r a s in g le in je c t io n o f

z in c , and th e low ered u r in a ry calcium e x c re tio n a f t e r bo th a cu te and

h igh d ie ta r y supplem ent o f z in c a re evidence o f a z in c -ca lc iu m

in te r a c t io n . This e f f e c t tended to enhance th e e f f e c t o f cadmium.

Z inc-ca lc ium in te r a c t io n s in b io lo g ic a l system s have n o t been re p o r te d

in d e t a i l , though z in c has been shown to in h ib i t th e up take o f calc ium

by r a b b i t a o r t i c s t r i p s (S ahag ian and S p rarag en , 1972), and z in c ,

l i k e cadmium, may compete w ith calc ium fo r b in d in g s i t e s on th e v ita m in D-

induced calcium b in d in g p ro te in ( I n g e r s o l l and Wasserman, 1971). The

supp lem en ta tion o f anim als w ith low er doses o f z in c f o r lo n g e r p e r io d s '

o f tim e d id not produce any o f th e se e f f e c t s , presum ably because

excess le v e ls o f th e m eta l were n o t a v a i la b le .

These experim ents have f u r th e r i l l u s t r a t e d th e com plex ity o f th e

r o le o f z in c in cadmium to x ic i t y . Zinc may a b o lish some o f th e

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cadmium-induced changes, as in d ic a te d by th e p re v e n tio n o f th e d ec rease

in bone a lk a l in e pho sp h atase , y e t i t may enhance o th e r e f f e c t s such as

th e d ecreased e x c re tio n o f calc ium in th e u r in e . In a d d it io n , z in c

may a f f e c t copper m etabolism in th e bone. A lthough th e re was no e v i­

dence f o r t h i s in th e experim en t, th e p o s s ib i l i t y should no t be excluded .

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CHAPTER 5

THE USE OF CHELATING AGENTS IN

THE THERAPY OF CADMIUM TOXICITY

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CONTENTS

Page

5.1 In tro d u c tio n 158

5.2 E xperim ental 160

M a te r ia ls ; ex perim en ta l desig n

a n a ly t ic a l methods

5.3 R e su lts 164

E ffe c t o f c h e la t in g ag en ts on th e

d i s t r i b u t io n and e x c re tio n o f a s in g le

dose o f cadmium; e f f e c t s o f re p e a te d

a d m in is tra tio n o f c h e la t in g ag en ts on

th e d i s t r i b u t io n and e x c re tio n o f

p a r e n te r a l ly ad m in is te red cadmium;

e f f e c t s o f c h e la t in g ag en ts on th e

d i s t r i b u t io n and e x c re t io n o f cadmium

a f t e r d ie ta r y a d m in is tra t io n ;

in f lu e n c e o f sodium s e le n i t e and Puchel

on th e d i s t r i b u t io n o f cadmium.

5 .4 D iscu ssio n 174

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5 .1 INTRODUCTION

There i s no e f f e c t iv e th e ra p y fo r th e tre a tm e n t o f a c c id e n ta l

cadmium p o iso n in g , d e s p ite th e in c re a se d in c id en ce o f bo th acu te -an d

ch ro n ic cases o f c l i n i c a l l y id e n t i f i a b le cadmium t o x ic i t y (F l ic k

e t a l . , 1971). S ince most b io lo g ic a l e f f e c t s o f to x ic m etal io n s a re

p robab ly due to th e b in d in g o f th e se m eta ls to m e ta b o lic a lly im p o rtan t

g roups, c o n s id e ra b le i n t e r e s t has a r is e n in th e a b i l i t y o f c h e la t in g

ag en ts to promote th e m o b il is a t io n and e x c re tio n o f to x ic m eta ls

which have been d ep o sited in v a r io u s body com partm ents. The e f f e c t iv e ­

ness o f such complexing a g e n ts , however, in re v e rs in g o r p re v e n tin g

cadmium-induced to x ic e f f e c t s i s m inim al, and d a ta a re l im ite d to

ex p erim en ta l in v e s t ig a t io n s on ly .

M etal c h e la t io n in b io lo g ic a l system s has s e v e ra l im p o rtan t

im p lic a tio n s . M etal c o -o rd in a tio n complexes a re invo lved in e s s e n t i a l

b iochem ical p ro cesses such as m eta l t r a n s p o r t , and in m etalloenzym es

and m acrom olecular conform ations fo r in s ta n c e . C onverse ly , th e

phenomenon o f c h e la t io n may produce n o n -e s s e n tia l e f f e c t s such as th e

a l t e r a t i o n o f th e re sp o n se to heavy m eta ls due to th e p resen ce o f

c h e la t in g ag en ts in th e environm ent. The p o te n t ia l v a lu e o f com plexing

agen ts f o r d e to x ify in g m eta l ions was however in d ic a te d a c o n s id e ra b le

tim e ago (K ety and L e to n o ff , 1941).

C h e la tio n occurs when a m eta l io n combines w ith lig a n d s o f a

complexing agen t which fu n c tio n s as an e le c t r o n - p a ir donor, w ith th e

fo rm atio n o f a h e te ro c y c lic r in g . Such a complex i s v e ry s t a b l e , and

th e c h e la te d m etal th e re fo re does no t e x h ib it th e chem ical r e a c t io n s

c h a r a c t e r i s t i c o f th e f r e e m etal io n . , S y n th e tic o rg an ic c h e la te s

hav ing m u ltip le p o la r groups have been developed fo r th e rem oval o f

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to x ic e lem en ts . The v a lu e o f th e polyam inocarboxylic ac id s such as

e th y le n e d ia m in e te tra a c e tic a c id (EDTA) and d ie th y le n e tr ia m in e p e n ta a c e tic

ac id (DTPA) as w e ll as th e su lp h u r-c o n ta in in g compounds such as

2 ,3 -d im ercap to p ro p an o l (B r i t is h -A n t i-L e w is i te : BAL) and p e n ic i lla m in e ,

has been w idely re p o r te d f o r th e tre a tm e n t o f t o x i c i t y in humans caused

by a v a r ie ty o f m eta ls in c lu d in g le a d , mercury and plutonium

( e .g . Foreman, 1953; K itz m il le r e t a l . , 1954; Chisholm , 1968). This

has been th o ro u g h ly review ed by C atsch and Harmuth-Hoerie, 1979).

However, th e re a re no lig a n d s which r e a c t s p e c i f i c a l ly w ith on ly

one p a r t i c u la r m eta l io n and t h i s la c k o f s e l e c t i v i t y o f most c h e la t in g

ag en ts r e s u l t s in a decreased e f f e c t iv e n e s s . The second problem i s

t h a t th e c h e la t in g agen ts them selves may possess some in h e re n t t o x i c i t y ,

which again may d ecrease th e th e ra p e u tic e f f e c t iv e n e s s . The s y n th e t ic

po lyam ino -carboxy lic ac id s have been s tu d ie d e x te n s iv e ly from th e se

p o in ts o f view . EDTA and DTPA bo th form s ta b le c h e la te s w ith ca lc iu m ,

which a re l e s s to x ic th an t h e i r sodium form s. However, even though

DTPA e x h ib its a h ig h e r t o x i c i t y th a n EDTA, i t has been su g g ested th a t

because o f i t s h ig h e r s t a b i l i t y c o n s ta n ts , DTPA should be used in

p re fe re n ce to EDTA fo r th e ra p e u tic purposes (C atsch and Harmuth-Hoene,

1979). The m o b ilis in g e f fe c t iv e n e s s o f DTPA and r e la t e d compounds i s

s e v e re ly decreased by delayed tre a tm e n t because o f t h e i r d i s t r i b u t i o n

alm ost e x c lu s iv e ly in th e e x t r a c e l lu la r sp ace , making i t more d i f f i c u l t

to m o b ilise m eta ls such as cadmium d e p o sited w ith in th e c e l l . One

a ttem p t to o b ta in h ig h c o n c e n tra tio n s o f c h e la t in g ag en ts in th e i n t r a ­

c e l l u l a r compartment was made u sin g more l ip o p h i l i c e s t e r i f i e d p o ly ­

am inocarboxylic a c id s (C a tsch and Harmuch-Hoene, 1975). W ith a s im i la r

aim , th e N a tio n a l R ad io lo g ica l P ro te c tio n B oard, H arw ell, have syn­

th e s is e d a novel c h e la t in g a g e n t, "P u ch e!" , which i s a d e r iv a t iv e o f

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DTPA. This compound p o ssesses l ip o p h i l i c f a t t y a c id s id e ch a in s and

i t i s a n t ic ip a te d th a t th e l ip o p h i l i c p o r tio n o f th e m olecule w i l l

enab le i t to e n te r th e c e l l and th u s remove in t r a c e l l u l a r m eta l con­

ta m in a n ts , which th e co rrespond ing h y d ro p h ilic calcium c h e la te cannot

do. Puchel has been shown to enhance th e c le a ra n c e o f p lu tonium from

exp erim en ta l an im als , bu t th e e f fe c t iv e n e s s o f th e compound in rem oving

cadmium has n o t been in v e s t ig a te d . C onsequently , s tu d ie s were p e r­

formed to examine th e a b i l i t y o f Puchel and v a rio u s o th e r d e r iv a t iv e s ,

to remove cadmium from th e body, and to compare th e e f f ic a c y o f such

l ip o p h i l i c compounds w ith co n v en tio n a l h y d ro p h ilic c h e la t in g a g e n ts .

The fo llo w in g a b b re v ia tio n s a re used in t h i s c h ap te r:

BAL - B r i t i s h A n ti-L ew is ite - 2 ,3 -d im ercap top ropano l

DMS - m eso -2 ,3 -d im ercap to su cc in ic ac id

DTPA - D ie th y le n e tr ia m in e p e n ta a c e tic a c id

Puchei-cys - Puchel b is c y s te in ei ~ i

DDDC - Disodium j D ie th y ld ic y s te d ic ac id j

BH-DTPA - B ishexylam ido DTPA

BD-DTPA - B is-decylam ido DTPA

5.2 EXPERIMENTAL

M ate ria ls

P u ch e l, o th e r d e r iv a t iv e s o f DTPA and DDDC were p rov ided by th e

N a tio n a l R ad io lo g ica l P ro te c tio n B oard, H arw ell. DMS and BAL were

o b ta in ed from Sigma Chemical Company L td . , P oo le , D o rse t, and sodium

s e le n i t e from B.D.H. Chemicals L t d . , P oo le , D o rse t.

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E xperim ental D esign

Male W ista r a lb in o r a t s , body w eight 100 - 200g were used

th roughou t th e se s tu d ie s . In any g iven experim ent anim als had a

w eight range o f ^ lO g, and were p rov ided w ith s tan d a rd ro d en t d i e t

and w ater ad l ib i tu m .

