Toxicology, 71 (1992) 63-68 Elsevier Scientific Publishers Ireland Ltd.
63
Effect of the fungicide benomyl on xenobiotic metabolism in rats Ramesh R. Dalvi Toxicology Laboratory, Department of Physiology, Pharmacology and Toxicology, School of Veterinary Medicine, Tuskegee University, Tuskegee, A L 36088 ( U.S. A. ) (Received July 22nd, 1991; accepted October 1st, 1991)
Summary The effect of benomyl administered orally (p.o.) and intraperitoneally (i.p.) on the activity of hepatic microsomal mixed-function oxidases (MFOs) was studied in rats. A dose of 100 mg/kg given i.p. reduced the activities of several hepatic drug-metabolizing enzymes 24 h followingthe treatment. A similar reduction in the activities of the MFOs was also noted 24 h following oral benomyl administration at a dose of 500 mg/kg. Furthermore, in vivo inhibition of drug metabolism by benomyl was demonstrated by increased pentobarbital sleeping-time 24 h after p.o. as well as i.p. dosing. No alterations were found in the serum sorbitol dehydrogenase (SDH) at 24 h after i.p. or oral benomyl indicating a lack of hepatotoxic effect. These results indicate that benomyl shows a route-independent effect on MFOs and is not toxic to the liver.
Key words: Benomyl; Microsomal enzymes; Metabolism; Hepatotoxicity
Introduction Benomyl (methyl 1-(butylcarbamoyl)benzimidazole-2-carbamate), is a fungicide registered for fruits, vegetables, flowers, o r n a m e n t a l crops a n d others. It is also used as a pasture dressing for the c o n t r o l of Pithomyces chartarum a n d Epichloe typhina, the fungi responsible for facial eczema in sheep a n d cattle [1] a n d s u m m e r fescue toxicity in cattle, respectively [2]. Its fungicidal property is attributed to its ability to interfere with mitosis [3], specifically by b i n d i n g to t u b u l i n [4] and preventing t u b u l i n polymerization. Despite its effective fungitoxicity, b e n o m y l has low acute toxicity in animals probably because of its rapid m e t a b o l i s m a n d excretion. In the rat, dog, cow a n d chicken, b e n o m y l is p r e d o m i n a n t l y metabolized to methyl 2 - b e n z i m i d a z o l e c a r b a m a t e (MBC) which is excreted in the urine as g l u c u r o n i d e a n d / o r sulfate conjugate [6]. Metabolic studies in mice, rabbits a n d sheep indicated that b e n o m y l is hydrolyzed a n d hydroxylated in the liver to different metabolites a n d that the h y d r o x y l a t i o n is catalyzed
Correspondence to: Ramesh R. Dalvi. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
64 by hepatic microsomal mixed-function oxidases. [1] The present study was conducted to examine the effect of benomyl on the liver and the activity of various microsomal mixed-function oxidases including cytochrome P-450 in rats. Materials and methods
Chemicals Benomyl (95% purity) was obtained from E.I. Du Pont (Wilmington, DE). Cytochrome c and standard kit for sorbitol dehydrogenase (SDH) were obtained from Sigma (St. Louis, MO). Glucose-6-phosphate, glucose-6-phosphate dehydrogenase and nicotinamide adenine dinucleotide phosphate (NADP) were bought from Boehringer Mannheim (Indianapolis, IN). All other chemicals used in these studies were of analytical reagent grade.
