Mutagenesis, Vol. 16, No. 6, 479-486,
November 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
N-Benzylimidazole for preparation of S9 fraction with multi-induction of metabolizing enzymes in short-term genotoxicity assays
Laboratory of Radiochemistry, Gifu Pharmaceutical University, 6-1 Mitahora-higashi 5-chome, Gifu 502-8585, Japan
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To evaluate the usefulness of N-benzylimidazole (BI) as an inducer with wide spectrum detection of precarcinogens in short-term bioassays, hepatic levels of cytochrome P-450 (CYP) and mutagenic activation of various carcinogens in Wistar and Sprague–Dawley rats orally treated with BI and BI plus ethanol or acetone were compared with those in the same strains of rats treated with 3-methylcholanthrene (MC), phenobarbital (PB) and polychlorobiphenyls (PCB). Immunoblot analyses for microsomal CYP proteins revealed a marked induction by BI in the levels of CYP1A1, CYP2B1 and constitutive CYP1A2 (~11-fold), 2B2 (~21-fold), 2E1 (1.5-fold) and 3A2 (4-fold) in rats of both strains. These levels were comparable with those induced by MC and PB, but were less than the CYP1A1/2 and 2B1 levels induced by PCB, while CYP2B2 was at the same level. In contrast, the level of CYP2E1 was clearly higher in BI-treated rats. The combinations of BI and acetone or ethanol specifically induced CYP2E1 (4-fold) and 2B1 (1.7-fold) levels when compared with BI alone in Wistar rats. The combined treatments also elevated mutagenic activities of eight heterocyclic amines (HCAs), aflatoxin B1 (AFB1), benzo[a]pyrene and 2-aminofluorene in strain TA98 up to 14.3-, 5.1-, 2.8- and 2.1-fold above the untreated group, respectively, and those of five N-nitrosamines in strain TA100 up to 19.1-fold. Induction of specific CYP species responsible for activation of HCAs, AFB1 and N-nitrosamines was confirmed by application of several CYP inhibitors. In addition, BI induced activities of both MC- and PB-inducible UDP-glucuronyltransferases towards 4-nitrophenol and testosterone. These results demonstrate that BI has a bifunctional action, with wide spectrum induction of phase I and II enzymes, and combined treatment with ethanol or acetone would be a pertinent inducer for metabolic enzymes in in vitro bioassays, the potential being comparable with or superior to other typical ones.
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Introduction |
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Most carcinogens and mutagens require metabolic activation by cytochrome P-450 (CYP) for the expression of carcinogenicity or mutagenicity. Polychlorobiphenyls (PCB), phenobarbital (PB), ß-naphthoflavone (ß-NF) and a combination of PB and ß-NF have been widely used as CYP inducers in in vitro genotoxicity assays (Matsushima et al., 1976
; Maron and Ames, 1983; Mori et al., 1986; Callander et al., 1995; Franco et al., 1999). Because specific CYP enzymes are involved in the activation of different kinds of carcinogens (Mori et al., 1985; Yang et al., 1987; Degawa et al., 1988; Buetler et al., 1996), it is important to obtain S9 fractions with induction of multiple CYP species for detection of genotoxic compounds. Paolini and Cantelli-Forti (1997) have pointed out the necessity of in future adopting a `superinduced' S9 fraction containing a wide spectrum of CYP enzymes induced by PB, ß-NF, isosafrol, ethanol and pregnenolone 16
-carbonitrile. However, since mice are given each inducer for different periods of time to reach maximal induction in this method, experimental conditions are complicated and the same laboratory animals have to be i.p. injected 19 times over 6 days.
Previously we have shown that in the Salmonella test N-benzylimidazole (BI) treatment once daily for 3 days markedly enhances the mutagenic activation of various carcinogens which are mainly activated by CYP1A and 2B (Mori et al., 1993). Furthermore, BI is known to induce metabolic activities specific for hepatic CYP1A1, 2B1 and 3A2, but not CYP2E1, in rats (Papac and Franklin, 1988) and hepatic CYP1A and 2B enzymes in rats (Magdalou et al., 1988; Kobayashi et al., 1994). However, no studies have compared the levels of CYP proteins induced by BI with those by PB, 3-methylcholanthrene (MC), ß-NF or PCB. On the other hand, Aroclor 1254, MC and PB plus ß-NF clearly suppress hepatic levels of CYP2E1 protein and metabolic activity in rats (Thomas et al., 1987; Papac and Franklin, 1988; Borlakoglu et al., 1993; Burke et al., 1994; Goasduff et al., 1995). Human and rodent CYP2E1, 2A and 3A play important roles in the metabolic activation of carcinogenic N-nitrosamines (Garro et al., 1981; Yamazaki et al., 1992; Burke et al., 1994) and AFB1 (Buetler et al., 1996). These findings suggest the necessity for confirmation of CYP2E1, 2A1/2 and 3A2 induction in BI-treated rats and the recommendation of a combined treatment with BI and a CYP2E1 inducer for preparation of the S9 fraction in short-term genotoxicity assays.
