Pharmacological Research Communications, VoL 13, No. 8, 1981 731

DECREASE OF THE IN VITRO DRUG-METABOLIZING ACTIVITY OF THE

HEPATIC MIXED FUNCTION OXIDASE SYSTEM IN RATS INFECTED

EXPERIMENTALLY WITH FASCIOLA HEPATICA: PHARMACOLOGICAL

IMPLICATIONS.

Roberto MAFFEI FACINO °, Marina CARINI °, Raffaele BERTULETTI °,

Claudio GENCHI °o and Alessandra MALCHIODI °°.

o Istituto di chimica Farmaceutica e Tossicologica

Viale AbrUZzi 42, 2Oi31 Milan, Italy.

oo Istituto di Patologia generale Veterinaria

Via Celoria 10, 20133 Milan, Italy.

Received in final form 19 May 1981

AOSTRACT

The e f f e c t s o f experimental i n f e c t i o n w i t h F a s c i o l a h e p a t i c a

on the in v i t r o drug-metabol iz ing a c t i v i t y of the hepatic

mixed funct ion oxidase (MFO) system have been studied in the

r a t . D e c r e a s e s i n c y t o c h r o m e P - 4 5 0 - d e p e n d e n t m e t a b o l i z i n g

activities (p-nitroanisole O-demethylase, aminopyrine

N-demethylase, hexobarbital oxidase, aniline hydroxylase

and zoxazolamine hydroxylase) are observed throughout the

infection period and are maximal in t~e acute stage.

The decreases are consequent to decreased cytochrome P-450

and NADPH-cytochrome P-450 (cytochrome c) reductase.

The depressed drug-metabol iz ing a b i l i t y of the l i v e r

monooxygenase system might alter the steroids and fatty

acids catabolism in the liver cell and decrease the

safety margin for those flukicidal agents that are

de tox i f i ed by the monooxyoenase pathway.

0031-6989/81/080731-12/$02.00/0 ©1981TheltahanPharmacologicalSociely

732 Pharmacol~alResearchCommun~at~ns, VoL13, No. 8,1981

INTRODUCTION

I t has been e s t a b l i s h e d f o r t h e mouse and t h e r a t ( T h o r p e ,

l g 6 5 ; Rahko , 1971 ) t h a t t h e i n t r a h e p a t i c m i g r a t i o n o f

F a s c i o l a h e p a t i c a , an u b i q u i t o u s h e l m i n t h p a r a s i t e o f

mammals, d u r i n g t h e i n c u b a t i o n p e r ~ o d ( 2 - 1 7 d a y s ) and

t h e a c u t e p e r i o d ( 1 7 - 3 2 d a y s ) o f t h e i n f e c t i o n i n d u c e s

s e v e r a l k i n d s o { m o r p h o l o g i c a l damage t o t h e l i v e r c e l l ,

i n c l u d i n g a l t e r a t i o n s o f t h e e n d o p l a s m i c r e t i c u l u m ( R z h k o ,

1 0 7 1 ) .

The smooth e n d o p l a s m i c r e t i c u l u m o f t h e mammal ian l i v e r

c e l l c o n t a i n s t h e c y t o c h r o m e P - 4 5 0 - d e p e n d e n t m i x e d f u n c t i o n

o x i d a s e (MFO) s y s t e m , an e l e c t r o n t r a n s p o r t p a t h w a y w h i c h

plays a key role in the oxidative metabolism of a variety

o? exogenous compounds (EstabrooK and Werringloer, 1977)

and is involved in the physiological oxidation of steroids,

fatty acids and cholesterol (Okit& et al., lgTg; Bjorkheim,

1 0 7 2 ) .

The p r e s e n t s t u d y w a s u n d e r t a k e n to e x a m i n e t h e e f f e c t s

o f an e x p e r i m e n t a l F a s c i o l & h e p a t i c a i n f e c t i o n on t h e

i n v i t r o d r u g - m e t a b o l i z i n g a c t i v i t y o f t h e l l e p a t i c NIFO

s y s t e m i n t h e r a t .

r.!ATE~IALS

All the substrates used were analytical orade.

NAOPII and c y t o c h r o m e c v:ere p u r c h a s e d f r o m 0 o e h r i n g e r

L ; i o c h e m i a R o b i n ( L ; i l a n , I t a l y } , 0 p e o t r o p h o t o m e t r i c s t u d i e s

v~cre c a r r i e d o u t i n ~ beckl.~&n D~-GT s p e c t r o p h o t o m e t e r .