Animals and experimental design Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 200-250 g were used. They were housed in standard stainless steel cages placed in a temperature controlled room (25°C) on a 12-h light-dark cycle and allowed free access to water and feed. In the first experiment, 16 rats were divided randomly into two equal groups. Benomyl (suspended in Mazola brand corn oil) was injected i.p. to rats in the second group at a dose of 100 mg/kg. The first group received an equivalent amount of corn oil i.p. and served as a control group. Twenty-four hours following the treatment, half of the animals from each group were injected with pentobarbital (50 mg/kg, i.p.) to determine sleeping-time while the remaining animals were used to measure the activities of microsomal and serum enzymes. The second experiment included 16 rats which were divided randomly into two equal groups. Rats from the second group were given a single dose (500 mg/kg) of benomyl orally, whereas the first group received an equivalent amount of corn oil and served as a control group. As in the first experiment, animals from each group were divided into two groups and used for the determination of sleeping-time, microsomal enzymes and serum SDH. At the end of the 24-h treatment period, rats were killed and blood samples collected. Livers were removed, weighed and perfused with ice-cold 1.15'¼, KCI solution containing 0.05 mM EDTA. Perfused livers were homogenized with three volumes of ice-cold 0.25 M sucrose solution. The homogenates were centrifuged at 9 000 x g for 10 min in a refrigerated Sorvall centrifuge and microsomes were isolated [7]. The microsomal pellets were washed once with KCI solution and immediately resuspended in an appropriate buffer for determining following components of the MFOs. Cytochrome P-450 and cytochrome b5 contents of the microsomes were determined by the method of O m u r a and Sato [8]. The activities of N A D P H - c y t o c h r o m e c reductase, aminopyrine N-demethylase and aniline hydroxy!ase were determined as described earlier [9]. Cytosolic glutathione S-transferase activity with 1-chioro2,4-dinitrobenzene (CDNB) was measured spectrophotometrically according to the method followed by Gawai and Pawar [10]. Protein concentration in all samples was determined using the Biuret method modified to include deoxycholate [7].
65
Blood samples were centrifuged to obtain serum which was analyzed for S D H by the method described in Sigma Technical Bulletin no. 50-UV. The data were analysed by using Student's t-test. Significance of mean differences was based on a P value of less than or equal to 0.05. Results
The data in Table I show that pretreatment with benomyl at a dose of 100 mg/kg given i.p. did not cause a significant change in the relative liver weight of treated rats as compared to the untreated controls. On the other hand, benomyl treatment produced a significant decrease in the levels of cytochrome P-450 (26%), aminopyrine N-demethylase (44%) and aniline hydroxylase (50%), as compared to the respective control values. In contrast, there was no change in microsomal N A D P H - c y t o c h r o m e c reductase and cytochrome b5 or even in the cytosolic glutathione S-transferase activity (Table I). The results also indicate that pentobarbital sleeping-time was significantly increased (125%) over the respective control value. On the other hand, the activity of S D H in the serum of the treated rats showed no significant difference from that of the control value indicating a possible absence of liver toxicity. Although there was no change in the relative liver weights, a significant decrease
TABLE 1 EFFECT OF BENOMYL (100 mg/kg, i.p.) ON RELATIVE LIVER WEIGHTS, MICROSOMAL ELECTRON TRANSPORT COMPONENTS, D R U G - M E T A B O L I Z I N G ENZYMES, CYTOSOLIC G L U T A T H I O N E S-TRANSFERASE, SERUM SORBITOL D E H Y D R O G E N A S E A N D PENTOBARBITAL SLEEPING-TIME IN RATS Results are expressed as the mean ± S.E.M. Parameter Relative liver wt. (% body wt.) Cytochrome P-450 a Cytochrome b5 a NADPH-cytochrome c reductase b Aminopyrine N-demethylase b Aniline hydroxylase b Glutathione S-transferase b Sorbitol dehydrogenase (Sigma Units/ml) Sleeping-time (min)
Control
Benomyl
4.5 + 0.9 0.509 + 0.03 0.162 ± 0.01
4.2 + 0.4 0.375 ± 0.03* 0.128 ± 0.02
101 ± 3
102 ± 3
8.975 ± 0.48
5.067 ± 0.39*
0.403 ± 0.04
0.200 ± 0.03*
1.70 ± 0.06
1.90 ± 0.10
676 ± 56 54 ± 3
780 ± 130 122 ± 5*
aExpressed as nmoles/mg protein. bExpressed as nmoles of product formed/min per mg protein. *Difference between groups was statistically significant (P < 0.05).
