2-Amino-3,4-dimethylimidazo[4,5-f]quinoline induces and inhibits cytochrome P450 from the IA subfamily in chick and rat hepatocytes.

Several heterocyclic amines, found in cooked food, are powerful mutagens in the Ames Salmonella mutagenicity test system. One of these, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) is one of the most mutagenic chemicals tested in this assay. In primary cultures of chick and rat hepatocytes, MeIQ, by itself, induced cytochrome P450 from the IA subfamily but was a weak inducer compared to 3-methylcholanthrene. However, in both chick and rat hepatocytes in culture, MeIQ decreased the amount of 3-methylcholanthrene-induced ethoxyresorufin deethylase activity, which is catalyzed by cytochrome P450 IA. The protein moiety of cytochrome P450 IA was decreased at MeIQ concentrations of 2.5 micrograms/ml or greater in chick hepatocytes and 25 micrograms/ml in rat hepatocytes. In hepatic microsomes from methylcholanthrene-treated chicks and rats, MeIQ was a competitive inhibitor of both ethoxyresorufin deethylase activity, a reaction catalyzed mainly by rodent cytochrome P450 IA1, and uroporphyrinogen oxidation, a reaction catalyzed by rodent P450 IA2. In cultured chick hepatocytes, MeIQ also decreased cytochrome P450-mediated oxidation of uroporphyrinogen by intact cells. The ability of MeIQ to inhibit as well as to induce cytochrome P450s of the IA subfamily may be important in assessing the mutagenic and carcinogenic effects of MeIQ in mammals.     

Expression and induction of cytochrome P450 isozymes in hyperplastic nodules of rat liver

The change of cytochrome P450 (P450) isozymes in a early stageof hepatocarcinogenesis in male F344 rats has been studied.Liver microsomes were prepared from normal rats (group 1), ratstreated with diethylnitrosamine (DEN) alone, which developedno hyperplastic nodules (group 2), and rats treated with DENplus 2-acetylaminofluorene, which developed many hyperplasticnodules (group 3). The amount and activity of P450IA1 and P450IA2expressed in the liver were analyzed by several immunologicalmethods using monoclonal antibodies against the P450 isozymesand a mutagenicity test. In the group 2 and 3 rats, the totalamount of P450 and the amount of P450IA2 were much smaller thanthose in the group 1 rats, and P450IA1 was detected only fromthe group 3 rats. As observed by immunohistochemistry, P450IA1was prominent in hyperplastic nodules developed in the group3 rats, and the distribution of P45OIA1⁺ cells in individualnodules was heterogeneous. When the rats were treated with aP450 inducer, 3-methoxy-4-aminoazobenzene or 3-methylcholanthrene,both P450IA1 and P450IA2 were induced in all groups of rats;however, the induction rates of the P450 isozymes, especiallythat of P450IA2, in the group 3 rats were smaller than thosein the group 1 and 2 rats. The present work demonstrated thatP450IA1, which is responsible mainly for detoxication of aromaticamine carcinogens, increased in level along with the developmentof hyperplastic nodules, whereas P450IA2, which is responsiblefor mutagenic or carcinogenic activation of these carcinogens,decreased in its amount and inducibility.     

Metabolic deactivation of furylfuramide by cytothrome P450 in human and liver microsomes

