Effects of cigarette smoke and a heterocyclic amine,MeIQx on cytochrome P-450, mutagenic activation of various carcinogens and glucuronidation in rat liver

In order to elucidate the mechanism underlying enhancement by cigarette smoke (CS) of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced rat hepatocarcinogenesis, hepatic levels of cytochrome P-450 (CYP) enzymes, mutagenic activation of various carcinogens and UDP-glucuronyltransferase (UDPGT) activities were assayed in male F344 rats. Immunoblot analyses for microsomal CYP proteins revealed induction of CYP1A1 and constitutive CYP1A2 (2.3- to 2.7-fold), but not CYP2B1/2, 2E1 or 3A2, by CS exposure for 1, 12 or 16 weeks using a Hamburg type II smoking machine; the enhancement of CYP1A2 was 4.7-5.7 times that of CYP1A1. CS exposure also elevated the mutagenic activities of MeIQx and five other heterocyclic amines (HCAs) 1.4- to 3.7-fold, but not those of benzo[a]pyrene (BP) and aflatoxin B(1) in strain TA98 and N-nitrosodimethylamine, N-nitrosopyrrolidine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in strain TA100. In contrast, feeding 300 p.p.m. MeIQx in the diet for 1 or 16 weeks produced no significant alterations in the levels of these CYP species and mutagenic activities. However, i.g. administration of 50 or 100 mg/kg MeIQx in a single dose selectively increased CYP1A1 and 1A2 (2.6-fold) levels and mutagenic activities of five HCAs (1.7- to 3.3-fold), but not BP. On the other hand, feeding of MeIQx for 16 weeks enhanced UDPGT activities towards 4-nitrophenol and testosterone (2.9- and 1.5-fold, respectively), but not bilirubin, while CS exposure induced that towards 4-nitrophenol (1.6-fold); combined treatment with CS and MeIQx showed a summation effect on induction of UDPGT1A6 activity (3.5-fold). Consequently, these results demonstrate that CS and MeIQx have a bifunctional action, with similar induction patterns of specific CYP proteins, mutagenic activity and UDPGT activity. In conjunction with the finding of N-hydroxy-MeIQx being a poor substrate for rat liver UDPGT, our results clearly indicate that enhancement by CS of MeIQx-induced hepatocarcinogenesis in F344 rats can be attributed to an increase in metabolic activation of MeIQx by hepatic CYP1A2 during the initiation phase.     

Predicting the mutagenicity of tobacco-related N-nitrosamines in humans using 11 strains of Salmonella typhimurium YG7108, each coexpressing a form of human cytochrome P450 along with NADPH-cytochrome P450 reductase.

Tobacco, including snuff and chewing tobacco, contains N-nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine (NPYR), N-nitrosopiperidine (NPIP), N-nitrosomorpholine (NMOR), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NABS), and N-nitrosoanatabine (NATB). The role of human cytochrome P450 (CYP) in the metabolic activation of these tobacco-related N-nitrosamines was examined by a Salmonella mutation test using genetically engineered Salmonella typhimurium (S. typhimurium) YG7108 cells each expressing a form of human CYP (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, or CYP3A5) together with human NADPH-cytochrome P450 reductase. Mutagen production from NNK was catalyzed by CYP in the following order: CYP1A2, CYP1A1, CYP1B1, CYP2A6, CYP2C19, CYP3A4. The metabolic activation of one of the N-alkylnitrosamines, NDEA, was mediated by CYP2A6, followed by CYP2E1. Cyclic N-nitrosamines such as NPYR, NPIP, and NMOR were also primarily activated by CYP2A6, and to a lesser extent by CYP2E1. NNN, a pyridine derivative of NPYR, was activated by CYP1A1 at an efficiency similar to that of CYP2A6. NABS, a pyridine derivative of NPIP, was mainly activated by CYP3A4, followed by CYP1A1 and CYP2A6. Thus, the addition of a pyridine ring to NPYR or NPIP altered the forms of CYP primarily responsible for mutagenic activation. NATB was metabolically activated solely by CYP2A6, whereas the genotoxicity of NATB was much lower than that of NNN or NPYR. Based on these data, we conclude that CYP2A6 was responsible for the mutagenic activation of essentially all tobacco-related N-nitrosamines tested in the present study.     