The G e t a c e r c a r i a e u~ed t o i n f e c t t h e r a t s ~ e r e r e c o v e r e d

f r o ~ L/~m~ea t r u n c a t u l a , m a i n t a i n e d by a r t i f i c i a l c u l t u r e ,

Pharmacolog~alResearchCommun~atmns, VoL13, No.~ 1981

~IETHODS

733

Experimental i n f e c t i o n

30 ~ale Sprague-Dawley rats (Charles River) weighing 120 +

I0 g were infected orally with 25 metacercariae each.

The in fected animals and ti le con t ro l s were fed "ad

l i b i t u m " wi th commercial complete dry food and 6 animals

of each group were k i l l e d &t 10 day i n t e r v a l over a 40

dab, per iod and at the 60th day a f t e r the i n f e c t i o n .

The l i v e r s were immediately removed and weigl~ed and the

f lukes removed by d i ssec t i on of the parenshyma and b i l e

ducts of the infected animals. Liver tissue homogenates

were subsequently s u b j e c t e d to d i f f e r e n t i a l c e n t r i f u g a t i o n

(Raffaeli et El., 1977) .

Tissue preparat ion

61icrosomes from in fec ted and con t ro l l i v e r s were prepared

by the methods p rev ious l y described (Ra f f ae l i et a l . , 1377)

and suspended in 0. I~ phosphate buffer, pH 7.4, to a final

concentration of 2.0 mg protein/ml for the assay of the

monooxygenase activities.

Microsomal protein was determined by tile method of Lowry

et al. (1951); cytochrome P-450 and cytochrome b 5 by the

method of Omura and Sato (1Q64).

~nzyme agsays

The monooxygenase activities were determined by the methods

reported in l i t e r a t u r e : p - n i t r o a n i s o l e O-demethylase

( N e t t e r and S e i d e l , 1964)., a m i n o p y r i n e N - d e m e t h y l a s e (Coch in

and Axelrod, 1950), hexobarb i ta l oxidase (Cooper and Urodie,

IC,55), a n i l i n e hydroxylase (Imai et a l . , 1966) and zoxazolamine

hydroxylase (Conney et a l . , 19G0).

734 PharmacologicalResearchCommunications, Vol. 13,No.& 1981

The m o n o o x y g e n a s e a c t i v i t i e s were e x p r e s s e d i n nmo les p r o d u c t

f o r m e d ( p - n i t r o p h e n o l , f o r m a l d e h y d e and p - a m i n o p h e n o l ) o r

substrate disappearing (hexobarbital and zoxazolamine)/min/

mg microsomal protein.

NADPH-cytocllrome c reductase a c t i v i t y was assayed by

the method of P h i l l i p s and Langdon (Ig62) with cytochrome

c as the f i n a l e lec t ron accepter.

Contro l values were determined whenevee Fasciqla heAatica

in fec ted animals were k i l l e d because of we l l - recogn ized,

day-to-day v a r i a t i o n s in hepat ic microsomal enzyme a c t i v i t y

(Valer ino et a l . , 1074).

RESULTS

The body w e i g h t s ( T a b l e I ) o f F a s c i o l a h e p a t i c a i n f e c t e d rats

did not significantly vary from those of uninfected controis.

Absolute liver weiohts increased at the 20th day, and the

variation was maximai at the 30th day (TabIe I); after this

period, when the fiukes begin to localize in the main bile

ducts of the host , the absolute l i v e r weights tend to decrease

to the con t ro l values. The increase in the absolute l i v e r

weight observed in the acute phase of the i n f e c t i o n might

be due, as p rev ious ly demonstrated fo r the mouse and the nat,

to an increase in the p r o l i f e r a t i v e a c t i v i t y of l i v e r

parenchymal c e l l s , in reply to the l i v e r t issue damage

induced by the Fascio la hepatica migra t ion {Thorpe, 1965;

Rahko, 1971).

20-30 days a f t e r the i n f e c t i o n , ra ts sllow a decrease in

microsomal p ro te in content (mg/g fresh l i v e r ) , which tends

to become corrected a f t e r the 40th day (Table I ) .

TABLE I

Oo

dy

, li

ve

r w

eig

hts

a

nd

microso~nal

pro

tein

in

rats

in

fec

ted

e

xp

eri

me

nta

lly

wit

h

Fa

sc

iola

h

ep

ati

ca

a

t th

e

va

rio

us

po

sti

nfe

cti

on

al

da

ys

.

c~

DAY

10

20

30

~0

GO

BO

DY

~

EIG

t{T

(g

) L

IVE

R

V!E

IGI{

T

(g)

Controls

Infected

p

17

5.0

+

8

.0

17

7.5

+

9

.3

n.s

,

24

0.3

+

8

.3

23

7.0

+

9

.8

n.s

.

