Changes in the quantity and activity of cytochrome P-450 isozymes in primary cultured rat hepatocytes

Hepatocytes from male Sprague-Dawley rats pretreated with a cytochrome P-450 inducer, 3-methoxy-4-aminoazobenzene (3-MeO-AAB), 3-methylcholanthrene (MC) or phenobarbital (PB), were cultured in vitro, and changes in the quantity and activity of microsomal cytochrome P-450 isozymes in the cells were determined by means of immunochemical methods and a bacterial mutation test, respectively. The results of enzyme-linked immunosorbent assay using monoclonal antibodies against rat P-450 isozymes revealed that the amount of cytochrome P-450d induced by 3-MeO-AAB or MC declined rapidly during culture and fell to 10 to 15% of the initial value after 24 h. A similar tendency was observed with PB-induced cytochrome P-450b/e. By contrast, cytochrome P-450c in MC-induced hepatocytes declined more slowly than cytochrome P-450d and remained at 45 to 60% of the initial value after 24 h. Similar quantitative changes of the individual cytochrome P-450 isozymes in culture were also observed by immunoblotting using the anti-cytochrome P-450 monoclonal antibodies. Changes in the activities of individual cytochrome P-450 isozymes in hepatocytes by culture were in accordance with the quantitative changes of the cytochromes, as determined by a mutation test using Salmonella typhimurium TA 98 and carcinogenic aromatic amines. These results indicate that microsomal cytochrome P-450c in primary cultured rat hepatocytes is more stable in culture, in terms of both quantity and activity, than cytochrome P-450d and P-450b/e.     

Induction of cytochrome P-450 and 5-aminolevulinate synthase activities in cultured rat hepatocytes

Cytochromes P-450IIB1 and P-450IIB2 were recently shown to be inducible in rat hepatocyte cultures maintained on a reconstituted extracellular tumor matrix (Matrigel) as indicated by increases in P-450IIB1 and -IIB2 mRNAs and immunoreactive proteins (J. Cell. Physiol., 134: 309-323, 1988). Here we show that treatment of cultured rat hepatocytes with phenobarbital and other compounds known to induce P-450IIB1/2 in vivo increased spectral cytochrome P-450, immunoreactive proteins, and benzyloxy- and pentoxy-resorufin dealkylases, activities known to be specific for cytochrome P-450IIB1/2. These increases were observed when cells were cultured on either Matrigel or collagen matrix in Williams E medium. Cytochrome P-450III was also increased by phenobarbital and dexamethasone on either matrix. Propoxycoumarin depropylase activity, which has been proposed as a specific activity catalyzed by cytochrome P-450III, was increased 3-4-fold more by treatment with 3-methylcholanthrene than by phenobarbital or dexamethasone. The activity catalyzed by P-450III could be distinguished from that catalyzed by other P-450 forms using the specific inhibitor triacetyloleandomycin. Benzoyloxyresorufin dealkylase was also increased in these cells by treatment with 2,4,5,2',4',5'-hexachlorobiphenyl, glutethimide, or mephenytoin. Treatment with phenobarbital or 2-allyl-2-isopropylacetamide slightly induced 5-aminolevulinate synthase activity. 5-Aminolevulinate synthase activity was slightly increased in cells treated with phenobarbital or 2-allyl-2-isopropylacetamide. Succinyl acetone also induced 5-aminolevulinate synthase activity and, in combination with either of the other two drugs, synergistically increased the enzyme activity regardless of whether cells were cultured on collagen or Matrigel. These results indicate that with simple and economical enzyme assays for holocytochrome P-450 and 5-aminolevulinate synthase, the rat hepatocyte culture system can be used for studies of the interrelationships between phenobarbital induction of cytochrome P-450 and heme metabolism.     