Glucuronidation is generally considered one of the detoxification reactions in the metabolism of various carcinogens and in the rat hepatic UDP-glucuronyltransferases (UDPGTs) 1A1, 1A6 and 2B1 are found as major enzymes which are inducible by clofibrate, MC and PB, respectively (Narayanan et al., 2000). However, there are conflicting reports on these activities in BI-treated rats: (i) UDPGT activities for bilirubin (specific for UDPGT1A1) and 4-methylumbelliferone (1A6) are clearly enhanced, while those for 1-naphthol (1A6) and morphine (2B1) are unchanged (Magdalou et al., 1988); (ii) those for 4-nitrophenol (1A6) and morphine are markedly induced, while no significant effect is observed in the case of testosterone (2B1) (Papac and Franklin, 1988).
In an attempt to evaluate CYP induction by BI and to compare the activities of phase I and II enzymes with those in conventional or superinduced S9, hepatic levels of microsomal CYP1A1/2, 2A1/2, 2B1/2, 2E1 and 3A2, mutagenic activation of different carcinogen substrates and UDPGT activities towards bilirubin, 4-nitrophenol and testosterone were assayed in Wistar and Sprague–Dawley (SD) rats treated with BI alone and BI plus acetone or ethanol.
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Chemicals
BI, metyrapone, furafylline, orphenadrine and 2-aminofluorene (2-AF) were obtained from Aldrich Chemical Co. (Milwaukee, WI) and AFB1 was from Makor Chemicals (Jerusalem, Israel). Hydrochlorides of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-aminopyrido[1,2-a:3',2'-d]imidazole (Glu-P-2) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), acetates of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA
C), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), benzo[a]pyrene (BP), N-nitrosodimethylamine (DMN), MC, PB, PCB (mixture of hexa-isomers), 4-methylpyrazole, 7,8-benzoflavone (7,8-BF), acetone, 4-nitrophenol, testosterone, bilirubin and UDP-glucuronic acid were purchased from Wako Pure Chemicals (Osaka, Japan). UDP-[14C(U)]glucuronic acid was purchased from American Radiolabeled Chemicals (St Louis, MO) and glucose 6-phosphate (G6P) dehydrogenase (G6PDH), G6P, NADP+, NADPH, NADH and ATP were from Oriental Yeast Co. (Tokyo, Japan). All other commercial products were of the purest grade available. N-Nitrosobis(2-hydroxypropyl)amine (BHP) and N-nitroso-2,6-dimethylmorpholine (NDMM) were synthesized in our laboratory as described previously (Mori et al., 1985) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was kindly provided by Dr Akiyoshi Nishikawa (National Institute of Health Sciences, Tokyo, Japan).
Animal treatment and tissue preparation
Male 6-week-old Wistar (143–149 g) or SD (183–194 g) rats (Japan SLC, Hamamatsu, Japan) were administered, orally via a stomach tube, BI (75 mg/kg) suspended in 0.5 ml of 0.5% Tween 20, once daily for 3 days, and decapitated 48 h after the last dose. Male Wistar rats were similarly given 25 or 100% acetone or ethanol at a dosage of 5 ml/kg body wt on the fourth day and the animals were starved for 12 h before decapitation. Alternatively, SD rats were given PCB i.p. as a single injection at a dose of 500 mg/kg and then killed 5 days later. Wistar rats were similarly injected once a day with PB (80 mg/kg) or MC (20 mg/kg) for 3 days and the animals killed 24 h after the last administration. All the animals treated with PCB, PB and MC were starved for 24 h before decapitation.