66 TABLE II EFFECT OF BENOMYL (500 mg/kg, p.o.) ON RELATIVE LIVER WEIGHTS, MICROSOMAL ELECTRON TRANSPORT COMPONENTS, DRUG-METABOLIZING ENZYMES, CYTOSOLIC GLUTATHIONE S-TRANSFERASE, SERUM SORBITOL DEHYROGENASE AND PENTOBARBITAL SLEEPING-TIME IN RATS Results are expressed as the mean 4- S.E.M. Parameter Relative liver wt. (% body wt.) Cytochrome P-450a Cytochrome b5a NADPH-cytochrome c reductaseb Aminopyrine N-demethylaseb Aniline hydroxylaseb Glutathione S-transferaseb Sorbitol dehydrogeanse (Sigma Units/ml) Sleeping-time (min)
Control
Benomyl
3.9 4- 0.4 0.75 ± 0.04 0.33 ± 0.01
4.3 ± 0.3 0.42 4- 0. 05* 0.25 ± 0. 02*
139 4- 93
137 ± 11
5.91 4- 0.32
4.17 m 0. 32*
0.36 4- 0.04
0.30 + 0.04
2.10 ± 0.19
1.80 4- 0.19
476 ± 72 55 4- 1
558 ± 44 95 ± 17'
aExpressed as nmoles/mg protein. bExpressed as nmoles of product formed/min per mg protein. *Difference between groups was statistically significant (P < 0.05).
of cytochrome P-450 (44%), c y t o c h r o m e b5 (25%) a n d a m i n o p y r i n e N-demethylase (30%) was f o u n d when b e n o m y i was given orally at a dosage level o f 500 mg/kg (Table II). In contrast, aniline hydroxylase, N A D P H - c y t o c h r o m e c reductase a n d cytosolic g l u t a t h i o n e S-transferase showed n o significant difference from their corr e s p o n d i n g controls. O n the other h a n d , b e n o m y l - t r e a t e d rats showed a significant increase in sleeping-time after p e n t o b a r b i t a l injection. Similar to the results of the experiment in which b e n o m y l was a d m i n i s t e r e d i.p., no significant change in the activity o f S D H was f o u n d with the oral dose. Discussion
Even though a single dose o f b e n o m y l administered orally or i n t r a p e r i t o n e a l l y did n o t produce a n y overt toxicity, a significant decrease in the activity of several comp o n e n t s of the hepatic d r u g - m e t a b o l i z i n g enzyme system was observed consistently in this study. While there was n o m a r k e d change in N A D P H - c y t o c h r o m e c reductase a n d cytochrome b5 levels, the activities o f a m i n o p y r i n e N-demethylase a n d aniline hydroxylase were significantly lower than those for the respective untreated controls. Benomyl is metabolized enzymatically in the liver to a m a j o r metabolite M B C which is subsequently hydroxylated to 5-HBC by a hepatic microsomal mixed-function o x -
67 idase system [1]. It is likely that binding of MBC to cytochrome P-450 resulting in the loss of this hemoprotein may have reduced the metabolism of aminopyrine and aniline. The increased pentobarbital sleeping-time caused by benomyl pretreatment also suggests that hydroxylation and subsequent deactivation of pentobarbital may have been impaired by the interaction of benomyl and its metabolite MBC with microsomal enzymes. Alternatively, the benomyl-induced decrease of mixedfunction oxidases may be attributed to the highly reactive degradation product of benomyl, namely, n-butylisocyanate which has been implicated in the inhibition of synthesis of a number of macromolecules such as enzymes and nucleic acids [5]. Similar to benomyl, antitumor agents chloroethylnitroso ureas have also been reported to biotransform under physiological conditions to reactive organic isocyanates which are capable of rapid and irreversible binding to several macromolecules such as enzymes and nucleic acids [! 1]. Additional explanations for the decreased activity of the liver enzymes may be found in the ability of benomyl and its metabolites to reduce ATP production and D N A synthesis since these compounds have been reported to inhibit yeast respiration and incorporation of thymidine into D N A molecules [12,51. Thus, some of these mechanisms of toxic action of benomyl should account, at least in part, for the observed depression of the activity of liver microsomal enzymes. Furthermore, the fact that only certain liver enzymes are affected and others such as CDNB-specific glutathione Stransferase are not affected suggests that benomyl and its me~abolites may be involved in site-directed inactivation of these enzymes and not in the inhibition of protein synsthesis. The change in the activity of serum SDH has been specifically used to assess liver injury in rats caused by chemicals. The results of this study indicate that benomyl given orally or intraperitoneally at dosage levels of 500 mg/kg and 100 mg/kg, respectively, is not toxic to liver as reflected in unchanged activity of the serum sorbitol dehydrogenase. This is consistent with the fact that benomyl possesses very low mammalian toxicity, the oral LDs0 in rats being 10 g/kg [13].