Hexachlorobenzene and substituted pentachlorobenzenes (X-C6Cl5) as inducers of hepatic cytochrome P-450-dependent mono-oxygenases

Hexachlorobenzene (HCB) and 2,3,4,4',5-pentachlorobiphenyl induced a similar spectrum of cytochrome-P-450-dependent mono-oxygenase activities in the rat, including 4-dimethylaminoantipyrine N-demethylase, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD). Levels of individual cytochrome P-450 isozymes and various mono-oxygenase activities in liver microsomes from rats treated with substituted pentachlorobenzene (X-C6Cl5) and 4'-substituted-2,3,4,5-tetrachlorobiphenyl (X-C12 H5Cl4) analogues demonstrated the remarkable effects of substituent structure on induction activities. The chloro- and bromopentachlorobenzenes induced hepatic microsomal 4-dimethylaminoantipyrine N-demethylase, AHH and EROD; the iodo-, fluoro-, acetamino- and nitropentachlorobenzene analogues together with pentachlorobenzene weakly induced both AHH and EROD (approximately 2-fold or less); and the remaining substituted pentachlorobenzenes tested (X = CH3, OCH3 and OH) were relatively inactive as inducers of microsomal mono-oxygenases. Substituent effects were observed for the induction of liver microsomal cytochromes P-450a, P-450b + e, P-450c, P-450d and total cytochrome P-450 by the X-C6Cl5 and X-C12H5Cl4 analogues. The chloro- and bromopentachlorobenzene analogues in both series induced total cytochrome P-450 and cytochromes P-450a to P-450d, whereas the hydroxy-, methyl- and methoxy-substituted analogues in both series were relatively inactive as inducers of cytochrome P-450. Iodo-, fluoro- and nitropentachlorobenzene were weak 3-methylcholanthrene-type inducers and, in contrast to the corresponding biphenyl analogues, had little or no effect on the induction of cytochromes P-450a, P-450c and P-450d.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate specificity and alkyl group selectivity in the metabolism of N-nitrosodialkylamines

Metabolic activation may be a key step in determining the tissue specificity of carcinogenic nitrosamines. In previous work, we characterized P450IIE1 (an acetone/ethanol-inducible form of cytochrome P-450) as the major enzyme for the metabolic activation of N-nitrosodimethylamine. In this work, we investigated the metabolism of other N-nitrosodialkylamines in rat liver microsomes and in reconstituted monooxygenase systems containing purified cytochrome P-450 isozymes. The enzyme specificities in the metabolism of N-nitrosoethylmethylamine and N-nitrosodiethylamine were similar to those of N-nitrosodimethylamine; i.e., these substrates were more efficiently metabolized by acetone- or ethanol-induced microsomes than by other types of microsomes. However, substituting one methyl group with a benzyl or butyl group, as in N-nitrosobenzylmethylamine or N-nitrosobutylmethylamine (NBMA), substantially changed the enzyme specificity. P450IIE1 efficiently catalyzed the demethylation but not the debutylation of NBMA, whereas P450IIB1 (a phenobarbital-inducible form) efficiently catalyzed both the debutylation and demethylation reactions. In the demethylation of NBMA by P450IIE1, the addition of cytochrome b5 markedly increased the activity at low but not at high substrate concentrations, suggesting a decrease in Km value. This effect, however, was not observed in the debutylation of NBMA by P450IIE1 or P450IIB1, and in the demethylation of NBMA by P450IIB1. These studies demonstrate the substrate specificity and alkyl group selectivity in the metabolism of nitrosamines by cytochrome P-450 isozymes.     

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.     

On the identity of the xenobiotic-metabolizing form(s) of cytochrome P-450 in endocrine organs

The adrenal cortex, the testes and the ovary metabolize polycyclic aromatic hydrocarbons (PAHs), e.g., 7,12-dimethylbenz[a]anthracene (DMBA). These activities have previously been shown to involve the cytochrome P-450 monooxygenase system [18-20]. In attempt to identify the form(s) of cytochrome P-450 involved, microsomes from these endocrine organs were subjected to SDS-gel electrophoresis, followed by immunochemical analysis using the Western blot technique. Antisera raised against the purified rat hepatic cytochrome P-450 isozymes a, b + e, c, d and PB-PCNE were tested. It was concluded that the electrophoretic mobilities of the immunoreactive bands obtained were not identical to the mobilities of the purified isozymes cytochromes P-450 a, b, c, d, e or PB-PCNE. These results indicate that the PAH-metabolizing monooxygenase(s) in these endocrine organs may involve a novel form(s) of cytochrome P-450.     