Metabolic deactivation of furylfuramide by human and rat livermicrosomal cytochrome P450 enzymes has been investigated ina system measuring induction of umu gene expression responsein Salmonella typhimurium TA1535/pSK1002. Both human and ratliver microsomes catalyzed the metabolism of furylfuramide toinactive form(s) that are incapable of inducing umu gene expressionin the tester strain. The reaction required an NADPH-generatingsystem and molecular oxygen and was inhibited by carbon monoxide,suggesting that a cytochrome P450-linked monooxygenase systemis prerequisite for the deactivation reaction. With liver microsomesfrom variously pretreated rats, 3-methylcholanthrene was foundto be a powerful inducer for the furylfuramide-metabolizingactivity, and antibodies raised against rat P450IA1(BNF-B, c)and P450IA2(ISF-G, d) inhibited the microsomal activity. Humanliver microsomal furylfuramide-metabolizing activity was alsoinhibited significantly by anti-P450IA2 lgG but weakly by anti-P450IA2IgG. In liver microsomes prepared from seven different humansamples, the activities of deactivation of furylfuramide werefound to correlate with the amounts of immunoreactive proteinrelated to rat P450IA2 and with the monooxygenase activitiesof metabolic activation of 2-amino-3,4-dimethyl-imidazo [4,5-]quinoline(MeIQ) and of ethoxyresorufin O-deethylation. These resultssuggest that P450IA1 and P450IA2 in rats, and P45OPA (IA2, thephenacetin O-deethylase and ortholog of rat P450IA2) in humansare the major enzymes involved in the deactivation of furylfuramidein liver microsomes. The metabolic studies involving HPLC analysisof products followed by spectrophotometric examination havealso suggested that furylfuramide can be degraded very rapidlythrough the aerobic metabolism by liver microsomes.     

Roles of different cytochrome P450 enzymes in bioactivation of the potent hepatocarcinogen 3-methoxy-4-aminoazobenzene by rat and human liver microsomes

The potent hepatocarcinogen 3-methoxy-4-aminoazobenzene (3-MeO-AAB) has been reported to be bioactivated to mutagenic intermediates by rat liver microsomal cytochrome P450 (P450) and to be a selective inducer of rat P450IA2. In this study we have further investigated the roles of individual rat and human P450 enzymes in the bioactivation of this hepatocarcinogen in a Salmonella typhimurium TA1535/pSK1002 system where umu response is indicative of DNA damage. 3-MeO-AAB was found to be bioactivated by liver microsomal enzymes from rats and humans in this assay system. The liver microsomal activities are increased by pretreatment of rats with various P450 inducers such as phenobarbital (PB), beta-naphthoflavone (BNF), dexamethasone (DEX), acetone, ethanol, isoniazid (INH), diphenylhydantoin and valproic acid, and can be inhibited considerably by SKF-525A and metyrapone. alpha-Naphthoflavone (ANF) is also an inhibitor for the reaction catalyzed in BNF-treated rats, but stimulated the microsomal activity in DEX-treated rats. Evidence has also been obtained that specific antibodies raised against P450IIB1, P450IA1 or IA2, P450IIE1, and P450IIIA2 inhibited the activation in liver microsomes from rats pretreated with PB, BNF, INH and DEX respectively, suggesting the possible roles of several P450 enzymes in the bioactivation of 3-MeO-AAB. The results obtained with reconstituted monooxygenase systems containing various rat P450 enzymes are highly supportive of this conclusion. Human liver microsomal activation of 3-MeO-AAB was also inhibited to various extents by antibodies raised against P450IA2, P450MP, P450IIE1 and P450IIIA4. In a reconstituted system containing purified forms of human P450, P450IA2 was the most active in catalyzing 3-MeO-AAB, followed by P450IIIA4 and P450MP. ANF, a known activator of P450IIIA-catalyzed reactions, caused an increase in activation of 3-MeO-AAB in human liver microsomal and P450IIIA4- and P450MP-containing reconstituted systems. From these results it is concluded that multiple P450 enzymes in rat and human liver microsomes are involved in the bioactivation of 3-MeO-AAB, regardless of its selective induction of the rat P450IA2 gene.     

Species difference among experimental rodents in induction of P450IA family enzymes by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

Rats, mice, hamsters and guinea pigs were given an i.p. injection of 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP), a protein-derived pyrolysate component present in cooked foods, and inductions of cytochrome P450 (P450) in the liver and kidney of these animals were examined. The activity and amount of P450s corresponding to the rat P450IA1 and P450IA2 were assessed by means of a bacterial mutation test using 3 carcinogenic heterocyclic aromatic amines including PhIP as substrates and by Western blotting with a monoclonal antibody reactive with both P450IA1 and P450IA2. In rats, PhIP induced P450IA1, P450IA2 and a new but unspecified P450 isozyme in the liver, and induced P450IA1 in the kidney. However, PhIP induced none of these P450 isozymes in mice, hamsters and guinea pigs.     