Acknowledgements This work was supported by an N I H / R C M I Grant no. G12 RR03059-01AI. Technical assistance provided by P.S. Terse is gratefully acknowledged.
References 1 P.G.C. Douch, The metabolism of benomyl in mammals. Xenobiotica, 3 (1973) 367-380. 2 J.A. Jackson, R.W. Hemken, J.A. Boling, R.J. Harmon, R.C. Buckner and L.P. Bush, Loline alkaloids in tall fescue hay and seed and their relationship to summer fescue toxicosis in cattle. J. Dairy Sci. 67 (1984) 104-109. 3 R.S. Hammerschlagand H.D Sisler, Benomyland methyl-2-benzimidazolecarbamate (MBC): Biochemical, cytological and chemical aspects of toxicity to Ustilago maydis and Saccharomyces cerevisiae. Pestic. Biochem. Physiol., 3 (1973)42-54. 4 L.C. Davidse and W. Flach, Differential binding of methyl benzimidazole-2-ylcarbamateto fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans. J. Cell Biol., 72 (1977) 174-193.
68 5
B. Hellman and D. Laryea, Inhibitory effects of benomyl and carbendazim on the [3H]thymidine incorporation in various organs of the mouse - - Evidence for a more pronounced action of benomyl. Toxicology, 61 (1990) 161-169. 6 J.A. Gardiner, J.J. Kirkland, H.L. Klopping and H. Sherman, Fate ofbenomyl in animals. J. Agric. Food Chem., 22 (1974) 419-427. 7 A. Peeples and R.R. Dalvi, Toxicological studies of N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide (Captan): Its metabolism by rat liver drug metabolizing enzyme system. Toxicology, 9 (1978) 341-351. 8 T. Omura and R. Sato, The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem., 239 (1964) 2370-2378. 9 R.R. Dalvi and P.S. Terse, Induction of hepatic microsomal drug metabolizing enzyme system by levamisole in male mice. J. Pharm. Pharmacol., 42 (1990) 58-59. 10 K.R. Gawai and S.S. Pawar, Purification and characterization of glutathione S-transferase from liver cytosol of phenobarbital-treated rabbits. Xenobiotica, 14 (1984) 605-607. 11 C. Dive, P. Workman and J.W. Watson, Novel dynamic flow cytoenzymological determination of intracellular esterase inhibition by BCNU and related isocyanates. Biochem. Pharmacol., 36 (1987) 3731-3738. 12 M. Chiba, A.W. Brown and D. Danic, Inhibition of yeast respiration and fermentation by benomyl, carbendazim, isocyanates and other fungicidal chemicals. Can. J. Microbiol., 33 (1987) 157-161. 13 J.P. Seiler, Toxicology and genetic effects of benzimidazole compounds. Mutat. Res., 32 (1975) 161-168.
63
Effect of the fungicide benomyl on xenobiotic metabolism in rats Ramesh R. Dalvi Toxicology Laboratory, Department of Physiology, Pharmacology and Toxicology, School of Veterinary Medicine, Tuskegee University, Tuskegee, A L 36088 ( U.S. A. ) (Received July 22nd, 1991; accepted October 1st, 1991)
Summary The effect of benomyl administered orally (p.o.) and intraperitoneally (i.p.) on the activity of hepatic microsomal mixed-function oxidases (MFOs) was studied in rats. A dose of 100 mg/kg given i.p. reduced the activities of several hepatic drug-metabolizing enzymes 24 h followingthe treatment. A similar reduction in the activities of the MFOs was also noted 24 h following oral benomyl administration at a dose of 500 mg/kg. Furthermore, in vivo inhibition of drug metabolism by benomyl was demonstrated by increased pentobarbital sleeping-time 24 h after p.o. as well as i.p. dosing. No alterations were found in the serum sorbitol dehydrogenase (SDH) at 24 h after i.p. or oral benomyl indicating a lack of hepatotoxic effect. These results indicate that benomyl shows a route-independent effect on MFOs and is not toxic to the liver.
