a quantitative protozoan bio-assay method for determining venom potencies - johnson, tullar, stahnke...

8/19/2019 A Quantitative Protozoan Bio-Assay Method for Determining Venom Potencies - Johnson, Tullar, Stahnke - Toxicon -…

1/4

Tua~cnn

1966, Vol

3

pp

297 100

Per~anwn

Press Ltd

Pri nted

i n Gr cet Ar i t ai n

QUANTITATIVE PROTOZO NBIO-ASSAY

METHOD

FORDETERMNNG

VENOM

POTENCIES

BosDJ ol l xs ox, J oxxCTULLAR

and

H

L

STAxxx

Ar i zona

State

Uni versi t y,

Tempe

Ar i zona

Accept ed j or

pebl l cat l on

30J ul y 1965

Abstract A

si mpl e,

r a pi d

pr ot ozoan

assay

method requi r i ng

smal l

amounts of venom s

descri bed

L e t h a l i t i e s

of

Crot al es

atrox, Cr otal es

s c u t u l a t u s

Cent r ur of des

s c ul p t r v a t e s

Hadrurus a r l z o ne ns i s and Hel oder ma

suspect um

venom deter m ned by

t h i s

method

are

compared

w t h those obtai ned

i n

m t x

INTRODU TION

STATISTICALLY

V a l i d

t o x i c i t y

t e s t s

usi ng

mammal s

r e qu i r e r e l a t i v e l y l a r g e amount s of

venom

The

use of

smal l q u a n t i t i e s of venomo

det er m ne

pot ency

s

necessary i n t e s t i n g r a r e

venoms

and

venom

r a c t i o ns

PH LPOTT [1-4] u t i l i z e d

protozoa

i n measur i ng t he r e s i s t a n ce and

potency

of venoms

and

t o x i n s

and t h e i r

ant i bodi es

LACALLADE [ 5] measur ed

venom

potency

l

t erms of Par ameci um caudat unt Ehr enber g c y t o l y s i s I n c o n t r a s t MNTON

[ 6]

used

P c a u d a t t t t t l

s u r v i v al t i me s t o determ ne venomp ot e nc i e s : These s t u d i e s

wer e

sem - quant i t at i ve

Thep o s s i b i l i t y of

s i m l a r r esponses bet ween protozoa

and

met azoa t o ot her

t o xi c ol o gi c al

s t i mu l i such as car ci nogens

and

a n t i h i s t a m n es

has

a l s o

mot i vat ed

t he

use

of

protozoa

i n

scr eeni ng

t e s t s

measur i ng r e l a t i v e

potency,

determ ni ng

mode, and s i t e

of a ct i o n

and

i d e n t i f y i n g

s i de

r e a c t i o ns [ 7 9 ] I n t e r p r e t at i o n of

dat a

i n

t erms

of l i m t a t i o n s andval ues

of

t o x i c ol o gi c al pr otozool ogy

s

di scussed by HuTxER

[10]

W

r e p o r t

here

a r e l a t i v e l y

r api d pr ot ozoan assay

met hod desi gned

t o measur e l e t h a l i t y

L e t h a l i t i e s measur ed

by

t h i s assay met hod are compar ed

w t h

those

obt ai ned i n

m ce

M TER LS NMETHODS

Sampl es

f r om

l a r g e pool s of l y o p h i l i z e d

venom

f r om t he r a t t l es n ak es Cr o t a l u s atrox

Bai r d andG r ard, and Cr otal us s c ut ul a t u s

Kenni cot t , andf r o mt he

G l a

monst er

Hel oder ma

s u s p e c t t n n

Cope,

and

f r om the scorpi ons Hadr t r r us a r i z on ens i s Ew ng,

and

Cent r ur oi des

s c u l p t t t r a t u s

Ew ng,

were used

Sampl e wei ght

was

cal cul at ed by aver agi ng wei ght s f r o m

t h r e e

consecut i ve wei ghi ngs at 30- m n i n t e r v a l s usi ng a Met t l er

a na l y t i c al

bal ance The

venoms

wer e

r e c on s t i t u t e d

i n

s t e r i l e wheat bro th medi umor d i s t i l l e d wat er i mmedi at el y

pr ecedi ng

each

exper i ment

Al l

LD60 wer e c al c ul a t e d by t he Thompson

and

Wei l

t echni que The Par ameci um

n t u l t i m c r o t : ucl eat umPower s andM tchel l LDaO

at 6 hr

expr essed as

mg

per m of medi um

was

deter m ned by

i nocul at i ng

f o u r

par ameci a i n t o

each

dosage l e ve l

of venom

e co ns t i t ut e d

i n

s t e r i l e

wheat

bro th

The

mouse

LDo at 24 hr expr essed as mgof venom

e co ns t i t u t e d

Suppor t ed

byNat i onal I n s t i t u t e s

of Heal th Gr ant

NB 02330

297

G


2/4

? ~l HO

t )