Livers were perfused in situ with ice-cold sterile 1.15% KCl and liver S9 and microsomal fractions were prepared using established procedures (Mori et al., 1985). Total CYP content in microsomes was spectroscopically determined by the method of Omura and Sato (1964).
Western blotting
Goat anti-rat polyclonal antibodies for CYP1A1/2, 2B1/2, 2E1 and 3A2 and NADPH-CYP reductase (Daiichi Pure Chemicals Co., Tokyo, Japan) and sheep anti-human polyclonal antibody for CYP2A6 (Chemicon International, Temecula, CA) were used as primary antibodies. Gel electrophoresis and blot analysis were performed as described in detail previously (Koide et al., 1999), according to the established methods of Laemmli (1970) and Towbin et al. (1979), respectively.
Mutation assay
All tests were carried out by the Ames preincubation assay (Yahagi et al., 1977). The N-nitrosamines were dissolved in 100 µl of water and the other carcinogens in 50 µl of dimethyl sulfoxide. The mutagenicity of MeIQ (dose, 0.006 µg/plate), Trp-P-2, Glu-P-1 and IQ (0.03 µg), Trp-P-1 (0.3 µg), Glu-P-2 (33 µg), MeAC (10 µg), PhIP and BP (5 µg), 2-AF (2 µg), AFB1 (1 µg), NNK (0.5 mg) and the other N-nitrosamines (1 or 10 mg) was checked in the presence of liver S9, according to the methods described by Smith et al. (1992), Edwards et al. (1994), Maron and Ames (1983), Ueno et al. (1978), Lee et al. (1996) and Mori et al. (1985). The amount of liver S9 fraction was 10 µl/plate for the HCA and AFB1, 50 µl for BP, 2-AF and NNK and 150 µl for the other N-nitrosamines. Salmonella typhimurium TA100 and TA98 tester strains were employed for the N-nitrosamines and all the other carcinogens, respectively. The S9 mix contained the cofactors 4 mM NADPH, 4 mM NADH, 0.5 U G6PDH, 5 mM G6P and 5 mM ATP, except for the N-nitrosamines, where 4 mM NADP+ and 5 mM G6P were used. One millimolar coumarin (Negishi et al., 1989) and metyrapone (Ullrich et al., 1973), 0.1 mM 4-methylpyrazole (Newton et al., 1995) and 0.2 mM 7,8-BF (Ullrich et al., 1973), furafylline (Manchee et al., 1996) and orphenadrine (Shu and Hollenberg, 1997) were preincubated with carcinogen substrate and S9 mix in the experiments for CYP inhibition.
Assay of UDPGT activity
UDPGT activities towards 4-nitrophenol and bilirubin in liver microsomes were assayed according to the methods described by Isselbacher et al. (1962) and Heirwegh et al. (1972), respectively, and that towards testosterone was determined using UDP-[14C(U)]glucuronic acid as described by Matern et al. (1994).
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Total CYP contents in liver microsomes from Wistar rats and the same strain of rats treated with MC, PB and BI were 0.78 ± 0.11 (mean ± SD), 1.00 ± 0.15, 2.98 ± 0.10 and 2.65 ± 0.10 nmol/mg, respectively, and those from untreated and BI- or PCB-treated SD rats were 0.57 ± 0.09, 2.41 ± 0.08 and 1.64 ± 0.05 nmol/mg, respectively. The differences between untreated rats and PB-, BI- and PCB-treated rats (P < 0.01) and those between BI- and PCB-treated rats (P < 0.01) were statistically significant. The combined treatment of Wistar rats with BI plus 25% ethanol, 25% acetone or 100% acetone resulted in upto 1.5-fold higher CYP content compared with rats treated with BI alone (P < 0.05).