( i ) The in f lu e n c e o f s in g le doses o f c h e la tin g agen ts fo llo w in g th e

acu te p a re n te r a l a d m in is tra t io n o f cadmium

R a ts , in groups o f f i v e , were in je c te d subcu taneously w ith 300 yg

2+ . . .o f Cd as CdCl^ in 0 .2 ml s a l in e . One hour l a t e r anim als w ere in je c te d

in t r a p e r i to n e a l ly w ith a v a r ie ty o f c h e la t in g agen ts as shown below.

Compounds were made up in aqueous s o lu t io n excep t fo r BAL which was

d is so lv e d in co rn o i l , and g iven in a 0 .2 ml volume.

Treatm ent Dose (mg/kg)

s a l in e -

Puchel 100

Puchel 200

DTPA 100

DDDC 100

P uchel-cys 100

DMS 25

BAL 25

BH-DTPA 100

BD-DTPA 5

BD-DTPA-ester 100

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Animals were housed in d iv id u a l ly in g la s s m etabolism cages fo r

c o l le c t io n o f u r in e and faeces in a c id washed c o n ta in e rs . A fte r

24 h o u rs , venous b lood was c o l le c te d , anim als were k i l l e d , and l i v e r

and k idneys were removed. Samples were s to re d a t -20°C fo r subsequent

m etal d e te rm in a tio n s .

( i i ) The in f lu e n c e o f re p e a te d doses o f c h e la t in g ag en ts fo llo w in g

th e a cu te p a re n te r a l a d m in is tra tio n o f cadmium

R a ts , in groups o f f iv e , were in je c te d subcu taneously w ith 300 yg

2+ . .o f Cd as CdCl^ m 0 .2 ml s a l i n e . 24 hours l a t e r anim als were g iven

d a i ly in t r a p e r i to n e a l in je c t io n s o f c h e la t in g agen ts (0 .2 ml) fo r

5 c o n secu tiv e d ay s, as shown below:

Treatm ent Dose (mg/kg)

s a l in e —

Puchel 100

Puchel-cys 100

DDDC 100

Animals were housed in d iv id u a l ly in .g la s s m etabolism cages f o r

c o l le c t io n o f u r in e and fa e c e s . At th e te rm in a tio n o f th e experim en t,

24 hours fo llo w in g th e l a s t in je c t io n o f c h e la t in g a g e n t, a venous

blood sample was ta k e n . Animals were k i l l e d and l i v e r and k idneys

were removed. Samples were s to re d a t -20°C f o r subsequent m eta l

d e te rm in a tio n s .

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( i i i ) The in f lu e n c e o f r e p e a te d d o ses o f c h e la t in g a g e n ts f o l lo w in g

th e d ie t a r y a d m in is tr a t io n o f cadmium

2+R ats were prov ided w ith a d ie ta r y supplem ent o f 150 p .p .m . Cd

as CdCl^ f o r 8 weeks. A fte r t h i s - tim e anim als were w ithdrawn from th e

cadmium-supplemented d i e t , and fed s tan d a rd ro d en t d ie t fo r 7 days.

They were g iven d a i ly in t r a p e r i to n e a l in je c t io n s o f c h e la tin g ag en ts

(0 .2 ml) fo r 5 co n secu tiv e d ays, as shown below:

Treatm ent no. o f anim als Dose (mg/kg)

s a l in e 10 —

Puchel 5 100

DTPA 5 100

DDDC 5 100

P uchel-cys 5 100

DMS 5 25

DMS & Puchel 5 25 + 100

Animals were housed in d iv id u a l ly in g la ss m etabolism cages fo r

c o l le c t io n o f u r in e and fa e c e s . At th e te rm in a tio n o f th e exp erim en t,

a venous b lood sample was ta k e n . Animals were k i l l e d and l i v e r and

kidneys were removed. Samples were s to re d a t -20°C fo r subsequen t

m eta l d e te rm in a tio n s .

( iv ) The in f lu e n c e o f s in g le doses o f sodium s e le n i t e and Puchel

fo llo w in g th e acu te p a re n te r a l a d m in is tra tio n o f cadmium

R a ts , in groups o f f iv e , were in je c te d subcu taneously w ith 300 yg Cd

as CdC^ in 0 .2 ml s a l in e .

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Treatm ent Dose (mg/kg)

100

2.5

2 .5 + 100

Selenium , as Na2Se0^ was in je c te d su b cu tan eo u sly , im m ediately a f t e r

cadmium a d m in is tra tio n . One hour l a t e r . Puchel was ad m in is te red

in t r a p e r i to n e a l ly as shown above. 24 hours l a t e r , a venous b lood

sample was ta k e n . Animals were k i l l e d and l i v e r and kidneys removed.

Samples were s to re d a t -2 0 °C f o r subsequent m etal d e te rm in a tio n s .

A n a ly tic a l Methods

Cadmium was determ ined in whole b lo o d , u r in e , fa ece s and t i s s u e s

by atom ic a b so rp tio n sp ec tro sco p y as d e sc rib e d in Appendix I I .

5 .3 RESULTS

The E f fe c t o f C h e la tin g Agents on th e D is t r ib u t io n and E x c re tio n

o f a S in g le Dose o f Cadmium2+

A fte r th e a d m in is tra tio n o f a s in g le dose o f 300 yg o f Cd to

male r a t s , approx im ate ly 150 yg and 6 yg o f th e m eta l accum ulated in

th e l i v e r and k idneys r e s p e c t iv e ly a t 24 h o u rs . M easurable concen­

t r a t i o n s o f cadmium were d e te c te d in whole b lo o d , and th e m eta l was

e x c re te d in th e faece s (2.7% o f d o se ) , and u r in e (0.06% o f dose)

(T ab les 5 .1 and 5 .2 ) .

s a l in e

Puchel

Selenium

Selenium & Puchel

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T ab le 5 .1 A cute P a r e n te r a l A d m in is tr a tio n o f Cadmium: E f f e c t o f

S in g le Doses o f C h e la tin g Agents on th e Tissue C ontent

and Blood C o n cen tra tio n o f Cadmium

L iv e r Kidney Blood2+ 2+ 2+ yg Cd yg Cd yg Cd /100 ml

C ontro ls 149.7 ± 3 .4 6 .0 ± 0 .2 4 .9 ± 0 .1

Puchel: 100 mg/kg 52.4 + 1.0*** 3 .2 + 0.4*** 3 .2 + 0.3***

P uchel: 200 mg/kg 55.1 + 2 ^ rfWWc 3 .4 ± 0.3*** 3 .6 ± Ui

DTPA 135.7 + 5 .5 5.3 + 0 .1* 3 .0 ± 0.1***

DDDC 102.3 ± 3.3*** 9 .6 ± 0.5*** 4 .2 ± 0 .3*

P uchel-cys 53.4 + 5 e 2*** 3 .7 + 0.2*** 3 .2 + 0.2***

DMS 127.7 + 6.2* 6 .2 + 1 .0 4 .3 + 0 .1

BAL 121.8 + 4 .9** 8 .2 Hr 0.3*** 5 .8 + 0 .2**

BH-DTPA 119.7 + 3.9*** 4 .4 ± 0.3** 4 .4 ± 0 .2 *

BD-DTPA 135.8 ± 6.3 5 .1 ± 0 .3* 3 .6 ± 0 .2***

BD-DTPA-ester 131.3 + 9 .9 4 .5 + 0.4*^' 3 .3 + 0 .1***

A ll v a lu es a re means - S.E.M. o f 5 an im als.

S t a t i s t i c a l l y s ig n i f i c a n t d if f e r e n c e s between t e s t groups and c o n tro ls

a re shown as fo llo w s: ***p < 0 .0 0 1 ; * * p < 0 .0 1 ; * p < 0 .0 5 .

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T able 5 .2 A cute P a r e n te r a l A d m in is tr a tio n o f Cadmium: E f f e c t o f

S in g le Doses o f C h e la tin g Agents on th e E x c re tio n o f

Cadmium

2+Cd e x c re tio n

yg /24 h

Treatm ent Faeces U rine

C o n tro ls 8 .1 + 1 .0 0.17 ± 0.02

P uchel: 100 mg/kg 95.1 + 6.9*** 0.72 + 0.12**

Puchel: 200 mg/kg 94 .5 + 8.0*** 0.61 + 0.07***

DTPA 10.4 + 1 .3 2.44 + 0,28***

DDDC 27.4 + 2,7?’.*** 1.60 + 0.18***

Puchel-cys 94 .5 + 6.7*** 0 .90 + 0.19**

DMS 12.9 + 2 .0 1 .82 + 0.32***

BAL 19.0 ± 0.6*** 10.87 ± 1.42***

BH-DTPA 8.7 + 0 .7 14.32 + 1.82***

BD-DTPA 11.3 + 1 .9 2.53 + 0.25***

BD-DTPA-ester 12.0 + 1.2* 27.11 + 1 . 53***

A ll v a lu es a re means - S.E.M. o f 5 an im als.

S t a t i s t i c a l l y s ig n i f ic a n t d if f e re n c e s betw een t e s t groups and

c o n tro ls a re shown as fo llo w s: ***p < 0 .0 0 1 ; * * p < 0 .0 1 ; * p < 0 .0 5

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Puchel a t a dose le v e l o f 100 mg/kg s ig n i f ic a n t ly decreased th e

d e p o s it io n o f cadmium in both l i v e r and kidneys so th a t a t 24 hours

on ly 19% o f th e in je c te d dose was re ta in e d in th e se t i s s u e s . The

m etal c o n c e n tra tio n in blood was a lso d ec rea sed , w hile a g r e a te r p ro ­

p o r t io n o f th e in je c te d dose o f cadmium was ex cre ted v ia faeces (31.7%)

and u r in e (0.24% ). In c re a s in g th e dose o f Puchel d id no t in c re a s e

th e e f fe c t iv e n e s s o f th e compound in removing cadmium. The c y s te in e -

d e r iv a t iv e o f Puchel a lso s ig n i f i c a n t ly decreased th e c o n c e n tra tio n

o f cadmium in l i v e r , k idneys and blood w h ile in c re a s in g fa e c a l and

u r in a ry e x c re tio n o f cadmium, b u t was no more e f f e c t iv e th a n Puchel

i t s e l f .

Of th e o th e r compounds, on ly Bishexylam ido DTPA s ig n i f i c a n t ly

decreased th e cadmium c o n c e n tra tio n in bo th l i v e r and k id n ey s. DMS

and BAL decreased th e l i v e r cadmium co n ten t only w h ile DTPA, BD-DTPA

and th e e s t e r o f BD-DTPA decreased only th e kidney co n ten t o f cadmium.