JOHNSON JOHNc t ULLARand

H

1 STAHNKE

i n de- i oni zed

d i s t i l l e d

wat er per

kg of

mouse,

was

determned

by

i nt raperi t oncal I P I

i nj ect i ons [11-13]

1 he

c l onal cul t ures [ 14J

of P

nur l r i nl i r r o r J UCl ec r l um wer e

mai ntai ned1

i n : mucl avcJ

wheat broth

medi um

i nocul at ed w t h Pse~udurr rurr cr s ueru ~nusa

p hi s b r o t } i consi sts of

h

wheat

grai ns

per 100

m of

synt heti c

spri ng wat er

I S]

Asepsi s

of

cul t ure

medi um

was

mai nt ai ned

up t o

t he

poi nt of

subcul t uri ng Al l

cul tur i ng

and

t e s t i n g of parameci a w~~s

conduct ed

a t

24- 25 Al l

gl assware was

c l eaned accord i ng

t o

procures out l i ned by

SoNN_twttN [ l 6 ] and aut ocl aved bef ore use

Pr i or

t o

each

exper i ment t he

P r u r r l t i -

n~ crorurcl eatum

wer e washed f our t i mes

i n

s t e r i l e wheat broth by cent ri f ugat i on at sl ow

speeds f or 6

mn After washi ng,

m of concent rated washedparamec i a was pi pet t ed

i nto : ~

Maxi mov

depressi on

s l i d e

Wt hi n

3 mn

these

par ameci a usual l y

aggregat ed

i n t o

a

r i ng

j ust

bel ow

t he

sur f ace

near t he

edge

of t he medi um Rapi d

transfer of 4 par ameci a from

t h i s aggregate t o u depressi on s l i d e

cont ai ni ng

0

~2 m

of

t e s t

medi um

w~ s done

w t h a

n t i c rol oop [ 1 7 j

Each

bi o-assay consi sted of

a

mni mum

of 4

dosage

l e v e l s

w t h 3

r e p l i -

cat i ons per l e ve l I nocul at i on of

each venom

dosage l e ve l was

compl et ed

before i nocul at i on

of

t he

next

hi gher

l e ve l

was

begun

Thus,

64

paramec i a

( 4

per

s l i d e )

were used

i n

each

bi o-

assay Two s e t s of cont rol s

wer e used

The l owest

dosage l e ve l i n whi chno

par ameci a

di ed

served as t he t e s t cont rol s

Paramec i a

i n venomdi l uent

s t e r i l e

wheat broth) served as

cont rol s of experi ment al condi t i ons

Resul t s wer e recorded af t er a 6 hr i ncubat i on

peri od

Four heal t hy

al bi no

mce

( 2 mal es

and

2

f emal es)

wei ghi ng

from

15- 23 g

wer e

used

per

dosage l e ve l

i n determni ng

t he t

. v

b The

mce

had

no

food

f or

24 hr ~t n d no wat er f or 2 hr

bef ore

wei ghi ng

After

wei ghi ng,

t he

mce wer e

i nj ect ed ( i P)

I nocul at i ons

wer e

made

fromvenomstock sol ut i ons of 5 mg per m Food and wat er were made avai l abl e 2 hr

a f t e r

i nj ec t i on

Observat i ons of mce a f t e r

i nj ect i on

wer e

f or

24 hr

These

exper i ment s

wer e

conduct ed

at

24- 2i ° and 30 per cent r e l a t i v e

humdi t y

RESULTS

Mce

responded t o

ol eos

venom

w t h rapi d i ur c e d respi rati on and hypoact i vi t y

Edema at t he

s i t e of

i nj ec t i on

was

observed

Autopsy showed t i s s u e dest ruct i on and

hemor r hagi ng

P

rr ur l t i rn i cranucl eat~urc responded

t o

t h i s venomw t h avoi di ng react i ons

f ol l owed by

c y t o l y s i s

and c e l l f r agmentat i on

I n both

par ameci a and mce, C scutul atus

venomcaused responses

si ml ar

t o

those

produced by C atro. ~

venom Less

venom

however ,

was

needed t o

produce

t he responses

M ce became hypoact i ve a f t e r

i n j e c t i o n

w t h H

srrspechrm

venom

aut opsy showed

venous di stensi on and hemor r hagi c l ungs

No

noti ceabl e par ameci a

responses wer e

observed except

w t h hi gh concent r at i ons of t h i s

venom

These concent r nt i ons of

venom

caused

hypoact i vi t y

;