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.      

Biotransformation of N,N',N'-triethylenethiophosphoramide: oxidative desulfuration to yield N,N',N'-triethylenephosphoramide associated with suicide inactivation of a phenobarbital-inducible hepatic P-450 monooxygenase

Oxidative metabolism of the polyfunctional alkylating agent N,N',N'-triethylenethiophosphoramide (thio-TEPA) was studied in isolated rat liver microsomes and purified, reconstituted cytochrome P-450 (P-450) enzyme systems in order to elucidate the pathways of drug oxidation and to identify the possible contributions of individual P-450 enzymes to the bioactivation of this chemotherapeutic agent. Rat liver microsomes were found to catalyze conversion of thio-TEPA to its oxo metabolite, N,N',N'-triethylenephosphoramide (TEPA), in a P-450-dependent reaction that was markedly stimulated by prior in vivo treatment with drug inducers of hepatic P-450 subfamily IIB (phenobarbital), but not by pretreatment with inducers of P-450 subfamilies IA (beta-naphthoflavone) or IIE (isoniazid). Thio-TEPA depletion and TEPA formation catalyzed by phenobarbital-induced liver microsomes were both inhibited by greater than 90% by antibodies selectively reactive with P-450 PB-4 (gene product IIB1), the major phenobarbital-inducible rat liver microsomal P-450 form, but not by antibodies inhibitory toward 7 other rat hepatic P-450s. Oxidation of thio-TEPA to TEPA was also catalyzed by purified P-450 PB-4 (Km (app) 19 microM; Vmax (app) = 11 mol thio-TEPA metabolized/min/mol P-450 PB-4) following reconstitution of the cytochrome with NADPH P-450 reductase in a lipid environment. Metabolism of thio-TEPA by P-450 PB-4 was associated with a suicide inactivation of the cytochrome characterized by kinactivation = 0.096 min-1, KI = 24 microM, and a partition ratio of 136 +/- 28 (SD) mol thio-TEPA metabolized/mol P-450 inactivated. The thio-TEPA metabolite TEPA, however, did not inactivate the cytochrome, nor was it subject to further detectable metabolism. In microsomal incubations, metabolism of thio-TEPA led to the inactivation of P-450 PB-4 (steroid 16 beta-hydroxylase) as well as P-450 IIIA-related enzymes (steroid 6 beta-hydroxylase) and the P-450-independent enzyme steroid 17 beta-hydroxysteroid:NADP+ 17-oxidoreductase, as demonstrated by use of the P-450 form-selective steroidal substrate androst-4-ene-3,17-dione. In contrast, little or no inactivation of microsomal P-450 IIA-related enzymes (steroid 7 alpha-hydroxylase) or microsomal NADPH P-450 reductase was observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Microsomal cytochrome P-450-linked monooxygenase systems and lipid composition of human hepatocellular carcinoma

The tissues of hepatocellular carcinoma were operatively resected from six patients. All four components of the systems of microsomal cytochrome P-450-linked monooxygenase of the tissues were investigated and compared to those of normal liver tissue. The concentrations of cytochromes P-450, P-420 and b5 were measured optically and the concentrations of all components except cytochrome P-450 were measured by the Western blotting method followed by immunochemical staining. In microsomes of hepatocellular carcinoma tissues, there was as much cytochrome P-450 and other redox components as in the normal liver tissues, but cytochrome P-450 in liver cancer tissues was unstable and easily converted to cytochrome P-420. The specific activities of NADPH- and NADH-ferricyanide and cytochrome c reductase of each sample were also measured. In the microsomes of the cancer tissues, the specific activities were remarkably reduced compared with those of normal liver tissues. The lipid compositions of the microsomes and the phospholipid/cholesterol ratios (w/w) were 13.1 +/- 3.13 in the cancer tissues and 43.0 +/- 6.74 in normal liver tissues. This difference of the lipid composition elucidates the instability of cytochrome P-450 molecules and the inefficiency of the electron transport of cytochrome P-450-linked monooxygenase systems.     

