Metabolic oxidation of the carcinogens 4-aminobiphenyl and 4,4'-methylene-bis(2-chloroaniline) by human hepatic microsomes and by purified rat hepatic cytochrome P-450 monooxygenases

Metabolic N-oxidation and ring-oxidation of carcinogenic arylamines by hepatic cytochromes P-450 are generally regarded as critical activation and detoxification pathways, respectively. Two arylamines with known human exposure, 4-aminobiphenyl (ABP) and 4,4'-methylene-bis(2-chloroaniline) (MOCA), have been examined as substrates for 10 different purified rat hepatic cytochromes P-450 and for human liver microsomal preparations from 22 individuals. Metabolites were analyzed by high-performance liquid chromatography and flow scintillation techniques. As reported for certain other carcinogenic arylamines, the isosafrole-inducible isozyme, P-450ISF-G, had the highest catalytic activity for ABP N-oxidation (13.6 nmol/min/nmol P-450), but P-450BNF-B, P-450UT-A, P-450UT-F, and P-450PB-B also showed appreciable activity. Ring-oxidation of ABP occurred only to a minor extent. In contrast, N-oxidation of MOCA was preferentially catalyzed by the phenobarbital-inducible enzymes, P-450PB-B and P-450PB-D (9.0 and 6.6 nmol/min/nmol P-450, respectively). MOCA ring-oxidation and methylene carbon oxidation showed varied cytochrome P-450 selectivity and accounted for 14 to 79% of total oxidation products. There was a 44-fold variation in rates of ABP N-oxidation in the 22 human liver microsomal preparations, while rates of N-oxidation of MOCA varied only 8-fold. Ring/methylene carbon-oxidation of MOCA accounted for 6-19% of total oxidation products in the case of the human microsomal preparations, whereas ring-oxidation of ABP accounted for less than 7% of total oxidation. In addition, there was a strong correlation (R = 0.90) between rates of ABP N-oxidation and phenacetin O-deethylation, which is considered a human genetic polymorphism. Moreover, both the ABP N-oxidation and phenacetin O-deethylation activities of human liver microsomes showed a good correlation (R = 0.72) with the levels of cytochrome P-450 immunochemically related to rat P-450ISF-G. These data indicate that specific inducible and constitutive cytochromes P-450 are involved in the metabolic activation and detoxification of the carcinogens ABP and MOCA. Therefore, individual profiles of cytochromes P-450, affected by both environmental and genetic factors, may be significant determinants of individual susceptibility to arylamine carcinogenesis.     

Metabolic activation of phenacetin and phenetidine by several forms of cytochrome P-450 purified from liver microsomes of rats and hamsters

Metabolic activation of phenacetin by liver microsomes proceeds via both phenetidine and N-hydroxyphenacetin to direct-acting mutagens, i.e., N-hydroxyphenetidine and p-nitrosophenetole. Five different molecular species of cytochrome P-450 have been purified from liver microsomes of drug-pretreated Wistar rats or Syrian hamsters and their abilities to activate phenetidine and phenacetin were compared using reconstituted microsome systems. High-spin forms of cytochrome P-450 purified from 3-methylcholanthrene-pretreated rats (MC-P-448-H) or hamsters (P-488 ham-II) showed higher catalytic activity for N-hydroxylation of phenetidine than three other low-spin forms of cytochrome P-450 purified from the same animals or from phenobarbital-pretreated rats. MC-P-448-H and P-488 ham-II required the presence of cytochrome b5 for their maximum activities in the reconstituted system. The five forms of cytochrome P-450, however, exhibited no measurable activity for N-hydroxylation of phenacetin either with or without cytochrome b5. The mutagenicity of phenacetin and phenetidine toward Salmonella typhimurium TA100 was generated when the reconstituted microsomes containing MC-P-488-H or P-488 ham-II were used as activating enzymes. From these results, it was suggested that high-spin forms of cytochrome P-450 (MC-P-448-H and P-448 ham-II) played an important role in the metabolic activation of phenacetin to the direct-acting mutagens.     

