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Modulation of Snake Hepatic Cytochrome P450 by 3-Methylcholanthrene and Phenobarbital
Comp. Biochem. Physiol. Vol. 119C, No. 2, pp. 143–148, 1998 Copyright 1998 Elsevier Science Inc. All rights reserved.
ISSN 0742-8413/98/$19.00 PII S0742-8413(97)00201-6
Modulation of Snake Hepatic Cytochrome P450 by 3-Methylcholanthrene and Phenobarbital Marie-He´le`ne Bani,1 Morio Fukuhara,1 Masanobu Kimura,2 and Fusao Ushio3 1
Department of Pharmaceutical Sciences and 2Department of Veterinary Public Health, National Institute of Public Health, Shirokanedai, Minato-ku, Tokyo 108, Japan; and 3Department of Food Hygiene and Nutrition, Tokyo Metropolitan Laboratory of Public Health, Hyakunincho, Shinjuku-ku, Tokyo 169, Japan ABSTRACT. Induction mode of the hepatic drug-metabolizing enzymes was studied in Corn snake (Elaphe guttata emoryi). Treatment of snakes with 3-methylcholanthrene or phenobarbital produced no effects on liver weight and total content of cytochromes P450 and b5. Treatment with 3-methylcholanthrene significantly induced the activities of arylhydrocarbon hydroxylase, 7-ethoxyresorufin O-deethylase and 7-pentoxyresorufin Odealkylase, whereas those of ethoxycoumarin O-deethylase, benzphetamine N-demethylase, erythromycin Ndemethylase and testosterone hydroxylases were not affected. 3-Methylcholanthrene-induced activities of 7ethoxyresorufin O-deethylase and 7-pentoxyresorufin O-dealkylase were inhibited by 20 µM α-naphthoflavone by 98% and 73%, respectively. Phenobarbital-treatment caused a significant induction of the activities of erythromycin N-demethylase and testosterone 6β-hydroxylase, but did not affect those of the other phase I enzymes and the other testosterone hydroxylases. The activities of UDP-glucuronyltransferase and glutathione S-transferase were not affected by either 3-methylcholanthrene or phenobarbital administration. Immunoblotting showed that 3-methylcholanthrene-treatment induced a protein band related to hamster CYP1A2, and decreased the intensity of the two bands detected with anti-rat CYP2B1. Phenobarbital-treatment did not affect the intensity of CYP2B-related proteins. The results suggest that snake liver has multiple forms of cytochrome P450, notably those inducible by 3-methylcholanthrene. comp biochem physiol 119C;2:143–148, 1998. 1998 Elsevier Science Inc. KEY WORDS. 3-Methylcholanthrene, cytochrome P450, induction, phase I and II enzymes, phenobarbital, reptiles, snake Elaphe guttata emoryi
INTRODUCTION In vertebrates, the hepatic biotransformation system has been shown to be responsible for the majority of xenobiotic biotransformations through a two-steps process which includes the phase I enzymes, represented by the cytochrome P450 (P450) enzymes (12). Among the numerous compounds metabolized by this system are endogenous substrates such as fatty acids and steroids and exogenous substrates such as drugs, carcinogens and pesticides (5,13). The majority of the P450s is selectively induced by a specific group of compounds which differ from species to species (23), while other P450s are constitutively expressed. Most of the information on xenobiotic biotransformation Address reprint requests to: M.-H. Bani, Department of Pharmaceutical Sciences, National Institute of Public Health, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan. Tel. (81) 3-3441-7111; Fax (81) 3-3446-4314. Abbreviations–3MC, 3-Methylcholanthrene; AHH, arylhydrocarbon hydroxylase; α NF, α-naphthoflavone; β NF, β-naphthoflavone; BzND, benzphetamine N-demethylase; ECOD, ethoxycoumarin O-deethylase; ErND, erythromycin N-demethylase; EROD, 7-ethoxyresorufin O-deethylase; GST, Glutathione S-transferase; P450, cytochrome P450; PB, phenobarbital; PROD, 7-pentoxyresorufin O-dealkylase; UDPGT, UDP-glucuronyltransferase. Received 29 May 1997; accepted 30 October 1997.
in vertebrates is for mammals and especially on P450s, consisting of 26 gene subfamilies (22). Direct knowledge of P450 genes in non-mammalian taxa is essential to properly evaluate the factors contributing to the diversification of P450 genes and their regulatory systems in vertebrates. Such knowledge also is essential to understand species differences in susceptibility or resistance to toxic chemicals. P450 proteins have been purified or identified from nonmammalian vertebrates including fishes, reptiles and birds. Among these, fishes have attracted attention with respect to their high responsiveness to polychlorinated biphenyl- or polyaromatic hydrocarbon-type inducers (29). These xenobiotics were shown to induce the CYP1A1 protein, mRNA and its related catalytic activities (7-ethoxyresorufin O-deethylase, EROD, and arylhydrocarbon hydroxylase, AHH) in the fish (15). However, only limited studies have been reported on P450 monooxygenases in reptiles such as lizards, snakes, turtles or alligators (17,25,26). Ectotherm vertebrates have been found to have lower metabolic rates compared to birds and mammals. Moreover, the studies on the effects of P450 inducing agents in reptiles have received very little attention as compared to those in mammals and fishes or birds (11,14,17,27).