Both DDDC and BAL s ig n i f i c a n t ly in c re ase d th e kidney co n ten t o f

cadmium and in B A L-treated an im a ls , th e blood c o n c e n tra tio n o f cad­

mium was a lso in c re a se d . Compounds o th e r th a n Puchel and i t s c y s te in e

d e r iv a t iv e caused a le s s s ig n i f i c a n t in c re a se in th e f a e c a l e x c re t io n

o f cadmium, bu t in a l l cases a much more s ig n i f ic a n t in c re a s e in th e

u r in a ry e x c re tio n o f th e m e ta l. The in c re a se in u r in a ry cadmium was

d ram atic w ith th e e s t e r o f BD-DTPA.

The E f fe c ts o f Repeated A d m in is tra tio n o f C h e la tin g Agents on th e

D is t r ib u t io n and E x c re tio n o f P a re n te ra l ly A dm inistered Cadmium

2+S ix days a f t e r th e a d m in is tra t io n o f 300 pg o f Cd to male r a t s ,

app rox im ate ly 150 yg o f th e m eta l were to be found in th e l i v e r , b u t

th e p e rcen tag e o f th e dose accum ulating in th e k idney was s l i g h t l y

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h ig h e r th a n a t 24 h o u rs . When th e c h e la tin g ag en ts were ad m in is te red

24 hours a f t e r th e dose o f cadmium, th e e f f e c t o f th e compounds on th e

r e t e n t io n o f th e m eta l in th e l i v e r and k idneys was no t s ig n i f ic a n t

(T ab le 5 .3 ) . The f a e c a l and u r in a ry e x c re tio n o f cadmium was s i g n i f i ­

c a n tly in c re a se d over th e 5 day tre a tm e n t p e rio d (T ab le 5 .4 ) . This

in c re a se in e x c re t io n , however, became sm a lle r w ith th e d u ra tio n o f

tre a tm e n t. At th e te rm in a tio n o f th e experim en t, th e blood c o n c e n tra tio n

o f cadmium was s ig n i f i c a n t ly in c re a se d by a l l compounds, p a r t i c u la r ly

DDDC.

The E f fe c ts o f C h e la tin g Agents on th e D is t r ib u t io n and E x c re tio n o f

Cadmium a f t e r D ie ta ry A d m in is tra tio n. . . 2+The d ie ta r y a d m in is tra t io n o f 150 p .p .m . of Cd fo r 8 weeks to

male r a t s caused th e accum ulation o f approx im ate ly 366 yg and 111 yg o f

cadmium in th e l i v e r s and kidneys r e s p e c t iv e ly . Two weeks a f t e r cadmium

exposure had ceased , th e se anim als had a blood c o n c e n tra tio n o f cadmium

o f about 3 .5 yg/100 ml (T ab le 5 .5 ) . Table 5 .6 shows th e f a e c a l and

u r in a ry e x c re tio n o f cadmium over a 5 day p e r io d . There was a t re n d

tow ards d e c rea s in g cadmium e x c re tio n v ia bo th r o u te s , w ith tim e . T reatm ent

w ith c h e la t in g ag en ts d id n o t s ig n i f i c a n t ly a l t e r th e accum ulation o f

cadmium in th e l i v e r b u t in anim als t r e a te d w ith DMS o r DMS & P uchel,

th e re was a s ig n i f i c a n t ly h ig h e r amount o f th e m etal in th e k id n ey s.i

Treatm ent w ith P uche l, DTPA, DDDC and th e c y s te in e d e r iv a t iv e o f Puchel

s ig n i f i c a n t ly in c re a se d th e c o n c e n tra tio n o f cadmium in th e b lo o d .

The f a e c a l e x c re tio n o f cadmium was in c re ase d by P uchel, DDDC, th e

c y s te in e d e r iv a t iv e o f Puchel and DMS w hile th e u r in a ry e x c re t io n o f

m etal was on ly c o n s is te n t ly in c re a se d by P uch e l, DDDC, P u c h e l-c y s te in e

and DTPA.

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T ab le 5 .3 A cu te P a r e n te r a l A d m in is tr a tio n o f Cadmium: E f f e c t o f

R epeated Doses o f C h e la tin g Agents on th e T issu e C ontent

and Blood C o n cen tra tio n o f Cadmium

Treatm entL iv er

yg Cd2+

Kidney

yg Cd2+

Blood

yg Cd2+/100 ml

C o n tro ls 148.2 - 6.0 8 .4 ± 0 .3 3 .7 ± 0 .2

Puchel 127.8 ± 7 .9 7 .6 ± 0 .3 6 .5 ± 0.3***

P uchel-cys 129.6 ±11.1 7 .9 ± 0 .6 6 .6 ± 0.3***

DDDC 129.3 ± 7 .2 7 .9 ± 0 .4 10 .1 ± 0.4***

A ll v a lu es a re means - S.E.M. o f 5 an im a ls .

S t a t i s t i c a l l y s ig n i f ic a n t d if f e re n c e s betw een t e s t groups and

c o n tro ls a re shown as fo llo w s: ***p < 0 .001 .

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Tabl

e 5.4

A

cute

P

aren

tera

l A

dmin

istr

atio

n of

Cadm

ium

: E

ffec

t of

Rep

eate

d D

oses

of

Che

latin

g A

gent

s on

***p

<

o.00

1;

**p

<0.

01;

*p <

0.05

.

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T ab le 5 .5 D ie ta r y A d m in is tr a tio n o f Cadmium: E f f e c t o f R epeated

Doses o f C h e la tin g Agents on th e T issue C ontent and Blood

C o n cen tra tio n o f Cadmium

L iv er Kidney Blood

yg Cd2+ yg Cd2+ yg Cd2+/100 ml

C on tro ls 366 ± 23 111 ± 4 3 .5 ± 0 .2

Puchel 322 + 37 96 + 12 5.3 + 0.2***

DTPA 384 + 21 96 + 9 5.0 + 0 ,4***

DDDC 385 + 18 98 + 13 6 .3 + 0.2***

Puchel-cys 351 + 18 100 + 3 4 .9 + 0.2***

DMS 414 + 24 123 + 4* 3 .6 + 0 .1

DMS & Puchel 418 + 17 139 + 12* 3 .6 ± 0 .2

A ll v a lu es a re means - S.E.M. o f 5 an im als, excep t c o n tro ls (10 a n im a ls) .

S t a t i s t i c a l l y s ig n i f ic a n t d if f e re n c e s between t e s t groups and c o n tro ls

a re shown as fo llo w s: ***p < 0 .0 0 1 ; * p < 0 .0 5 .

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Tabl

e 5.6

D

ieta

ry

Adm

inis

trat

ion

of Ca

dmiu

m:

Eff

ect

of R

epea

ted

Dos

es

of C

hela

ting

Age

nts

on th

e

/

Sta

tist

ical

ly

sign

ific

ant

diff

eren

ces

betw

een

test

gr

oups

an

d co

ntro

ls

are

show

n as

foll

ows:

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The I n f lu e n c e o f Sodium S e l e n i t e and P uchel on th e D is t r ib u t io n

o f Cadmium

Table 5 .7 shows th e cadmium co n ten t o f l i v e r , k idneys and blood

a f t e r tre a tm e n t w ith cadmium, sodium s e le n i te and Puchel. Cadmium

tre a tm e n t a lone caused an accum ulation o f th e m eta l in th e l i v e r and

k id n ey s, amounting to about 50% o f th e in je c te d dose. Puchel t r e a te d

anim als showed a s ig n i f i c a n t ly low er amount o f cadmium in th e s e organs

and a decreased c o n c e n tra tio n o f th e m etal in th e b lood . Selenium

caused a v e ry s ig n i f ic a n t in c re a se in th e blood c o n c e n tra tio n o f cadmium

and a decreased cadmium co n ten t in th e l i v e r and kidneys was found.

The a d m in is tra tio n o f s e le n i t e and Puchel to g e th e r produced r e s u l t s

s im ila r to th o se f o r s e le n i t e a lo n e .

Table 5 .7 Acute P a re n te ra l A d m in is tra tio n o f Cadmium: E f fe c t o f

Puchel and Sodium S e le n ite on th e T issu e C ontent and

Blood C o n cen tra tio n o f Cadmium

L iv e r Kidney BloodTreatm ent

PS Cd2+ PS Cd2+ pg Cd2+/100 ml

C o n tro ls 139.7 ± 6 . 7 5.3 ± 0 .2 5 .8 ± 0 .4

Puchel 69.2 - 6.0*** 2 .6 ± 0.2*** 3 .6 ± 0.2***

Sodium s e le n i te 74 .7 ± 4.4*** 4 .2 ± 0 .2** 96 .5 ± 5***

Sodium s e le n i te & Puchel 70 .8 ± 4.3*** 4 .5 - 0 .2* 97 .6 ± 4 * * *

A ll v a lu es a re means - S.E.M. o f 5 an im als.

S t a t i s t i c a l l y s ig n i f ic a n t d if f e r e n c e s between t e s t groups and c o n tro ls

a re shown as fo llo w s: ***p< 0 .0 0 1 ; **p< 0 .0 1 ; *p< 0 .0 5 .

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5 .4 DISCUSSION

The a b i l i t y o f a c h e la tin g agen t to m o b ilise a to x ic m eta l depends

upon th e c o n d itio n a l s t a b i l i t y c o n s ta n t , which can be d e fin ed m athe­

m a tic a l ly , and th e pharm acok inetics o f th e compound (C atsch and

Harmuth-Hoene, 1975). However, th e th e ra p e u tic p o te n t ia l o f th e

c h e la t in g agent w i l l depend upon s e v e ra l f a c to r s :

1 . The m etal c h e la te should be r e a d i ly e x c re te d .

2. During th e m etabolism o f th e c h e la te , to x ic e f f e c t s

r e s u l t in g from th e r e le a s e o f th e m etal o r due to

in c reased uptake in c r i t i c a l organs should n o t be

- observed .

3 . The c h e la tin g agen ts should have a s tro n g e r a f f i n i t y

fo r th e to x ic m etal th an o th e r e s s e n t ia l m e ta ls .

4 . The compounds used should be r e l a t i v e l y n o n -to x ic .

Thus, th e m o b ilis in g e f fe c t iv e n e s s o f any compound fo r cadmium w i l l

depend p r im a r i ly on th e a b i l i t y o f t h a t compound to remove cadmium

from m e ta l lo th io n e in , o r o th e r p r o te in s , and th e c a p a c ity o f th e com­

pound to be tak en up in to th e t i s s u e s where cadmium i s accum ulated ,

b e fo re i t s rem oval from th e body.