Key words: Benomyl; Microsomal enzymes; Metabolism; Hepatotoxicity
Introduction Benomyl (methyl 1-(butylcarbamoyl)benzimidazole-2-carbamate), is a fungicide registered for fruits, vegetables, flowers, o r n a m e n t a l crops a n d others. It is also used as a pasture dressing for the c o n t r o l of Pithomyces chartarum a n d Epichloe typhina, the fungi responsible for facial eczema in sheep a n d cattle [1] a n d s u m m e r fescue toxicity in cattle, respectively [2]. Its fungicidal property is attributed to its ability to interfere with mitosis [3], specifically by b i n d i n g to t u b u l i n [4] and preventing t u b u l i n polymerization. Despite its effective fungitoxicity, b e n o m y l has low acute toxicity in animals probably because of its rapid m e t a b o l i s m a n d excretion. In the rat, dog, cow a n d chicken, b e n o m y l is p r e d o m i n a n t l y metabolized to methyl 2 - b e n z i m i d a z o l e c a r b a m a t e (MBC) which is excreted in the urine as g l u c u r o n i d e a n d / o r sulfate conjugate [6]. Metabolic studies in mice, rabbits a n d sheep indicated that b e n o m y l is hydrolyzed a n d hydroxylated in the liver to different metabolites a n d that the h y d r o x y l a t i o n is catalyzed
Correspondence to: Ramesh R. Dalvi. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
64 by hepatic microsomal mixed-function oxidases. [1] The present study was conducted to examine the effect of benomyl on the liver and the activity of various microsomal mixed-function oxidases including cytochrome P-450 in rats. Materials and methods
Chemicals Benomyl (95% purity) was obtained from E.I. Du Pont (Wilmington, DE). Cytochrome c and standard kit for sorbitol dehydrogenase (SDH) were obtained from Sigma (St. Louis, MO). Glucose-6-phosphate, glucose-6-phosphate dehydrogenase and nicotinamide adenine dinucleotide phosphate (NADP) were bought from Boehringer Mannheim (Indianapolis, IN). All other chemicals used in these studies were of analytical reagent grade.
Animals and experimental design Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 200-250 g were used. They were housed in standard stainless steel cages placed in a temperature controlled room (25°C) on a 12-h light-dark cycle and allowed free access to water and feed. In the first experiment, 16 rats were divided randomly into two equal groups. Benomyl (suspended in Mazola brand corn oil) was injected i.p. to rats in the second group at a dose of 100 mg/kg. The first group received an equivalent amount of corn oil i.p. and served as a control group. Twenty-four hours following the treatment, half of the animals from each group were injected with pentobarbital (50 mg/kg, i.p.) to determine sleeping-time while the remaining animals were used to measure the activities of microsomal and serum enzymes. The second experiment included 16 rats which were divided randomly into two equal groups. Rats from the second group were given a single dose (500 mg/kg) of benomyl orally, whereas the first group received an equivalent amount of corn oil and served as a control group. As in the first experiment, animals from each group were divided into two groups and used for the determination of sleeping-time, microsomal enzymes and serum SDH. At the end of the 24-h treatment period, rats were killed and blood samples collected. Livers were removed, weighed and perfused with ice-cold 1.15'¼, KCI solution containing 0.05 mM EDTA. Perfused livers were homogenized with three volumes of ice-cold 0.25 M sucrose solution. The homogenates were centrifuged at 9 000 x g for 10 min in a refrigerated Sorvall centrifuge and microsomes were isolated [7]. The microsomal pellets were washed once with KCI solution and immediately resuspended in an appropriate buffer for determining following components of the MFOs. Cytochrome P-450 and cytochrome b5 contents of the microsomes were determined by the method of O m u r a and Sato [8]. The activities of N A D P H - c y t o c h r o m e c reductase, aminopyrine N-demethylase and aniline hydroxy!ase were determined as described earlier [9]. Cytosolic glutathione S-transferase activity with 1-chioro2,4-dinitrobenzene (CDNB) was measured spectrophotometrically according to the method followed by Gawai and Pawar [10]. Protein concentration in all samples was determined using the Biuret method modified to include deoxycholate [7].