Species Difference among Experimental Rodents in Induction of P450IA Family Enzymes by 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

Rats, mice, hamsters and guinea pigs were given an i.p. injection of 2-amino-1-methyl-6-phenyl-imidazo[4,5- b ]pyridine (PhIP), a protein-derived pyrolysate component present in cooked foods, and inductions of cytochrome P450 (P450) in the liver and kidney of these animals were examined. The activity and amount of P450s corresponding to the rat P4S0IA1 and P450IA2 were assessed by means of a bacterial mutation test using 3 carcinogenic heterocyclic aromatic amines including PhIP as substrates and by Western blotting with a monoclonal antibody reactive with both P4S0IA1 and P450IA2. In rats, PhIP induced P450IA1, P450IA2 and a new but unspecified P450 isozyme in the liver, and induced P450IA1 in the kidney. However, PhIP induced none of these P450 isozymes in mice, hamsters and guinea pigs.     

Differential rates of metabolic activation and detoxication of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by different cytochrome P450 enzymes

Rat liver microsomes metabolized the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to the genotoxic metabolite 2-hydroxamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (2-hydroxamino-PhIP) and to the detoxified product 2-amino-4'-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP). A 25-fold higher rate of metabolism was measured in microsomes from polychlorinated-biphenyl-treated rats (94 nmol/mg proteins/30 min) in comparison with those from untreated rats. Other effective inducers of PhIP metabolism were beta-naphthoflavone and isosafrole (ISF), whereas phenobarbital was ineffective. About twice as much 2-hydroxamino-PhIP as 4'-hydroxy-PhIP was formed in microsomes irrespective of the inducer the rats had been treated with. The metabolism was dependent on NADPH and was abolished by the cytochrome P450 inhibitor alpha-naphthoflavone. In a reconstituted enzyme system purified rat cytochrome P450 IA2 (P450ISF-G) had the highest N-hydroxylation rate (30 nmol/nmol P450/30 min) closely followed by the rat cytochrome P450 IA1 (P450BNF-B). Less activity was seen with rat P450 IIC11 (P450UT-A) and rabbit P450 IA2 (P450 LM4). Rat P450 IIE1 (P450j), P450 IIB1 (P450PB-B) and rabbit P450 IIB4 (P450 LM-2) and P450 IIE1 (P450 LM3a) were essentially inactive. Rat P450 IA1 (P450BNF-B) produced five times more 4'-hydroxy-PhIP (32 +/- 2 nmol/nmol P450/30 min) than did P450 IA2 (P450ISF-G). Hence, the measured ratio of activation to detoxication for rat P450 IA2 (P450ISF-G) enzyme was 7-fold higher than that of the other active P450 enzymes.     

Cytochrome P450IA expression in adult and fetal human liver.

A monoclonal antibody has been produced that recognizes the cytochrome P450 form, cytochrome P450IA1, but not cytochrome P450IA2 in rats and recognizes a single protein band of similar mol. wt on immunoblots of human liver microsomes. Immunohistochemical studies have been carried out with this antibody to investigate the localization and distribution of cytochrome(s) P450 of the P450IA family in human liver. Cytochrome P450IA was identified in both adult and fetal liver and in each case it was localized predominantly to hepatocytes. In adult liver there was a heterogeneous distribution of cytochrome P450IA immunoreactivity with cytochrome P450IA mainly present in zone 3 hepatocytes of the liver acinus. Within fetal liver there was a uniform distribution of cytochrome P450IA immunoreactivity with no apparent zonal distribution. Bile duct epithelium did not show definite immunostaining for cytochrome P450IA in either adult or fetal liver.     

Effects of the food mutagens MeIQx and PhIP on the expression of cytochrome P450IA proteins in various tissues of male and female rats.