Changes in the Quantity and Activity of Cytochrome P-450 Isozymes in Primary Cultured Rat Hepatocytes

Hepatocytes from male Spragne-Dawley rats pretreated with a cytochrome P-450 inducer, 3-methoxy-4-aminoazobenzene (3-MeO-AAB), 3-methylcholanthrene (MC) or phenobarbital (PB), were cultured in vitro , and changes in the quantity and activity of microsomal cytochrome P-450 isozymes in the cells were determined by means of immunochemical methods and a bacterial mutation test, respectively. The results of enzyme-linked immunosorbent assay using monoclonal antibodies against rat P-450 isozymes - test using Salmonella typhimurium TA 98 and carcinogenic aromatic amines. These results indicate that microsomal cytochrome P-450c in primary cultured rat hepatocytes is more stable in culture, in terms of both quantity and activity, than cytochrome P-450d and P-450b/e.     

Positional specificity for methyl-n-amylnitrosamine hydroxylation by cytochrome P-450 isozymes determined with monoclonal antibodies

Inhibitory monoclonal antibodies (MAbs) were used to determine the contribution of epitope-specific cytochrome P-450 isozymes in rat liver microsomes to hydroxylation of the esophageal carcinogen methyl-n-amylnitrosamine. These P-450-catalyzed reactions form 2-, 3-, 4-, and 5-hydroxymethyl-n-amylnitrosamine, formaldehyde (demethylation), and pentaldehyde (depentylation). With uninduced microsomes from male rats, MAb 1-68-11 inhibited 4-hydroxylation by 73% and demethylation by 46%. This indicated the major contribution of constitutive male-specific P-450 IIC11 to the metabolism. Inhibition studies with MAbs 2-66-3 and 1-91-3 indicated that P-450 IIB1 contributed 19% and IIE1 35% to demethylation. With uninduced microsomes from females, MAb 1-68-11 produced similar inhibitions to those in male rats, indicating that female-specific P-450 IIC12 (which is closely related to IIC11) also catalyzed 4-hydroxylation and demethylation. With microsomes from 3-methylcholanthrene-induced male rats, P-450 IA1 and/or IA2 were responsible for 60% of 3-hydroxylation and 40% of depentylation. With microsomes from phenobarbital-treated rats, P-450 IIB1 and IIB2 catalyzed all 6 reactions but especially 4-hydroxylation and depentylation, which were 50-75% inhibited by MAb 2-66-3. Microsomes from Aroclor-induced males behaved as if they were induced by both 3-methylcholanthrene and phenobarbital. After treatment with isoniazid (a P-450 IIE1 inducer), inhibition by MAb 1-91-3 indicated a 45% contribution of P-450 IIE1 to demethylation, and both P-450 IIE1 and IIB1 (or IIB2) appear to have been induced. A major finding with uninduced microsomes was the high specificity of MAb 1-68-11 for inhibiting 4-hydroxylation, indicating that P-450 IIC11 and IIC12 catalyzed most of this omega-1-hydroxylation. In microsomes from induced rats, the MAb inhibitions showed the role of the induced P-450 IA1 (or IA2), IIB1 (or IIB2), and IIE1 in methyl-n-amylnitrosamine hydroxylation at different positions, as well as the presence of P-450 IIC11. This study illustrates the usefulness of inhibitory MAbs for defining the contribution of individual P-450s to position-specific metabolism.     

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.     

Selective mutagenic activation by cytochrome P3-450 of carcinogenic arylamines found in foods

Heterocyclic arylamines found in cooked foods including fish and beef are potent mutagens and carcinogens. The purpose of this investigation was to determine the specificity of cytochromes P1-450 and P3-450 toward the metabolic activation of these arylamines. We used a novel mutagenicity test system which combined human cells expressing either recombinant cytochrome P1-450 or P3-450 with Salmonella typhimurium to score mutants. Cytochrome P3-450, a single isoform of the cytochrome P-450 supergene family, bioactivated these food mutagens. Cytochrome P1-450 showed little or no activation of these arylamines but was the isoform predominantly responsible for the activation of the aromatic hydrocarbon benzo[a]pyrene-7,8-diol. This assay system should serve to define the specificities of individual cytochromes P-450 in the metabolic activation of carcinogens.     