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The present study was untertaken to investigate the inducibility of the snake xenobiotic biotransformation enzyme activities (phase I and phase II) with 3-methylcholanthrene (3MC), and phenobarbital (PB), two major inducing agents of P450s. MATERIALS AND METHODS Chemicals Testosterone, 7-ethoxyresorufin, 7-pentoxyresorufin, benzphetamine and erythromycin were purchased from Sigma Chemicals (St Louis, MO). NADPH, NADH, glucose 6phosphate, and glucose 6-phosphate dehydrogenase were obtained from Boehringer-Mannheim (Mannheim, Germany). Benzo[a]pyrene, 7-ethoxycoumarin, α-naphthoflavone (α NF) and other reagents were obtained from Wako Chemical (Osaka, Japan). The polyclonal antibodies directed against rat CYP2B1 were donated by Dr. Omata (University of Kurume, Kurume, Japan). The polyclonal antibodies against the hamster CYP1A2 were prepared in our laboratory as previously described (10). Renaissance Western Blot Chemiluminescence Reagent was purchased from DuPont NEN (Boston, MA). Bicinchoninic acid kit for the protein assay was obtained from Pierce Chemical (Rockford, IL). Other chemicals were of the highest grade commercially available. Animal Treatment and Hepatic Microsome Preparation Corn snakes from North America (Elaphe guttata emoryi) aged between 10–11 months and weighing 200–250 g were used. They were maintained and bred in our laboratory as previously described (9). The animals received intracoelomically either 3MC or PB at a dose of 25 mg/kg of body weight for 4 consecutive days. The dose was chosen as the dosage of barbiturates for the snakes advised previously was 15 to 30 mg/kg given intracoelomically and doses exceeding 50 mg/kg have been proved to be fatal in snakes or cause flaccidity (4). Control animals received an equal volume of olive oil in which the compounds were dissolved. On the 5th day after the first injection of the compounds, the snakes were cooled in crushed ice water to rapidly lower their body temperature to 4–5°C and they were killed by decapitation. The livers were quickly excised and hepatic microsomal fractions were prepared, as previously described (10), with slight modifications. The microsome suspension buffer contained 50 mM potassium phosphate, 1 mM EDTA, 1 mM dithiothreitol, 0.01% phenylmethylsulfonyl fluoride and 20% glycerol. Liver microsomes were stored at 280°C until use. Assays Microsomal protein concentration was determined by using the PIERCE kit with bicinchoninic acid (28) and with bo-
vine serum albumin as standard. Total amounts of P450 and cytochrome b5 were measured as previously described (24). The activities of EROD, 7-pentoxyresorufin O-dealkylase (PROD), ethoxycoumarin O-deethylase (ECOD), and AHH were determined fluorometrically as previously described (1,7,8,21). The activities of benzphetamine Ndemethylase (BzND) and erythromycin N-demethylase (ErND) were determined by the generation of formaldehyde as described (20,33). Glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDPGT) were assayed as previously described with 1-chloro-2,4-dinitro-benzene and pnitrophenol as substrate, respectively (2). The testosterone hydroxylase and oxidase activities were determined by the rate of formation of the corresponding products as described (34). The 11α-hydroxyprogesterone was used as an internal standard. The inhibitiory effects of α NF on the EROD and PROD activities were studied in microsomes from 3MCtreated animals by in vitro addition of α NF at concentration ranging from 0.2 µM to 20 µM to the assay system. α NF was dissolved in Me2SO and an equal volume of Me2SO was added to the control assays. SDS-PAGE and Immunoblot Analysis Microsomal proteins were electrophoresed on 10%-acrylamide gels in the presence of SDS (19). The resolved proteins were transferred onto nitrocellulose membranes and immunostained by the polyclonal antibodies directed against hamster CYP1A2 and rat CYP2B1. The polyclonal antibodies against hamster CYP1A2 were shown to crossreact with the rat CYP1A subfamily proteins (3). Isozyme protein bands were detected using Renaissance Western Blot Chemiluminescence Reagent. Statistics The values of control and treated groups were compared statistically using Student’s t-test. RESULTS Effects on Enzyme Activities No significant variation of the liver weight was observed by 3MC- or PB-treatment as compared to the control (Table 1). 