DRUG AND CHEMICAL TOXICOLOGY, 2(3), 245-255 (1979)
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PROTECTIVE EFFECT OF GLUTATHIONE ON THE IN VITRO INHIBITION OF HEPATIC CYTOCHROME P-450 B Y C m R. R. Dalvi and W. M. Ashley Toxicology Laboratory Department of Physiology and Pharmacology School of Veterinary Medicine Tuskegee Institute Tuskegee, Alabama 36088, U.S.A. ABSTRACT The in vitro effect of various concentrations of captan onhepatic microsomal cytochrome P-450 from phenobarbital-pretreated rats was studied. The 1-50 value, namely the concentration of the inhibitor necessary to produce 50% l o s s of cytochrome P-450 was determined from the plotted inhibition curve. The presence of ethylenediaminetetraacetic acid (EDTA) in microsomal incubations prior to the addition of captan failed to prevent the loss of cytochrome P-450 by captan. In contrast, reduced glutathione (0.5 mM) added to microsomal incubations before captan (0.1 mM) afforded almost complete protection of cytochrome P-450 from captan inhibition. These data indicate that the inhibitory effect of captan on vitally important drugmetabolizing enzyme system, of which cytoehrome P-450 is a major component, can be prevented by prior presence of reduced glutathione (GSH) but not of EDTA. INTRODUCTION Captan (N-trichloromethylthio-4-cyclohexene-l,?dicarboximide) is a broad-spectrum, nonpersistent fungicide widely used for the control of various fungus diseases of seeds, plants, and fruits.
It is also used
as an industrial fungicide in paints, plastics, leather, 245 Copyright 0 1979 hy Marcel Dekker. Inc All Rights Reserved Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher
DALVI AND ASHLEY
246
an5 ce;tain
s o a p s and shampoos 1
.
A l t h o u g h many r e p o r t s
have b e e n p u b l i s h e d on c a p t a n as a n e f f e c t i v e f u n g i -
Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
c i d e , l i t t l e i s known c o n c e r n i n g i t s mechanism o f t o x i c a c t i o n i n a n i m a l s a n d humans. S t r u c t u r a l l y , captan i s c l o s e l y r e l a t e d t o t h a l i d omide which h a s b e e n f o u n d t o b e a p o t e n t t e r a t o g e n i n humans.
Whether c a p t a n i s t e r a t o g e n i c i n humans i s n o t
knc;wn; h o w e v e r , some d a t a i n d i c a t e t h a t i t i s n o t a t e r a t o g e n i n l a b o r a t o r y animals' oxhers
i t is t e r a t o g e n i c 4
3
y 3
while according t o
Similarly, conflicting
5.
r e p o r t s about t h e mutagenic e f f e c t s of c a p t a n have been ?dbli~hed~-~ A . r e c e n t s t u d y h a s shown t h a t c h r o n i c e x 7 o s u r e of mice t o t h i s compound i n d u c e d t u m o r s i n t h e duodenum, t h u s l a b e l i n g c a p t a n t o b e a c a r c i n o g e n
1
.
A c u t e t o x i c i t y of c a p t a n i s r e l a t i v e l y low when g i v e n orally; h o w e v e r , i t i s a h i g h l y t o x i c m a t e r i a l i f administered i n t r a p e r i t o n e a l l y
8
.
A t t h e molecular l e v e l ,
i t h a s been found t o u n c o u p l e o x i d a t i v e p h o s p h o r y l a t i o n ,
thus inhibiting c e l l u l a r respiration 9
.
Similarly,
T r u h a u t e t a 1 . I u o b s e r v e d i n h i b i t i o n of m i c r o s o m a l d r u g m e t a b o l i z i n g enzymes i n r a t s g i v e n c a p t a n i p a t s u b 1C.thal l e v e l s for t h r e e s u c c e s s i v e d a y s .
Preliminary
s t u d i e s c a r r i e d o u t i n our l a b o r a t o r y h a v e a l s o s u g ~ ~ s t te hda t c a p t a n is a n i n h i b i t o r o f t h e h e p a t i c
m i c r ~ ~ ; c m aeln z y m e s y ~ t e m 8.
I n contrast, t h i s fungicide
w h e n fed t o r a t s i n h i g h p r o t e i n d i e t h a s b e e n r e p o r t e d
to be a weak i n d u c e r of t h e m i c r o s o m a l enzymes''.
In
24 7
PROTECTIVE EFFECT OF GLUTATHIONE
light of these observations, the work reported herein was undertaken to examine the -in_ vitro effect of this __ important fungicide on rat liver microsomal cytochrome Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
P-450 and to study whether GSH and EDTA alter the action of captan on the cytochrome P-450. MATERIALS AND METHODS Captan, 98.6% purity, was obtained from Stauffer Chemical Company, Richmond, California.
Reduced
glutathione was purchased from Sigma Chemical Company, St. Louis, Missouri.
All other chemicals used were
analytical or reagent grade. Male Sprague-Dawley rats (180-220 g ) obtained from Southern Animal Farms, Prattville, Alabama, were used in these studies.