Metabolic oxidation of carcinogenic arylamines by rat, dog, and human hepatic microsomes and by purified flavin-containing and cytochrome P-450 monooxygenases

Hepatic N-oxidation and aryl ring oxidation are generally regarded as critical activation and detoxification pathways for arylamine carcinogenesis. In this study, we examined the in vitro hepatic metabolism of the carcinogens, 2-aminofluorene (2-AF) and 2-naphthylamine (2-NA), and the suspected carcinogen, 1-naphthylamine (1-NA), using high-pressure liquid chromatography. Hepatic microsomes from rats, dogs, and humans were shown to catalyze the N-oxidation of 2-AF and of 2-NA, but not of 1-NA; and the rates of 2-AF N-oxidation were 2- to 3-fold greater than the rates of 2-NA N-oxidation. In each species, rates of 1-hydroxylation of 2-NA and 2-hydroxylation of 1-NA were comparable and were 2- to 5-fold greater than 6-hydroxylation of 2-NA or 5- and 7-hydroxylation of 2-AF. Purified rat hepatic monooxygenases, cytochromes P-450UT-A, P-450UT-H, P-450PB-B, P-450PB-D, P-450BNF-B, and P-450ISF/BNF-G but not P-450PB-C or P-450PB/PCN-E, catalyzed several ring oxidations as well as the N-oxidation of 2-AF. Cytochromes P-450PB-B, P-450BNF-B, and P-450ISF/BNF-G were most active; however, only cytochrome P-450ISF/BNF-G, the isosafrole-induced isozyme, catalyzed the N-oxidation of 2-NA. The purified porcine hepatic flavin-containing monooxygenase, which was known to carry out the N-oxidation of 2-AF, was found to catalyze only ring oxidation of 1-NA and 2-NA. No activity for 1-NA N-oxidation was found with any of the purified enzymes. These data support the hypothesis that 1-NA is probably not carcinogenic. Furthermore, carcinogenic arylamines appear to be metabolized similarly in humans and experimental animals and perhaps selectively by a specific form of hepatic cytochrome P-450. Enzyme mechanisms accounting for the observed product distributions were evaluated by Hückel molecular orbital calculations on neutral, free radical, and cation intermediates. A reaction pathway is proposed that involves two consecutive one-electron oxidations to form a paired substrate cation-enzyme hydroxyl anion intermediate that collapses to ring and N-hydroxy products.     

Metabolism of arylhydrazines by cytochrome P-450 mixed function oxidases and prostaglandin(H)synthase from mouse lungs

The arylhydrazines 4-methylphenylhydrazine hydrochloride, N'-acetyl-4-methylphenylhydrazine and N'-acetyl-4-hydroxymethylphenylhydrazine (HMPH) were metabolized by ram seminal vesicle prostaglandin(H)synthase (P(H)S) and by cytochrome P-450- and P(H)S-dependent enzymes from mouse lung. Based on the Km-values, the cytochrome P-450 enzymes were the most efficient, suggesting that they would be responsible for the metabolic activation of these compounds in vivo. Cytochrome P-450-dependent metabolism was inhibited by metyrapone and SKF-525A, ruling out the involvement of flavin mono-oxygenase. Agaritine, an arylhydrazide, was poorly metabolized by both systems.     

The role of highly purified cytochrome P-450 isozymes in the activation of 4-aminobiphenyl to mutagenic products in the Ames test

The role of cytochromes P-450 and P-447 in the activation of4-aminobiphenyl to mutagens in the Ames test was studied usingS9 preparations and highly purified isozymes. S9 preparationsfrom ß-naphthoflavone-pretreated rats were more efficientin converting 4-aminobiphenyl to mutagens than the correspondingpreparations from phenobarbitone-pretreated animals. Similarly,reconstituted systems comprising purified cytochrome P-447 weretwice as efficient as cytochrome P-450 in activating the carcinogen.Of all the known Phase I metabolites of 4-aminobiphenyl, onlythe N-hydroxy-derivative was mutagenic in the Ames test. Thesefindings indicate that arylamine N-hydroxylase is a cytochromeP-450 dependent enzyme, and the nature of the isozyme of thecytochrome is an important determinant of its mutagenicity.     