3MC-treatment at a daily dose of 25 mg/kg for 4 days did not affect the levels of P450 and cytochrome b5, nor did it cause a 2 nm blue shift from 450 to 448 nm in the COdifference spectrum. PB-treatment at a daily dose of 25 mg/ kg for 4 days did not produce any overt toxicity (i.e. flaccidity), nor increase the content of P450 and cytochrome b5. Treatment with 3 MC resulted in an increase in the EROD activity by about 40-fold and also in the AHH and PROD activities which were enhanced over 7.7- and 4-fold, respectively as compared to the control group. There was no significant effect of 3MC on the activities of ECOD, BzND and ErND. PB-treatment caused a significant 4-fold
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TABLE 1. Effects of 3MC and PB on liver weight, P450, cytochrome b 5 and xenobiotic metabolizing enzyme activities in
snake livers Parameters
Control 2.71 6 0.30 a 0.80 6 0.08 0.24 6 0.07 0.098 6 0.021 0.17 6 0.06 1.07 6 0.44 0.116 6 0.040 0.677 6 0.010 0.091 6 0.038 0.187 6 0.045 1.11 6 0.20
Liver weight (g/100 b.w.) Total P450 (nmol/mg prot.) Cytochrome b 5 (nmol/mg prot.) EROD b AHH ECOD PROD BzND ErND UDPGT GST
3MC 2.90 0.77 0.14 3.900 1.30 0.97 0.473 0.699 0.170 0.160 1.03
6 6 6 6 6 6 6 6 6 6 6
0.62 0.09 0.03 0.875** 0.20** 0.18 0.130** 0.425 0.097 0.039 0.21
PB 3.85 1.06 0.18 0.202 0.16 0.75 0.195 0.874 0.432 0.190 1.11
6 6 6 6 6 6 6 6 6 6 6
1.25 0.36 0.02 0.077 0.08 0.31 0.088 0.328 0.167* 0.026 0.12
The values are the mean 6 SD for 3 (PB) and 4 (Control and 3MC) animals. b Enzyme activities are given in nmol/min/mg of protein. Liver cytosolic fractions (for GST) and microsomal fractions (for ECOD, PROD, BzND, ErND, EROD, AHH and UDPGT) were used for the assay of the enzymes. Significance of the differences versus the control group at *P , 0.05 and **P , 0.01. a
induction of the ErND and testosterone 6β-hydroxylase activities, but did not affect the other phase I enzyme activities. Neither 3MC nor PB had an effect on UDPGT and GST activities. The effects of 3MC- and PB-treatment on testosterone hydroxylase and oxidase activities are shown in Table 2. The major metabolite in all the groups was the 6βhydroxytestosterone. Hepatic microsomes from PB-treated snakes exhibited enhanced ability to hydroxylate testosterone at the 6β position by about 4-fold, while the hydroxylation at other positions was not significantly affected as compared to the control group. 3MC-treatment had no significant effects on the testosterone hydroxylase and oxidase activities.
Effects of aNF on 3MC-induced Activities of EROD and PROD The P450 induced by 3MC was characterized by an inhibitory effect of α NF on the EROD and PROD activities in liver microsomes (Fig. 1). Addition of α NF (0.2 µM to 20 µM) to the reaction mixture caused a marked dosedependent inhibition of EROD and PROD activities in the liver microsomes from 3MC-treated hamsters. The activities of EROD and PROD were inhibited by 98% and 60%, respectively, by α NF at 10 µM.
TABLE 2. Effects of 3MC and PB on testosterone hydroxylase activities in snake livers
Control 3MC PB (pmol/min/ (pmol/min/ (pmol/min/ mg prot.) mg prot.) mg prot.) Testosterone metabolites a 2α-OH 2β-OH 6α-OH 6β-OH 7α-OH 15α-OH 15β-OH 16α-OH 16β-OH Androstenedione a
17.0 6 5.9 b 15.7 6 4.5 7.2 6 5.2 209 6 70 ND 17.7 6 2.7 ND 31.7 6 11.9 15.0 6 3.2 240 6 21
17.5 6 3.7 20.2 6 6.2 7.2 6 2.9 272 6 28 ND 15.7 6 5.0 ND 18.5 6 9.8 16.1 6 3.0 180 6 69
19.7 39.0 10.3 849 1.3 22.0 2.3 20.7 29.1 218
6 6 6 6 6 6 6 6 6 6
4.9 17.7 0.6 150* 1.5 12.2 2.5 8.5 12.0 48
The abbreviations denote the hydroxylated testosterone metabolites. Androstenedione is the product of the testosterone 17-oxidase activity. b The values are the mean 6 SD for 3 (PB) and 4 (Control and 3 MC) animals. Significance of the differences versus the control group at *P , 0.01. ND: not detected.
FIG. 1. Inhibition by a NF of the activities of EROD and
PROD of hepatic microsomes from snakes treated with 3MC. Results are expressed as % of the control values. Values are the means 6 SD of 4 determinations. *Significantly different (P , 0.01) from the control values.