The promutagenic 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) found in cooked food are converted to their active forms mainly by cytochrome P450 forms IA1 and IA2. By induction of these isoenzymes the food mutagens could thus influence their own rate of activation. Male and female Wistar rats were given MeIQx, PhIP, beta-naphthoflavone (BNF) or saline i.p. at 50 mg/kg body wt on three consecutive days. On the fourth day the rats were killed and lungs, kidneys, liver and intestines taken. The microsomal fraction from each organ was prepared as well as 9000 g supernatant from the liver. The induction of cytochrome P450IA was measured at the protein level by enzymatic assays (ethoxyresorufin-O-deethylation, Ames' mutagenicity test) and immunoassays (Western blot) and at the pretranslational level by RNA hybridization (Northern blot). The binding affinities of MeIQx, PhIP and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-receptor were studied by evaluation of the competition with 3H-labelled TCDD for specific binding. Ethoxyresorufin-O-deethylase (EROD) activity was significantly increased in the liver (males 2.1-fold, females 3.3-fold), kidneys (males 2.1-fold, females 1.8-fold) and lungs (males 4.3-fold, females 3-fold) of the MeIQx-treated rats. Furthermore, the levels of cytochrome P450IA proteins were increased in these animals. It was not possible, however, to detect the corresponding mRNA. In the case of the PhIP-treated animals a significantly increased EROD activity (2.7-fold) and an increased cytochrome P450IA protein level were seen only in the male lungs. Only a very weak TCDD-receptor affinity was observed for PhIP, whereas MeIQx or IQ did not appear to compete significantly with [3H]TCDD for binding to the TCDD-receptor. It is concluded that MeIQx is a weak inducer of cytochrome P450IA in several organs of the rat, while PhIP induced these isoenzymes only in the male lungs. More work is needed to clarify the mechanism(s) whereby this induction occurs.     

Smoking and peripheral type of cancer are related to high levels of pulmonary cytochrome P450IA in lung cancer patients.

A specific member of the cytochrome P450 superfamily of enzymes, designated P450IA (including 2 isozymes, P450IA1 and P450IA2), which is involved in the metabolic activation of polycyclic aromatic hydrocarbons and aromatic amines, was studied in lung tissue from 25 lung cancer patients by immunohistochemistry. The pulmonary activity of a P450IA1-dependent enzyme, aryl hydrocarbon hydroxylase (AHH), from the same patients was also measured. Cytochrome P450IA was localized principally in the peripheral airways in alveolar epithelium of types I and II and in ciliated columnar and cuboidal bronchiolar epithelium. The amount of P450IA in the bronchial wall was minimal and was localized mainly in the capillary endothelium and the epithelium of the bronchial glands. Smoking was the most important factor related to the presence of P450IA and the AHH activity in lung tissue. None of the 10 ex-smokers, but all except I of the current smokers had detectable level of P450IA. The localization of the cancer was also correlated with the presence of cytochrome P450IA. Peripheral lung tissue stained positively in all patients with a peripheral adenocarcinoma who currently smoked (8/8) but in less than half of those with a bronchial cancer who were smokers (3/7). Our data suggest that the smokers who have an inducible cytochrome P450IA are especially at increased risk of developing lung cancer of the peripheral adenocarcinomatous type.     

Effect of dietary fiber on cytochrome P450IA1 induction in rat colonic mucosa.

The effect of dietary fiber on the induction of cytochrome P450IA1 in rat colonic mucosa after a single intragastric injection of 3-methylcholanthrene (3MC, 20 mg/kg) was investigated by examining the drug-metabolizing enzyme activity, immunoblotting for cytochrome P450IA1 and immunohistochemistry. 7-Ethoxycoumarin-O-deethylase activities were approximately 20-fold higher in microsomes from both proximal and distal portions of the colonic mucosa of control diet-fed 3MC-treated rats compared with those of control diet-fed untreated rats. Strong immunofluorescence for cytochrome P450IA1 was localized in the cytoplasm of the colonic mucosa surface epithelium from the control diet-fed 3MC-treated rats. 7-Ethoxycoumarin-O-deethylase activity and cytochrome P450IA1 content determined by immunoblotting were significantly lower in wheat bran-fed 3MC-treated rats than in control diet-fed 3MC-treated rats. Immunohistochemical analysis showed much weaker immunofluorescence for cytochrome P450IA1 in the surface epithelium of the colonic mucosa of the wheat bran-fed 3MC-treated rats. These observations suggested that dietary fiber can affect the induction of cytochrome P450IA1 in colonic mucosa by dietary inducers or carcinogens.     