29

5.5

+

9

.8

30

0.4

+

7

0 n

,s.

34

7.0

+

8

.5

35

0.0

+

9

.7

n.s

.

45

0.5

+

1

2.0

4

60

.0

+

9.5

n

.s.

Controls

Infected

p

6.4

5

4-

0.3

4

6.7

+

0

.35

n

.s.

7.3

8

+

0.2

8

9.9

+

0

.27

~

&~

8.8

2

+

0.3

5

13

.7

+

1.1

0

~&

9.4

0

+

0.3

6

11

.3

+

0.4

5

~

14

.9

+

0.7

2

15

.2

+

0.5

0

n.s

.

RE

LA

TIV

E

/.ilC

RO

SO

[,IA

L

PR

OT

EIN

(mg

/g

liv

er)

--

..

.,

.

Co

ntr

ols

In

fec

ted

p

20

,75

~

0

.35

2

0.1

5

+

0.2

8

n.s

.

22

.25

~

0

.36

1

8.2

5

+

0.3

0

~

25

,80

~

0

.38

1

2.6

0

+

0.1

6

~$

~

26

.60

4-

0

.42

2

0.7

3

+

0.4

0

~,

Each value represents

the mean _+ S.E.

relative

to six animals.

P<

0

.00

27

.44

+

0

.32

2

6.7

0

+

0.4

0

~

c) }

~ P

<

0.0

1

,~ ~

P< 0.001

n.s.

not sionifican t

~4

736

F I G . I

Pharmacological Research Communications, VoL f 3, No. 8. 198f

12

o 08

EF 0.

" - ~ - " e6 •

C 10 20 30 40 O0

days

250

200 • &

100 - ~E

50 ~_~

Z~

C y t o c h r o m e P - 4 5 0 , b 5 and N A O P H - c y t o c h r o m e c r e d u c t a s e

activity in r a t s experimentally infected w i t h

Fasciola hepatica.

C y t o c h r o m e P-450 (- ); cytochrome b 5 ( - . - - - ) ;

NADPH-cytochrome c reduct&se a c t i v i t y ( - - - - - ) .

C r e p r e s e n t s t h e c o n t r o l v a l u e , mean + S . E . o f tt~e

values _+ ., o.E. r e l a t i v e to each .group of uninfected

animals.

& P{ O. 05

~ P ( O . 0 1

~ P<O. OO1

C y t o c h r o m e P-4DO, c y t o c h p o m e b_ and N A D P H - c y t o c h r o m e c

r e d u c t a s e a c t i v i t y

C y t o e h r o m e P - 4 5 0 and N A D P H - c y t o c h r o m e C r e d u c t a s e a c t i v i t y

in parasitized liver are slightly but significantly decreased

( P < O . O 5 ) on t h e 1Oth day a f t e r the i m f e c t i o n ; t h e two m a i n

c o m p o n e n t s of the e lect ron t ranspor t chain continue to

decrease u n t i l the gOth gay, at which time the leve ls ape

on1~y about bO'i of the cont ro l values ( F i g . l ) .

Cytoehrome P-450 and NADPl4-cytochrome c .reductase a c t i v i t y

Pharmacological Research Commun/cat/ons, Vo/. 13, No. 8, 1981

a r e s t i l l b e l o w t i l e c o n t r o l v a l u e s (85,% and gO~) on t h e

00th day of the infection.

C y t o c h r o m e b 5 c h a n g e s i n t h e same way as c y t o c h r o m e P - 4 5 0 ,

a l t h o u g h l e s s s t r i k i n g l y : 7 0 % on t h e 3 0 t h day and 95~,; on t h e

OOtl~ day (Fig.l).

737

C y t o g h r o m e . . P - 4 5 0 c . d e p e n d e p t m e . t a b o l i z . i n g _ ~ q t i v i t i e s

As r e p o r t e d i n F i g . 2A, on t h e l O t h day o f t h e i n f e c t i o n a l l

t h e c y t o c h r o m e P - A i O - d e p e n d e n t m e t a b o l i z i n g a c t i v i t i e s were

s i g n i f i c a n t l y d e c r e a s e d : t h e h e × o b a r b i t a l o x i d a s e , p - n i t r o -

a n i s o l e O - d e m e t h y l a s e and a m i n o p y r i n e N - d e m e t h y l a s e a c t i v i t i e s

were t h e more s e n s i t i v e t o Fa.§ .c io la h e p a t i c a damage (45-50%

o f t h e c o n t r o l v a l u e s ] .