65
Blood samples were centrifuged to obtain serum which was analyzed for S D H by the method described in Sigma Technical Bulletin no. 50-UV. The data were analysed by using Student's t-test. Significance of mean differences was based on a P value of less than or equal to 0.05. Results
The data in Table I show that pretreatment with benomyl at a dose of 100 mg/kg given i.p. did not cause a significant change in the relative liver weight of treated rats as compared to the untreated controls. On the other hand, benomyl treatment produced a significant decrease in the levels of cytochrome P-450 (26%), aminopyrine N-demethylase (44%) and aniline hydroxylase (50%), as compared to the respective control values. In contrast, there was no change in microsomal N A D P H - c y t o c h r o m e c reductase and cytochrome b5 or even in the cytosolic glutathione S-transferase activity (Table I). The results also indicate that pentobarbital sleeping-time was significantly increased (125%) over the respective control value. On the other hand, the activity of S D H in the serum of the treated rats showed no significant difference from that of the control value indicating a possible absence of liver toxicity. Although there was no change in the relative liver weights, a significant decrease
TABLE 1 EFFECT OF BENOMYL (100 mg/kg, i.p.) ON RELATIVE LIVER WEIGHTS, MICROSOMAL ELECTRON TRANSPORT COMPONENTS, D R U G - M E T A B O L I Z I N G ENZYMES, CYTOSOLIC G L U T A T H I O N E S-TRANSFERASE, SERUM SORBITOL D E H Y D R O G E N A S E A N D PENTOBARBITAL SLEEPING-TIME IN RATS Results are expressed as the mean ± S.E.M. Parameter Relative liver wt. (% body wt.) Cytochrome P-450 a Cytochrome b5 a NADPH-cytochrome c reductase b Aminopyrine N-demethylase b Aniline hydroxylase b Glutathione S-transferase b Sorbitol dehydrogenase (Sigma Units/ml) Sleeping-time (min)
Control
Benomyl
4.5 + 0.9 0.509 + 0.03 0.162 ± 0.01
4.2 + 0.4 0.375 ± 0.03* 0.128 ± 0.02
101 ± 3
102 ± 3
8.975 ± 0.48
5.067 ± 0.39*
0.403 ± 0.04
0.200 ± 0.03*
1.70 ± 0.06
1.90 ± 0.10
676 ± 56 54 ± 3
780 ± 130 122 ± 5*
aExpressed as nmoles/mg protein. bExpressed as nmoles of product formed/min per mg protein. *Difference between groups was statistically significant (P < 0.05).
66 TABLE II EFFECT OF BENOMYL (500 mg/kg, p.o.) ON RELATIVE LIVER WEIGHTS, MICROSOMAL ELECTRON TRANSPORT COMPONENTS, DRUG-METABOLIZING ENZYMES, CYTOSOLIC GLUTATHIONE S-TRANSFERASE, SERUM SORBITOL DEHYROGENASE AND PENTOBARBITAL SLEEPING-TIME IN RATS Results are expressed as the mean 4- S.E.M. Parameter Relative liver wt. (% body wt.) Cytochrome P-450a Cytochrome b5a NADPH-cytochrome c reductaseb Aminopyrine N-demethylaseb Aniline hydroxylaseb Glutathione S-transferaseb Sorbitol dehydrogeanse (Sigma Units/ml) Sleeping-time (min)
Control
Benomyl
3.9 4- 0.4 0.75 ± 0.04 0.33 ± 0.01
4.3 ± 0.3 0.42 4- 0. 05* 0.25 ± 0. 02*
139 4- 93
137 ± 11
5.91 4- 0.32
4.17 m 0. 32*
0.36 4- 0.04
0.30 + 0.04
2.10 ± 0.19
1.80 4- 0.19
476 ± 72 55 4- 1
558 ± 44 95 ± 17'
aExpressed as nmoles/mg protein. bExpressed as nmoles of product formed/min per mg protein. *Difference between groups was statistically significant (P < 0.05).