Metabolic Activation of Pyrolysate Arylamines by Human Liver Microsomes; Possible Involvement of a P-448-H Type Cytochrome P-450

Metabolic activating capacity of human livers for carcinogenic heterocyclic arylamines has been studied using a Salmonella mutagenesis test. A large individual variation was observed among 15 liver samples in the capacities of activation of Glu-P-1(2-amino-6-methyldipyrido[1,2-α:3',2'- d ]imidazole), IQ (2-amino-3-methylimidazo[4,5- f ]quinoline) and MeIQx (2-amino-3,8-dimethyl-3 H -imidazo[4,5- f ]quinoxaline). The average numbers of revertants induced by the three heterocyclic arylamines were nearly the same or rather higher in the presence of hepatic microsomes from human than those from rat. In high-performance liquid chromatography, formation of N-hydroxy-Glu-P-1 was detected and accounted for more than 80% of the total mutagenicity observed in the human microsomal system with Glu-P-1, indicating that, similarly to experimental animals, N-hydroxylation is a major activating step for heterocyclic arylamines in human. Addition of flavone or 7,8-benzoflavone to human liver microsomes showed effective inhibition of the mutagenic activation of Glu-P-1, although the treatment rather enhanced microsomal benzo[ a ]pyrene hydroxylation in human livers. Mutagenic activation of Glu-P-1 by human liver microsomes was also decreased by the inclusion of anti-rat P-448-H IgG, and was well correlated with the content of immunoreactive P-448-H in livers, suggesting the involvement of a human cytochrome P-450, which shares immunochemical and catalytic properties with rat P-448-H, in the metabolic activation of heterocyclic arylamines in human livers.     

Specificity of rabbit cytochrome P-450 isozymes involved in the metabolic activation of the food derived mutagen 2-amino-3-methylimidazo[4,5-f] quinoline

The involvement of rabbit liver cytochromes P-450 in the activation of the food derived heterocyclic amine mutagen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), was assessed using the Ames/Salmonella test. The number of revertants induced by IQ per microgram of control rabbit liver microsomes was 1872 +/- 50 (SD, n = 3), and this increased to 3690 +/- 239 when microsomes from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) animals were used as the metabolic activation source. Microsomes from phenobarbital, rifampicin, and acetone pretreated rabbits were less efficient the controls at activating IQ to a mutagen. Cytochrome P-450 Forms 4 and 6, which are induced by TCDD, were found to be efficient activators of IQ to a mutagen in reconstitution experiments. Form 4 was 7.7-fold more active than Form 6 and produced 1702 revertants/0.125 pmol with a 20-min preincubation step in the Ames test. Anti-Form 4 IgG inhibited the activation of IQ in control and TCDD induced microsomes by 78 and 79%, respectively. The contents of cytochrome P-450 Form 4, determined by Western blot analysis, in control and phenobarbital, acetone, rifampicin, and TCDD pretreated microsomes were 0.55 +/- 0.19, 0.63 +/- 0.34, 0.5 +/- 0.27, 0.28 +/- 0.16, and 2.19 +/- 0.43 (n = 3) nmol/mg protein, respectively. A highly significant statistical correlation existed between the capacity of the above microsomes to activate IQ to a mutagen and their cytochrome P-450 Form 4 content (r = 0.96; r2 = 0.92). The content of cytochrome P-450 Form 6 in the above microsomes was also highly correlated with their capacity to activate IQ (r = 0.92; r2 = 0.85). Based on these results and the tissue distribution of cytochrome P-450 Forms 4 and 6, the former obviously contributes most toward the activation of IQ in the liver, whereas Form 6 would be expected to be primarily involved in this process in extrahepatic tissues.     