The m o n o o x y g e n a s e a c t i v i t i e s p r o g r e s s i v e l y . . d e c r e a s e a u n t i l

t h e 3 0 t h d a y , a t w h i c h t i m e t h e h e x o b a r b i t a l o x i d a s e ,

p-nitroanisole O-demethylase and aminopyrine N-demethylase

had f a l l e n t o 10~ o f t h e c o n t r o l v a l u e s ,

The aniline and zoxazolamine hydroxylase activities were

less affected by Fasciola hepatica infection (90~ on the 10tl~

d a y ; 00~6 and 50% o f t h e c o n t r o l v a l u e s on t h e 3 0 t h day

o f t h e i n f e c t i o n ) ( F i g . 2 B ) .

The r e s t o r a t i o n o f t h e m o n o o x y g e n a s e a c t i v i t i e s had begun

by t h e 4 0 t h day o f t h e i n f e c t i o n . We w i s h to e m p h a s i z e t h a t

on t h e 5 0 t h d a y , when t he m i g r a t o r y s t a g e i n t o tl~e l i v e r

o a r e n c h y m a had l o n g beam f i n i s h e d and a l l t h e f l u k e s were

l o c a l i z e d i n t h e b i l e d u c t s ( T h o r p e , 1 9 6 5 ) , a l l o f t h e

m o n o o x y g e n a s e a c t i v i t i e s , e s p e c i a l l y I ~ e x o b a r b i t a l o x i d a s e

and p - n i t r o a n i s o l e O ~ d e m e t h y l a s e Were s t i l l l o w .

DISCUSSION

The m i x e d f u n c t i o n o x i d a s e o f t h e e n d o p l a s m i c r e t i c u l u m i s

r e s p o n s i b l e f o r t h e d e t o x i f i c a t i o n m e c h a n i s m s o r t h e l i v e r

738 Pharmacolopical Research Communications, Vol. 13, No. 8, 1981

0 ~ ~2 Z

I0

~ o6 g~ 4~ o,

~ 04

6 o,

A

,

l i t

B

k - . i ~ 1 l o p

120

I00 .%.,

40 ~&

,o f,:

C I0 20 30 40 60

cloys

FIG.2 Cytochrome P-450-dependent metabo l i z ing a c t i v i t i e s in

r a t s e x p e r i m e n t a l l y i n fec ted w i th Fa.scigla ' h..epatica.

A) I I/~xobanbital oxidase ( - - ) ; p - n i t r o a n i s o l e O-demethylase

( - - - - - ) ; aminopyr ine N-demethylase ( . . . . . ).

3) A n i l i n e hydroxy lase ( ..... ); zoxazolamine hydroxy lase

( - - - - - ) . C represents the c o n t r o l va lue, mean + S.E.

of t l le values _+ S.E. r e l a t i v e to each group of

un in fec ted animals.

P< O. O5

• P<O. 01

~ $ P<O.O01

ceil, and for the physiological oxidation of steroids and

f a t t y ac ids.

The f unc t i on of the e l ec t r on t r anspo r t chain of the ~F0

system depends on the f u n c t i o n of the f l a v o p r o t e i n , NADPH-

cytochrome P-450 (cytochrome e) reductase, respons ib le in

t l le endoplasmic r e t i c u l u m fo r the d i r e c t t r a n s f e r of

electrons from I~ADP}t to cytochrome(~) P-450, a family

PharmacologicalResearchCommun~ation~ Vol. 13, No.~ 1981

o f h e m o p r o t e i n s w h i c h s e r v e as t e r m i n a l o x i d a s e s ,

The p r e s e n t s t u d y d e m o n s t r a t e s t h a t d u r i n g t h e i n c u b a t i o n

period (2-17 days) the intrahepatic migration of Fasciola

hepatica induces a remarkable loss in the metabolic activity

of the MFO system on its typical substrates: this loss is

quite dram&tic in the acute period.

The beginning of the biliary stage (40th day) coincides with

a slight recovery of the MFO activity.

The activity is however still significantly depressed in the

chronic phase of the infection (40-60 days), when the absolute

liver weight and the microsomal proteins have already returned

to r ange of the con t ro l values.