N-benzylimidazole for preparation of S9 fraction with multi-induction of metabolizing enzymes in short-term genotoxicity assays.

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 (approximately 11-fold), 2B2 (approximately 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 B(1) (AFB(1)), 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, AFB(1) 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.     

Effect of cigarette smoke on mutagenic activation of environmental carcinogens by cytochrome P450 2A8 and inactivation by glucuronidation in hamster liver

To elucidate the mechanism underlying suppression of N-nitrosobis(2-oxopropyl)amine (BOP)-induced hamster pancreatic carcinogenesis by cigarette smoke (CS), hepatic levels of microsomal cytochrome P450 (CYP) enzymes, mutagenic activation of environmental carcinogens and three types of uridine diphosphate-glucuronyltransferase (UDPGT) and sulphotransferase (ST) activities were assayed in male Syrian golden hamsters and F344 rats exposed to CS. Immunoblot analyses of microsomal CYP proteins revealed induction of constitutive CYP1A2 (2.6-fold increase) and 2A8 (4.0-fold increase) and induction of CYP1A1 and constitutive CYP1A2 (3.9-fold increase) in rats following exposure to CS for 4 weeks using a Hamburg type II smoking machine. CS exposure enhanced mutagenicities of four heterocyclic amines in the presence of liver S9 in both species, whereas the mutagenicities of aflatoxin B(1) (AFB(1)), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were significantly increased by CS in hamsters but not in rats. However, no CS-induced alterations in the mutagenic activities of other carcinogens, including BOP and other pancreatic carcinogens, were observed in either species. Application of several CYP inhibitors revealed that the mutagenic activities of MeAαC, AFB(1) and NNK in the hamster liver S9 were partly associated with CYP2A8, whereas those of the three pancreatic carcinogens were selectively associated with CYP2B. CS enhanced UDPGT activities towards 4-nitrophenol (4-NP) (1.9- to 2.0-fold) but did not affect those of bilirubin, testosterone UDPGTs and three STs in both species. Together with the previous findings that BOP does not induce tumourigenesis in rats and that the glucuronidation of β-oxypropylnitrosamines is higher in rats than in hamsters, suppression of BOP-induced pancreatic carcinogenesis by CS might be attributed to increased detoxification by 4-NP UDPGT and not decreased CYP2B activation. This is the first demonstration of the induction of CYP2A protein by CS; CYP2A protein polymorphisms have been associated with oral and pulmonary carcinogenesis in smokers.     

Cancer prevention mediated by caffeic acid phenethyl ester involves cyp2b1/2 modulation in hepatocarcinogenesis

Studies of cancer chemoprevention with caffeic acid phenethyl ester (CAPE) in the resistant hepatocyte model of hepatocarcinogenesis have shown the participation of CYP drug metabolizing enzymes. To prevent neoplastic and preneoplasic lesions, we must specifically identify which CYP activities are modified in the mechanism of action of CAPE. Male Fischer-344 rats were pretreated with CAPE twelve hours before administration of diethylnitrosamine (DEN) and were sacrificed twelve hours after CAPE and twelve hours, twenty-four hours, twenty-four days, and twelve months after DEN. Other rats were treated with the CYP inhibitors α-naphthoflavone or SKF525A and sacrificed twenty-four hours and twenty-four days after DEN. Microsomes were obtained from livers to quantify protein using Western blot. Diethylnitrosamine metabolism was measured based on nitrite formation and liver histology using GGT histochemistry. Caffeic acid phenethyl ester diminished the protein levels of CYP1A2 and CYP2B1/2. The inhibition of CYP2B1/2 prevented the appearance of preneoplastic lesions. Microsomal assays demonstrated that CAPE interfered with DEN activation diminishing nitrites similar to SKF525A and probably mediated by CYP2B1/2 inhibition. A single dose of CAPE before DEN treatment reduced the appearance of tumors by 43%. These results confirmed that CAPE is a promising agent to confer chemoprotection in liver cancer and should be considered for human therapies.     