Metabolism of carcinogenic heterocyclic and aromatic amines by recombinant human cytochrome P450 enzymes

The N-hydroxylation of carcinogenic arylamines represents an initial step in their metabolic activation. Animal studies have shown that this reaction is catalyzed by the cytochrome P450 (P450) enzymes P450 1A1 and P450 1A2. In this study, utilizing enzymes expressed in Escherichia coli (and purified) or in human B-lymphoblastoid cells, the catalytic activities of recombinant human P450 1A1, P450 1A2, and P450 3A4 for N- hydroxylation of several carcinogenic arylamines were determined. P450 1A2 from both expression systems catalyzed the N-hydroxylation of 4- aminobiphenyl and the heterocyclic amines, 2-amino-3-methylimidazo[4,5- f/quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Rates were similar, with values of 1.1-7.8 nmol/min/nmol P450. In contrast, P450 1A1 catalyzed N-hydroxylation of only PhIP, and no activity was observed with P450 3A4. Further kinetic analysis with purified P450 1A2 showed similar Km and Vmax values for N-hydroxylation of the arylamines. Furafylline and fluvoxamine, inhibitors of P450 1A2 activity in human liver microsomes, were found to be inhibitory of the recombinant P450 1A2 N-hydroxylation activity. Results from this study are supportive of a major role for human P450 1A2 in the metabolic activation of arylamines.      

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.     

The role of the human acetylation polymorphism in the metabolic activation of the food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ).

The metabolic activation of the heterocyclic food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) by two human cytochrome P450 monoxygenases (P4501A1 and P4501A2) and two human N-acetyltransferases (NAT1 and NAT2) was investigated. Various combinations of these enzymes were functionally expressed in COS-1 cells. DNA adducts resulting from the activation of IQ were assayed quantitatively by the 32P-postlabeling procedure. The highest adduct frequency was observed in cells expressing both CYP1A2 and NAT2. CYP1A2 in combination with NAT1 was 3-6 times less active. When expressed alone these enzymes gave rise to low adduct frequencies. Experiments with N-acetyl-IQ as substrate suggest that NAT1 and NAT2 in addition to their known role in N-acetylation display arylhydroxamic acid N, O-acetyltransferase (AHAT) activity. Quantitative differences in adduct formation between IQ and N-acetyl-IQ indicated that metabolic activation of these arylamines preferentially occurs by P4501A2-catalyzed N-hydroxylation followed by O-acetylation mediated through NAT1 and/or NAT2. These data, in combination with the known genetic polymorphism of NAT2, may explain the clinical observation that the acetylation polymorphism constitutes a risk factor in the carcinogenic activation of environmental mutagens.     

Alkylation of cellular macromolecules and target specificity of carcinogenic nitrosodialkylamines: metabolic activation by cytochromes P450 2B1 and 2E1