Altered drug metabolizing potential of acinar cell lesions induced in rat pancreas by hydroxyaminoquinoline 1-oxide

Foci of atypical acinar cells observed in male rats 1 year after a single injection of hydroxyaminoquinoline 1-oxide (HAQO) were assessed immunohistochemically for altered expression of a number of enzyme forms considered to play important roles in drug metabolism. The pancreatic lesions, classified as of either basophilic or eosinophilic type on histological appearance, demonstrated distinctive patterns of altered enzyme phenotype. On the one hand, the basophilic foci composed of enlarged cells/nuclei with very prominent nucleoli were characterized by increase in GST-P, G6PD and P450 PB1 and MC2 forms. The eosinophilic type, in contrast, comprised smaller cells demonstrating elevated P450 MC1 and PB1 but not MC2, normal G6PD and strong GST-P binding limited only to a proportion of the nuclei. Both shared decreased GGT and almost total lack of GST-B positive connective tissue and ductular elements. Apparent islet cell lesions and normal islet tissue were characterized by a distinct enzyme phenotype strongly positive for all P450 species investigated. The results indicate that HAQO-induced putative preneoplastic pancreatic lesions, like equivalent carcinogen associated with focal populations in liver, kidney and ductular pancreas, demonstrate a non-random altered expression of specific drug metabolizing enzyme species.     

Differences in the modulation of P450IA1 and epoxide hydratase expression by benz[a]anthracene and 2,3,7,8-tetrachlorodibenzo-p-dioxin in mouse embryo versus mouse hepatoma-derived cell lines

Polycyclic aromatic hydrocarbon (PAH)-induced C3H/10-T1/2/CL8 mouse embryo fibroblasts (10T1/2) and mouse hepatoma-derived Hepa 1c1c7 cells (Hepa-1), exhibit comparable total cytochrome P450 levels and total PAH-metabolizing activities but very different distributions of PAH metabolites. Based on anti-P450IA1-IgG inhibition data, P450IA1 contributes essentially all PAH metabolism in Hepa-1 microsomes but is not involved in PAH metabolism by 10T1/2 cells. In addition, the microsomal epoxide hydratase (EHm) in Hepa-1 cells is far less effective in dihydrodiol (diol) formation compared to that in 10T1/2 microsomes [Pottenger, L.H. and Jefcoate, C.R. Carcinogenesis, 11, 321-327 (1990)]. In the present study, the levels of expression of P450IA1 and EHm proteins and the corresponding mRNAs, both prior to and following exposure to benz[a]anthracene (BA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have been correlated with microsomal PAH metabolism by each cell type. In 10T1/2 cells, P450IA1 protein (56 kd) and mRNA (2.6 kb) were detectable at extremely low levels in only two of five cell preparations and then only after maximum induction by TCDD and BA. Thus although 10T1/2 cells contain functional Ah receptors, their capacity to induce P450IA1 is highly suppressed, representing at most 2% of the total P450. TCDD (10 nM) was 4-fold more effective than BA (10 microM) in inducing P450IA1 mRNA, while the levels of immunodetectable protein were comparable. An even greater discrepancy between P450IA1 mRNA and protein levels was seen in BA-induced Hepa-1 cells, where a 4-fold increase in mRNA was paralleled by a 20-fold increase in protein. This difference is probably due to the greater effect of BA depletion on mRNA compared to protein levels. In 10T1/2 cells, BA and TCDD were equally effective at increasing expression of an unidentified 1.9 kb mRNA sequence that blotted very weakly with the P450IA1 cDNA probe. The expression of this mRNA was independent from that of P450IA1. A similar band was visible in Hepa-1 cells less than 1% of the P450IA1 mRNA. EHm mRNA was almost 3-fold higher in 10T1/2 compared to Hepa-1 cells and was unaffected by cell treatments. In Hepa-1 cells, BA and TCDD elevated EHm protein and hydrating activity to levels comparable to those expressed in 10T1/2 cells. It is, therefore, suggested that the relative ineffectiveness of Hepa-1, compared to 10T1/2 EHm, to hydrate low levels of PAH-epoxides is due to differences between the two proteins or their disposition in the microsomal membrane.     