Characterization of the CPY isozyme profile induced by cyclohexanol

In a previous report we described the ability of cyclohexanolto induce CYP activity. In order to characterize this inductionwe tested the capacity of liver S9 from rats orally treatedwith cyclohexanol for 5 days, to activate several carcinogenicnitrosamines into mutagens in the Salmonella typhimurium TA100test system. Additionally, Western blot analysis of hepaticmicrosomes from the same treated animals were analysed withspecific antibodies against P450 protein families 1A1/A2, 2B1/B2and 2E1. Cyclohexanol-S9 mixture was more efficient in activatingthe following nitrosamines: N-nitrosodimethylamine (NDMA), N-nitrosodipropylamine(NDPA), N-nitrosomethylpropylamine (NMPA), N-nitrosodibutylamine(NDBA), and N-nitrosopyrrolidine (NPYR) into bacterial mutagensthan S9 from non-treated animals. The mutagenicity of N-nitrosodiethylamine(NDEA) was not modified in the presence of S9 from cyclohexanol-treatedanimals. Since the main metabolic pathway leading to the productionof mutagenic intermediates of NDMA and NPYR is catalysed byisozyme CYP2E1 and that of NDPA, NMPA and NDBA by CYP2B1/B2,mutagenicity experiments predicted that cyclohexanol inducesthese two P450 isozyme families. Western blot analysis confirmedthe results of the mutagenicity assay, showing an increase inthe intensity of CYP2E1 and CYP2B1/B2 protein bands in hepaticmicrosomes from cyclohexanol treated rats in comparison withnon-treated controls. Bacterial mutagenicity tests with specificpro-mutagens were good predictors of the P450 induction propertiesof cyclohexanol. ³To whom correspondence should be addressed at: Instituto de Investigaciones Biomédicas, Universidad Nacional Autonoma de Mexico, Apartado Postal 70228, 04510 Mexico, D.F., México     

S9 induction by the combined treatment with cyclohexanol and albendazole.

Cyclohexanol (CH) is an industrial solvent capable of inducing cytochrome P450 (CYP) enzymes including the CYP2E and CYP2B subfamilies. S9 from CH treated rats is able to activate several N-nitrosamines that are poorly activated by Aroclor 1254, phenobarbital/beta-naphthoflavone (PB/NF) or 3-methylcholanthrene S9 fractions into mutagens detected by the Salmonella typhimurium Ames test. Additionally, albendazole (ABZ) is a widely used anthelmintic drug and a potent inducer of the CYP1A subfamily. Since CYP1A, -2B and -2E subfamilies are implicated in the activation of several environmental mutagens/carcinogens, we studied CYP induction in the rat liver by the combined effect of these two compounds, and used S9 derived from it in the Salmonella/microsome assay to compare with S9 obtained from Aroclor or PB/NF treated rats. Total CYP content in hepatic microsomes was induced by Aroclor, but not by any of the other chemical combinations. Western blot and enzymatic activity analysis revealed quantitative but not qualitative differences in the CYP subfamilies present in the different microsomal fractions; all of the chemicals used increased the levels of CYP1A1/2, CYP2B1/2 and CYP2E1 with respect to control microsomes. CYP3A was not modified by the different treatments. When tested in the Ames test, Aroclor S9 and PB/NF S9 were the most effective in the activation of benzo[a]pyrene and 3-methylcholanthrene which are metabolized mainly by CYP1A1; additionally, the highest mutagenic potency of 2-aminofluorene and N-nitrosodipropylamine, which are activated by CYP1A2 and CYP2B, respectively, were obtained with PB/NF S9. All these compounds were also activated when CH/ABZ S9 was used as the exogenous source of metabolism. Mutagens like N-nitrosopyrrolidine and N-nitrosodimethylamine, activated by CYP2E1, were detected only when CH/ABZ S9 was used, and the effectiveness of the different S9 fractions in activating cyclophosphamide decreased in the following order: Aroclor = PB/NF > CH/ABZ > control. From these experiments we can conclude that the individual CYP- inducing properties of ABZ and CH complement each other when the two compounds are administered in conjunction and that the resulting S9 fraction is able to activate several known mutagens in the Ames test.     