The alkylation of DNA, RNA and protein by labeled metabolites of [alpha- 14C]nitrosodimethylamine (NDMA), [alpha-14C]nitrosodipropylamine (NDPA) and [alpha-14C]nitrosodibutylamine (NDBA) was determined as a measure of the metabolic activation of these nitrosamine carcinogens in vitro using microsomes prepared from freshly isolated rat hepatocytes as well as in intact cells using primary cultured rat hepatocytes. The abilities of these nitrosodialkylamines to alkylate cellular macromolecules were significantly affected by pretreatment of rats with inducers of cytochrome P450 and were related to the specific activities of cytochrome P450 2B1 or 2E1 in rat hepatocytes. Pretreatment of rats with phenobarbital (PB) substantially increased the catalytic activity of pentoxyresorufin (PR) O-depentylase, an activity catalyzed by cytochrome P450 2B1, in rat hepatocytes. The increase in the PR O- depentylase activity was associated with a significant increase in the alkylation of DNA or RNA by NDPA, and in alkylation by NDBA, particularly of proteins. However, induction of cytochrome P450 2B1 resulted in a significant decrease in alkylation of cellular macromolecules by NDMA in all cases. In contrast, enhancement of the catalytic activity of the p-nitrophenol (pNP) hydroxylase (P450 2E1) due to pretreatment of rats with pyridine (PYR) resulted in a significant increase in the alkylation of cellular DNA by NDMA. The induction of cytochrome P450 2E1 also increased the alkylation of DNA and RNA by NDPA, but to a lesser extent. Inhibition studies using the chemical inhibitors orphenadrine (OP) and diethyldithiocarbamate (DDC), which are specific for cytochromes P450 2B1 and 2E1, respectively, indicated that cytochrome P450 2B1 was not involved in the metabolic activation of NDMA and that cytochrome P450 2E1 was not responsible for the bioactivation of NDBA. The results presented here demonstrate the substrate specificity and important role of cytochromes P450 2B1 and 2E1 in the bioactivation of nitrosodialkylamines, and suggest that multiple mechanisms may be involved in carcinogenesis induced by nitrosodialkylamines.      

Enzymatic mechanisms in the metabolic activation of N-nitrosodialkylamines

The metabolism of several N-nitrosodialkylamines was studied using rat liver microsomes and purified cytochrome P450 isozymes in a reconstituted monooxygenase system. With purified acetone/ethanol-inducible cytochrome P450 (P450ac), high N-nitrosodimethylamine (NDMA) demethylase activity was observed. Cytochrome b5 was also involved in NDMA metabolism by decreasing the Km of NDMA demethylase. A close relationship between the demethylation and denitrosation of this substrate was observed. P450ac was also active in the metabolism of N-nitrosoethylmethylamine (NEMA), but was less active than phenobarbital-inducible cytochrome P450 (P450b) in the metabolism of N-nitrosobutylmethylamine (NBMA), especially in catalysing the debutylation reaction. Similar substrate specificity was demonstrated with liver microsomes from rats treated with other inducers. With different P450 isozymes and microsomes, a close relationship between metabolism and activation of nitrosamines to mutagens to V79 cells was demonstrated. DNA alkylation by NDMA in vitro was correlated with the rate of metabolism of these compounds, whereas DNA alkylation in vivo was more complex and was dose-dependent. The work demonstrates the importance of knowledge of the substrate specificity of cytochrome P450 isozymes in understanding the mechanisms of the metabolic activation of nitrosamines.     

Aryl hydrocarbon hydroxylase activity and microsomal cytochrome content of human fetal tissues

Aryl hydrocarbon hydroxylase (AHH) and microsomal cytochromes were measured in tissues of human features aborted by prostaglandins. Cytochrome P-450 concentrations and AHH activity were about 4 times higher in adrenal glands than in liver. AHH was present in testes, ovaries, and vagina and uterus at levels equal to or greater than those in the liver. In lung, kidney, and intestines it was present at levels lower than those in the liver. Mean hepatic AHH and hepatic and adrenal cytochromes P-450 and b5 were not significantly different in prostaglandin and hysterotomy abortuses; mean adrenal AHH was significantly lower in prostaglandin abortuses, but the ranges in both groups were overlapping. Neither fetal sex nor maternal cigarette smoking affected hepatic or adrenal AHH activity or microsomal cytochrome concentrations or difference spectra. Hepatic and adrenal AHH activities were concentrated in microsomes and were correlated with microsomal P-450 content. These findings are constant with P-450 mediation of AHH in human fetal tissues. The data demonstrate the utility of prostaglandin abortuses for studies of fetal tissue mixed-function oxidase activity and point to the endocrine glands and target tissues in addition to the liver as potential sites for activating chemical carcinogens and cytotoxins in the human fetus.     