sw mmng

gradual l y decreased

unt i l

death

C

scul pt rr ratus venomcaused convul si ons

and

copi ous sal i vat i on i n

mce

No

gross

t i s s u e dest ruct i on was

observed after

aut opsy

The

responses of P r r rul t i mc ronuc

l eatum

t o

t h i s venom

wer e

t he

f ormat i on

of l engt hy

prot opl asmc

ext rusi ons

and

an e r r a t i c swmmng

behavi or f ol l owed

by cessat i on of movement

Subsequent

mechani cal

sti mul at i on

i nduced

br i e f r esumpt i on

of swmmng

I n mce,

l l

ar i z onc r rs i s venomcaused

spasms,

i ncreased sal i vat i on, and

paral ysi s

of

l i mbs ;

aut opsy

showedhent orrhagi c l ungs

P

rncr l t i r ni c roni r c l eatur r t responded

w t h

avoi di ng

react i ons,

e r r a t i c

sw mmng behavi or, and f i n a l l y ,

f r agment at i on

I n Tabl e

l

venom

e t h a l i t i e s

f or

mce

are

compared

w t h

those obt ai ned by usi ng P

rnrr l t i mcrornecl eatr ur~

I n

mce, t he order of decr easi ngvenome t h a l i t i e s

i s

Csctt l pt t rratus,

C


3/4

Quant i tat i ve Prot ozoan Bi o- assay

Methodf or Det enm ni ngVenomot enci es 299

s c ut ul a t u : suspectum a t r o x and a r i z o ne ns i s Wt hP mul t i m c r o nuc l e at ur n

t he

order of decreasi ng

l e t h a l i t i e s

i s

a r i z o ne ns i s

s c ut u l a t u s

a t r o x

s c ul p t u r a t u s

and

suspect um

Tnar a 1

Coedrnrurt va v s r ~ o e t

rsr rt wurn s

P m

r

. n

and I

ar e i nmgper m ; musecn and I ar e

i n

mgper kg

t

Parameci ummul t i mcr onucl eat urn

DS USSION

Due t o t he hygroscopi c

nat ure of

l y o p h i l i z e d

venoms t he wei ghi ngs

wer e

per f ormed

i n a

l ow

humdi t y chamber

t o

mni mze

wei ght f l uc t u at i ons

I n

both

mce

and parameci a Cs c ut u l a t u s

venom

wasmor e l e t ha l

t han

atrox

venom

Except f or t h i s

r e l a t i on s hi p no c o r r e l a t i o n amng a t r o x C s c ut u l a t u s suspectun

s c ul p t u r a t u s and

a r i z on ens i s

venome t h a l i t i e s

i n parameci a and

mce seems

evi dent

At

p r e s e n t

i t woul d be

pr esumpt uous

t o

concl ude why

t h e s e

condi t i ons

e x i s t

However

t he

s i mp l i c i t y

of

t echni ques

i n vo l v ed and t he hi gh consi st ency of r e s u l t s obt ai ned

duri ng

t he

p a s t

year w t h

t h i s

prot ozoan assay met hod seemt o i n d i c a t e i t s val ue i n f u t u r e s t udi e s

concerned

w t h

determ ni ng

modes of acti on

and

l o c a t i n g s i t e s of act i ons of

venoms

SUMM RY

1 e l a t i v e l y

rapi d

prot ozoan

assay

met hod

was

devel oped

and

used to q u a n t i t a t i v e l y

measur e

a t r o x

s c ut u l a t us a r i z o ne ns i r

s c r r l p t

: r r a t i t

s

and

s u s p e c t t t m venom

p ot e nc i e s

2 O

t he 5 venoms s t u di e d s c ul p t u r a t u s

venom

was

t he mos t

l e t h a l t o

mce

and

N

ar i z o ne ns i s

venom

was t he l eas t l et hal

3

I n

parameci a

a r i z o ne ns i s

venomwas

t he

mos t

l e t h a l

whi l e s c ul p t n r a t u s

venomwas onl y

moderat el y l e t h al

4

H

suspect um

venom

was r e l a t i v e l y nonl et hal t o P

mul t i m c r o nu c l e at u r n

~I ckrrow edgeneent- W

t hank N Ser

. saNO

f or hi s advi ce

Our

appreci at i on goes t o

M

T t r t t

A t t

HRJ OHNSONandGL Lrr t s f or t h ei r a s s i s t a n c e

REFEREN ES

I l l PampoTr E f f o c t

of

t oxi ns

andvenomoa prot ozoa

J

exp Zool

56 167 1930

[ 2 ]