Liver tumor-promoting effect of beta-naphthoflavone, a strong CYP 1A1/2 inducer, and the relationship between CYP 1A1/2 induction and Cx32 decrease in its hepatocarcinogenesis in the rat

Interrelationships among induction of cytochrome P-450 (CYP) 1A1/2, decrease in connexin 32 (Cx32), and liver tumor-promoting activity by beta-naphthoflavone (BNF) in the promotion stage were examined in a 2-stage liver carcinogenesis model. A total of 20 male Fischer 344 rats were initiated with a single intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone. Starting 2 weeks later, they were fed a diet containing 2%, 1%, or 0% BNF for 6 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. Absolute and relative liver weights were significantly increased in the DEN+BNF groups as compared to the DEN-alone group. Diffuse hepatocellular hypertrophy with cytoplasmic eosinophilia, sometimes accompanied by development of adenoma-like hepatic foci, was observed in the BNF-treated rats. Remarkable induction of cytochrome CYP 1A1/2 and significant increase in CYP 2E1 were noted in the DEN+BNF groups, and positive immunohistochemical staining for both was observed diffusely. The areas of Cx32-positive spots per hepatocyte in the centrilobular areas of livers of the BNF-treated rats were significantly decreased, but no changes were observed in periportal areas. The numbers and areas of foci positive for glutathione S-transferase placental form were increased in the BNF-treated groups. These results suggest that BNF is a liver tumor promoter that, unlike phenobarbital, does not induce CYP 2B1/2 isozymes, and there seems to be no direct relationship between CYP 1A1/2 induction and Cx32 reduction in BNF hepatocarcinogenesis.     

Lack of change in the levels of liver and kidney cytochrome P-450 isozymes in p53+/- knockout mice treated with N-butyl-N-(4-hydroxybutyl)nitrosamine

We have previously shown that p53(+/-) knockout mice are highly sensitive to urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in spite of a lack of effects of p53 heterozygosity on N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN) excretion in urine. To determine the influence of p53 deficiency on in vitro formation of BCPN, mutagenicity of BBN and BCPN and levels of several cytochrome P450 (CYP) isozymes, groups of five p53(+/-) knockout and wild-type mice (littermates), as well as animals of the C57BL/6 parental strain, were administered 0.025% BBN in their drinking water for 4 weeks. The livers and kidneys were then used for analyses of BBN metabolism, western immunoblotting and Ames liquid incubation. BBN treatment caused a slight decrease in BCPN formation in the livers of C57BL/6 mice, but there was no significant difference between p53 knockout, wild-type and C57BL/6 mice. In kidney BCPN formation in p53 knockout mice was 33-46% less than that in their wild-type counterparts. Using anti-rat CYP antibodies, CYP1A2, 2B9/10, 2E1 and 3A11/13 were constitutively detected in liver microsomes and CYP2E1 and 3A11/13 in the kidney. Densitometric determination of these CYP proteins revealed no significant variation in levels detected in both tissues among the four groups of mice. BBN and BCPN were not mutagenic for Salmonella typhimurium TA100 in either the absence or presence of liver S9 from untreated mice and rats and from p53 knockout mice treated with BBN. In conclusion, p53 deficiency and BBN had no enhancing effects on metabolism of BBN to BCPN and expression of the CYP isozymes typically responsible for activation of environmental carcinogens, including both of the N-nitrosamines tested, and their mutagenicity, indicating that the high susceptibility of p53(+/-) knockout mice is not attributable to metabolic activation in liver and kidney by CYP isozymes or urinary excretion of BCPN.     

Liver tumor promoting effects of fenbendazole in rats.