Figure 1 shows immunoblots and levels (pmol/mg protein) of microsomal CYP proteins and NADPH-CYP reductase in Wistar and SD rats. There were no significant differences in hepatic levels of all the CYP proteins determined between the two strains of rats, either untreated (lanes 1 and 5) or BI-treated (lanes 4 and 6). Treatment of Wistar rats with BI markedly induced hepatic CYP1A1 and constitutive CYP1A2 (11.4-fold above control) to the same extent, but the CYP1A1 level was slightly less than that induced by MC (lane 2). In SD rats PCB induced levels of both CYP1A1 and 1A2 >2.5-fold above BI (lane 7). CYP2B1 and constitutive CYP2B2 (18.9 or 21.2-fold) were also induced and the CYP2B2 level was equal to that induced by PB (lane 3) and PCB, however, the CYP2B1 level was about half that induced by PB and PCB. CYP3A2 level was increased approximately four times each control, but the level was slightly less than that observed in the PB- and PCB-treated groups. In addition, BI induced a slight but significant increase (upto 1.5-fold, P < 0.01) in CYP2E1 level, despite neither PB nor PCB having a significant effect. In contrast, MC decreased the levels of CYP2E1, 3A2 and 2B2 by 63, 20 and 57%, respectively. Immunoreactive bands with an anti-CYP2A6 antibody could not be found in any rats of the seven groups (Figure 1E) and no treatment exerted an enhancing or suppresive effect on the level of NADPH-CYP reductase (Figure 1F).
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The effects of combination with a CYP2E1 inducer on CYP expression were compared with those of BI alone. As shown in Figure 2
, there were no significant differences in any CYP levels among Wistar rats treated with BI plus 25% ethanol, 25% acetone or 100% acetone. CYP2E1 and 2B1 proteins were more intensely stained (4.4- and 1.7-fold higher, respectively) in rats treated with the combinations than with BI alone, while the other four proteins were at the same levels. No immunoreactive band with an anti-CYP2A6 antibody was observed in these BI-induced livers, as shown in Figure 2E.
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To clarify the mixed-type induction characteristics of BI plus acetone or ethanol in the Salmonella test, sixteen carcinogens known to be metabolically activated by specific CYP enzymes were assayed in the presence of liver S9 from Wistar rats. As shown in Figure 3
, the mutagenic activities of AFB1, 2-AF, BP and eight HCAs in strain TA98 were clearly increased in rats treated with BI plus 25% ethanol, 25% acetone and 100% acetone to almost the same extents. However, carcinogen specificity was observed in the enhancing effect; the activities of 2-AF, BP and MeAC were increased up to 3.0-fold above each control, those of AFB1, Trp-P-1 and PhIP 4.5–6.5-fold and those of the other HCAs 21–14.3-fold. Table I shows the abilities of the liver S9 fractions to activate five N-nitrosamines to mutagens in strain TA100. In the presence of liver S9 from rats treated with BI plus a CYP2E1 inducer the mutagenic activities of DMN and DEN at 1 and 10 mg doses were increased up to 8.8- and 19.1-fold above the untreated group, respectively, and that of 10 mg BHP was up to 5.9-fold above control, while 1 mg BHP showed no clear mutagenicity. Similarly, the mutagenic activities of NNK and NDMM in the treated groups were increased up to 5.4-fold above each control.
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) and from three each rats treated with BI + 25% acetone (
), BI + 100% acetone (
) and BI + 25% ethanol (
). Each bar represents the mean ± SD (4–8 plates) after subtraction of spontaneous revertants (18–24).
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To confirm the extent of participation of each CYP species in mutagenic activation of several carcinogens, specific CYP inhibitors were preincubated with liver S9 from Wistar rats treated with BI plus 25% acetone or ethanol. As shown in Figure 4A
–C, 7,8-BF caused a marked inhibition of the mutagenic activation of Glu-p-1, Trp-P-2 and MeAC and furafylline inhibited the mutagenic activities of Glu-P-1 and Trp-P-2 by up to 86 and 50%, respectively, but not that of MeAC. On the other hand, metyrapone and 4-methylpyrazole caused 51 and 61% inhibition, respectively, of the mutagenic activity of DEN (Figure 4E). 4-Methylpyrazole showed a marked inhibition of the activity of DMN, while no significant effect was seen with metyrapone. The opposite was the case for BHP. As shown in Figure 5, metyrapone and orphenadrine inhibited the mutagenic activity of AFB1 by 66 and 43%, respectively, in both rats treated with acetone and those treated with ethanol, while 7,8-BF and furafylline had no inhibitory effect on mutagenicity. 7,8-BF, furafylline and metyrapone caused 83, 69 and 68% inhibition, respectively, of the mutagenic activity of NNK, while there were no significant changes in the presence of orphenadrine, 4-methylpyrazole and coumarin.