The r e s u l t s o f th e se experim ents show q u i te c le a r ly th a t th e

e f fe c t iv e n e s s o f th e c h e la tin g ag en ts to in c re a s e th e e x c re t io n o f

cadmium and th e reb y reduce th e body burden o f th e m etal was on ly s ig n i ­

f i c a n t when th e compounds were ad m in is te red soon a f t e r cadmium tre a tm e n t

A nalysis o f th e d a ta r e v e a ls t h a t th e compounds most e f f e c t iv e in

b r in g in g about th i s low ering o f t i s s u e cadmium, a lso caused a g re a te r

in c re a s e in th e f a e c a l e x c re tio n o f cadmium, r a th e r th a n u r in a ry cadmium

However, most co n v en tio n a l c h e la t in g agen ts depend upon th e p ro p e r ty

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o f r a p id e lim in a tio n v ia th e k idneys due to t h e i r h igh h y d ro p h il ic i ty ,

fo r t h e i r mode o f a c t io n , and a re l i k e ly th e re fo re to be l e s s e f f e c t iv e

in rem oving cadmium.

A bsorp tion o f DTPA fo llo w in g p a re n te ra l a d m in is tra tio n i s f a s t ,

and th e re i s a ra p id c le a ra n c e from th e plasm a. DTPA forms c h e la te s

which have been shown to be a lm ost com pletely ex cre ted in th e u r in e .

90% o f DTPA has been shown to be e lim in a ted in th e u r in e o f r a t s 3 hours

fo llo w in g in je c t io n (Foreman e t a l . , 1953). There i s a lso ev idence th a t

th e compound can enhance th e b i l i a r y e x c re tio n o f plutonium (S m ith , 1958)

This c h a r a c te r i s t i c has been enhanced by th e p ro p e r t ie s o f Puchel and

i t s c y s te in e d e r iv a t iv e . The b a s ic s t r u c tu r e o f Puchel i s t h a t o f DTPA,

bu t i t has a s id e c h a in , th e v a lu e of n being about 10 (F ig u re 5 .1 ) .

Thus Puchel and Puchel-cys a re much more l ip o p h i l i c .

Puchel has been shown to be more e f f i c i e n t th an DTPA fo r th e

rem oval of in te r n a l ly d e p o sited plutonium from both th e l i v e r and lungs

o f ro d e n ts (Bulman e t a l . , 1977; S ta th e r e t a l , , 1977). In th e p re se n t

ex perim en ts, Puchel and i t s c y s te in e d e r iv a t iv e were th e most e f f e c t iv e

compounds te s te d f o r low ering th e body burden o f cadmium a f t e r th e

a cu te a d m in is tra tio n o f th e m e ta l. S in g le doses o f th e c h e la t in g ag en ts

decreased~ the p ercen tag e o f th e dose o f cadmium re ta in e d in th e l i v e r

and k idneys to around 20% compared w ith 50% in c o n tro l an im a ls . This

re d u c tio n in th e organ burden o f cadmium was achieved by g re a t ly

in c re a s in g fa e c a l e l im in a tio n o f th e m e ta l, so th a t over 30% o f th e

dose o f m eta l was c le a re d by way o f th e g a s t r o - in t e s t i n a l t r a c t w ith in3

24 h o u rs . S tu d ies w ith H -puchel have shown th a t 64% o f th e compound

i s e x c re ted in faece s 24 hours a f t e r an in trav en o u s in je c t io n in ham sters

compared to 17% in th e u r in e (Craw ley e t a l . , 1978). Most o f th e

e x c re tio n in to th e faece s occurs v ia th e b i l e (p e rso n a l com m unication).

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F ig u re 5 .1

S tru c tu re o f D ie th y le n e tr ia m in e p e n ta a c e tic a c id and D e riv a tiv e s

r1oc. ch2 ch2cooh C ^ .C O ^

NCH2CH2NCH2CH2N(

R2OC.CH2/ CH2 .COR2

Compound R^ R2

DTPA -OH -OH

Puchel -NH(CH2) 1C)C00H -OH

Puchel b is c y s te in e -NH(CH2) 1()C00H -NHCHCH2SH

Bishexamido DTPA -NH(CH2) 5CH3 -OH

cooc2h5

B isdecylam ido DTPA -NH(CH2) n CH3 -OH

B isdecylam ido ( .DTPA-ester -0(CH2) 11CH3 -OH

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The b i l e i s g e n e ra lly reg a rd ed as o f m inor s ig n if ic a n c e fo r th e excre­

t io n o f cadmium, th e m ajor p ro p o r tio n o f th e m etal in th e fa e ce s being

d e riv e d from o th e r g a s t r o - in te s t in a l s e c re t io n s ( s e e s e c t io n 1 .2 ) .

However, th e p resen ce o f p o la r groups on th e Puchel-cadmium c h e la te ,

and th e f a c t t h a t th e complex has a g re a te r m olecu lar w eigh t, may f a c i ­

l i t a t e th e rem oval o f cadmium by t h i s ro u te . The in c o rp o ra tio n o f

c y s te in e re s id u e s to th e s t r u c tu r e d id no t in c re a se th e m o b ilis in g

e f fe c t iv e n e s s o f th e compound.

The e f fe c t iv e n e s s o f th e o th e r th io l - c o n ta in in g compounds, DDDC,

DMS and BAL in d ec rea s in g th e body burden o f cadmium was in s ig n i f ic a n t

compared to Puchel and i t s c y s te in e d e r iv a t iv e . E a rly in v e s t ig a t io n s

w ith BAL have shown th a t in r a t s , th e m ajor p o r tio n o f th e drug i s

e x c re te d r a p id ly in th e u r in e (P e te rs e t a l , , 1947) and th e r e s u l t s o f

th e p re se n t s tu d y su pport t h i s s in c e more th a n 3.5% o f an in je c te d

dose o f cadmium was removed v ia th i s ro u te . This amounts to a 6 0 -fo ld

in c re a s e over c o n tro ls . DDDC i s p a r t i a l l y l ip o p h i l i c on account o f

th e e th y l groups on th e c a rb o x y lic groups o f c y s te in e (F ig u re 5 .2 ) .

This in c re a se d l i p o p h i l i c i t y compared to DTPA i s r e f le c te d in th e

in c re a se d m o b ilis in g e f fe c t iv e n e s s o f th e compound.

F ig u re 5 .2

S tru c tu re of! D ie th y ld ic y s te d ic ac id

HOOCCH2 CH COOH

nch2ch2n

H5C200CHCNH.0CCH2 CH C0.NHCHC00C2H5

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More in te r e s t i n g , however, i s th e e f f e c t o f bo th DDDC and BAL in

cau sin g an in c re a se d cadmium co n ten t o f th e k id n ey s. O ther t h i o l -

c o n ta in in g compounds have been shown to have a s im ila r e f f e c t . P e n ic i l l ­

am ine, 2 ,3 -D im ercap topropane-su lphonate and c y s te in e have been shown

to cause an in c re a se d r e n a l uptake o f cadmium (N eim ier, 1967, c i te d in

C atsch and Harmuth-Hoene, 1978; Gunn e t a l . , 1968). Such compounds may

th e re fo re a c tu a l ly p o te n t ia te th e to x i c i t y o f cadmium, and t h i s s ev e re ly

l im i t s t h e i r use as th e ra p e u t ic a g e n ts . This c h a r a c te r i s t i c o f t h i o l -

c o n ta in in g compounds i s analagous to th e e f f e c t o f m e ta l lo th io n e in .

As d e sc rib ed in s e c t io n 1 .3 , cadmium when ad m in is te red as cadm ium -thionein

shows an in c re a se d uptake in to th e k idney and enhanced n e p h ro to x ic ity

(C h erian e t a l . , 1976).

The r e la t io n s h ip betw een in c re a s in g l i p o p h i l i c i t y and in c re a se d

a b i l i t y to promote fa e c a l e x c re tio n o f cadmium, which i s dem onstrated

fo r Puchel, P uchel-cys and DDDC, does n o t however app ly to b is h e x y l-

amido-DTPA and th e b isdecy lam ido d e r iv a t iv e s o f DTPA. Only b is h e x y l-

amido-DTPA produced a s ig n i f i c a n t d ec rea se in bo th l i v e r and k idney

c o n te n t , though th e e f f e c t s on th e p e rcen tag e o f th e dose r e ta in e d in

th e s e two organs by a l l th r e e compounds i s m inim al. D esp ite t h e i r

h ig h ly l ip o p h i l i c n a tu re ( s e e F ig u re 5 .3 d e sc r ib in g o rd e r o f l ip o p h i ­

l i c i t y ) , . f a e c a l e lim in a tio n o f cadmium was n o t g r e a t ly in c re a s e d , b u t

u r in a ry cadmium e lim in a tio n was v e ry s ig n i f i c a n t . In th e case o f

bishexylamido-DTPA and th e e s t e r o f bisdecyl-DTPA, th e in c re a s e in th e

u r in a ry e x c re tio n o f cadmium was d ra m a tic , an 8 0 -fo ld and 1 5 0 -fo ld

in c re a s e being re c o rd ed . This meant th a t about 5% and 9% o f th e

in je c te d dose o f cadmium re s p e c t iv e ly was removed v ia th e k id n e y s ,

and th e re fo re any low ering o f th e t i s s u e cadmium c o n ten t may have been

produced by t h i s e f f e c t .

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F ig u r e 5 .3

Order o f L ip o p h i l ic i ty o f C h e la tin g Agents

i l B isdecylam ido DTPA

B isdecylam ido DTPA-ester

Puchelin c re a s in g

Puchel biscysteinel i p o p h i l i c i t y

D ie th y ld ic y s te d ic ac id |

B ishexylam ido DTPA

DTPA

Expected o rd e r o f l i p o p h i l i c i t y from a c o n s id e ra tio n o f

th e Hansch param eters (Bulman, R .A ., p e rso n a l com m unication).

The d is c u s s io n so f a r has a ttem pted to r e l a t e th e s t r u c tu r e o f

th e se c h e la t in g ag en ts to t h e i r a c t i v i t y . The a c tu a l mechanisms invo lved

in th e m o b ilis a tio n and removal o f cadmium from th e body a re perhaps more

obscu re . When th e c h e la t in g ag en ts were g iven alm ost im m ediately a f t e r

an acu te dose o f cadmium, a l l th e compounds t e s t e d , w ith th e ex ce p tio n

o f DMS, produced a low ering o f blood cadmium. C onverse ly , when th e

compounds were g iven 24 hours o r lo n g e r , a f t e r th e a d m in is tra t io n o f

cadmium, th e c h e la t in g agen ts t e s t e d (P u ch e l, Puchel-cys and DDDC) tended

to in c re a se th e b lood c o n c e n tra tio n o f cadmium, and under th e s e c o n d it io n s ,

a tre a tm e n t o f 5 co n secu tiv e doses o f th e drugs d id no t s ig n i f i c a n t ly

a f f e c t th e cadmium co n ten t o f th e l i v e r s and k idneys. F aeca l and

u r in a ry e x c re t io n o f cadmium were in c re a s e d , though to a much l e s s e r

degree th a n in th e p rev io u s experim en t, and th e e f f e c t was g re a te r

i n i t i a l l y , a f t e r th e f i r s t dose o f compound.