Metabolic activation of pyrolysate arylamines by human liver microsomes; possible involvement of a P-488-H type cytochrome P-450

Metabolic activating capacity of human livers for carcinogenic heterocyclic arylamines has been studied using a Salmonella mutagenesis test. A large individual variation was observed among 15 liver samples in the capacities of activation of Glu-P-1 (2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole), IQ (2-amino-3-methylimidazo[4,5-f]quinoline) and MeIQx (2-amino-3,8-dimethyl-3 H-imidazo[4,5-f]quinoxaline). The average numbers of revertants induced by the three heterocyclic arylamines were nearly the same or rather higher in the presence of hepatic microsomes from human than those from rat. In high-performance liquid chromatography, formation of N-hydroxy-Glu-P-1 was detected and accounted for more than 80% of the total mutagenicity observed in the human microsomal system with Glu-P-1, indicating that, similarly to experimental animals, N-hydroxylation is a major activating step for heterocyclic arylamines in human. Addition of flavone or 7,8-benzoflavone to human liver microsomes showed effective inhibition of the mutagenic activation of Glu-P-1, although the treatment rather enhanced microsomal benzo[a]pyrene hydroxylation in human livers. Mutagenic activation of Glu-P-1 by human liver microsomes was also decreased by the inclusion of anti-rat P-448-H IgG, and was well correlated with the content of immunoreactive P-448-H in livers, suggesting the involvement of a human cytochrome P-450, which shares immunochemical and catalytic properties with rat P-448-H, in the metabolic activation of heterocyclic arylamines in human livers.     

Decrease in the Metabolic Activating Capacities of Arylamines in Livers Bearing Hyperplastic Nodules: Association with the Selective Changes in Hepatic P-450 Isozymes

The mechanism of the alteration in carcinogenic arylamine-activating capacities in livers bearing pre-neoplastic (or hyperplastic) nodules induced by the Solt-Farber protocol was investigated in relation to the changes in hepatic cytochrome P-450 isozymes. In the Salmonella mutagenesis test, the numbers of revertants induced with 2-amino-3-methylimidazo[4,5- f ]quinoline and 2-aminofluorene were significantly lower in the presence of microsomes of nodule-bearing livers than of control livers. A similar tendency was also observed with another heterocyclic arylamine, 2-amino-6-methyldipyrido-[1,2- a :3',2'-d]imidazole. In Western blots using specific antibodies against 5 different forms of cytochrome P-450, hepatic contents of P-450-male (a main constitutive form) and P-450b (a main phenobarbital-inducible form) were decreased in the livers with hyperplastic nodules to 63% and 35% of the corresponding controls, while no significant decrease was observed in the contents of P-448-H (a main 3-methylcholanthrene-inducible form), P-450_6β-1 (testosterone 6β-hydroxylase) and P-450e (a phenobarbital-inducible form). In accordance with the reduction in P-450-male, capacities for microsomal 16α- and 2α-hydroxylations, but not 6β-hydroxylation, of testosterone were decreased in the livers with hyperplastic nodules. Although P-448-H has higher capacities for the activation of arylamines than does P-450-male, the hepatic content of P-450-male is more than ten-fold higher than that of P-448-H in both normal and nodule-bearing livers. These results indicate that the selective decrease in hepatic content of P-450-male is likely to be a main cause of the decrease in arylamine metabolic activating capacities in livers with hyperplastic nodules.     

Role of cytochrome P-450 from the human CYP3A gene family in the potentiation of morpholino doxorubicin by human liver microsomes.