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) or ethanol (
). Each bar represents the mean ± SD (4–6 plates) and all mutagenic activities were compared with incubations in the absence of CYP inhibitor, as shown in Figure 3
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) or ethanol (
). Each bar represents the mean ± SD (4–6 plates) expressed as a percentage of the control. Control activities are shown in Figure 3Table II
summarizes the effects of MC, PB, BI and BI plus 25 or 100% acetone on three kinds of UDPGT activities in liver microsomes from Wistar rats. UDPGT activities towards 4-nitrophenol and testosterone in BI-treated rats were increased up to 2.4-fold above the respective control, being almost equal to those in MC- and PB-treated rats. That is to say, acetone did not significantly affect induction by BI, although that towards testosterone was slightly less in rats treated with BI alone than with PB (P < 0.01). On the other hand, there were no significant differences in UDPGT activity towards bilirubin among the five groups.
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No strain differences between Wistar and SD rats were observed in hepatic levels of several CYP enzymes induced by BI (Figure 1
). This is consistent with our earlier findings that there is no significant difference in mutagenic activation of various carcinogens between BI-induced rats of both strains (Mori et al., 1993, 1994). BI is known to enhance hepatic activities of ethoxyresorufin (EROD), pentoxyresorufin (PROD), erythromycin and ethylmorphine (ETD) dealkylases, but not those of p-nitrophenol and aniline hydroxylases, in mice and rats (Papac and Franklin, 1988; Manning and Franklin, 1992). This is in agreement with the present findings of a marked induction of hepatic CYP1A1/2, 2B1/2 and 3A2 and a slight induction of CYP2E1. PCB enhanced hepatic levels of these CYP proteins except CYP2E1, in accordance with previous findings on CYP protein levels and their metabolic activities (Thomas et al., 1987; Callander et al., 1995), and induction by PCB of CYP1A1/2 and 2B1 was more than double that by BI. However, it has been shown that in the presence of liver S9 from BI-treated rats mutagenic activities of various carcinogens which are known to be metabolically activated by these CYP species are almost the same as those observed with PCB-treated rats (Mori et al., 1993). It has also been reported that no significant differences between PB plus ß-NF- and Aroclor 1254-induced rat liver S9 fractions are found in mutagenic activation of seven genotoxins, despite Aroclor 1254 having a greater effect than PB plus ß-NF on CYP enzyme profiles and CYP-mediated monooxygenase activities (Callander et al., 1995). Accordingly, it can be concluded that the CYP protein levels induced by BI, PB plus ß-NF and PCB represent an effective excess in terms of mutagenic activation, indicating that BI may be considered an adequate alternative to either PB plus ß-NF or PCB in in vitro genotoxicity assays.
In the mouse hepatic activities of EROD, PROD and phenacetin O-dealkylase are 1.7–4.3-fold higher in superinduced S9 than in PB plus ß-NF-induced S9 (Paolini et al., 1991). In the rat Aroclor 1254 enhances the activities of EROD, PROD and methoxyresorufin O-deethylase 1.5–4.5-fold relative to PB plus ß-NF (Callander et al., 1995). BI and Aroclor 1254 show equal effects in induction of the activities of EROD, PROD and p-nitroanisole O-demethylase (Papac and Franklin, 1988). Therefore, it is suggested that BI-induced S9 might have equal ability to superinduced S9 in induction of metabolic activities specific for CYP1A and 2B enzymes.