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In th e f i r s t hours a f t e r cadmium exposure , th e m eta l i s lo c a te d

m ainly in th e plasm a ( s e e s e c t io n 1 .2 ) b e fo re ra p id c lea ra n c e and a

slow er reap p ea ran ce o f th e m eta l in th e e ry th ro c y te s . I t would appear

th e re fo re t h a t Puchel and o th e r compounds a c t m ainly by complexing

cadmium in th e plasm a a t a tim e b e fo re m e ta llo th io n e in sy n th e s is i s

ad eq u a te ly i n i t i a t e d . However, because o f th e l ip o p h i l i c n a tu re o f

some o f th e compounds, th e re i s th e p o s s i b i l i t y th a t th e y may a lso

become d i s t r ib u te d in th e i n t r a c e l l u l a r enivronm ent. The a b i l i t y o f

such compounds to m o b ilise cadmium w i l l th e n a lso depend upon t h e i r

a f f i n i t y f o r th e m eta l compared to th e b in d in g a f f i n i t y o f th io n e in

and o th e r p ro te in s fo r cadmium. The r e s u l t s suggest t h a t once th io n e in

sy n th e s is i s p ro g re s s in g and ad eq u a te ly se q u e s te r in g th e m e ta l, th e

c h e la t in g ag en ts do n o t have th e a b i l i t y to remove th e m eta l from th e

b in d in g s i t e s on th e p ro te in .

There i s ev idence th a t Puchel may have a g re a te r h a l f - l i f e th a n

3 . .DTPA s in c e H -Puchel, fo llo w in g pulmonary in tu b a tio n i n th e r a t i s

14c le a re d much more slow ly from th e lu n g th a n C-DTPA. However, m v e s -3 .t ig a t io n s have dem onstrated t h a t H-Puchel i s c le a re d from r a t plasm a

3much more q u ic k ly th a n from ham ster plasm a, and th a t H-Puchel accumu­

l a t e s in ham ster l i v e r to a much g re a te r e x te n t th an r a t l i v e r

(Craw ley e t a l . , 1978).

C an n u la tio n o f th e b i le - d u c t o f th e r a t , f o r in s ta n c e , has shown

th a t 84% o f th e in je c te d a c t i v i t y i s c le a re d in to th e b i l e w ith in

60 m in. I t could be expected th e re fo re t h a t any e f f e c t o f Puchel on

m o b ilis in g cadmium would occur soon a f t e r in je c t io n o f th e compound.

T his i s in accordance w ith th e r e s u l t s showing th a t p ro longed tre a tm e n t

w ith Puchel does n o t in c re a se th e e f fe c t iv e n e s s o f th e d ru g , and th e

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g r e a te s t in c re a s e in e x c re tio n o f cadmium occurs w ith in th e f i r s t

24 hours ( s e e T able 5 .4 ) . From th e se c h a r a c te r i s t i c s o f th e pharmaco­

k in e t ic s o f P u ch e l, i t cou ld be expected th a t th e compound may be le s s

e f f e c t iv e in th e r a t th a n in th e ham ster.

The la c k o f e f fe c t iv e n e s s o f Puchel in d e c rea s in g th e body burden

o f cadmium i s ev id en t a f t e r bo th p a re n te ra l and o ra l a d m in is tra t io n

o f th e m etal to r a t s , though th e in c re a se d blood le v e l s o f cadmium

a f t e r c h e la tin g agen t tre a tm e n t does sug g est some m o b il is a t io n . W ith

DMS tre a tm e n t in r a t s exposed to d ie ta r y cadmium, ag a in th e re was e v i­

dence o f in c re a se d re n a l up take o f th e m e ta l. The com bination o f DMS

and Puchel was an a ttem p t to in v e s t ig a te th e concept forw arded by

Schubert (1972) th a t th e combined a d m in is tra tio n o f two c h e la t in g ag en ts

may in c re a se e f f e c t iv e n e s s . I t has been suggested th a t in th e p resen ce

o f two lig a n d s , th e fo rm atio n o f a mixed lig a n d complex may be fav o u red ,

and such a complex may be more s ta b le . For in s ta n c e , th e e f f ic a c y o f

DTPA can be p o te n t ia te d i f c i t r a t e i s ad m in is te red s im u ltan eo u s ly

(V o lf , 1974), and th i s enhancement o f e f fe c t iv e n e s s i s due to th e forma­

t io n o f mixed lig a n d complexes as p o s tu la te d by S ch u b ert. However, th e

p resen ce o f two ty p es o f l ig a n d as p rov ided by a m ix tu re o f DMS and

Puchel d id n o t in c re a s e th e m o b ilis a tio n o r e lim in a tio n o f cadmium. In

f a c t i t caused a d ecrease in th e amount o f m etal e x c re te d v ia th e fa e ce s

and a n e g l ig ib le in c re a se in u r in a ry cadmium e x c re tio n .

An a l te r n a t iv e approach to in c re a s in g th e e f fe c t iv e n e s s o f a

c h e la t in g agen t m ight be to a l t e r th e k in e t ic s o f cadmium i t s e l f . I t

has been known f o r some tim e th a t selen ium compounds w i l l p r o te c t a g a in s t

some o f th e t o x i c i t y o f cadmium ( e .g . Gunn e t a l . , 1966). More r e c e n t ly

experim ents have been conducted to in v e s t ig a te th e e f f e c t o f selen ium

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on th e k in e t ic s o f cadmium. Chen e t a l . (1975) have re p o r te d th a t

r a t s p re tre a te d w ith selen ium showed a decreased c o n c e n tra tio n o f cad­

mium in l i v e r and k idney , w h ile th e c o n c e n tra tio n in c re a se d in th e

blood and t e s t i s . Selenium ac ted by d iv e r t in g cadmium from th e low

m o lecu lar w eigh t p ro te in s to h ig h e r m o lecu lar w eight p ro te in s (mwt.

about 115 ,000). The most pronounced change appears to be in th e plasma

where a 30 - 5 0 -fo ld in c re a se in cadmium le v e ls has been re p o r te d

(G asiew icz and Sm ith , 1978). I f Puchel a c ts p r im a r i ly by complexing

cadmium in th e plasm a b e fo re i t s subsequent d i s t r i b u t io n and t i s s u e

r e te n t io n , th e n th e e f fe c t iv e n e s s o f th e compound may have been

in c re a se d by selen ium r a i s in g th e le v e l o f cadmium in th e b lood .

However, a lth o u g h e x p e rim en ta lly a v e ry s ig n i f ic a n t in c re a se in blood

cadmium was observed , selen ium d id n o t in f lu e n c e th e a c t io n o f Puchel.

The a d m in is tra t io n o f Puchel a lo n e , in accordance w ith p rev ious e x p e r i­

m ents, co n s id e ra b ly decreased th e l i v e r and kidney co n ten t o f cadmium,

and a ls o in agreem ent w ith re p o r te d e f f e c t s , s e l e n i t e had a s im i la r

though le s s s ig n i f i c a n t e f f e c t . Thus i t appears t h a t d e s p ite th e

change in b in d in g k in e t ic s o f cadmium from th e low m olecu lar w eigh t

p ro te in to h ig h e r m olecu lar w eight com ponents, th e b in d in g o f cadmium

by P u ch e l“was n o t in c re a se d .

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CHAPTER 6

GENERAL DISCUSSION

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A v a s t l i t e r a t u r e has been pu b lish ed concern ing th e m etabolism

and to x ic o lo g y o f cadmium in experim en ta l an im als, and th e h e a l th e f f e c t s

in humans. N e v e rth e le s s , our und ers tan d in g o f th e b io lo g ic a l changes

produced by exposure to cadmium i s incom plete. The experim ents des­

c r ib e d h e re were designed to p ro v id e more in fo rm atio n on th e d ia g n o s is ,

mechanisms and tre a tm e n t o f cadmium po iso n in g . Cadmium was ad m in is te red

to anim als by both e n te r a l and p a re n te ra l ro u te s in o rd e r to produce a

range o f organ c o n c e n tra tio n s o f th e m eta l. The b iochem ical resp o n se

to cadmium could th e re fo re be in v e s t ig a te d in th e fo llo w in g s i tu a t io n s :

1 . I n i t i a l exposure to cadmium, i . e . th e accum ulation o f th e m etal

a t le v e ls below th e c r i t i c a l c o n c e n tra tio n in k idney ( s in g le

p a re n te r a l a d m in is tr a t io n ) .

2 . Repeated exposure to cadmium a t le v e ls s u f f i c i e n t to exceed th e

c r i t i c a l c o n c e n tra tio n in k idney (re p e a te d p a re n te r a l a d m in is tr a t io n ) .

3. R epeated exposure to cadmium a t low er l e v e l s , a llow ing th e g radual

accum ulation o f c r i t i c a l c o n c e n tra tio n s ( d ie ta r y a d m in is tr a t io n ) .

The D iagnosis o f Cadmium P o ison ing

D esp ite in te r - in d iv id u a l v a r i a t io n s , i t may be concluded th a t

measurement o f enzymes in th e u r in e p rov ides a u se fu l anim al model fo r

fo llo w in g th e developm ent o f cadmium-induced re n a l damage. The f in d in g s

in th e r a t m e rit a s im i la r s tu d y in human w orkers exposed to th e m e ta l,

to a s c e r ta in i f th e d e te c t io n o f in c re a se s in u r in a ry enzymes i s a

more s e n s i t iv e p re d ic t iv e in d ic a to r o f cadmium n e p h ro to x ic ity . However,

b e fo re adequate co n clu s io n s can be made, v a rio u s a sp e c ts o f u r in a ry

enzyme e x c re tio n re q u ir e f u r th e r in v e s t ig a t io n . The o r ig in o f enzymes

in th e u r in e has not been s tu d ie d f u l l y , a lthough d ecreases in th e

r e n a l a c t i v i t y o f enzymes have been dem onstrated to occur in r a b b i t s

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a f t e r cadmium tre a tm e n t (A xelsson and P is c a to r , 1966). The measurement

o f r e n a l isoenzymes in th e u r in e , w herever p o s s ib le , could p rov ide

u s e fu l ev idence and may be more s e n s i t iv e th a n th e measurement o f t o t a l

enzyme a c t i v i t y . I t would a lso be u se fu l to compare th e developm ent o f

p r o te in u r ia , e s p e c ia l ly th e $2"ra c ro S lo t)u lin , w ith enzym uria, and th e

o ccu rrence o f enzym uria needs to be r e la te d to h is to lo g ic a l and b io ­

chem ical changes in th e k idney . This would perhaps h e lp to e x p la in

th e s ig n if ic a n c e o f p ro te in u r ia and enzym uria in r e l a t i o n to changes in

organ fu n c tio n . There appears to be a need fo r b e t t e r methods o f

a s se s s in g kidney fu n c tio n , no t on ly w ith re s p e c t to cadmium, b u t to

o th e r m e ta ls , and indeed to a l l to x ic chem ica ls . I t i s c r u c ia l to in d i ­

c a te t h a t s in ce tu b u la r damage caused by cadmium may be i r r e v e r s ib l e ,

param eters r e f l e c t i n g t h i s damage, however e a r ly , may n o t be adequate

to p re d ic t w ith s u f f i c i e n t s a f e ty th e r i s k o f po ison ing o c cu rrin g in

cadmium-exposed in d iv id u a ls . This problem i s perhaps th e most im portan t

one to be ad d ressed .