In order to examine whether fenbendazole has tumor-promoting activity, a total of 70 male Fischer 344 rats were initiated with a single intraperitoneal injection of 100 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone; beginning 1 wk later, rats were given a diet containing 3,600; 1,800; 600; 200; 70; or 0 ppm of fenbendazole for 8 wk. Subgroups of 5 rats each from the DEN+ 1,800; DEN+0; 1,800; and 0 ppm groups were euthanatized after 1 wk of fenbendazole treatment, and the remaining animals were euthanatized at 8 wk. After 1 wk, relative liver weights (ratios to body weights) were significantly increased in the DEN+ 1,800 and 1,800 ppm groups, and based on light microscopy, periportal hepatocellular hypertrophy was evident in these groups. After 8 wk, relative liver weights were significantly increased in the groups given > or =600 ppm with or without DEN initiation. Periportal hepatocellular hypertrophy, characterized by a marked increase in smooth endoplasmic reticulum, was observed in the groups given > or =600 ppm with or without DEN initiation. Induction of cytochrome P-450 (CYP) 1A2, 2B1, or 4A1 was noted in the fenbendazole-treated groups with or without DEN initiation; that associated with CYP 1A2 was most marked. Positive immunostaining for anti-CYP 1A1/2 or CYP 2B1/2 was observed diffusely in the livers of animals in the DEN+1,800 and DEN+3,600 ppm groups. The numbers and areas of connexin 32 (Cx32)-positive spots per square centimeter in centrilobular hepatocytes were significantly decreased in an almost dose-dependent manner with fenbendazole treatment after DEN initiation. In situ hybridization for Cx32 mRNA revealed a remarkable decrease in its expression in the centrilobular hepatocytes in the DEN+70 ppm group. The numbers of glutathione S-transferase placental-form positive single cells (plus mini foci) were significantly increased in the DEN+ 1,800 and DEN+3,600 ppm groups. Since those agents that induce CYP 2B1/2 isozymes and reduce Cx32 in centrilobular hepatocytes have been suggested to be liver tumor promoters, the present results indicate that fenbendazole may be a liver tumor promoter.     

Effects of alpha-naphthyl isothiocyanate and a heterocyclic amine, PhIP, on cytochrome P-450, mutagenic activation of various carcinogens and glucuronidation in rat liver

To elucidate the mechanism underlying suppression by -naphthylisothiocyanate (ANIT) of mammary carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP), we evaluated hepatic levels of cytochrome P-450 (CYP)enzymes, mutagenic activation of environmental carcinogens andUDP-glucuronyltransferase (UDPGT) activities in female Sprague–Dawleyrats fed a high fat diet. Immunoblot analyses revealed inductionof CYP1A1, newly found 51 and 53 kDa proteins and constitutiveCYP1A2 and 2B2 by intragastric treatment with 85 mg/kg PhIPeight times for 11 days. Although the extents of induction werenot as high as in the case of PhIP, 3 weeks feeding of 400 p.p.m.ANIT induced CYP1A1 and the 51 and 53 kDa proteins. CP1A2 levelwas decreased by the feeding of ANIT. The mutagenicity in strainTA98 of PhIP, four other heterocyclic amines (HCAs) and benzo[a]pyrenewas greatly enhanced in the presence of liver S9 mix preparedfrom rats pretreated with PhIP but not with ANIT. The mutagenicitiesof these five HCAs were significantly decreased in the presenceof liver S9 from rats pretreated with a combination of PhIPand ANIT as compared with that pretreated with PhIP alone. Thelevel of hepatic CYP1A2, which is known to be involved in themetabolic activation of PhIP, was consistently decreased inliver microsomes from rats administered PhIP plus ANIT as comparedwith that from rats administered PhIP alone. On the other hand,UDPGT activity towards 4-nitrophenol (4-NP) was enhanced usingliver microsomes prepared from rats pretreated with a combinationof PhIP and ANIT as compared with those pretreated with PhIPor ANIT alone. These results show that chemoprevention by ANITagainst PhIP-induced rat mammary carcinogenesis can be explainedby a dual action mechanism, i.e. a reduction in metabolic activationby hepatic CYP1A2 and an enhancement of detoxification by 4-NPUDPGT. The role of the newly found 51 and 53 kDa proteins inactivation of HCAs is also discussed. ^* To whom correspondence should be addressed. Tel: +81 58 237 3931; Fax: +81 58 237 5979; Email: ymori{at}gifu-pu.ac.jp     