The cytotoxicity of the morpholino derivative of doxorubicin (MRA) can be potentiated 50- to 100-fold by human liver microsomes and NADPH (J. Natl. Cancer Inst., 81: 1034, 1989). This metabolic potentiation is inhibited by carbon monoxide or hypoxia, indicating that it is cytochrome P-450-dependent. The potentiation is also inhibited by the cytochrome P-450 inhibitors, SKF-525A and cimetidine. The metabolism by the microsomes is substrate-specific, varying markedly with alterations of either the morpholino or anthracycline ring substituents. No potentiation occurred with doxorubicin itself, or the cyanomorpholinyl, methoxypiperidinyl, N-hydroxyethyl or the O-bridged cyanomorpholinyl analogues of doxorubicin. We utilized a panel of human liver microsomes and cytochrome P-450 type-specific antibodies to further identify the isoform(s) of cytochrome P-450 that potentiated the cytotoxicity of MRA. The potentiation correlates well with the benzyloxyresorufin assay (r2 = 0.98) and aflatoxin B1 metabolism (r2 = 0.98), both assays that are relatively specific for CYP3A proteins. Correlations were also observed for the expression of protein(s) cross-reacting with an antibody against rat cytochrome P-450 CYP3A1 (r2 = 0.97) and MRA metabolism. This antibody against the rat cytochrome P-450 CYP3A isoform(s) inhibited more than 90% of the potentiation of the cytotoxicity by human liver microsomes. Antibodies against the CYP1A2, CYP2C6, and CYP2B2 isoforms produced no inhibition, nor did their expression by Western blotting correlate with MRA potentiation. Complete inhibition of the potentiation of MRA by human liver microsomes was found when the CYP3A substrates cyclosporin A and erythromycin were used in the reaction system. These data indicate that the CYP3A isoform(s) of cytochrome P-450 play a major role in the metabolism of MRA in vitro to a more active species.     

Stable expression of human cytochrome P450IIE1 in mammalian cells: metabolic activation of nitrosodimethylamine and formation of adducts with cellular DNA.

To introduce cytochrome P450IIE1 DNA stably into the chromosomal DNA of mammalian cells, we constructed recombinant retroviruses containing the full-length complementary DNA for human cytochrome P450IIE1 and a selectable neo gene. Rat and mouse cells were infected with these viruses, and clones expressing the neo marker gene product were selected in G418-containing medium. Analysis of the DNA of the clones by Southern blotting showed that the viral DNA was integrated into the cellular DNA. Enzymatic analysis of the clones showed that the transduced DNA directed the expression of enzymatically active cytochrome P450IIE1. Treatment of the cells with the carcinogen [14C]-nitrosodimethylamine and analysis of the cellular DNA by CsCl equilibrium density gradients showed incorporation of the label into DNA, indicating the formation of covalent adducts with the cell DNA. Construction of recombinant cell lines constitutively expressing cytochrome P450IIE1 provides a permanent source for this enzyme and can aid in the analysis of its catalytic properties, such as the metabolic activation of chemical mutagens/carcinogens, and the consequent cytotoxic and genotoxic effects of these compounds.     

Metabolic activation of a protein pyrolysate promutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by rat liver microsomes and purified cytochrome P-450

The enzymatic activation of a promutagenic pyrolysate, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx), was studied using the Ames mutagenesis test system.The enzyme catalyzing the mutagenic activation of MeIQx is mainlylocalized in the microsomal fraction. A large number of revertantswas observed in the presence of hepatic microsomes obtainedfrom 3-methylcholanthrene (3-MC)- or polychlorinated biphenyl(PCB)-treated rats but only a minimal number with the hepaticmicrosomes from untreated or phenobarbital (PB)-treated rats.In addition, the microsomal activation was reduced efficientlyby known inhibitors of cytochrome P-450- mediated reactionssuch as 7,8-benzoflavone, ellipticine and flavone. Among fiveforms of purified rat cytochrome P-450, the highest sp. act.(no. of revertants induced/nmol cytochrome P-450) for the activationof MeIQx was observed with a high-spin form of cytochrome P-450,P-448-H, followed by the low-spin form, P-448-L, and to a lesserextent by PB-inducible forms, P-450b and P-450e. P-450-male,which is a main constitutive form of cytochrome P-450 In malerat livers, showed considerable catalysis for the mutagenicactivation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) andMeIQx. These results Indicate that the metabolic activationof MeIQx is catalyzed mainly by two forms of cytochrome P-450,P-448-H and P-488-L, in the livers of PCB- or 3-MC-treated rats,but also that P-450-male may play an important role in the activationin livers of Intact male rats.     