The depression in rAFO a c t i v i t y is the consequence of decreases

in the monooxygenase enzymes, cytochrome P-450, cytochrome b b

and NADPH-cytochrome P-450 (cytochrome c) red,uctase.

The Fa l l in monooxygenase p ro te in might be lhe consequence

of d i r e c t l i v e r c e l l damage induced by Fasciola .hePatica

migra t ion - - u l t r a s t r u c t u r a l a l t e r a t i o n s of endoplasmic

re t i cu lum have been observed (Rahko, 1971).

I t might also be the r e s u l t of an increased p r o l i f e r a t i v e

response of the damaged t i ssue (see the increase in absolute

l i v e r weights) , which i s in accord wi th previous s tud ies

demonstrating tha t hepat ic monooxygenase enzymes are

decreased in any s i t u a t i o n in which there is an increased

rate of c e l l u l a r p r o l i f e r a t i o n , as in regenerat ive growth

a f t e r p a r t i a l hepatectomy, in f avou r ,o f spec ia l i zed p ro te ins

of the m i t o t i c sp ind le (Henderson and Kersten, 1969).

Ooth hypoteses are being i nves t i ga ted .

The decrease in cytochrome P-450-dependent metabol iz ing

a c t i v i t i e s in the pa ras i t i zed l i v e r c e l l s is of remarkable

i n t e r e s t , since i t might lead to an accumulation of

endogenous ( s t e r o i d s , f a t t y acids) and exogenous substrates

in the liver cell, with obvious physiological and toxicological

739

740 PharmacologicaI Research Communications. VoL 13, No. 8. 1981

c o n s e q u e n c e s . I n t h i s c o n n e c t i o n , i f t h e phenomenon (and

p r e l i m i n a r y e x p e r i m e n t s s6em t o c o n f i r m i t ) a l s o o c c u r s

i n a n i m a l s p e c i e s o£ e c o n o m i c i m p o r t a n c e ( s h e e p and c a t t l e ) , I

f l u k i c i d a l ' d r u g s w h i c h a r e e x t e n s i v e l y d e t o x i f i e d by

c y t o c h r o m e P - 4 5 0 (Gram e t a l . , 1 0 7 7 ) , mus t be g i v e n w i t h

c a u t i o n d u r i n g t h e a c u t e p e r i o d o f i n f e c t i o n , s i n c e

a~cumul&tion of the flukicid&l in the liver cell might

decrease its safety margin.

In addition, tl~ose flukieidal agents that, as untoward

effect, inhibit monooxygenase activities should be also

avoided, i.e., hexachlorophene, which has been shown

i n v i t r o to' c a u s e a l o s s o f c y t o c h r o m e P - 4 5 0 f r om r a t

l i v e r m i c r o s o m e s and t o i n h i b i t t h e m o n o o x y g e n a s e a c t i v i t i e s

b o t h i n v i t r o and i n v i v o ( G a n d o l f i e t a l . , 1974; C o n d i e

and G u h l e r , l g 7 g ) . i

For these reasons, the need for studies of\i_n vitro

metabolisr.~ of the commonest flukicidal agents, to determine

the enzyme system involved in their degradation, becomes

of striking importance.

In addition, the remarkable sensitivity of hexobarbital

m e t a b o l i s m i n v i t r o i n a l l s t a g e s o f t h e d i s e a s e , makes

t h i s compound s u i t a b l e f o r t e s t i n g f l u k e h e p a t o t o x i c i t y

i n v i v o (Conney e t a l . , 1960; Hansen and F o u t s , 1968;

t ~ a n n e r i n g , 1 9 6 8 ) .

P r o l o n g a t i o n o f t h e h e x o b a r b i t a l s l e e p i n g t i m e o r o f t h e

p l a s m a h e x o b a r b i t a l h a l f - l i f e i n F a s c i o l a h e p a t i c a i n f e c t e d

r a t s m i g h t be an i n v i v o e x p r e s s i o n o f t h e d e c r e a s e d

h e x o b a r b i t a l m e t a b o l i s m o b s e r v e d i n v i t r o .

This might serve as an index of the severity of the liver

damage caused by Fasciola hepatica, while the recovery

toward normal of these parameters would be an index of

the t l ;erapeut ic e f fec t iveness and safety of tl~e f l u k i c i d a l

a,lcnt,

Pharmacological Research Commun~ations, VoL 13, No. 8, 1981

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- B J O R K H E I M I . , E u r . . ) . 8 i o c h e m . , 2 7 , 354 ( 1 9 7 2 ) .

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105 ( 1 9 5 9 ) .

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( 1 9 7 9 ) .

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