Liver microsomes from phenobarbital-
treated rats were isolated following the procedure established in our laboratory12 The washed microsomes
.
were suspended in 0.1 M phosphate buffer, pH 7.4, and incubated in Erlenmeyer flasks in a final volume of 5 ml.
Each incubation contained 2 mg microsomal protein
per ml of the reaction media.
After 5 min temperature
equilibrium, appropriate concentration of captan in dimethyl sulfoxide (DMSO) was added to the appropriate flasks.
Subsequently, the reaction mixtures were
incubated at
37°C for 10 min at a shaking rate of 300 rpm
in a gyrotory water bath shaker. The reactions were terminated by placing the flasks in a water-ice bath.
DALVI AND ASHLEY
248 After c o o l i n g a p p r o x i m a t e l y t o 4OC,
cytochrome P-450
c o n t e n t i n e a c h o f t h e i n c u b a t i o n s was dcatermined i n a d u a l beam Beckman Acta C I I I s p e c t r o p h o t o m e t e r e q u i p p e d Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
with s c a t t e r e d transmission accessory13. t r a t i o n o f cytochrome P-450
The c o n c e n -
i n t h e microsomes w a s cal-
c u l a t e d by t h e method o f Omura a n d S a t 0
14
.
The p r o t e i n c o n t e n t o f t h e s a m p l e s was d e t e r m i n e d by t h e b i u r e t method m o d i f i e d t o i n c l u d e 0 . 1 m l of 1% d e o x y c h o l a t e i n e a c h sample
15
.
Data were a n a l y z e d u s i n g s t a n d a r d t - t e s t .
Signifi-
c a n c e of t r e a t m e n t mean d i f f e r e n c e s w a s b a s e d o n a p v a l u e of 0 . 0 1 . RESULTS AND DISCUSSION
________
P r e v i o u s work from our l a b o r a t o r y d e m o n s t r a t e d t h a t a d m i n i s t r a t i o n of a s i n g l e o r a l d o s e ( 6 5 0 mg/kg) of c a p t a n r e s u l t s i n t h e i n h i b i t i o n of h e p a t i c m i c r o s o m a l enzymes i n t h e r a t
8
.
However, when t h e r a t s were g i v e n
a n i p d o s e of ( 2 0 mg/kg) c a p t a n d a i l y f o r t h r e e s u c c e s s i v e d a y s , no s i g n i f i c a n t change i n t h e a c t i v i t y
of t h e d r u g - m e t a b o l i z i n g enzymes w a s o b s e r v e d .
Under
similar e x p e r i m e n t a l c o n d i t i o n s , o t h e r workers found c a p t a n t o be i n h i b i t o r y of t h e microsomal enzymes
10
.
The p r e s e n t wGrk c l e a r l y i n d i c a t e s t h a t i n c u b a t i o n of c a p t a n w i t h r a t l i v e r microsomes r e s u l t s i n t h e i n a c t i v a t i o n of microsomal c y t o c h r o m e P - 4 5 0 ,
the terminal
o x i d a s e o f t h e d r u g - m e t a b o l i z i n g enzyme s y s t e m w h i c h
PROTECTIVE EFFECT OF GLUTATHIONE
249
m e t a b o l i z e s a v a r i e t y of d r u g s , s t e r o i d s , f a t t y a c i d s and o t h e r f o r e i g n compounds. I n o r d e r t o determine t h e c o n c e n t r a t i o n of captan Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
n e c e s s a r y t o produce 5 0 % l o s s of cytochrome P - 4 5 0 ,
liver
microsomes i s o l a t e d from p h e n o b a r b i t a l - p r e t r e a t e d r a t s were i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s (0-100 uM) of c a p t a n .
The r e s u l t s a r e p l o t t e d as cytochrome P - 4 5 0
c o n c e n t r a t i o n ( p e r c e n t of c o n t r o l ) a g a i n s t c a p t a n concentration.
The 1 - 5 0 v a l u e f o r c a p t a n f o r t h e
i n h i b i t i o n o f cytochrome P-450 was t h e n d e t e r m i n e d from t h e p l o t t e d i n h i b i t i o n c u r v e ( F i g u r e 1).