MC, PB plus ß-NF and PCB are known to exert suppressive effects on expression of CYP2E1 in rat liver (Thomas et al., 1987; Borlakoglu et al., 1993; Burke et al., 1994; Goasduff et al., 1995) and combined administration of ethanol with MC also decreases enhanced levels of CYP2E1 protein and mRNA (Goasduff et al., 1995). In contrast, BI showed a slight induction of CYP2E1 protein and the level was further increased up to 4.4-fold by acetone or ethanol, relative to BI alone (Figure 2). In addition to CYP2E1, acetone and ethanol caused an induction of CYP2B1 protein (1.7-fold), consistent with the finding of Johansson et al. (1988) that both inducers increase CYP2B protein and mRNA levels. CYP2E1 has a remarkable capacity to activate low molecular weight N-nitrosamines such as DMN (Garro et al., 1981), DEN (Yamazaki et al., 1992) and N-nitrosopyrrolidine (Burke et al., 1994). This is confirmed in the present study on mutagenicity of several N-nitrosamines with BI plus a CYP2E1 inducer. The inhibitory effect of metyrapone on mutagenic activities was in order BHP > DEN > DMN, while 4-methylpyrazole markedly inhibited the activity of DMN rather than BHP. Enhancement of the mutagenic activities of DMN and DEN at a 10 mg dose was more than double that at 1 mg, in accord with the much higher induction of CYP2B1/2 rather than CYP2E1. These results are in line with previous findings that CYP2B1/2 and CYP2E1 are differentially involved in activation of N-nitrosodialkylamines, depending on the length of the alkyl chain and the dose of substrate (Mori et al., 1985; Yoo and Yang, 1985; Yang et al., 1987; Kawanishi et al., 1992; Shu and Hollenberg, 1996). When compared with the results for BI alone (Mori et al., 1993), the mutagenic activity of 10 mg DMN is much higher (~5-fold) in the case of BI plus a CYP2E1 inducer, while those of NDMM and other carcinogens are almost the same. Furthermore, the mutagenic activities at 1 and 10 mg doses of DMN are up to 7.9- and 2.4-fold higher, respectively, than those observed in superinduced S9. Accordingly, a combination of BI and acetone or ethanol is an excellent inducer for preparation of S9 fractions in short-term bioassays, with multi-induction of CYP species, especially CYP2E1 and 2B1/2.
NNK is a specific N-nitrosamine occurring in cigarette smoke (International Agency for Research of Cancer, 1986) and is metabolically activated by CYP1A2, 2A1 and 3A2, but not by CYP1A1, 2B1, 2C11 and 2E1 (Guo et al., 1992). Although MC, ß-NF, PB and PCB are known to induce CYP2A1 protein (Chang and Waxman, 1996), the effect of BI on CYP2A induction has not been elucidated. The mutagenic activity of NNK was increased by the combined treatments and this increase was markedly inhibited by 7,8-BF, furafylline and metyrapone, but not by orphenadrine, 4-methylpyrazole and coumarin, indicating the involvement of CYP1A2 but not CYP1A1, 2B1 and 2E1 in mutagenic activation. Metyrapone is a potent inhibitor of CYP2A1, 2A5 and 2A6 (Mukhtar et al., 1987; Mäenpää et al., 1991, 1993; Draper et al., 1997) and CYP3A2 (Parkinson et al., 1982), in addition to CYP2B1/2. 7,8-BF is also reported to specifically inhibit the metabolic activity of CYP2A6 by ~40% (Draper et al., 1997). Since CYP2A5, 2A6 and 2A8 can metabolize coumarin to 7-hydroxycoumarin (Mäenpää et al., 1991; Pelkonen et al., 1993; Draper et al., 1997) but CYP2A1 cannot (Fentem and Fry, 1991; Peters et al., 1991), it is reasonable that coumarin could not inhibit the mutagenicity of NNK. These findings indicate the involvement of CYP2A1 and 3A2, in addition to CYP1A2, in NNK mutagenic activation and suggest induction of CYP2A1 protein by BI. However, no bands were found in any samples of rat liver with sheep anti-CYP2A6 antibody. It has been reported that CYP2A1 in liver microsomes from Wistar and SD rats treated with MC or PB are not detected using rabbit anti-CYP2A5 and 2A8 antibodies (Raunio et al., 1988; Fukuhara et al., 1989; Lang et al., 1989; Lai and Chiang, 1990). This may be attributed to the relatively low amino acid sequence identity between CYP2A1/2 and CYP2A5, 2A6 and 2A8 (Phillips et al., 1985; Nagata et al., 1987; Lindberg et al., 1989; Lai and Chiang, 1990), indicating that it is necessary to prepare anti-CYP2A1 antibody (Arlotto et al., 1989; Pearce et al., 1992) for this study. Accordingly, induction of hepatic CYP2A1/2 protein by BI is not clear and remains to be demonstrated.