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FIG. 2. Western blots of snake liver microsomes using polyclonal antibodies against hamster CYP1A2 (A), and rat CYP2B1 (B). Microsomal proteins from control (lane 1), 3MC-treated (lane 2) and PB-treated (lane 3) snakes were applied on the gel in panel A (5 mg protein) and B (2.5 mg protein). Microsomes (0.25 mg protein) from 3MC-treated hamster and PB-treated rat were applied (lane 4) in panel A and B, respectively.
Immunoblot Analysis Figure 2 shows representative Western blots of hepatic microsomes of the snake. Treatment with 3MC caused an apparent increase in the intensity of a protein band immunorelated to hamster CYP1A2, while no difference in this band intensity was observed in the microsomes of PBtreated animals compared to that of the controls (Fig. 2A). In the control animals, two bands were detected which immunoreacted with antibodies to rat CYP2B1. The intensity of these protein bands was significantly decreased by 3MC-treatment (Fig. 2B). Treatment with PB did not affect the intensity of the CYP2B1-related proteins. DISCUSSION In the present report, we studied the inducibility by 3MC and PB of the hepatic biotransformation system of a reptile and found differences from studies reported so far on the other non-mammalians vertebrates, but also from those on the other snake species. 3MC-treatment failed to induce the P450 content and related enzyme activities in the livers of snakes. This is in accordance with previous results which reported that intraperitoneal injection of β-naphthoflavone (β NF) at a dose of 100 mg/kg for 22 hr had no effects on garter snakes (Thamnophis sp.) and painted turtles (Chrysomyces picta) in P450 content, and EROD and ECOD activities (14). However, the inability of 3MC observed here might be due to the length of treatment time, which may have been too short to elicit any changes in the enzyme activity. This was previously reported that, even though a high level of 3MC was present in the snake liver 24 hr after the last dose, induction of P450 did not occur until 5 days after the last dose of 3MC (27). The shift in the Soret maximum of the reduced CO spectrum from 450 to 448 nm was not observed in the snake microsomes after 3MC administration. This finding is in agreement with a previous study on garter snakes (27), while the shift was reported for an alligator (Alligator mississippiensis) (17). The results of the catalytic and immunochemical studies
suggest that 3MC induced a snake P450 isozyme belonging to the CYP1A subfamily. This was shown by the increase in the activities of EROD and AHH, the selective activities of fish and rodent CYP1A1 (15), by the inhibitory effect of α NF, a specific inhibitor of CYP1A1, on EROD activity and by the increase in the protein band related to hamster CYP1A2. Moreover, we obtained an inhibition by α NF on the 3MC-induced PROD activity in the microsomes obtained from the 3MC-treated snakes. In rats, this enzymatic activity is considered to be related to the CYP2B1/2B2 isozymes. Whether the PROD activity is related to the CYP1A subfamily in the snake needs to be further characterized. PB-treatment to the snake failed to induce P450 content and the proteins immunorelated to rat CYP2B1 but increased the ErND and testosterone 6β-hydroxylase activities, both correlated to the CYP3A subfamily in rodents (16,33). However, no proteins immunorelated to rat CYP3A were detected in the microsomes of control and PB-treated animals (data not shown). In mammals, PB was also shown to induce the CYP3A1 isozyme (32). In fishes, putative CYP3A have been reported to have steroid 6βhydroxylase activity (29). Whether ErND and testosterone 6β-hydroxylase activities are related to CYP3A proteins in the snake remains to be characterized. To our knowledge, only one previous study detailed the effects of PB-treatment on the P450-dependent monoxygenases of snakes (27) in which no inductive effects of PB on P450 systems was produced by several doses of the compound and different PB-exposure times. Extensive studies suggest that fishes and quails are resistant to PB-type induction (i.e. total P450 content and CYP2B induction) (18,30). However, these results are not entirely consistent. Ethylmorphine N-demethylase was induced in a fish (Poecilia reticulata) by a two weeks-treatment with PB (31). To date, little attention has been directed toward the induction of phase II enzymes in non-mammalian vertebrates. In the present study, we did not observe any effects of 3MCand PB-treatment on UDPGT and GST activities. However in fish, β NF was shown to induce these conjugation enzyme activities (2) and β NF and 2,3,7,8-tetrachlorodibenzo-p-dioxin were shown to induce to a similar extent
Induction of Snake Cytochrome P450
the UDPGT activity (6). It was reported that UDPGT and GST response to β NF lasted longer than the response of the P450 monooxygenase activities (2). This could be one of the reasons for the lack of induction of UDPGT and GST in the snake under our experimental conditions. In summary, the present study showed that a snake species (Elaphe guttata emoryi) responded to 3MC-treatment in a similar manner as in fishes and rodents in the induction of CYP1A, while the snake was refractive to PB-like induction (i.e. CYP2B induction). Knowledge of the species differences in phase I and II enzyme systems in reptiles from other species could provide fundamental insights into the evolution of P450 proteins. Studies on animals living in ecosystems could also be useful in the research for biological indicators for monitoring environmental pollution.