of cytochrome P-450 (44%), c y t o c h r o m e b5 (25%) a n d a m i n o p y r i n e N-demethylase (30%) was f o u n d when b e n o m y i was given orally at a dosage level o f 500 mg/kg (Table II). In contrast, aniline hydroxylase, N A D P H - c y t o c h r o m e c reductase a n d cytosolic g l u t a t h i o n e S-transferase showed n o significant difference from their corr e s p o n d i n g controls. O n the other h a n d , b e n o m y l - t r e a t e d rats showed a significant increase in sleeping-time after p e n t o b a r b i t a l injection. Similar to the results of the experiment in which b e n o m y l was a d m i n i s t e r e d i.p., no significant change in the activity o f S D H was f o u n d with the oral dose. Discussion
Even though a single dose o f b e n o m y l administered orally or i n t r a p e r i t o n e a l l y did n o t produce a n y overt toxicity, a significant decrease in the activity of several comp o n e n t s of the hepatic d r u g - m e t a b o l i z i n g enzyme system was observed consistently in this study. While there was n o m a r k e d change in N A D P H - c y t o c h r o m e c reductase a n d cytochrome b5 levels, the activities o f a m i n o p y r i n e N-demethylase a n d aniline hydroxylase were significantly lower than those for the respective untreated controls. Benomyl is metabolized enzymatically in the liver to a m a j o r metabolite M B C which is subsequently hydroxylated to 5-HBC by a hepatic microsomal mixed-function o x -
67 idase system [1]. It is likely that binding of MBC to cytochrome P-450 resulting in the loss of this hemoprotein may have reduced the metabolism of aminopyrine and aniline. The increased pentobarbital sleeping-time caused by benomyl pretreatment also suggests that hydroxylation and subsequent deactivation of pentobarbital may have been impaired by the interaction of benomyl and its metabolite MBC with microsomal enzymes. Alternatively, the benomyl-induced decrease of mixedfunction oxidases may be attributed to the highly reactive degradation product of benomyl, namely, n-butylisocyanate which has been implicated in the inhibition of synthesis of a number of macromolecules such as enzymes and nucleic acids [5]. Similar to benomyl, antitumor agents chloroethylnitroso ureas have also been reported to biotransform under physiological conditions to reactive organic isocyanates which are capable of rapid and irreversible binding to several macromolecules such as enzymes and nucleic acids [! 1]. Additional explanations for the decreased activity of the liver enzymes may be found in the ability of benomyl and its metabolites to reduce ATP production and D N A synthesis since these compounds have been reported to inhibit yeast respiration and incorporation of thymidine into D N A molecules [12,51. Thus, some of these mechanisms of toxic action of benomyl should account, at least in part, for the observed depression of the activity of liver microsomal enzymes. Furthermore, the fact that only certain liver enzymes are affected and others such as CDNB-specific glutathione Stransferase are not affected suggests that benomyl and its me~abolites may be involved in site-directed inactivation of these enzymes and not in the inhibition of protein synsthesis. The change in the activity of serum SDH has been specifically used to assess liver injury in rats caused by chemicals. The results of this study indicate that benomyl given orally or intraperitoneally at dosage levels of 500 mg/kg and 100 mg/kg, respectively, is not toxic to liver as reflected in unchanged activity of the serum sorbitol dehydrogenase. This is consistent with the fact that benomyl possesses very low mammalian toxicity, the oral LDs0 in rats being 10 g/kg [13].
Acknowledgements This work was supported by an N I H / R C M I Grant no. G12 RR03059-01AI. Technical assistance provided by P.S. Terse is gratefully acknowledged.
References 1 P.G.C. Douch, The metabolism of benomyl in mammals. Xenobiotica, 3 (1973) 367-380. 2 J.A. Jackson, R.W. Hemken, J.A. Boling, R.J. Harmon, R.C. Buckner and L.P. Bush, Loline alkaloids in tall fescue hay and seed and their relationship to summer fescue toxicosis in cattle. J. Dairy Sci. 67 (1984) 104-109. 3 R.S. Hammerschlagand H.D Sisler, Benomyland methyl-2-benzimidazolecarbamate (MBC): Biochemical, cytological and chemical aspects of toxicity to Ustilago maydis and Saccharomyces cerevisiae. Pestic. Biochem. Physiol., 3 (1973)42-54. 4 L.C. Davidse and W. Flach, Differential binding of methyl benzimidazole-2-ylcarbamateto fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans. J. Cell Biol., 72 (1977) 174-193.
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