It is known that CYP3A2, 3A4 and 3A13 are involved in metabolic activation of AFB1 (Ueng et al., 1995; Buetler et al., 1996; Yanagimoto et al., 1994). However, conflicting results have been reported for the involvement of rat CYP enzymes in mutagenic activation of AFB1: (i) activation by CYP1A2, CYP2B1/2 and CYP2C11 (Kawajiri et al., 1980; Ishii et al., 1986); (ii) activation by CYP3A2 and CYP2C11, but not CYP1A2 (Hayes et al., 1991; Buetler et al., 1996). BI induced CYP3A2 protein and mutagenic activity of AFB1 to ~4- and 5-fold above the respective controls. The enhanced activity of AFB1 was inhibited slightly more by metyrapone than by orphenadrine, but 7,8-BF and furafylline did not show significant effects. Together with the inhibitory effect of metyrapone on CYP3A2, the present results indicate that CYP3A2 may be involved in the mutagenic activation of AFB1, in addition to the clear contribution of CYP2B1, but possible involvement of CYP1A species can be excluded.
BI increased the mutagenic activities of 2-AF, BP and the eight HCAs, especially the HCAs, and the effect was different for each carcinogen and each HCA, although induction of CYP1A1 and 1A2 by BI was almost equal. Further, inhibition by 7,8-BF and furafylline was different for Glu-P-1, Trp-P-2 and MeAC, indicating that these HCAs are activated selectively by CYP1A2, almost equally by both CYP1A1 and 1A2 and predominantly by CYP1A1, respectively. These results are in line with previous findings that the comparative contributions of hepatic CYP1A1 and 1A2 to mutagenic activation differs with individual HCA in MC-treated rats (Degawa et al., 1988) and in Hep G2 cell lysate containing cDNA-expressed mouse CYP1A1 and 1A2 (Aoyama et al., 1989). On the other hand, the mechanism of CYP1A1 induction by BI is known to be different from that by MC and ß-NF; BI fails to bind with high affinity to the cytosolic aryl hydrocarbon (Ah) receptor in rat liver (Magdalou et al., 1988). Actually, BI induces EROD activity and CYP1A1 level in both Ah-non-responsive DBA/2N and Ah-responsive C57BL/6N mice, while MC and ß-NF induce those in Ah-responsive mice rather than Ah-non-responsive mice (Manning and Franklin, 1992; Gradelet et al., 1997). Omeprazole, a benzimidazole compound, is another CYP1A1 inducer from Ah receptor ligands, including BP and 2,3,7,8-tetrachlorodibenzo-p-dioxin (Daujat et al., 1992). It appears to induce CYP1A1 by initiating a protein tyrosine kinase-mediated signal transduction pathway (Kikuchi et al., 1998), suggesting a mechanism for induction of CYP1A1 by BI.
BI induced the hepatic UDPGT activities for 4-nitrophenol and testosterone to almost the same levels as those observed with their representative inducers, MC and PB, and acetone showed no further induction. Reports of the action of BI on rat hepatic UDPGT1A6 and 2B1 activities show that they differ depending on the substrate used (Magdalou et al., 1988; Papac and Franklin, 1988). Since the present results for the effect of BI on 4-nitrophenol are in agreement with previous findings on the same substrate and 4-methylumbelliferone, we conclude that BI has inductive capacity for rat UDPGT1A6 activity. On the other hand, the present results for testosterone are consistent with findings for morphine but do not agree with previous ones for testosterone. The activity against bilirubin is reported to be induced by ethanol (Ideo et al., 1971) and acetone (Braun et al., 1998), in addition to BI. Anyway, BI and acetone did not induce UDPGT1A1 activity in the present study. Nevertheless, there are no reports of the involvement of CYP4A1 and UDPGT1A1 in carcinogen activation and detoxification, although clofibrate (Orton and Parker, 1982) and BI (Magdalou et al., 1988) are known to induce hepatic CYP4A1 protein. The reasons for these discrepancies with testosterone and bilirubin are not clear, but it is suggested that the differences might be due to experimental conditions, such as substrate concentration, BI treatment method, detection method, etc. However, the present results with three UDPGT activities are consistent with the findings of Lilienblum et al. (1982), that Aroclor 1254 does not stimulate hepatic activity for bilirubin whereas it enhances those for 1-naphtol, 4-methylumbelliferone and morphine.
In conclusion, we have demonstrated that BI has a bifunctional action with wide spectrum induction of phase I and phase II enzymes and in combination with ethanol or acetone would be a pertinent inducer for metabolic enzymes in in vitro bioassays. The potential is superior to other metabolic enzyme inducers.
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1 To whom correspondence should be addressed. Tel: +81 58 237 3931; Fax: +81 58 237 5979; Email: ymori{at}gifu-pu.ac.jp
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Received on March 26, 2001; accepted on June 22, 2001.
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