Detection of mutagenic activity in the urine of rodents treated with p-rosaniline

p-Rosaniline was fed to male and female Fischer 344 rats and B6C3F1 mice at doses of 1,000 and 2,000 ppm for male rats and 500 and 1,000 ppm for female rats and mice of both sexes. Urine was collected overnight at 1-wk intervals over a 4-wk treatment period and frozen until its use in the mutagenicity assay. The neat urine was tested in triplicate without S-9 on Salmonella tester strains TA98, TA100, TA1535, and TA1537 at 0.75, 0.5, 0.2, and 0.05 ml per plate. When sufficient urine was available, samples were tested on TA100 in the presence of S-9. Either urine samples were pretreated for 18 hr at 37 degrees C with beta-glucuronidase, or the deconjugating enzyme was added to the top agar at the time of plating in the mutagenicity assay (non-pretreatment). Direct-acting mutagenic activity was detected on TA98 in the urine from male mice, but only when using the non-pretreatment deconjugation method. No direct-acting mutagenic activity was detected in the urine of male and female rats and female mice; however, in the presence of S-9, mutagenic activity was observed in the urine of male rats and in the urine of male and female mice regardless of the deconjugation method used. The non-pretreatment method was superior for detecting direct acting mutagenic activity, and the pretreatment method was superior for detecting mutagenic activity requiring metabolic activation by S-9.     

Human CYP2E1-dependent mutagenicity of benzene and its hydroxylated metabolites in V79-derived cells: Suppression and enhancement by ethanol pretreatment

Benzene is a human carcinogen that requires metabolic activation. We previously observed that benzene and its hydroxylated metabolites induce micronuclei in mammalian cells expressing human CYP2E1. This study was initially aimed to study another endpoint, the induction of gene mutations by those compounds in the same cell models. A V79-derived cell line expressing human CYP2E1 and sulfotransferase (SULT) 1A1 (V79-hCYP2E1-hSULT1A1) pretreated with ethanol (a CYP2E1 stabilizer) was used in the hprt gene mutagenicity assay. Phenol, hydroquinone, catechol, and 1,2,4-trihydroxybenzene all induced gene mutations, while they were inactive, or only weakly positive (hydroquinone), in parental V79-Mz cells. Unexpectedly, benzene was non-mutagenic in both cell lines, but it became positive in V79-hCYP2E1-hSULT1A1 cells using regimes of short exposure/long recovery without ethanol pretreatment, for both gene mutations and micronuclei formation. In silico molecular simulation showed binding energies and positions favorable for each compound to be oxidized by human CYP2E1, benzene demonstrating the highest affinity. By tunnel analysis, ethanol binding did not limit benzene to pass tunnel S, which was specifically active for benzene. However, its end product, acetic acid, decreased the occurrence of tunnel S from 5.4 to 2.2% and extended the length of its bottleneck from 5.5 to 9.0 Å. With residual ethanol molecules still being present in CYP2E1 for a period of time after benzene exposure, the acetic acid formed could limit the entrance of benzene, thus inhibit its metabolic activation. In summary, ethanol may interfere with the activation of benzene to mutagenic metabolites, at least in cultured cells.     

Effects of pregnancy on CYPs protein expression in rat liver

A body of evidence suggests that pregnancy may be responsible for the depression in the microsomal enzyme activity and the reduction in the total content of cytochrome P450 (CYP) in the rat liver. However, changes in expression of individual CYP isozyme remain poorly known. The current study was designed to examine the changes in CYPs protein expression in the liver of F344 rats in midpregnancy and late pregnancy by Western blot analysis and immunohistochemistry. Total nine antirat CYPs antibodies (CYP1A1, CYP2B1/CYP2B2, CYP2C6, CYP2C12, CYP2D1, CYP2D4, CYP2E1, CYP3A1, and CYP4A1) were used. In comparison with age-matched nonpregnant control rats, there were significant decreases in hepatic levels of CYP2B2, CYP2C6, and CYP4A1 in midpregnancy (day 13) and CYP2B2, CYP2C6, CYP4A1, CYP1A1, CYP2B1, and CYP2E1 in late pregnancy (day 19). The expression of CYP2C12, CYP2D1, and CYP 3A1 did not differ between nonpregnant and pregnant rats, and CYP2D4 was not detectable in microsomal proteins obtained from nonpregnant and pregnant rats at a protein loading of 20 μg total protein per lane. Immunohistochemistry showed that there were no differences in the distribution and degree of immunostainability for the abovementioned antibodies to nine CYPs between pregnant and nonpregnant rats.     