It i s e v i d e n t
t h a t 1 2 VMconcentration of c a p t a n i s r e q u i r e d t o c a u s e 5 0 % d e s t r u c t i o n o f t h e cytochrome P-450
indicating that
t h i s compound i s a s t r o n g i n h i b i t o r of t h e hemoprotein in vitro. --
The o b s e r v e d i n h i b i t i o n of t h e cytochrome
P - 4 5 0 may be a t t r i b u t e d t o t h e b i n d i n g o f c a p t a n or i t s
d e g r a d a t i o n p r o d u c t ( s ) i n t h e s l i g h t l y a l k a l i n e medium (pH 7 . 4 )
t o t h e hemoprotein t h u s i n t e r f e r i n g w i t h t h e
a b i l i t y of t h e heme group t o complex w i t h c a r b o n monoxide t h a t i s used t o d e t e r m i n e t h e amount of cytochrome P - 4 5 0 . There a r e two p o s s i b l e e x p l a n a t i o n s : e i t h e r t h a t t h e b i n d i n g of c a p t a n or i t s h y d r o l y t i c p r o d u c t p r e v e n t s t h e r e d u c t i o n o f t h e heme i r o n of cytochrome P - 4 5 0 s o t h a t carbon monoxide can no l o n g e r complex w i t h i t , or t h e b i n d i n n of t h e i n h i b i t o r s t e r i c a l l y i n t e r f e r e s w i t h t h e approach and b i n d i n g of c a r b o n monoxide t o t h e r e d u c e d
DALVI AND ASHLEY
250 100
--
80
0
4
C
Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
i
: L
6o
u 0 d in
40
e
z
u
Y 0
20 U
0
I
25
I
1
50
75
Captan
[ gM
I
I00
3
FIGURE 1 In Vitro Effect of Captan on the Concentration of Hepatic Microsomal Cytochrome P - 4 5 0 .
heme iron16 .
Both of these possible explanations would
also account for the decreased activity of the microsoma1 drug-metabolizing enzyme system. Since it has been shown that EDTA stablizes microsomal drug-metabolizing enzymes by inhibiting lipid peroxidation17 and that GSH can protect the enzyme system from toxic compounds12, the ability of these agents to prevent the destruction of cytochrome P-450 by captan was studied.
As can be seen from Table 1,
Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
PROTECTIVE EFFECT OF GLUTATHIONE
m
251
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CJ
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Fc
a,
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a,
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x
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Lo
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-rl
+
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c a +J
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252
DALVI AND ASHLEY
t h e p r e s e n c e o f EDTA i n t h e i n c u b a t i o n s p r i o r t o t h e a d d i t i o n o f c a p t a n w a s n o t a b l e t o p r e v e n t t h e l o s s of cytochrome P-450 by c a p t a n .
Thus t h e f a i l u r e o f EDTA
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t o p r o t e c t t h e a c t i v i t y o f t h e d r u g - m e t a b o l i z i n g enzymes
from c a p t a n s u g g e s t s t h a t l i p i d p e r o x i d a t i o n o f m i c r o soma1 membranes may n o t b e t h e m a j o r mechanism t h r o u g h which c a p t a n c a u s e s i n a c t i v a t i o n o f c y t o c h r o m e P-450.
I n c o n t r a s t , GSH w a s c o m p l e t e l y e f f e c t i v e i n p r o t e c t i n g t h e c y t o c h r o m e P-450 from i n h i b i t i o n by c a p t a n s u g g e s t i n g h i g h a f f i n i t y o f c a p t a n t o w a r d GSH.
I n f a c t , it has
been r e p o r t e d t h a t t h i o l s r e a d i l y r e a c t w i t h c a p t a n t o p r o d u c e t h i o p h o s g e n e 8 y 1 8,19. T h e s e r e s u l t s , t h e r e f o r e
,
i n d i c a t e t h a t it i s t h e captan molecule t h a t i s respons i b l e for t h e d e s t r u c t i o n o f c y t o c h r o m e P-450 and n o t
i t s degradation product thiophosgene.
Thiophosgene
p e rs e h a s l i t t l e e f f e c t on t h e h e m o p r o t e i n , b u t i t s m e t a b o l i s m by t h e m i c r o s o m a l enzymes i n t h e p r e s e n c e o f a c o f a c t o r NADPH l e a d s t o t h e d e s t r u c t i o n o f c y t o c h r o m e 8
P-450
.
Since the i n c u b a t i o n mixtures d i d n o t c o n t a i n
NADPH, t h e l i k e l i h o o d t h a t t h e d e g r a d a t i o n p r o d u c t of c a p t a n m i g h t h a v e c a u s e d t h e loss o f c y t o c h r o m e P-450 may be r u l e d o u t .
In conclusion, captan appears t o be
a s t r o n g i n h i b i t o r o f microsoma1 c y t o c h r o m e P-450 -in vitro and t h a t p r i o r p r e s e n c e o f GSH a f f o r d s a l m o s t c o m p l e t e protection against the inhibition.
253
PROTECTIVE EFFECT OF GLUTATHIONE
ACKNOWLEDGMENT This work %as supported in part by USPHS, NIH Grant No. 5T01-GM02264-02 and by DHEW NIH Grant No. Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
RR-08091, REFERENCES 1.
Anonymous.
1977.
Carcinogensis Bioassay of
Captan for Possible Carcinogenicity. CAS No. 133-06-2, NCI-CG-TR-15.
National Cancer Institute
Technical Report, Series # 1 5 . 2.
NCI, Bethesda, Md.
G. Kennedy, 0. E. Fancher, and J. C. Calandra. An investigation of the teratogenic potential of captan, folpet, and difolatan.
Toxicol. Appl. Pharmacol.,
13, 420 (1968). 3.
J. F. Vondruska, 0. E. Fancher, and J. C. Calandra.
An investigation into the teratogenic potential of captan, folpet, and difolatan in nonhuman primates. Toxicol. Appl. Pharmacol., 4.