Cytochrome P-450 3A4 (nifedipine oxidase) is responsible for the C-oxidative metabolism of 1-nitropyrene in human liver microsomal samples.

The nitrated polycyclic aromatic hydrocarbon 1-nitropyrene is a ubiquitous environmental pollutant. The role of cytochromes P-450 in the human metabolism of [3H]-1-nitropyrene was investigated using human liver microsomes. The range of microsomal metabolism from 16 individual liver specimens was 0.13 to 0.99 nmol/min/mg protein. Using 3 microsomal samples exhibiting different maximal velocities, the Km of 1-nitropyrene metabolism was 3.3 +/- 0.5 microM, indicating that perhaps a single or similar cytochromes P-450 was involved in the metabolism of 1-nitropyrene in these samples. The P-450 3A inhibitor triacetyloleandomycin inhibited 86 +/- 8% of the microsomal metabolism of 1-nitropyrene. Further evidence for the role of P-450 3A in human microsomal metabolism of 1-nitropyrene was gained using inhibitory anti-P-450 3A antibodies. Using 3 separate microsomal samples, antibody conditions that inhibited approximately 90% of the metabolism of the P-450 3A4-specific substrate nifedipine inhibited approximately 60-70% of the metabolism of 1-nitropyrene. Human liver microsomes demonstrated a preference for 1-nitropyren-3-ol formation over 1-nitropyren-6-ol or 1-nitropyren-8-ol, which is in contrast to that noted in rodents where the 6-ol and 8-ol are preferentially formed over the 3-ol, yet in agreement with earlier studies on the metabolism of 1-nitropyrene using Vaccinia-expressed human cytochromes P-450. These results indicate that the human hepatic metabolism of 1-nitropyrene is carried out by at least two or more P-450s including those in the P-450 3A subfamily. These studies also suggest that the metabolism of this compound by humans may differ from that in rodents in both the cytochromes that are involved and the specific metabolites that are formed.     

Phosphorylation of cytochrome-P-450-dependent monooxygenase components

Most chemical carcinogens require activation by polysubstratemonooxygenase. The phosphorylation of essential components ofthis cytochrome P-450 monooxygenase system, isolated from rabbitliver microsomes, cytochrome P-450 (LM₂) and cytochrome reductase,was tested using two different protein kinases. One of the kinases,a cyclic AMP-independent phosvitin kinase (kinase P), was inactivein all systems tested. However, the catalytic subunit of a cyclicAMP-dependent protein kinase (kinase C) catalyzed phosphorylgroup transfer to both proteins, but to different extents. CytochromeP-450 was phosphorylated when added as sole component and alsowhen in the presence of P-450 reductase and phosphatidylcholine.In contrast, the weak phosphorylation of P-450 reductase wasreduced considerably in a complete reconstituted system containingP-450 and phosphatidylcholine. The inclusion of kinase P didnot alter these results which excludes the possibility thatthese kinases participate in a sequential phosphorylation mechanism.The monooxygenase constituents themselves were without kinaseactivity. When hepatic microsomes were isolated in presenceof the phosphatase inhibitor sodium fluoride no significantchange in monooxygenase (7-ethoxycoumarin O-deethylation) activitywas observed, whilst after preincubation with either acid oralkaline phosphatase a significant reduction in monooxygenaseactivity was measured. Thus, cytochrome P-450 (LM₂) is phosphorylatableby protein kinase C and the catalytic activity of polysubstratemonooxygenase decreases after preincubation of microsomes withphosphatases.