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12. 13. 14.
15.
16. 17.
This work was supported by the Human Sciences Foundation of Japan. 18.
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ISSN 0742-8413/98/$19.00 PII S0742-8413(97)00201-6
Modulation of Snake Hepatic Cytochrome P450 by 3-Methylcholanthrene and Phenobarbital Marie-He´le`ne Bani,1 Morio Fukuhara,1 Masanobu Kimura,2 and Fusao Ushio3 1
Department of Pharmaceutical Sciences and 2Department of Veterinary Public Health, National Institute of Public Health, Shirokanedai, Minato-ku, Tokyo 108, Japan; and 3Department of Food Hygiene and Nutrition, Tokyo Metropolitan Laboratory of Public Health, Hyakunincho, Shinjuku-ku, Tokyo 169, Japan ABSTRACT. Induction mode of the hepatic drug-metabolizing enzymes was studied in Corn snake (Elaphe guttata emoryi). Treatment of snakes with 3-methylcholanthrene or phenobarbital produced no effects on liver weight and total content of cytochromes P450 and b5. Treatment with 3-methylcholanthrene significantly induced the activities of arylhydrocarbon hydroxylase, 7-ethoxyresorufin O-deethylase and 7-pentoxyresorufin Odealkylase, whereas those of ethoxycoumarin O-deethylase, benzphetamine N-demethylase, erythromycin Ndemethylase and testosterone hydroxylases were not affected. 3-Methylcholanthrene-induced activities of 7ethoxyresorufin O-deethylase and 7-pentoxyresorufin O-dealkylase were inhibited by 20 µM α-naphthoflavone by 98% and 73%, respectively. Phenobarbital-treatment caused a significant induction of the activities of erythromycin N-demethylase and testosterone 6β-hydroxylase, but did not affect those of the other phase I enzymes and the other testosterone hydroxylases. The activities of UDP-glucuronyltransferase and glutathione S-transferase were not affected by either 3-methylcholanthrene or phenobarbital administration. Immunoblotting showed that 3-methylcholanthrene-treatment induced a protein band related to hamster CYP1A2, and decreased the intensity of the two bands detected with anti-rat CYP2B1. Phenobarbital-treatment did not affect the intensity of CYP2B-related proteins. The results suggest that snake liver has multiple forms of cytochrome P450, notably those inducible by 3-methylcholanthrene. comp biochem physiol 119C;2:143–148, 1998. 1998 Elsevier Science Inc. KEY WORDS. 3-Methylcholanthrene, cytochrome P450, induction, phase I and II enzymes, phenobarbital, reptiles, snake Elaphe guttata emoryi
INTRODUCTION In vertebrates, the hepatic biotransformation system has been shown to be responsible for the majority of xenobiotic biotransformations through a two-steps process which includes the phase I enzymes, represented by the cytochrome P450 (P450) enzymes (12). Among the numerous compounds metabolized by this system are endogenous substrates such as fatty acids and steroids and exogenous substrates such as drugs, carcinogens and pesticides (5,13). The majority of the P450s is selectively induced by a specific group of compounds which differ from species to species (23), while other P450s are constitutively expressed. Most of the information on xenobiotic biotransformation Address reprint requests to: M.-H. Bani, Department of Pharmaceutical Sciences, National Institute of Public Health, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan. Tel. (81) 3-3441-7111; Fax (81) 3-3446-4314. Abbreviations–3MC, 3-Methylcholanthrene; AHH, arylhydrocarbon hydroxylase; α NF, α-naphthoflavone; β NF, β-naphthoflavone; BzND, benzphetamine N-demethylase; ECOD, ethoxycoumarin O-deethylase; ErND, erythromycin N-demethylase; EROD, 7-ethoxyresorufin O-deethylase; GST, Glutathione S-transferase; P450, cytochrome P450; PB, phenobarbital; PROD, 7-pentoxyresorufin O-dealkylase; UDPGT, UDP-glucuronyltransferase. Received 29 May 1997; accepted 30 October 1997.
in vertebrates is for mammals and especially on P450s, consisting of 26 gene subfamilies (22). Direct knowledge of P450 genes in non-mammalian taxa is essential to properly evaluate the factors contributing to the diversification of P450 genes and their regulatory systems in vertebrates. Such knowledge also is essential to understand species differences in susceptibility or resistance to toxic chemicals. P450 proteins have been purified or identified from nonmammalian vertebrates including fishes, reptiles and birds. Among these, fishes have attracted attention with respect to their high responsiveness to polychlorinated biphenyl- or polyaromatic hydrocarbon-type inducers (29). These xenobiotics were shown to induce the CYP1A1 protein, mRNA and its related catalytic activities (7-ethoxyresorufin O-deethylase, EROD, and arylhydrocarbon hydroxylase, AHH) in the fish (15). However, only limited studies have been reported on P450 monooxygenases in reptiles such as lizards, snakes, turtles or alligators (17,25,26). Ectotherm vertebrates have been found to have lower metabolic rates compared to birds and mammals. Moreover, the studies on the effects of P450 inducing agents in reptiles have received very little attention as compared to those in mammals and fishes or birds (11,14,17,27).