Mechanisms o f Cadmium T o x ic ity

The mechanism o f th e cadmium-induced k idney le s io n i s unknown.

The experim ents d e sc rib e d h e re have prov ided more in fo rm a tio n on th e

r e n a l e f f e c t s , b u t th e cause o f th e se changes re q u ire s f u r th e r s tu d y .

The im portance o f th e k idney in r e g u la t io n as w e ll as e x c re tio n

must n o t be overlo o k ed , s in c e i t se rv es to conserve e s s e n t ia l com ponents,

in a d d it io n to rem oving th e w aste p roducts o f m etabolism . The e x c re t io n

o f an a lk a l in e u r in e by r a t s g iven re p e a te d in je c t io n s o f cadmium

(C hap ter 2) su g g es ts some d is tu rb a n c e in a c id /b a se b a lan c e . The norm al

fu n c tio n in g o f m e tab o lic p ro cesses re q u ire s th e m aintenance o f th e com­

p o s i t io n o f th e in te r n a l environm ent, so any cadmium-induced derangem ent .

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o f a c id /b a s e r e g u la t io n could have im portan t consequences fo r th e i n t e ­

g r i t y o f th e organism . An a lk a l in e u rin e in d ic a te s a f a i l u r e to e x c re te+ —

H an d /o r an ex cessiv e lo s s o f HCO > which may le ad to a m etab o lic

a c id o s is (W ills , 1978). Hydrogen io n s e c re t io n and b ic a rb o n a te re a b so rp ­

t io n b o th ta k e p lace predom inan tly in th e proxim al tu b u le s , th e s i t e o f

th e cadmium-induced re n a l damage.

C arbonic anhydrase i s th e key enzyme invo lved in a c id /b a se r e g u la ­

t io n and a h igh a c t i v i t y i s found in th e k idney . D e s tru c tio n o f r e n a l

c e l l s by cadmium le a d in g to a lo s s o f t h i s enzyme in to th e u r in e may be

im p o rtan t, bu t i t i s a lso notew orthy th a t t h i s enzyme i s z in c -d ep en d en t.

Thus cadmium may in t e r f e r e w ith th e fu n c tio n o f carbon ic anhydrase e i th e r

by d i r e c t d isp lacem ent o f th e z in c , o r by d ec rea s in g th e a v a i l a b i l i t y

o f z in c .

The q u e s tio n o f w hether th e cadmium c a tio n o r cadm ium -thionein i s

th e n ep h ro to x ic agen t rem ains an i n t e r e s t in g problem . The p ro d u c tio n

o f r e n a l c e l l damage as in d ic a te d by enzym uria a f t e r th e a cu te adm inis­

t r a t i o n o f cadmium ( i . e . b e fo re in d u c tio n o f r e n a l m e ta l lo th io n e in ) ,

would in d ic a te t h a t th e cadmium io n was re s p o n s ib le . The a b o l i t io n o f

t h i s re sp o n se by th e p r e - in je c t io n o f z in c a t doses s u f f i c i e n t to induce

z in c - th io n e in sy n th e s is f u r th e r su p p o rts t h i s id e a ( s e c t io n 3 .3 ) .

Recent work by Suzuki e t a l . (1979) has shown th a t th e e x te n t o f cadmium-

induced re n a l n e c ro s is in th e r a t was c o r r e la te d w ith th e amount o f

cadmium in th e m e ta llo th io n e in , and n o t w ith th e amount o f p r o te in .

Webb and E tien n e (1977) have suggested t h a t th e t o x i c i t y o f cadmium-

. . . . . 2+th io n e m when in je c te d i s due to l i b e r a t i o n o f Cd d u rin g lysosom al

c a ta b o lism o f th e p ro te in . Cadm ium -thionein, when ad m in is te red o r a l ly

i s absorbed i n t a c t bu t i s com plete ly degraded in th e k idney (C h e ria n ,

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1979). O ther r e s u l t s su g g est t h a t i f cadm ium -thionein becomes e x tr a ­

c e l l u l a r , i t has a d i r e c t to x ic e f f e c t on c e l l membranes (C h erian

e t a l . , 1976).

W hile th e ex ac t fu n c tio n o f th e m e ta llo th io n e in s rem ains o b scu re ,

more in fo rm a tio n has r e c e n t ly been pub lished on th e m etabolism o f th e

p ro te in s . The m eta l sp e c ie s seems to be im portan t in de te rm in in g th e

m etabolism o f th io n e in , and d e g ra d a tio n may have a r o le in r e g u la t in g

th e i n t r a c e l l u l a r c o n c e n tra tio n o f th e p ro te in . The p ro cess o f degrada­

t io n has been co n sid ered in r e l a t i o n to th e developm ent o f c e l l damage

(Bremner e t a l . , 1978; Feldman e t a l . , 1 9 7 8 a ,b ). A c le a r e r u n d e rs tan d in g

o f th e f a t e o f m e ta llo th io n e in i s im portan t fo r e lu c id a tin g th e mechanisms

o f t o x i c i t y o f cadmium.

The r e s u l t s o f C hapter 3. show c le a r ly th a t cadmium may in t e r a c t

w ith z in c , copper and i ro n soon a f t e r a d m in is tra tio n , b u t i t i s n o t known

w hether th e se changes in e s s e n t ia l t r a c e m etal m etabolism a re a mani­

f e s t a t i o n o f th e to x ic re sp o n se to cadmium, o r w hether th e y a re funda­

m ental in e l i c i t i n g th e t o x i c i t y . P rocesses e x is t in th e organism to

m a in ta in a b a lance betw een b io a v a i l a b i l i ty and m etab o lic req u irem en ts

o f th e e s s e n t ia l m e ta ls , i t i s n o t s u rp r is in g th e re fo re t h a t any

derangem ent in th e hom eostasis o f z in c , copper and iro n may i n t e r f e r e

w ith v i t a l m etabo lic fu n c tio n s .

There appear to be two d i s t i n c t phases in th e developm ent o f

cadmium-induced to x i c i t y . Exposure to th e m eta l induces th e s y n th e s is

o f th io n e in , which consequen tly in c re a se s th e demand f o r z in c and copper.

During th e i n i t i a l phase , b e fo re c r i t i c a l c o n c e n tra tio n s a re re a ch e d ,

th e organism w i l l a ttem p t to m a in ta in th e i n t r a c e l l u l a r c o n c e n tra tio n s

o f z in c and copper n ece ssa ry f o r b iochem ical fu n c tio n s , by norm al

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ho m eo sta tic mechanisms such as in c re ase d uptake from th e g u t, and

decreased e x c re tio n . I f exposure to cadmium c o n tin u e s , th e se homeo­

s t a t i c c o n tro ls may become in s u f f i c i e n t and e v e n tu a lly m o b ilis a tio n o f

z in c and copper from t i s s u e s may become n e ce ssa ry .

During th e second phase , th e c r i t i c a l c o n c e n tra tio n is exceeded

and m e ta llo th io n e in sy n th e s is becomes in ad eq u a te . The d is ru p t io n in

th e normal in t e r - r e l a t io n s h ip s between th e e s s e n t ia l m eta ls may con­

t r i b u t e to th e m a n ife s ta tio n o f to x i c i t y . The developm ent o f r e n a l

damage w i l l f u r th e r enhance th e se d is tu rb a n c e s due to in c re a se d lo s s

o f m eta l ions in to th e u r in e . A s im ila r lo s s o f cadmium w i l l a f f e c t

i t s t i s s u e r e t e n t io n and could a l t e r i t s own k in e t ic s . The changes

le a d in g to fu n c tio n a l d is tu rb a n c e s a re summarised in F ig u re 6 .1 .

The im portance o f cadmium-induced d is tu rb a n c e s in t r a c e m etal

m etabolism i s seen in C hapter 4 , where th e e f f e c t o f cadmium on th e bone

was in v e s t ig a te d . The r e s u l t s su g g est th a t th e bone le s io n i s more

complex th a n o r ig in a l ly th o u g h t, and occurs as a r e s u l t o f cadmium

in te r f e r in g w ith s e v e ra l p ro c e sse s .

E xperiences in th e p a s t , e .g . I t a i - i t a i p a t ie n ts and cadmium-

exposed w orkers , have le d to th e b e l i e f th a t th e bone le s io n induced

by cadmium i s a ty p e o f o s teo m alac ia , o f te n c l a s s i f i e d as a Fanconi

Syndrome, and may a lso be accompanied by o s te o p o ro s is . This su g g es tio n

i s re a so n a b le s in c e th e b a s is o f Fanconi Syndrome i s a tu b u la r l e s io n

s im ila r to t h a t produced by cadmium. From a c o n s id e ra tio n o f th e

c l i n i c a l f e a tu re s o f o s teo m alac ia and o s te o p o ro s is , i t i s seen th a t

th e symptoms o f I t a i - i t a i d is e a s e commonly f a l l in to bo th c a te g o r ie s .

The advanced s k e le t a l d e fo rm it ie s , m uscle weakness and a c h a r a c t e r i s t i c

g a i t a re ty p ic a l o f o s teo m alac ia , w h ile th e m u ltip le p seu d o frac tu re s

a re u s u a lly a c h a r a c t e r i s t i c o f o s te o p o ro s is (D avidson e t a l . , 1975).

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Figure 6.1

CHANGES DURING THE COURSE OF CADMIUM-

INDUCED RENAL DAMAGE

Metallothioneinsynthesis

Enzymes Proteins Amino acids Glucose Phosphate Trace metals

CELL DAMAGE

CADMIUMEXPOSURE

FUNCTIONAL .DISTURBANCE

CRITICALCONCENTRATIONS

RENALACCUMULATION

2+OF Cd

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However, when th e b iochem ical changes o f bone d is e a se s a re compared

to th e cadmium-induced changes, th e s i tu a t io n i s con fusing (F ig u re 6 .2 ) .