Induction of cytochrome P450IA1 in rat colon and liver by indole-3-carbinol and 5,6-benzoflavone

It is known that consumption of cruciferous vegetables protects against the chemical induction of cancer in many organs. It has been suggested that this protection is mediated through an effect on the cytochrome P450 monooxygenase system. This system is responsible for the activation of a number of chemical carcinogens to their ultimate forms. In the present study, the effect of indole-3-carbinol (I3C) and 5,6-benzoflavone (5,6BF) on the expression of cytochrome P450IA1 in rat colon and liver has been investigated. Cytochrome P450IA1 mRNA was induced in colon following a single oral administration of I3C or 5,6BF. A biphasic induction profile was obtained with maxima at 4 and 16 h post-administration. Both inducers caused an approximately 2-fold increase in P450IA1 mRNA at 4 h and a 10-fold increase at 16 h. In contrast, both cytochrome P450IA1 and IA2 mRNAs was increased over the control between 4 and 24 h. The total amount of P450IA mRNAs in liver at 4 and 16 h was increased about 2- and 4-fold respectively by I3C; 5,6BF induced the P450IA mRNAs 4- and 5-fold respectively. The expression of cytochrome P450IA1 and IA2 is induced by I3C and several flavones present in cruciferous vegetables. This suggests that one of the protective effects of cruciferous vegetables in the reduction of chemically induced cancer may be regulation of cytochrome P450s involved in the metabolism of the chemical carcinogens.     

Effects of synthetic and naturally occurring flavonoids on benzo[a]pyrene metabolism by hepatic microsomes prepared from rats treated with cytochrome P-450 inducers

Activity-directed fractionation of Trifolium pratense resulted in isolation of the isoflavone biochanin A, a potent inhibitor of metabolic activation of the carcinogen benzo[a]pyrene (B[a]P) in cells in culture. To determine the structural features required for maximal inhibition of cytochrome P-450 mediated metabolism of B[a]P, the inhibitory potencies of 23 flavonoids on metabolism of B[a]P to water-soluble derivatives were examined in liver S-9 homogenate from rats induced with Aroclor 1254. Flavones were much more efficient inhibitors than their corresponding isoflavone or flavanone analogs. Most flavonols were as effective inhibitors as their flavone analogs with the exception of kaempferide. Flavones with two hydroxyl or two methoxyl groups at positions 5 and 7 were the most active. Although all eight flavonoids tested effectively inhibited B[a]P metabolism by beta-naphthoflavone-induced microsomes, none were very effective inhibitors of B[a]P metabolism by phenobarbitol-induced microsomes, and only three were effective inhibitors of B[a]P metabolism by microsomes from non-induced rats. These results indicate that flavones or flavonols that contain free 5- and 7-hydroxyls are potent inhibitors of P-450 induced by beta-naphthoflavone (P-450IA1 and/or P-450IA2) and may potentially be useful as chemopreventive agents against hydrocarbon-induced carcinogenesis.     

Effect of tamoxifen feeding on metabolic activation of tamoxifen by the liver of the Rhesus monkey: Does liver accumulation of inhibitory metabolities protect from tamoxifen-dependent genotoxicity and cancer?