J. F. Robens.
18, 619
(1971).
Teratogenic activity of several
phthalimide derivatives in the golden hamster. Toxicol. Appl. Pharmacol., 5.
B. A. Bridges.
16, 24
The mutagenicity of captan and
related fungicides. Mutat. Res., 6.
(1970).
32, 3
(1975).
G. L. Kennedy, D. Ii. Arnold, and M. L. Keplinger. Mutagenicity studies with captan, captafol, folpet, and thalidomide. (1975).
Food Cosmet. Toxicol.,
15, 55
254
7.
DALVI AND ASHLEY V.
r.
Simmons,
I).
C. Poole, and G. W.
Newell.
I n_ v_i _ t r o m e t a g e n i c s t u d i e s of t w e n t y p e s t i c i d e s .
37, 1 0 9
Toxicol. Appl. Pharmacol., Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
8.
A.
Peeples and R.
R.
Dalvi.
(1976).
T o x i c o l o g i c s t u d i e s of
hJ-trichlorornethylthio-4-cyclohexene-l,2I t s metabolism by r a t l i v e r
dicarboximide (Captan): drug-metabolizing
enzyme s y s t e m .
9, Toxicology, -
341
(1978). 3.
B.
D. N e l s o n , Jr.
Action of the f u n g i c i d e s captari
and f o l p e t on r a t l i v e r m i t o c h o n d r i a . Pharrnacol.,
LO.
20, 7 3 7
R . T r u h a u t , D.
Biochem.
(1971).
P . H i e r , a n d P. L . Nguyen.
Influence
of t h e a d m i n i s t r a t i o n of o r g a n o h a l o g e n p e s t i c i d e s a n d p o l y c h l o r i n a t e d b i p h e n y l s on t h e metabolism of zoxazolamine i n t h e rat.
D., 11.
B.
778, 3 5 0 3
C.
R. Acad. S c i . P a r i s S e r .
(1974).
IJrbanek-Karlowska.
T h e a c t i v i t y of microsomal
pnsyrnes o f r a t l i v e r i n r e l a t i o n t o d i e t a r y p r o t e i n
level a n d a d m i n i s t r a t i o n of s e l e c t e d p e s t i c i d e s .
Rocz. P a n s t w . Z a k l . I?.
R.
Hig., 28, 243 (1977).
R. Dalvi a n d T . J . R o b b i n s .
Comparative s t u d i e s
q n t h e e f f e c t of c a d m i u m , c o b a l t , l e a d , a n d s e l e n i u m
o n 1 , e p a t i c mi-rosomal m o n o o x y g e n a s e e n z y m e s a n d g l u t a t h i o n e l e v c l s i n mice. 'roxicol.,
1, 6 0 1 ( 1 9 7 8 ) .
,J. E n v i r o n . P a t h .
255
PROTECTIVE EFFECT OF GLUTATHIONE
13.
R. R. Dalvi and C. D. Howell.
Interaction of
parathion and malathion with hepatic cytochrome P-450 from rats treated with phenobarbital and Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
carbon disulfide. 14.
Drug Chem. Toxicol., 1, 191 (1978).
T. Omura and R. Sato.
The carbon monoxide-binding
I. Evidence for its
pigment of liver microsomes. hemoprotein nature. 15.
J. Biol. Chem., 239, 2370 (1964).
R. R. Dalvi and C. D. Howell.
Toxic effects of a
fungicide, 5-ethoxy-3-(tr~chloromethyl)-l,2,4thiadiazole (Terrazole), on the hepatic drugmetabolizing enzyme system in mice. Contam. Toxicol., 16.
17, 225
Bull. Environ.
(1977).
R. R. Dalvi, A. L. Hunter, and R. A. Neal. Toxicological implications of the mixed-function oxidase catalyzed metabolism of carbon disulfide. Chem.-Biol. Interact.,
17.
10, 349
T. Kamataki and H. Kitagawa.
(1975).
Effect of lipid
peroxidation on activities of drug-me-tabolizing enzymes in liver microsomes of rats. Pharmacol.,
18.
22, 3199
Biochem.
(1973).
R. J. Lukens and H. D. Sisler.
2-thiazolidinethione-
4-carboxylic acid from the reaction of captan with cysteine. 19.
Science,
R. J. Lukens.
127, 650
(1958).
The fungitoxicity of compounds
containing a trichoromethylthio-group. J. Agr. Food Chem.,
E,
365 (1966).
Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
PROTECTIVE EFFECT OF GLUTATHIONE ON THE IN VITRO INHIBITION OF HEPATIC CYTOCHROME P-450 B Y C m R. R. Dalvi and W. M. Ashley Toxicology Laboratory Department of Physiology and Pharmacology School of Veterinary Medicine Tuskegee Institute Tuskegee, Alabama 36088, U.S.A. ABSTRACT The in vitro effect of various concentrations of captan onhepatic microsomal cytochrome P-450 from phenobarbital-pretreated rats was studied. The 1-50 value, namely the concentration of the inhibitor necessary to produce 50% l o s s of cytochrome P-450 was determined from the plotted inhibition curve. The presence of ethylenediaminetetraacetic acid (EDTA) in microsomal incubations prior to the addition of captan failed to prevent the loss of cytochrome P-450 by captan. In contrast, reduced glutathione (0.5 mM) added to microsomal incubations before captan (0.1 mM) afforded almost complete protection of cytochrome P-450 from captan inhibition. These data indicate that the inhibitory effect of captan on vitally important drugmetabolizing enzyme system, of which cytoehrome P-450 is a major component, can be prevented by prior presence of reduced glutathione (GSH) but not of EDTA. INTRODUCTION Captan (N-trichloromethylthio-4-cyclohexene-l,?dicarboximide) is a broad-spectrum, nonpersistent fungicide widely used for the control of various fungus diseases of seeds, plants, and fruits.
It is also used
as an industrial fungicide in paints, plastics, leather, 245 Copyright 0 1979 hy Marcel Dekker. Inc All Rights Reserved Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher
DALVI AND ASHLEY
246
an5 ce;tain
s o a p s and shampoos 1
.
A l t h o u g h many r e p o r t s
have b e e n p u b l i s h e d on c a p t a n as a n e f f e c t i v e f u n g i -
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c i d e , l i t t l e i s known c o n c e r n i n g i t s mechanism o f t o x i c a c t i o n i n a n i m a l s a n d humans. S t r u c t u r a l l y , captan i s c l o s e l y r e l a t e d t o t h a l i d omide which h a s b e e n f o u n d t o b e a p o t e n t t e r a t o g e n i n humans.
Whether c a p t a n i s t e r a t o g e n i c i n humans i s n o t
knc;wn; h o w e v e r , some d a t a i n d i c a t e t h a t i t i s n o t a t e r a t o g e n i n l a b o r a t o r y animals' oxhers
i t is t e r a t o g e n i c 4
3
y 3
while according t o
Similarly, conflicting
5.
r e p o r t s about t h e mutagenic e f f e c t s of c a p t a n have been ?dbli~hed~-~ A . r e c e n t s t u d y h a s shown t h a t c h r o n i c e x 7 o s u r e of mice t o t h i s compound i n d u c e d t u m o r s i n t h e duodenum, t h u s l a b e l i n g c a p t a n t o b e a c a r c i n o g e n
1
.
A c u t e t o x i c i t y of c a p t a n i s r e l a t i v e l y low when g i v e n orally; h o w e v e r , i t i s a h i g h l y t o x i c m a t e r i a l i f administered i n t r a p e r i t o n e a l l y
8
.
A t t h e molecular l e v e l ,
i t h a s been found t o u n c o u p l e o x i d a t i v e p h o s p h o r y l a t i o n ,
thus inhibiting c e l l u l a r respiration 9
.
Similarly,
T r u h a u t e t a 1 . I u o b s e r v e d i n h i b i t i o n of m i c r o s o m a l d r u g m e t a b o l i z i n g enzymes i n r a t s g i v e n c a p t a n i p a t s u b 1C.thal l e v e l s for t h r e e s u c c e s s i v e d a y s .
Preliminary
s t u d i e s c a r r i e d o u t i n our l a b o r a t o r y h a v e a l s o s u g ~ ~ s t te hda t c a p t a n is a n i n h i b i t o r o f t h e h e p a t i c
m i c r ~ ~ ; c m aeln z y m e s y ~ t e m 8.
I n contrast, t h i s fungicide
w h e n fed t o r a t s i n h i g h p r o t e i n d i e t h a s b e e n r e p o r t e d
to be a weak i n d u c e r of t h e m i c r o s o m a l enzymes''.
In
24 7
PROTECTIVE EFFECT OF GLUTATHIONE
light of these observations, the work reported herein was undertaken to examine the -in_ vitro effect of this __ important fungicide on rat liver microsomal cytochrome Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
P-450 and to study whether GSH and EDTA alter the action of captan on the cytochrome P-450. MATERIALS AND METHODS Captan, 98.6% purity, was obtained from Stauffer Chemical Company, Richmond, California.
Reduced
glutathione was purchased from Sigma Chemical Company, St. Louis, Missouri.
All other chemicals used were
analytical or reagent grade. Male Sprague-Dawley rats (180-220 g ) obtained from Southern Animal Farms, Prattville, Alabama, were used in these studies.
Liver microsomes from phenobarbital-
treated rats were isolated following the procedure established in our laboratory12 The washed microsomes
.
were suspended in 0.1 M phosphate buffer, pH 7.4, and incubated in Erlenmeyer flasks in a final volume of 5 ml.
Each incubation contained 2 mg microsomal protein
per ml of the reaction media.
After 5 min temperature
equilibrium, appropriate concentration of captan in dimethyl sulfoxide (DMSO) was added to the appropriate flasks.
Subsequently, the reaction mixtures were
incubated at
37°C for 10 min at a shaking rate of 300 rpm
in a gyrotory water bath shaker. The reactions were terminated by placing the flasks in a water-ice bath.