F igu re 6 .2

B iochem ical Changes in th e D iagnosis o f Bone D isease

D isease

Plasma

Ca P i ALP

U rine

Ca P i

O steoporosis

O steom alacia

N N N N-f N

- n u t r i t i o n a l N-! t t- r e n a l g lom eru lar N-* t t * *

- r e n a l tu b u la r N-| t t-Fanconi Syndrome 1H I f t

H ypophosphatasia t t +

I t a i - i t a i N-| t N

I n d u s t r ia l Cd exposure N N4 N t N-|

P i: in o rg a n ic phosphate ; ALP: a lk a l in e phosphatase

N: norm al; t : an in c re a s e ; | : a d ec rea se .

R esu lts summarised from Adams e t a l . (1969);

F rib e rg e t a l . (1974); Itokaw a e t a l . (1975);

K azantz is e t a l . (1 9 6 3 ); K azan tz is (1978); Nogawa (1978);

and P o tts and D eftos (1974).

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The most s t r i k in g c h a r a c te r i s t i c s o f a Fanconi Syndrome a re a low

c o n c e n tra tio n o f plasm a in o rg a n ic phosphate and a h igh u r in a ry e x c re tio n

o f phosphate . H yperphosphaturia was n o t a common f in d in g in I t a i - i t a i

p a t ie n ts (F r ib e rg e t a l . , 1974), and was never seen in th e experim ents

d e sc rib e d in th i s t h e s i s . In a d d i t io n , th e fo rm ation o f r e n a l s to n es

and n e p h ro c a lc in o s is i s never encountered in Fanconi Syndrome (B rod, 1973),

but has been a common f in d in g in cadmium-exposed w orkers (K a z a n tz is , 1970;

F r ib e rg et_ a l . , 1974) .

The g e n e t ic a l ly determ ined d e fe c t o f hypophosphatasia i s i n t e r e s t in g

a ls o , s in c e th e c h a r a c t e r i s t i c f e a tu re i s a d e f ic ie n c y o f a lk a l in e phos­

p h a tase in v a r io u s t i s s u e s in c lu d in g th e bone. The fundam ental s k e le ta l

d e fe c ts in c lu d e d e f ic ie n t m in e ra l is a t io n , in c re a se d bone f r a g i l i t y and

m u ltip le p se u d o fra c tu re s . This a lso c lo s e ly resem bles th e fe a tu re s o f

I t a i - i t a i d is e a s e , a lth o u g h th e changes in calc ium hom eostasis a re

d is s im i la r .

On th e b a s is o f th e ex p erim en ta l evidence o b ta in e d , an a l t e r n a t iv e

mechanism f o r th e e f f e c t o f cadmium upon th e bone, in th e r a t , may be

proposed (F ig u re 6 .3 ) . Exposure to cadmium in b o th th e s h o r t and lo n g

term produces changes in th e t r a c e m etal c o n ten t o f th e bone, and may

occur in d ep en d en tly o f k idney damage. Such changes may a d v e rse ly a f f e c t

th e a c t i v i t y o f z in c and copper c o n ta in in g enzymes invo lved in bone

fo rm atio n . The e x is te n c e o f k idney damage may ex ace rb a te th e d ecreased

a v a i l a b i l i t y o f th e se e s s e n t ia l m e ta ls . The accum ulation o f cadmium in

th e k idney may in f lu e n c e calcium hom eostasis in s e v e ra l ways. I n t e r ­

fe re n ce in th e b io tra n s fo rm a tio n o f c h o le c a lc i f e r o l has been su g g ested

as a p o s s i b i l i t y (Feldman and C ousins, 1973). T his could be m ediated

v ia an e f f e c t upon cytochrome P-450, as su g gested in s e c t io n 4 .4 , b u t

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Figu

re

6.3

Effe

ct

of ca

dmiu

m

upon

th

e bo

ne

- Pr

opos

ed

mec

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sms

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i t i s i n t e r e s t in g to n o te th a t in r a t s , acu te system ic a c id o s is causes

an im pairm ent o f th e conversion o f 2 5 -h y d ro x y ch o le c a lc ife ro l to

1 ,2 5 -d ih y d ro x y c h o le c a lc ife ro l (Lee e t a l . , 1977). M etabolic a c id o s is

due to ch ro n ic r e n a l f a i l u r e i s a lso known to cause bone d is s o lu t io n ,

though u s u a lly accompanied by e x cessiv e lo s s o f calcium in th e u r in e

(W ills , 1978), which was n o t seen in th e p re se n t s tu d ie s .

I f a derangem ent in th e hom eostasis o f e s s e n t ia l m eta ls i s impor­

ta n t in th e a e tio lo g y o f cadmium to x i c i t y , th e n i t i s re a so n a b le to t r y

to a l l e v i a t e th e t o x i c i t y by r e s to r in g th e normal m etabolism and fu n c tio n

o f th e se m e ta ls . The r e s u l t s o f C hapters 3 and 4 in d ic a te t h a t su p p le ­

ments o f z in c may p rev en t some o f th e cadmium-induced changes. The

e f f e c t i s v e ry dependent upon th e dose o f z in c however, and a t h ig h

le v e ls z in c may produce adverse e f f e c t s i t s e l f . C onsidering th e i n t e r ­

r e la t io n s h ip s o f z in c , copper and i ro n , a combined supplem ent o f a l l

m eta ls may prove more u s e fu l .

Treatm ent o f Cadmium T o x ic ity

The experim ents d e sc rib e d in C hapter 5 were designed to in v e s t ig a te

th e a b i l i t y o f c h e la t in g a g e n ts , s p e c i f i c a l ly Puchel and i t s d e r iv a t iv e s ,

to m o b ilise and promote th e e x c re tio n o f cadmium, th e re b y p ro v id in g

an o th er means fo r t r e a t i n g cadmium p o iso n in g . There a re few re p o r te d

in v e s t ig a t io n s in th e l i t e r a t u r e where th e e f fe c t iv e n e s s o f c h e la t in g

agen ts f o r th e rem oval o f s y s te m ic a lly absorbed cadmium have been

s tu d ie d . I t was concluded by F r ib e rg (1956) t h a t th e re was i n s u f f i c i e n t

ev idence showing a b e n e f ic ia l e f f e c t o f EDTA in experim en ta l cadmium

p o iso n in g , a n d 'i t was prem ature th e re fo re to sug g est any p o s s ib le a p p l i ­

c a t io n fo r th e th e ra p y o f human cadmium p o iso n in g . O ther s tu d ie s u s in g

such compounds as DTPA, EDTA, BAL, e tc . have been review ed by

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F rib e rg e t a l . (1974) and C atsch and Harmuth-Hoene (1 978).

I t seems re a so n a b le to conclude th a t :

1 . There a re no sy s te m a tic r e p o r ts o f th e a c tio n o f v a r io u s

c h e la t in g ag en ts on th e d i s t r i b u t io n and e x c re tio n o f cadmium

a f t e r d i f f e r e n t exposures to th e m eta l.

2 . The ev idence a v a i la b le su g g es ts th a t compounds such as EDTA

and BAL o f f e r l i t t l e p o te n t ia l in th e tre a tm e n t o f cadmium

p o iso n in g .

The r e s u l t s re p o r te d h e re may h e lp to compare th e m o b ilis in g

e f fe c t iv e n e s s o f d i f f e r e n t ty p es o f compounds, and p ro v id e in fo rm a tio n

on th e a c t io n o f th e novel Puchel compounds fo r th e rem oval o f cadmium

in r a t s . A lthough Puchel and Puchel-cys were found to be th e most

e f f e c t iv e compounds te s t e d fo r d e c rea s in g th e l i v e r and k idney accumu­

l a t i o n o f cadmium, th e e f f e c t was only b e n e f ic ia l in cases o f a cu te

cadmium exposure. S ince most cases o f ex cessiv e human exposure to

cadmium a re l i k e ly to occur a f t e r prolonged in ta k e o f th e m e ta l, th e

v a lu e o f such compounds appears l im ite d to th o se cases o f a c c id e n ta l

acu te exposure.

B efore c l i n i c a l a p p lic a t io n s o f Puchel could be c o n s id e re d ,

however, c o n s id e ra b ly more in fo rm a tio n i s re q u ire d . C h e la tin g ag en ts

tend to form c o o rd in a tio n compounds w ith a number o f m e ta ls . The

e f f e c t o f Puchel on th e d i s t r i b u t io n and e x c re tio n o f e s s e n t i a l m e ta ls

such as z in c , copper and i ro n , needs to be in v e s t ig a te d , s in c e a d i s ­

tu rb an ce in t h e i r hom eostasis could le ad to to x i c i t y i t s e l f . The

acu te to x ic dose o f Ca-DTPA i s h ig h ( in t r a p e r i to n e a l L D ^ in mice i s

about 12 .5 mmol/kg) bu t DTPA-induced h is to lo g ic a l changes have been

re p o r te d in v a rio u s t i s s u e s in c lu d in g l i v e r , k idney and in t e s t i n e

(rev iew ed by C atsch and Harmuth-Hoene, 1978), bu t th e se changes may

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be t r a n s i e n t (Morgan and Sm ith , 1974). The to x i c i t y o f Puchel th e r e ­

fo re needs to be in v e s t ig a te d .

In t h i s t h e s i s , th e e f f e c t o f Puchel has only been examined in th e

l i v e r and kidney bu t th e p o s s i b i l i t y t h a t i t may cause some r e d i s t r i b u ­

t io n o f cadmium cannot be excluded . For in s ta n c e , due to th e in c re a se d

l i p o p h i l i c i t y o f th e complex, an in c reased uptake o f cadmium in to

b ra in and o th e r t i s s u e s may be caused . The most s u i ta b le dose o f Puchel

i s a ls o unknown, b u t in th e se ex p erim en ts , a dose o f 100 mg/kg was found

to be as e f f e c t iv e as 200 mg/kg. F u rth e r s tu d ie s to in v e s t ig a te th e

th e ra p e u tic e f f e c t iv e n e s s , i . e . th e e f f e c t o f Puchel on th e symptoms

o f cadmium to x i c i t y , r a th e r th a n th e m o b ilis in g e f f e c t iv e n e s s , may be

w orthw hile .