Tamoxifen induces hepatocellular carcinomas in rats and is convertedby rat hepatic cytochrome P450 enzymes into reactive metabolitescapable of forming adducts with nucleic acids, proteins andchromosomal aberrations. In rats tamoxifen has also been shownto induce liver cytochrome P450 enzymes, to stimulate its ownmetabolism leading to greater covalent binding and to inducea higher degree of unscheduled DNA synthesis. This suggeststhat, at least in the rat, a sensitive species, tamoxifen maycontribute significantly to its genotoxic and carcinogenic potential,by assisting its own metabolic activation. We have now investigatedthe effect of feeding tamoxifen to male and female Rhesus monkeys.A marked induction of the hepatic cytochrome(s) P450 is foundin the monkey but, in spite of this, the in vitro metabolismof 7-ethoxy-resorufin by microsomes from treated animals ismarkedly inhibited and so is the dealkylation of two other 7-alkoxy-resorufinsubstrates. Evidence is presented for the accumulation in theliver of monkeys treated with tamoxifen of a powerful inhibitorof drug metabolism, and the inhibitor is identified as a metaboliteof tamoxifen, its N,N-didesmethyl derivative. The level of ³²P-postlabelledDNA adducts was considerably higher in rats given tamoxifenthan in similarly treated monkeys. Also, whereas rats respondedto tamoxifen treatment with a marked increase in covalent bindingto microsomal protein, in the monkeys, where accumulation ofthe inhibitory metabolite in the microsomal fraction was alsoseen, convalent binding was not greater with microsomes fromtreated animals than in the corresponding controls. N, N-Didesmethyl-tamoxifen,added in vitro to human and rat microsomes, reduced significantlythe extent of covalent binding, suggesting that the accumulationof the metabolite observed in the liver of primates may discouragethe cytochrome P450-dependent conversion of tamoxifen into reactivederivatives and in this way protect against the formation ofadducts. This mechanism may also contribute to protecting theprimate against liver cancer.     

Metabolism of 2-acetylaminofluorene and benzo(a)pyrene and activation of food-derived heterocyclic amine mutagens by human cytochromes P-450

The human P-450 CYP1A1 gene and a P450IA2 complementary DNA have been expressed in Cos-1 cells and the expressed proteins were assayed for their capacity to metabolize the carcinogens 2-acetylaminofluorene (AAF), benzo(a)pyrene, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was determined. The expressed human P450IA1 and P450IA2 proteins, when run on a 7.5% sodium dodecyl sulfate-polyacrylamide gel, migrated with different mobilities, with the former displaying the lower molecular weight. In human liver microsomes from 18 subjects, only a protein band corresponding to P450IA2 was detectable. Cos-1 cell-expressed P450IA1 and P450IA2 were capable of N-hydroxylating AAF and these activities were inhibited by alpha-naphthoflavone. In human liver microsomes, a correlation of r = 0.76 (P less than 0.05; n = 18) was obtained between AAF N-hydroxylase activity and P450IA2 content. AAF N-hydroxylase activity of human liver microsomes was also strongly inhibited by alpha-naphthoflavone. Except in the case of PhIP, where both proteins exhibited similar activities, P450IA2 was at least an order of magnitude more efficient than P450IA1 in activating IQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline to mutagens as measured in the Ames test. Statistically significant correlations were obtained between IQ activation and P450IA2 content (r = 0.75, r2 = 0.56) and PhIP activation and P450IA2 content (r = 0.71, r2 = 0.5) in human liver microsomes. The activation of both IQ and PhIP by expressed proteins and human liver microsomes was strongly inhibited by alpha-naphthoflavone. The above data suggest a major role for P450IA2 in activation (N-hydroxylation) of aromatic amides and amines in human liver. When benzo(a)pyrene hydroxylase activity was determined, only Cos-1 cell-expressed P450IA1 exhibited appreciable activity. While alpha-naphthoflavone inhibited Cos-1 cell-expressed P450IA1 benzo(a)pyrene hydroxylase activity, it caused a marked stimulation of this activity in human liver microsomes, which lack P450IA1 protein. The lack of a role for P450IA proteins in benzo(a)pyrene metabolism is further supported by the poor correlation (r = 0.43, P greater than 0.05) between this activity and P450IA2 content of human liver microsomes. However, when P450IIIA3 content of the above human liver microsomes was determined by using the Western blot technique and correlated with benzo(a)pyrene metabolism, an r value of 0.70 (P less than 0.5) was obtained. These data suggest that human P450IIIA proteins are involved in benzo(a)pyrene metabolism.     