DALVI AND ASHLEY
248 After c o o l i n g a p p r o x i m a t e l y t o 4OC,
cytochrome P-450
c o n t e n t i n e a c h o f t h e i n c u b a t i o n s was dcatermined i n a d u a l beam Beckman Acta C I I I s p e c t r o p h o t o m e t e r e q u i p p e d Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
with s c a t t e r e d transmission accessory13. t r a t i o n o f cytochrome P-450
The c o n c e n -
i n t h e microsomes w a s cal-
c u l a t e d by t h e method o f Omura a n d S a t 0
14
.
The p r o t e i n c o n t e n t o f t h e s a m p l e s was d e t e r m i n e d by t h e b i u r e t method m o d i f i e d t o i n c l u d e 0 . 1 m l of 1% d e o x y c h o l a t e i n e a c h sample
15
.
Data were a n a l y z e d u s i n g s t a n d a r d t - t e s t .
Signifi-
c a n c e of t r e a t m e n t mean d i f f e r e n c e s w a s b a s e d o n a p v a l u e of 0 . 0 1 . RESULTS AND DISCUSSION
________
P r e v i o u s work from our l a b o r a t o r y d e m o n s t r a t e d t h a t a d m i n i s t r a t i o n of a s i n g l e o r a l d o s e ( 6 5 0 mg/kg) of c a p t a n r e s u l t s i n t h e i n h i b i t i o n of h e p a t i c m i c r o s o m a l enzymes i n t h e r a t
8
.
However, when t h e r a t s were g i v e n
a n i p d o s e of ( 2 0 mg/kg) c a p t a n d a i l y f o r t h r e e s u c c e s s i v e d a y s , no s i g n i f i c a n t change i n t h e a c t i v i t y
of t h e d r u g - m e t a b o l i z i n g enzymes w a s o b s e r v e d .
Under
similar e x p e r i m e n t a l c o n d i t i o n s , o t h e r workers found c a p t a n t o be i n h i b i t o r y of t h e microsomal enzymes
10
.
The p r e s e n t wGrk c l e a r l y i n d i c a t e s t h a t i n c u b a t i o n of c a p t a n w i t h r a t l i v e r microsomes r e s u l t s i n t h e i n a c t i v a t i o n of microsomal c y t o c h r o m e P - 4 5 0 ,
the terminal
o x i d a s e o f t h e d r u g - m e t a b o l i z i n g enzyme s y s t e m w h i c h
PROTECTIVE EFFECT OF GLUTATHIONE
249
m e t a b o l i z e s a v a r i e t y of d r u g s , s t e r o i d s , f a t t y a c i d s and o t h e r f o r e i g n compounds. I n o r d e r t o determine t h e c o n c e n t r a t i o n of captan Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
n e c e s s a r y t o produce 5 0 % l o s s of cytochrome P - 4 5 0 ,
liver
microsomes i s o l a t e d from p h e n o b a r b i t a l - p r e t r e a t e d r a t s were i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s (0-100 uM) of c a p t a n .
The r e s u l t s a r e p l o t t e d as cytochrome P - 4 5 0
c o n c e n t r a t i o n ( p e r c e n t of c o n t r o l ) a g a i n s t c a p t a n concentration.
The 1 - 5 0 v a l u e f o r c a p t a n f o r t h e
i n h i b i t i o n o f cytochrome P-450 was t h e n d e t e r m i n e d from t h e p l o t t e d i n h i b i t i o n c u r v e ( F i g u r e 1).
It i s e v i d e n t
t h a t 1 2 VMconcentration of c a p t a n i s r e q u i r e d t o c a u s e 5 0 % d e s t r u c t i o n o f t h e cytochrome P-450
indicating that
t h i s compound i s a s t r o n g i n h i b i t o r of t h e hemoprotein in vitro. --
The o b s e r v e d i n h i b i t i o n of t h e cytochrome
P - 4 5 0 may be a t t r i b u t e d t o t h e b i n d i n g o f c a p t a n or i t s
d e g r a d a t i o n p r o d u c t ( s ) i n t h e s l i g h t l y a l k a l i n e medium (pH 7 . 4 )
t o t h e hemoprotein t h u s i n t e r f e r i n g w i t h t h e
a b i l i t y of t h e heme group t o complex w i t h c a r b o n monoxide t h a t i s used t o d e t e r m i n e t h e amount of cytochrome P - 4 5 0 . There a r e two p o s s i b l e e x p l a n a t i o n s : e i t h e r t h a t t h e b i n d i n g of c a p t a n or i t s h y d r o l y t i c p r o d u c t p r e v e n t s t h e r e d u c t i o n o f t h e heme i r o n of cytochrome P - 4 5 0 s o t h a t carbon monoxide can no l o n g e r complex w i t h i t , or t h e b i n d i n n of t h e i n h i b i t o r s t e r i c a l l y i n t e r f e r e s w i t h t h e approach and b i n d i n g of c a r b o n monoxide t o t h e r e d u c e d
DALVI AND ASHLEY
250 100
--
80
0
4
C
Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
i
: L
6o
u 0 d in
40
e
z
u
Y 0
20 U
0
I
25
I
1
50
75
Captan
[ gM
I
I00
3
FIGURE 1 In Vitro Effect of Captan on the Concentration of Hepatic Microsomal Cytochrome P - 4 5 0 .
heme iron16 .