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The present study was untertaken to investigate the inducibility of the snake xenobiotic biotransformation enzyme activities (phase I and phase II) with 3-methylcholanthrene (3MC), and phenobarbital (PB), two major inducing agents of P450s. MATERIALS AND METHODS Chemicals Testosterone, 7-ethoxyresorufin, 7-pentoxyresorufin, benzphetamine and erythromycin were purchased from Sigma Chemicals (St Louis, MO). NADPH, NADH, glucose 6phosphate, and glucose 6-phosphate dehydrogenase were obtained from Boehringer-Mannheim (Mannheim, Germany). Benzo[a]pyrene, 7-ethoxycoumarin, α-naphthoflavone (α NF) and other reagents were obtained from Wako Chemical (Osaka, Japan). The polyclonal antibodies directed against rat CYP2B1 were donated by Dr. Omata (University of Kurume, Kurume, Japan). The polyclonal antibodies against the hamster CYP1A2 were prepared in our laboratory as previously described (10). Renaissance Western Blot Chemiluminescence Reagent was purchased from DuPont NEN (Boston, MA). Bicinchoninic acid kit for the protein assay was obtained from Pierce Chemical (Rockford, IL). Other chemicals were of the highest grade commercially available. Animal Treatment and Hepatic Microsome Preparation Corn snakes from North America (Elaphe guttata emoryi) aged between 10–11 months and weighing 200–250 g were used. They were maintained and bred in our laboratory as previously described (9). The animals received intracoelomically either 3MC or PB at a dose of 25 mg/kg of body weight for 4 consecutive days. The dose was chosen as the dosage of barbiturates for the snakes advised previously was 15 to 30 mg/kg given intracoelomically and doses exceeding 50 mg/kg have been proved to be fatal in snakes or cause flaccidity (4). Control animals received an equal volume of olive oil in which the compounds were dissolved. On the 5th day after the first injection of the compounds, the snakes were cooled in crushed ice water to rapidly lower their body temperature to 4–5°C and they were killed by decapitation. The livers were quickly excised and hepatic microsomal fractions were prepared, as previously described (10), with slight modifications. The microsome suspension buffer contained 50 mM potassium phosphate, 1 mM EDTA, 1 mM dithiothreitol, 0.01% phenylmethylsulfonyl fluoride and 20% glycerol. Liver microsomes were stored at 280°C until use. Assays Microsomal protein concentration was determined by using the PIERCE kit with bicinchoninic acid (28) and with bo-
vine serum albumin as standard. Total amounts of P450 and cytochrome b5 were measured as previously described (24). The activities of EROD, 7-pentoxyresorufin O-dealkylase (PROD), ethoxycoumarin O-deethylase (ECOD), and AHH were determined fluorometrically as previously described (1,7,8,21). The activities of benzphetamine Ndemethylase (BzND) and erythromycin N-demethylase (ErND) were determined by the generation of formaldehyde as described (20,33). Glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDPGT) were assayed as previously described with 1-chloro-2,4-dinitro-benzene and pnitrophenol as substrate, respectively (2). The testosterone hydroxylase and oxidase activities were determined by the rate of formation of the corresponding products as described (34). The 11α-hydroxyprogesterone was used as an internal standard. The inhibitiory effects of α NF on the EROD and PROD activities were studied in microsomes from 3MCtreated animals by in vitro addition of α NF at concentration ranging from 0.2 µM to 20 µM to the assay system. α NF was dissolved in Me2SO and an equal volume of Me2SO was added to the control assays. SDS-PAGE and Immunoblot Analysis Microsomal proteins were electrophoresed on 10%-acrylamide gels in the presence of SDS (19). The resolved proteins were transferred onto nitrocellulose membranes and immunostained by the polyclonal antibodies directed against hamster CYP1A2 and rat CYP2B1. The polyclonal antibodies against hamster CYP1A2 were shown to crossreact with the rat CYP1A subfamily proteins (3). Isozyme protein bands were detected using Renaissance Western Blot Chemiluminescence Reagent. Statistics The values of control and treated groups were compared statistically using Student’s t-test. RESULTS Effects on Enzyme Activities No significant variation of the liver weight was observed by 3MC- or PB-treatment as compared to the control (Table 1). 3MC-treatment at a daily dose of 25 mg/kg for 4 days did not affect the levels of P450 and cytochrome b5, nor did it cause a 2 nm blue shift from 450 to 448 nm in the COdifference spectrum. PB-treatment at a daily dose of 25 mg/ kg for 4 days did not produce any overt toxicity (i.e. flaccidity), nor increase the content of P450 and cytochrome b5. Treatment with 3 MC resulted in an increase in the EROD activity by about 40-fold and also in the AHH and PROD activities which were enhanced over 7.7- and 4-fold, respectively as compared to the control group. There was no significant effect of 3MC on the activities of ECOD, BzND and ErND. PB-treatment caused a significant 4-fold