Effect of pinocembrin isolated from Boesenbergia pandurata on xenobiotic-metabolizing enzymes in rat liver

Pinocembrin, 5, 7-dihydroxyflavanone, is one of the flavanones found in the rhizomes of Boesenbergia pandurata. Previous study demonstrated that pinocembrin was neither toxic nor mutagenic to male rats. This study evaluated the effects of pinocembrin on phase I and II xenobiotic-metabolizing enzymes in rat liver. It was found that heme oxygenase activity significantly increased in 10 and 100 mg/kg bw of pinocembrin treated groups (p<0.05). However, pinocembrin did not affect the activities of NADPH: cytochrome P450 reductase, NADPH: quinone reductase, UDP-glucuronosyltransferase and glutathione-S-transferase. It also did not affect the expression of phase I metabolizing enzymes, including CYP1A1, CYP2B1, CYP2C11, CYP2E1, CYP3A2, and NADPH: cytochrome P450 reductase. In conclusion, short-term treatment of pinocembrin in Wistar rats increased the activity of heme oxygenase but did not affect on the activities of other phase II xenobiotic-metabolizing enzymes or the expression of cytochrome P450 enzymes.     

Promotion of chemically induced rat esophageal tumorigenesis with post-initiation ethanol modification.

Post-initiation ethanol modification on N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal carcinogenesis model was investigated in male, 6-week-old, F344 rats that received s.c. injections, 3 times per week, of 0.5 mg/kg NMBA for the first 5 weeks and then were treated with 0% (Group 1), 3.3% (Group 2), and 10% (Group 3) ethanol in the drinking water for up to 20 weeks. Group 4 received 10% ethanol without NMBA administration and Group 5 was maintained without any chemical treatment. There were no statistical differences in the incidence and multiplicity of esophageal tumors among Groups 1 to 3. However, the multiplicity of hyperplasias was statistically greater in Group 3 than in Groups 1 or 2. Esophageal epithelia of all rats in Groups 4 and 5 demonstrated a normal histology. BrdU labelling indices of tumors and hyperplasias in NMBA-treated groups were essentially similar, although cycline D1 was overexpressed to a greater extent in tumors and also hyperplasias of Group 3 than in Groups 1 or 2. The results indicated ethanol to exert weak promotion effects through cycline D1 overexpression on rat esophageal tumorigenesis initiated with NMBA.     

Oxidative damage and induced mutations in m13mp2 phage DNA exposed to N-nitrosopyrrolidine with UVA radiation

N:-Nitrosopyrrolidine (NPYR) is carcinogenic in rodents and undergoes alpha-hydroxylation upon microsomal CYP450 metabolism, giving rise to mutations. Previously, we reported the direct mutagenicity of NPYR, under ultraviolet A (UVA) irradiation, towards Salmonella typhimurium and phage M13mp2. In the present study, we measured the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in a replicative form of M13mp2 DNA exposed to NPYR plus UVA. Formation of 5-hydroxy-2'-deoxycytidine in calf thymus DNA treated with NPYR plus UVA was also observed. Singlet oxygen is likely to account for the formation of 8-oxodGuo. We analyzed the spectrum of mutations in lacZalpha of M13mp2 phages produced on transfecting Escherichia coli with the replicative form of phage DNA that had been treated with NPYR plus UVA. The role of oxidative DNA damage in mutagenesis was explored using mutM-proficient and -deficient E.coli strains as the hosts. A higher level of mutation was observed with the mutM-deficient host than with the -proficient host. Base substitutions at GC pairs predominated in both mutM-proficient and -deficient hosts. With the mutM-deficient host, we observed an overall increase in the percentage of GC-->TA transversions. In addition we noted that there were fewer GC-->AT transitions than in the mutM-proficient host. With these hosts, different hot spots were observed and a new GC-->TA hot spot was produced. The formation of 8-oxodGuo in DNA, which is known to induce GC-->TA transversion, may contribute to mutagenesis by NPYR plus UVA.     