Both of these possible explanations would
also account for the decreased activity of the microsoma1 drug-metabolizing enzyme system. Since it has been shown that EDTA stablizes microsomal drug-metabolizing enzymes by inhibiting lipid peroxidation17 and that GSH can protect the enzyme system from toxic compounds12, the ability of these agents to prevent the destruction of cytochrome P-450 by captan was studied.
As can be seen from Table 1,
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PROTECTIVE EFFECT OF GLUTATHIONE
m
251
a
m ( D
9-L-
cu
m
0
0
0 r . I n 0 0
v)
0
0
0
0
0
0
0
0
0
+
+I
+I
+I
+I
+
ri
m
b
f
cu
L D O
. . . .
W W N
3
d
In
N
N
N
N N
ri
d
ri
0
I n L o
. o.
07
m Fi
[I)
c
0 -4 -I-’
rQ
> L
a, v)
n 0
(I]
rl 0
Fc
tJ
c
0
u M
c
.rl
a c
-P
44 0
Cf
r i
0 1 .Ti a
0
a 0
a,
Fc Fc
2
0
w
E 0 Fc
0
+I c rd
2
a,
5
: a a,
v) (0
CJ
ccc
n
rl 0 0
V
a v -P
c
a,
Fc
a,
ccc a, ccc .ri Fc a a x
h
x
E
Lo
rl
0 Fc
-I-’
c
0
V
0
a
O - P
cna
8 s
W
a,
h
x
z
d
V
Lo
v)
-rl
+
0
c a +J
a
a
2n 3n
w
w
h
E
rd
+J
v
5:
cn W
+Jc
rd U
ccc
*rl
c
M
252
DALVI AND ASHLEY
t h e p r e s e n c e o f EDTA i n t h e i n c u b a t i o n s p r i o r t o t h e a d d i t i o n o f c a p t a n w a s n o t a b l e t o p r e v e n t t h e l o s s of cytochrome P-450 by c a p t a n .
Thus t h e f a i l u r e o f EDTA
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t o p r o t e c t t h e a c t i v i t y o f t h e d r u g - m e t a b o l i z i n g enzymes
from c a p t a n s u g g e s t s t h a t l i p i d p e r o x i d a t i o n o f m i c r o soma1 membranes may n o t b e t h e m a j o r mechanism t h r o u g h which c a p t a n c a u s e s i n a c t i v a t i o n o f c y t o c h r o m e P-450.
I n c o n t r a s t , GSH w a s c o m p l e t e l y e f f e c t i v e i n p r o t e c t i n g t h e c y t o c h r o m e P-450 from i n h i b i t i o n by c a p t a n s u g g e s t i n g h i g h a f f i n i t y o f c a p t a n t o w a r d GSH.
I n f a c t , it has
been r e p o r t e d t h a t t h i o l s r e a d i l y r e a c t w i t h c a p t a n t o p r o d u c e t h i o p h o s g e n e 8 y 1 8,19. T h e s e r e s u l t s , t h e r e f o r e
,
i n d i c a t e t h a t it i s t h e captan molecule t h a t i s respons i b l e for t h e d e s t r u c t i o n o f c y t o c h r o m e P-450 and n o t
i t s degradation product thiophosgene.
Thiophosgene
p e rs e h a s l i t t l e e f f e c t on t h e h e m o p r o t e i n , b u t i t s m e t a b o l i s m by t h e m i c r o s o m a l enzymes i n t h e p r e s e n c e o f a c o f a c t o r NADPH l e a d s t o t h e d e s t r u c t i o n o f c y t o c h r o m e 8
P-450
.
Since the i n c u b a t i o n mixtures d i d n o t c o n t a i n
NADPH, t h e l i k e l i h o o d t h a t t h e d e g r a d a t i o n p r o d u c t of c a p t a n m i g h t h a v e c a u s e d t h e loss o f c y t o c h r o m e P-450 may be r u l e d o u t .
In conclusion, captan appears t o be
a s t r o n g i n h i b i t o r o f microsoma1 c y t o c h r o m e P-450 -in vitro and t h a t p r i o r p r e s e n c e o f GSH a f f o r d s a l m o s t c o m p l e t e protection against the inhibition.
253
PROTECTIVE EFFECT OF GLUTATHIONE
ACKNOWLEDGMENT This work %as supported in part by USPHS, NIH Grant No. 5T01-GM02264-02 and by DHEW NIH Grant No. Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
RR-08091, REFERENCES 1.
Anonymous.
1977.
Carcinogensis Bioassay of
Captan for Possible Carcinogenicity. CAS No. 133-06-2, NCI-CG-TR-15.
National Cancer Institute
Technical Report, Series # 1 5 . 2.