P F I I L P 01 r Usa

of

prot ozoa i n measur i ng t he n e u t r a l i z i n g

val ue

of

cobra

ant i serum

Sci ence N

Y 74

157

1931

[ 3]

P t u t rorr

Rel at i vo

r e s i s t a nc e

of

fourteen

s p ec i e s of prot ozoa t o t ho act i on

of

Crotal es

a r r o r

and

cobra

venom Mart r e Bi o l Bul l Mar t he

Bl o l Lab

Woods Hol e 60 64 1931

Venom

T e s t

ani mal

r . n

95

Conf i dence

i n t e r v al

Route

of

i gj xt i on

Hadrurus

Pmt 030 0

25- 0

35 Medi um

a r l z on en si s

Mouse 168

0

99

0- 285

I P

Crotal us

P

. m 5

049- 0

55 Medi um

s c u t u l a t u s Mouse 1 8 1

2 25

I P

Crotal us

P

. m

0

67

062- 0

73 Medi um

atrox Mouse 6 54 67

I P

Cent rur ot des P. m 1 3 1 12- 1 5 Medi um

s c u l p t u r a t u s Mouse

1~5

1 1- 1 9 l

: P

Hel oderma P. m 24 7 20 8- 29 3 Medium

suspectum

Mouse

30 2

1- 4

2

I P


4/4

300 HOB JOHNSON

JOHN

CTULLAR

and

H

L STAHNKE

[ 4] PHLPUTT

CH Nat ural and acqui r ed r e s i s t a nc e

i n protozoa

t o

t he

a c t i o n o f

anti geni c

poi xrns

J

exp Zool

. 63, 553, 1932

[ 5]

L C I LL DE C

W

J r

The determ nati on

of

t he pot ency

of

bee

venomn v i t r o

nr

J

Phys

103,

251, 1933

6]

MNTON S A Some

propert i es

of north amer i can

pi t

v i p er venom and

t h e i r corr el at i on

w t h

phyl ogeny

Venonr s

E

E

BuctcLeY

and

NN

PORGFS

Eds

.

A

A

. A

. S

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Washi ngt on,

p

143 ,

1956

[ 7 ] Huu RW Usi ng parameci um assay t o screen carci nogeni c

hydr ocarbons

J Prot ozool

9, 1 8,

1962

[ 8] WEST R A J r B RBER PW

KoLnR

J Rand

MURRELL

CB The agar l a ye r method f or

determ ni ng t he a c t i v i t i e s of

di verse

materi al s a ga i n s t

s e l e c t e d

pr otozoa J Protozol

9,

6 5,

1962

[ 9] S NDERSMandNATHANH Protozoa a s pharmacol ogi cal t o o l s The anti hi stamnes J gcrr

M crubi ol

21, 264, 1959

[ 1 0 ] HUTNER S H Protozoa a s t o xi c ol o gi c al tool s

J

Protozcw

11 , I , 1964

[ l l ]

THOMPSON

R

Use o f movi ng

averages

and i n t e r p o l a t i o n t o

est i mate

medi an

e f f e c t i v e

dose

Bact

Rev

11,

1 1 5 ,

1947

[ 1 2 J

THOMPSON

W

R

and

WEI L C

S

On

t he

const ructi on o f t a b l e s

f or movi ng average

i nterpol at i on

Bi onretr i cs, 8,

51 ,

1952

[ 1 3 ] Wts

CS

Tabl es fo r

conveni ent

cal cul ati on of

medi an

e f f e c t i v e

dose

w Or

ED

T

and

i n s t r u c t i o n

i n

t h e i r

use

Bi onretr i cs, 8,

2 4 9 , 1952

l a WCHTERMAN

R

TheBi ol ogy

oj Parm rec i unr

Bl aki st on, NewYork,

1953, p 104

I S ]

K RBY

H

J l ~a t er i a l s

andAf elhorl s

i n t hr

Strrdy

ojProtozoa

Berkel ey

Uni versi ty

of

C a l i f o r n i a ,

p

14,

1950

[ 1 6 J

SONNEaORN T

M

Methods

i n

gener ; t l bi ol ogy

and

g en et i c s of Par urr recl wr r

a r r r e l i a

J Eaptl Zuol

113,

87 , 1964

[ 1 7 ] TULL R

J C JOHNSON

B

andST HNKe HL Rapi d

t r v t s f e r

of parameci um

by

M crol oopi ng

T i r r t o

x Nervs, 1966 I n

p r e s s

a quantitative protozoan bio-assay method for determining venom potencies - johnson, tullar, stahnke...

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