There a re no l ig a n d s which r e a c t s p e c i f i c a l ly w ith on ly one p a r t i ­

c u la r m eta l io n , however th e sy n th e s is o f m acropo lycyclic m o lecu les , o r

c r y p ta te s , may produce new p o s s i b i l i t i e s . C ry p ta te s c o n ta in i n t r a ­

m o lecu lar c a v i t ie s which possess b in d in g and r e a c t iv e s i t e s , and can

th u s form in c lu s io n com plexes. S e le c t iv e com plexation o f cadmium by

such compounds has been d e sc rib e d and th e th e ra p e u tic p o te n t ia l i s th e r e ­

fo re i n te r e s t in g (Lehn, 1978).

The e n c a p su la tio n o f DTPA w ith in liposom es has a lso been shown to

in c re a s e th e e lim in a tio n o f p lu tonium in .m ice compared to DTPA a lo n e

(Rahman e t a l . , 1973), Liposomes a re p robab ly phagocy tised by c e l l s ,

cau sin g a h ig h e r and lo n g e r - la s t in g r e te n t io n in organs th e re b y f a c i l i ­

t a t i n g m o b ilis a tio n o f th e i n t r a c e l l u l a r m e ta l.

A nother concept fo r th e in a c t iv a t io n o f a to x ic m etal i s c h e la t io n

in s i t u , such as th e fo rm ation o f a s ta b le in s o lu b le c h e la te o f a u r in -

2+t r i c a r b o x y l ic a c id (ATCA) w ith Be (S chubert e t a l . , 1952).

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M e ta llo th io n e in p a r t ly f u l f i l l s th e se requ irem en ts b u t i t s con tinued

adequate sy n th e s is i s l im ite d . I t appears t h a t a t p re sen t known

c h e la t in g ag en ts have a low er a f f i n i t y th an th io n e in fo r cadmium.

A means o f s t a b i l i s i n g m e ta llo th io n e in in s i t u , so th a t th e complex

would n o t be s u b je c t to d e g ra d a tio n , th e re fo re p rev en tin g m o b ilis a tio n

o f th e m e ta l, could p rov ide an i n te r e s t in g approach.

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APPENDICES

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APPENDIX I

C om p osition o f L ab oratory Animal D ie t

Chemical Com position %

M oisture 8 .8

E th e r e x t r a c t 3 .5Crude p ro te in 21 .5

Crude f i b r e 2 .7

T o ta l d ig e s t ib le n u t r i e n ts 78.0

V itam in and M ineral Com position

L ysine % 1 .1M ethionine % 0.39Calcium % 0 .9Phosphate % 0 .8

V itam in A i .u . / k g 11,000V itam in D i . u . / k g 1,200

a -to c o p h e ro l i . u . / k g 24V itam in Kg mg/kg 10

R ib o fla v in mg/kg 7

P y ridox ine mg/kg 1P an to th en ic a c id mg/kg 17N ic o tin ic ac id mg/kg 80

F o lic a c id mg/kg 0 .2C holine c h lo r id e mg/kg 450

Cyanocobalamin mg/ kg 15Manganese mg/kg 65

Iro n mg/kg 100Io d in e mg/kg 0 .5

Copper mg/kg 20

Zinc mg/kg 40

C obalt mg/kg 1

D ata su p p lie d .b y m anufactu rer

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APPENDIX I I

The P re p a ra tio n o f Samples f o r Measurement o f

M etals by Atomic A bsorp tion S pectrom etry

( i ) T issu es and Faeces

To 0 .5g q u a n t i t i e s o f sample in 50 ml c o n ica l f l a s k s , 5 .0 ml

o f HNOg ( s p .g r . 1 .4 2 ) and 1 .0 ml o f HCIO^ ( s p .g r . 1 .5 4 ) were added,

and allow ed to s tan d fo r 30 m in. .The f la s k s were th e n hea ted g e n tly

to 150°C on a h o tp la te , av o id in g a v igo rous r e a c t io n . A fte r th e

i n i t i a l r e a c t io n had subsided and a yellow -brow n s o lu t io n form ed, th e

sam ples were ev ap o ra ted to d ryness a t 200 - 300°C. When c o o l, th e

re s id u e s were made up w ith 5 .0 ml o f 4 .5 M-HC1 and p repared f o r m eta l

d e te rm in a tio n s as fo llo w s:

Cadmium

S tock S tandard S o lu tio n : CdCl2 1 mg/ml.

Working S tandard S o lu tio n : 10 jug/ml.

A ran g e o f s ta n d a rd s from 0 - 2 .5 ng/ml was p repared in 4 .5 M-HC1,

and samples d i lu te d w ith a c id as n e ce ssa ry .

Zinc and Copper

S tock S tandard S o lu tio n s : (CH^COO^Zn^H^O .i 10 mM

CuC12 .2H20 J

Working S tandard S o lu tio n s : 100 juM

A range o f s ta n d a rd s from 0 - 10 juM was p repared in 4 .5 M-HC1, and

sam ples d i lu te d w ith a c id as n e ce ssa ry .

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Iro n

S tock S tandard S o lu tio n : F eC l^ .611 0 50 mM

Working S tandard S o lu tio n : 500 /uM

A ran g e o f s tan d a rd s from 0 - 25 mM was p repared in equal volumes o f

4 .5 M-HC1 and sodium /potassium s o lu tio n (140 mM-Na, 5 mM-K). Samples

were d i lu te d w ith a c id as n e ce ssa ry .

( i i ) Blood and Plasma

Cadmium (m icrosam pling method)

S tock S tandard S o lu tio n : CdCl^ 1 mg/ml

Working S tandard S o lu tio n : 10 ng/ml

A range o f s tan d a rd s was p repared in d i s t i l l e d w a te r .

Samples were p rep ared in t r i p l i c a t e , in n ic k e l cups as fo llo w s:

working t e s ts tan d a rd blood

10 m1

10 Ml

Samples were d r ie d on a h o tp la te a t 150°C fo r 5 min. b e fo re flam in g .

Z inc and Copper

Plasma was d i lu te d 1 in 5 w ith d i s t i l l e d w a te r . At t h i s d i l u t i o n

in te r f e re n c e by th e plasma m a trix i s removed. S tan d ard s were s im i la r ly

p rep ared in w a te r .

S tock S tandard S o lu tio n : (CH,.C00)9Zn.2H90I 10 mM

CuC12 .2H20 I

Working S tandard S o lu tio n : 100 mM

A ran g e o f s ta n d a rd s from 0 - 10 mM was p rep a red .

sample normal d i s t i l l e dblood w ater

B lank 10 m! 10 m!

S tandard 10 m!

T est - 10 m1

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Ir o n

In te r f e r e n c e from n o n -v is ib le haem olysis was e lim in a ted by th e

rem oval o f haem oglobin by p ro te in p r e c ip i ta t io n . E rro r due to enhance­

ment o f iro n a b so rp tio n by sodium and potassium io n s was compensated

f o r by adding th e se ions to th e b lan k and s ta n d a rd s .

S tock S tandard S o lu tio n : F e C l^ ^ ^ O 50 mM

Working S tandard S o lu tio n : 500 mM

P ro te in P r e c ip i ta n t : T r ic h lo ro a c e t ic a c id (lOOg)

T h io g ly c o ll ic a c id ( 30 ml)

conc. HC1 (166 ml)

d i s t i l l e d w a te r to 1 l i t r e

A ran g e o f s tan d a rd s from 0 - 25 nH was p repared in equal volumes o f

p ro te in p r e c ip i ta t in g s o lu t io n and sodium /potassium s o lu t io n .

Samples were p repared by adding 0 .5 ml o f p ro te in p r e c ip i ta t in g

s o lu t io n to 0 .5 ml o f plasm a. These were mixed fo r 45 s e c , l e f t f o r

10 min and th e n c e n tr ifu g e d fo r 5 min. The su p e rn a ta n t was a s p ir a te d

f o r m eta l d e te rm in a tio n s .

Calcium

The “a d d i t io n o f a s o lu t io n o f lanthanum c h lo r id e removes i n t e r ­

fe re n c e from phosphate and p ro te in .

S tock s ta n d a rd s : CaCO 1 .5 - 5 .0 mM

Samples and s tan d a rd s were d i lu te d w ith 0.1% w/v lanthanum c h lo r id e

as re q u ire d .

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( i i i ) U rine

Cadmium

The m icrosam pling method as d e sc rib e d fo r blood was used .

U rine may be d i lu te d w ith d i s t i l l e d w a te r .

Z inc

In te r f e re n c e o f th e u r in e m a trix was avoided by u sing th e method

o f s tan d a rd a d d i t io n s , th e u r in e sam ples being used to p rep a re a s ta n ­

dard cu rve . Thus any m a trix e f f e c t was id e n t ic a l fo r s tan d a rd s and

sam ples.

S tock S tandard S o lu tio n : (CH^. COO) 2Zn. 21^0 10 mM

Working S tandard S o lu tio n : 2 .5 juM

A range o f s tan d a rd s from 0 - 50 /zM was p rep ared .

Copper

S tock S tandard S o lu tio n : C u C ^ .21^0 10 mM

Working S tandard S o lu tio n : 0 .1 mM

A range o f s tan d a rd s from 0 - 6 /zM was p repared in u r in e .

I ro n

S tock S tandard S o lu tio n : F eC l^^I^O 50 mM

Working S tandard S o lu tio n : 500 fM

A ran g e o f s tan d a rd s from 0 - 30 /zM was p repared in equal volumes

o f u r in e and sodium /potassium s o lu t io n .

Calcium

S tandards and samples were p rep ared as fo r plasm a.

Page 227: STUDIES ON THE TOXICITY OF CADMIUMepubs.surrey.ac.uk/848476/1/10804025.pdf · The Importance of Studies on Cadmium Toxicity The risk to human health; the diagnosis of cadmium toxicity;

PUBLICATIONS

B onner, F.W ., K ing, L .J . and P arke , D.V. (1979)

The t i s s u e d is p o s i t io n and u r in a ry e x c re tio n o f cadmium, z in c , copper

and iro n fo llo w in g re p e a te d p a re n te ra l a d m in is tra tio n o f cadmium to

r a t s ,

C hem .-B iol. I n te r a c t io n s , 27, 343-351.

Bonner, F.W ., K ing, L .J . and P ark e , D.V. (1980)

The e f f e c t o f d ie ta r y cadmium on z in c , copper and iro n le v e ls in th e

bone o f r a t s ,

Toxicology L e t t . , 5 , 105-108.

Bonner, F.W ., K ing, L .J . and P ark e , D.V.

Cadmium-induced re d u c tio n o f bone a lk a l in e phosphatase and i t s

p rev en tio n by z in c ,

C hem .-B iol. I n t e r a c t io n s , in p re s s .

B onner, F.W ., K ing, L .J . and P ark e , D.V.

The u r in a ry e x c re t io n o f enzymes fo llo w in g re p e a te d p a re n te ra l

a d m in is tra tio n o f cadmium to r a t s ,

E nviron . R e s ., in p re s s .