NCI, Bethesda, Md.
G. Kennedy, 0. E. Fancher, and J. C. Calandra. An investigation of the teratogenic potential of captan, folpet, and difolatan.
Toxicol. Appl. Pharmacol.,
13, 420 (1968). 3.
J. F. Vondruska, 0. E. Fancher, and J. C. Calandra.
An investigation into the teratogenic potential of captan, folpet, and difolatan in nonhuman primates. Toxicol. Appl. Pharmacol., 4.
J. F. Robens.
18, 619
(1971).
Teratogenic activity of several
phthalimide derivatives in the golden hamster. Toxicol. Appl. Pharmacol., 5.
B. A. Bridges.
16, 24
The mutagenicity of captan and
related fungicides. Mutat. Res., 6.
(1970).
32, 3
(1975).
G. L. Kennedy, D. Ii. Arnold, and M. L. Keplinger. Mutagenicity studies with captan, captafol, folpet, and thalidomide. (1975).
Food Cosmet. Toxicol.,
15, 55
254
7.
DALVI AND ASHLEY V.
r.
Simmons,
I).
C. Poole, and G. W.
Newell.
I n_ v_i _ t r o m e t a g e n i c s t u d i e s of t w e n t y p e s t i c i d e s .
37, 1 0 9
Toxicol. Appl. Pharmacol., Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
8.
A.
Peeples and R.
R.
Dalvi.
(1976).
T o x i c o l o g i c s t u d i e s of
hJ-trichlorornethylthio-4-cyclohexene-l,2I t s metabolism by r a t l i v e r
dicarboximide (Captan): drug-metabolizing
enzyme s y s t e m .
9, Toxicology, -
341
(1978). 3.
B.
D. N e l s o n , Jr.
Action of the f u n g i c i d e s captari
and f o l p e t on r a t l i v e r m i t o c h o n d r i a . Pharrnacol.,
LO.
20, 7 3 7
R . T r u h a u t , D.
Biochem.
(1971).
P . H i e r , a n d P. L . Nguyen.
Influence
of t h e a d m i n i s t r a t i o n of o r g a n o h a l o g e n p e s t i c i d e s a n d p o l y c h l o r i n a t e d b i p h e n y l s on t h e metabolism of zoxazolamine i n t h e rat.
D., 11.
B.
778, 3 5 0 3
C.
R. Acad. S c i . P a r i s S e r .
(1974).
IJrbanek-Karlowska.
T h e a c t i v i t y of microsomal
pnsyrnes o f r a t l i v e r i n r e l a t i o n t o d i e t a r y p r o t e i n
level a n d a d m i n i s t r a t i o n of s e l e c t e d p e s t i c i d e s .
Rocz. P a n s t w . Z a k l . I?.
R.
Hig., 28, 243 (1977).
R. Dalvi a n d T . J . R o b b i n s .
Comparative s t u d i e s
q n t h e e f f e c t of c a d m i u m , c o b a l t , l e a d , a n d s e l e n i u m
o n 1 , e p a t i c mi-rosomal m o n o o x y g e n a s e e n z y m e s a n d g l u t a t h i o n e l e v c l s i n mice. 'roxicol.,
1, 6 0 1 ( 1 9 7 8 ) .
,J. E n v i r o n . P a t h .
255
PROTECTIVE EFFECT OF GLUTATHIONE
13.
R. R. Dalvi and C. D. Howell.
Interaction of
parathion and malathion with hepatic cytochrome P-450 from rats treated with phenobarbital and Drug and Chemical Toxicology Downloaded from informahealthcare.com by UB Giessen on 10/30/14 For personal use only.
carbon disulfide. 14.
Drug Chem. Toxicol., 1, 191 (1978).
T. Omura and R. Sato.
The carbon monoxide-binding
I. Evidence for its
pigment of liver microsomes. hemoprotein nature. 15.
J. Biol. Chem., 239, 2370 (1964).
R. R. Dalvi and C. D. Howell.
Toxic effects of a
fungicide, 5-ethoxy-3-(tr~chloromethyl)-l,2,4thiadiazole (Terrazole), on the hepatic drugmetabolizing enzyme system in mice. Contam. Toxicol., 16.
17, 225
Bull. Environ.
(1977).
R. R. Dalvi, A. L. Hunter, and R. A. Neal. Toxicological implications of the mixed-function oxidase catalyzed metabolism of carbon disulfide. Chem.-Biol. Interact.,
17.
10, 349
T. Kamataki and H. Kitagawa.
(1975).
Effect of lipid
peroxidation on activities of drug-me-tabolizing enzymes in liver microsomes of rats. Pharmacol.,
18.
22, 3199
Biochem.
(1973).
R. J. Lukens and H. D. Sisler.
2-thiazolidinethione-
4-carboxylic acid from the reaction of captan with cysteine. 19.
Science,
R. J. Lukens.
127, 650
(1958).
The fungitoxicity of compounds
containing a trichoromethylthio-group. J. Agr. Food Chem.,
E,
365 (1966).