Reductive metabolism and protein binding of chromium(VI) by P450 protein enzymes

The cytochrome P450-dependent reduction of Cr(VI) using reconstituted phospholipid vesicles containing purified preparation of various forms of rabbit and rat liver microsomal cytochrome P450 has been investigated. The alcohol-induced form of the rat, P450IIE1, was the most efficient enzyme, 7.2 +/- 0.40 nmol Cr/nmol P450/min, whereas the corresponding rates for rat P450IA1, rat IIB1, rabbit IIB4, rabbit IA2 and rabbit IIE1 were 1.7 +/- 0.09, 2.5 +/- 0.08, 1.6 +/- 0.08, 2.5 +/- 0.15 and 1.6 +/- 0.08 nmol Cr/nmol P450/min respectively. NADPH-cytochrome P450 reductase had Cr(VI) reductase activity which was dependent on enzyme concentration. Below 0.15 nmol P450 reductase/ml the sp. act. was low and constant, while at a higher concentration the activity was markedly dependent upon the amount of enzyme present. In a quantitative binding assay it was shown that binding of [51Cr]Cr(VI) to the catalytic enzymes was proportional to the enzyme concentration up to 0.8 nmol P450/ml, which caused binding of 70% of the total radioactivity. Analysis by SDS-PAGE and autoradiography exhibited binding to the individual catalytic proteins of [51Cr]Cr. EDTA treatment removed the radioactivity from the bands matching P450 and P450 reductase, indicating that Cr(III) is bound to the proteins. The reducing activity of both P450 and P450 reductase was potently inhibited by oxygen. The inhibitory effect of oxygen is not due to reoxidation of the reduced Cr and redox cycling. Rat P450IA1 ethoxycoumarin O deethylase activity was inhibited after preincubation with chromate (CrO4(2-). The P450 reductase inhibitor 2'-AMP stimulated the anaerobic P450 reductase dependent Cr(VI) reductase rate approximately 2-fold. Both CO and CCl4 inhibited the different P450 enzymes to various extents. With rabbit P450IIE1 CCl4 stimulated the Cr(VI) reduction approximately 4-fold, whereas the activity of the other enzymes was inhibited when the reconstituted system was incubated with CrO4(2-) and CCl4 prior to NADPH addition. Neither CO nor CCl4 affected the Cr(VI) reducing activity of the P450 reductase. The difference in CrO4(2-) reducing activity of the P450 enzymes and binding to the enzymes may be important for in vivo endoplasmic catalytic metabolism of CrO4(2-).     

Analysis of peroxisome proliferator-induced preneoplastic and neoplastic lesions of rat liver for placental form of glutathione S-transferase and gamma-glutamyltranspeptidase

Structurally unrelated peroxisome proliferators induce altered areas (AA), neoplastic nodules (NN), and hepatocellular carcinomas (HCC) in rats and mice. In this study we have examined several AA, NN, and HCC induced by Wy-14,643 and ciprofibrate in rats for gamma-glutamyltranspeptidase (GGT) and the placental form of glutathione S-transferase (GST-P) by histochemical and immunohistochemical procedures, respectively. In Wy-14,643-treated animals 96-100% of NN and HCC was negative for both GGT and GST-P. Eighty-seven % of the AA was negative for both GGT and GST-P, and only 2% was positive for both the marker enzymes. In ciprofibrate-treated animals 52% and 75% of AA were negative for GST-P and GGT, respectively, and 16% was positive for both the enzymes. However, a large majority of NN and HCC (more than 95%) was devoid of both these marker enzymes. Thus these studies clearly indicate that the hepatic lesions induced by peroxisome proliferators display different phenotypic properties as compared to the lesions induced by commonly used classical liver carcinogens. We conclude that GGT and GST-P are not the ideal markers for identifying AA, NN and HCC induced by peroxisome proliferators.