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TABLE 1. Effects of 3MC and PB on liver weight, P450, cytochrome b 5 and xenobiotic metabolizing enzyme activities in
snake livers Parameters
Control 2.71 6 0.30 a 0.80 6 0.08 0.24 6 0.07 0.098 6 0.021 0.17 6 0.06 1.07 6 0.44 0.116 6 0.040 0.677 6 0.010 0.091 6 0.038 0.187 6 0.045 1.11 6 0.20
Liver weight (g/100 b.w.) Total P450 (nmol/mg prot.) Cytochrome b 5 (nmol/mg prot.) EROD b AHH ECOD PROD BzND ErND UDPGT GST
3MC 2.90 0.77 0.14 3.900 1.30 0.97 0.473 0.699 0.170 0.160 1.03
6 6 6 6 6 6 6 6 6 6 6
0.62 0.09 0.03 0.875** 0.20** 0.18 0.130** 0.425 0.097 0.039 0.21
PB 3.85 1.06 0.18 0.202 0.16 0.75 0.195 0.874 0.432 0.190 1.11
6 6 6 6 6 6 6 6 6 6 6
1.25 0.36 0.02 0.077 0.08 0.31 0.088 0.328 0.167* 0.026 0.12
The values are the mean 6 SD for 3 (PB) and 4 (Control and 3MC) animals. b Enzyme activities are given in nmol/min/mg of protein. Liver cytosolic fractions (for GST) and microsomal fractions (for ECOD, PROD, BzND, ErND, EROD, AHH and UDPGT) were used for the assay of the enzymes. Significance of the differences versus the control group at *P , 0.05 and **P , 0.01. a
induction of the ErND and testosterone 6β-hydroxylase activities, but did not affect the other phase I enzyme activities. Neither 3MC nor PB had an effect on UDPGT and GST activities. The effects of 3MC- and PB-treatment on testosterone hydroxylase and oxidase activities are shown in Table 2. The major metabolite in all the groups was the 6βhydroxytestosterone. Hepatic microsomes from PB-treated snakes exhibited enhanced ability to hydroxylate testosterone at the 6β position by about 4-fold, while the hydroxylation at other positions was not significantly affected as compared to the control group. 3MC-treatment had no significant effects on the testosterone hydroxylase and oxidase activities.
Effects of aNF on 3MC-induced Activities of EROD and PROD The P450 induced by 3MC was characterized by an inhibitory effect of α NF on the EROD and PROD activities in liver microsomes (Fig. 1). Addition of α NF (0.2 µM to 20 µM) to the reaction mixture caused a marked dosedependent inhibition of EROD and PROD activities in the liver microsomes from 3MC-treated hamsters. The activities of EROD and PROD were inhibited by 98% and 60%, respectively, by α NF at 10 µM.
TABLE 2. Effects of 3MC and PB on testosterone hydroxylase activities in snake livers
Control 3MC PB (pmol/min/ (pmol/min/ (pmol/min/ mg prot.) mg prot.) mg prot.) Testosterone metabolites a 2α-OH 2β-OH 6α-OH 6β-OH 7α-OH 15α-OH 15β-OH 16α-OH 16β-OH Androstenedione a
17.0 6 5.9 b 15.7 6 4.5 7.2 6 5.2 209 6 70 ND 17.7 6 2.7 ND 31.7 6 11.9 15.0 6 3.2 240 6 21
17.5 6 3.7 20.2 6 6.2 7.2 6 2.9 272 6 28 ND 15.7 6 5.0 ND 18.5 6 9.8 16.1 6 3.0 180 6 69
19.7 39.0 10.3 849 1.3 22.0 2.3 20.7 29.1 218
6 6 6 6 6 6 6 6 6 6
4.9 17.7 0.6 150* 1.5 12.2 2.5 8.5 12.0 48
The abbreviations denote the hydroxylated testosterone metabolites. Androstenedione is the product of the testosterone 17-oxidase activity. b The values are the mean 6 SD for 3 (PB) and 4 (Control and 3 MC) animals. Significance of the differences versus the control group at *P , 0.01. ND: not detected.
FIG. 1. Inhibition by a NF of the activities of EROD and
PROD of hepatic microsomes from snakes treated with 3MC. Results are expressed as % of the control values. Values are the means 6 SD of 4 determinations. *Significantly different (P , 0.01) from the control values.
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FIG. 2. Western blots of snake liver microsomes using polyclonal antibodies against hamster CYP1A2 (A), and rat CYP2B1 (B). Microsomal proteins from control (lane 1), 3MC-treated (lane 2) and PB-treated (lane 3) snakes were applied on the gel in panel A (5 mg protein) and B (2.5 mg protein). Microsomes (0.25 mg protein) from 3MC-treated hamster and PB-treated rat were applied (lane 4) in panel A and B, respectively.