Comparison of co-cultivation of V79 cells with rat hepatocytes and rat H4IIE hepatoma cells for studying nitrosamine-induced hprt gene mutations

The induction of mutations by nitrosamines in the hprt locusof V79 Chinese hamster cells was examined after metabolic activationin a co-cultivation system using either freshly isolated rathepatocytes or H4IIE rat hepatoma cells and the results obtainedwere compared with systems which employ the rat liver microsomalfraction (S9-mix). This study was also designed as a first approachto investigating the induction of point mutations by tobacco-specificnitrosamines in mammalian cells in order to obtain informationabout the significance of these compounds in connection withthe carcinogenicity of tobacco smoke. The mutagenicity of twotobacco-specific nitrosamines, 4-(methylnitroso)-1-(3-pyridol)-1-butanone(NNK) and N'-nitrosonornicotine (NNN), were investigated andcompared to two extensively investigated nitrosamines, i.e.dimethylnitrosamine (DMN) and diethylnitrosamine (DEN). DMNwas activated to mutagenic species by primary hepatocytes atµmolar concentrations, i.e. 1/100 of the concentrationsrequired for mutagenesis by DEN and NNK. NNN was not activatedto mutagenic species by liver S9 or primary hepatocytes. Thefindings shown here on the mutagenicities of NNK and NNN withliver preparations are in agreement with their relative carcinogenicpotencies. When the established liver cell line H4IIE was usedfor metabolic activation, DMN and was found to be mutagenic,whereas the results for NNN were borderline and for DEN andNNK were without effect. The fate of these compounds via differentmetabolic pathways is discussed in terms of systems for detectionof mutagenic metabolites and type of mutation induced.     

Modulation of rat liver cytochrome P450 by protein restriction assessed by biochemical and bacterial mutagenicity methods [corrected

Protein restriction (PR) significantly inhibits spontaneous and chemical carcinogenesis. Several factors seem to be involved in this effect, including a decrease in body weight, cellular proliferation and DNA damage and an increase in antioxidant defenses. The current study was designed to determine modifications in some hepatic cytochromes P450 (CYPs) due to a hypoproteic diet and to investigate its implications on chemical mutagenesis. Western blot analysis showed decreases of 73, 40 and 74% in CYP1A, CYP2B and CYP2E1 protein concentrations in hepatic microsomes from animals fed a protein-restricted (6% protein) diet for 6 weeks in comparison with microsomes from rats fed a 24% protein diet during the same period. In the same way, low protein fed animals showed a 3.5-fold decrease in hepatic CYP1A1-associated ethoxyresorufin O-deethylase activity, a 6-fold decrease in CYP1A2-associated methoxyresorufin O-demethylase activity, a 1.7-fold decrease in CYP2B1-associated penthoxyresorufin O-dealkylase activity, a 9-fold decrease in CYP2B2-associated benzyloxyresorufin O-dealkylase and, finally, a 3.4-fold decrease in CYP2E1-associated 4-nitrophenol hydroxylase activity. As a result of decreased CYP hepatic protein concentrations and enzymatic activities, liver S9 from rats fed a hypoproteic diet was less efficient in activating promutagens than S9 prepared from rats fed a 24% protein diet in the Ames test. Mutagenic potency obtained with protein-restricted S9 was reduced 25-fold for 2-aminoanthracene, 1.5-fold for N-nitrosodipropylamine, 12.5-fold for N-nitrosodibutylamine, 2-fold for cyclophosphamide and N-nitrosopyrrolidine and 71-fold for N-nitrosodimethylamine. However, the mutagenic potency of benzo[a]pyrene was the same (4 revertants/ microg) with S9 derived from rats fed either a 6 or 24% protein diet.     

Re-evaluation of the effect of ellagic acid on dimethylnitrosamine mutagenicity

Dimethylnitrosamine (DMN) is activated to mutagenic speciesin the Ames test (Salmonella typhimurium strain TA 100) by hamsterhepatic S9 preparation. This S9 activity is induced by administrationof ethanol to the animals. The organic solvents dimethylsulphoxide(DMSO) and N-methyl-2-pyrrolidinone (MP) inhibit this mutagenicity,apparently because they inhibit DMN demethylase activity (assayedas formaldehyde production). Ellagic acid, dissolved in DMSOor MP, had no inhibitory effect on DMN mutagenicity, beyondthe effect of the solvent vehicle. ²To whom correspondence should be addressed