Immunoblot Analysis Figure 2 shows representative Western blots of hepatic microsomes of the snake. Treatment with 3MC caused an apparent increase in the intensity of a protein band immunorelated to hamster CYP1A2, while no difference in this band intensity was observed in the microsomes of PBtreated animals compared to that of the controls (Fig. 2A). In the control animals, two bands were detected which immunoreacted with antibodies to rat CYP2B1. The intensity of these protein bands was significantly decreased by 3MC-treatment (Fig. 2B). Treatment with PB did not affect the intensity of the CYP2B1-related proteins. DISCUSSION In the present report, we studied the inducibility by 3MC and PB of the hepatic biotransformation system of a reptile and found differences from studies reported so far on the other non-mammalians vertebrates, but also from those on the other snake species. 3MC-treatment failed to induce the P450 content and related enzyme activities in the livers of snakes. This is in accordance with previous results which reported that intraperitoneal injection of β-naphthoflavone (β NF) at a dose of 100 mg/kg for 22 hr had no effects on garter snakes (Thamnophis sp.) and painted turtles (Chrysomyces picta) in P450 content, and EROD and ECOD activities (14). However, the inability of 3MC observed here might be due to the length of treatment time, which may have been too short to elicit any changes in the enzyme activity. This was previously reported that, even though a high level of 3MC was present in the snake liver 24 hr after the last dose, induction of P450 did not occur until 5 days after the last dose of 3MC (27). The shift in the Soret maximum of the reduced CO spectrum from 450 to 448 nm was not observed in the snake microsomes after 3MC administration. This finding is in agreement with a previous study on garter snakes (27), while the shift was reported for an alligator (Alligator mississippiensis) (17). The results of the catalytic and immunochemical studies
suggest that 3MC induced a snake P450 isozyme belonging to the CYP1A subfamily. This was shown by the increase in the activities of EROD and AHH, the selective activities of fish and rodent CYP1A1 (15), by the inhibitory effect of α NF, a specific inhibitor of CYP1A1, on EROD activity and by the increase in the protein band related to hamster CYP1A2. Moreover, we obtained an inhibition by α NF on the 3MC-induced PROD activity in the microsomes obtained from the 3MC-treated snakes. In rats, this enzymatic activity is considered to be related to the CYP2B1/2B2 isozymes. Whether the PROD activity is related to the CYP1A subfamily in the snake needs to be further characterized. PB-treatment to the snake failed to induce P450 content and the proteins immunorelated to rat CYP2B1 but increased the ErND and testosterone 6β-hydroxylase activities, both correlated to the CYP3A subfamily in rodents (16,33). However, no proteins immunorelated to rat CYP3A were detected in the microsomes of control and PB-treated animals (data not shown). In mammals, PB was also shown to induce the CYP3A1 isozyme (32). In fishes, putative CYP3A have been reported to have steroid 6βhydroxylase activity (29). Whether ErND and testosterone 6β-hydroxylase activities are related to CYP3A proteins in the snake remains to be characterized. To our knowledge, only one previous study detailed the effects of PB-treatment on the P450-dependent monoxygenases of snakes (27) in which no inductive effects of PB on P450 systems was produced by several doses of the compound and different PB-exposure times. Extensive studies suggest that fishes and quails are resistant to PB-type induction (i.e. total P450 content and CYP2B induction) (18,30). However, these results are not entirely consistent. Ethylmorphine N-demethylase was induced in a fish (Poecilia reticulata) by a two weeks-treatment with PB (31). To date, little attention has been directed toward the induction of phase II enzymes in non-mammalian vertebrates. In the present study, we did not observe any effects of 3MCand PB-treatment on UDPGT and GST activities. However in fish, β NF was shown to induce these conjugation enzyme activities (2) and β NF and 2,3,7,8-tetrachlorodibenzo-p-dioxin were shown to induce to a similar extent
Induction of Snake Cytochrome P450
the UDPGT activity (6). It was reported that UDPGT and GST response to β NF lasted longer than the response of the P450 monooxygenase activities (2). This could be one of the reasons for the lack of induction of UDPGT and GST in the snake under our experimental conditions. In summary, the present study showed that a snake species (Elaphe guttata emoryi) responded to 3MC-treatment in a similar manner as in fishes and rodents in the induction of CYP1A, while the snake was refractive to PB-like induction (i.e. CYP2B induction). Knowledge of the species differences in phase I and II enzyme systems in reptiles from other species could provide fundamental insights into the evolution of P450 proteins. Studies on animals living in ecosystems could also be useful in the research for biological indicators for monitoring environmental pollution.
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12. 13. 14.
15.
16. 17.
This work was supported by the Human Sciences Foundation of Japan. 18.
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