Depression of the hepatic cytochrome P-450 monooxygenase system by treatment of mice with the antineoplastic agent 5-azacytidine

The effects of 5-azacytidine (5-AC) administration on the hepatic cytochrome P-450 systems of mice were studied. A single i.p. dose of 5-AC (25 mg/kg) to male Swiss-Webster mice caused about a 50% depression of microsomal cytochromes P-450 and b5 and of ethylmorphine N-demethylase and ethoxycoumarin O-deethylase activities. Depression was greatest 24 h after treatment; by 48 to 72 h, cytochromes and drug metabolism had returned to near control values. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity was also depressed by 5-AC, whereas reduced nicotinamide adenine dinucleotide-cytochrome c reductase was not. Incubation of 5-AC with microsomes produced no effect on drug metabolism. The prolongation of hexobarbital sleeping time by 5-AC showed that drug metabolism is also impaired by 5-AC in vivo. These studies may have important clinical implications when certain drugs are coadministered with 5-AC.     

Genotoxicity of heterocyclic amines in the Salmonella/hepatocyte system

The Salmonella/hepatocyte system was employed to determine the mutagenicity in bacteria as well as the DNA damage induced in mouse hepatocytes following exposure to heterocyclic amines. With hepatocytes from C57BL/6N mice, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) showed a clear mutagenic effect in the Salmonella, while weak mutagenic effects were observed with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 2-amino-6-methyldipyrido[1,2-a:3',2'-b]imidazole (Glu-P-1), and 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2). All the compounds induced low levels of DNA damage in the hepatocytes. In vivo pretreatment of mice with the potent monooxygenase inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 50 micrograms/kg) clearly increased both the mutagenicity in the bacteria and the DNA damage induced in the hepatocytes in vitro. Glu-P-2 showed the lowest mutagenic effect but induced more DNA damage at low concentrations than the other compounds when TCDD-pretreated hepatocytes were used. These data indicate that the genotoxic potency of Glu-P-2 in the intact hepatocyte differs from that observed in the bacteria. Treatment of hepatocytes with a-naphthoflavone, a selective inhibitor of polycyclic hydrocarbon-inducible cytochrome P-450 form(s), prior to exposure to the heterocyclic amines completely inhibited the mutagenic effect in the bacteria. In vivo administration of all the heterocyclic amines 4 hr prior to isolating the hepatocytes resulted in DNA damage, and this effect was augmented by TCDD pretreatment of mice. Our data suggest that agents modulating the activity and composition of the cytochrome P-450 system may greatly influence both toxicity and carcinogenicity of these heterocyclic amines.     

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.     

Differential induction of fetal mouse liver and lung cytochromes P-450 by beta-naphthoflavone and 3-methylcholanthrene

Previous studies have shown that the incidences of liver and lung tumors in mice exposed transplacentally to 3-methyl-cholanthrene (MC) were significantly influenced by the sensitivity of both mothers and fetuses to induction of cytochrome(s) P-450 by polycyclic aromatic hydrocarbons. In order to delineate further the biochemical and molecular processes underlying the observed biological effects, the inductive effect of MC and beta-naphthoflavone (beta NF) on cytochrome P-450 was determined at the biochemical and molecular levels. C57BL/6 females were mated with DBA/2 males and treated i.p. on day 17 of gestation with olive oil alone, 150 mg/kg of beta NF or different doses of MC. At various times after injection the mothers were sacrificed and the fetuses removed for biochemical and molecular studies. MC caused maximal induction of aryl hydrocarbon hydroxylase (AHH) activity by 8 h in both the liver and lung. beta NF caused nearly maximal induction of AHH activity by 8 h in the lung but had little effect on liver AHH activity at this time. Maximal induction with beta NF occurred by 24 h in both organs. Addition of monoclonal antibody 1-7-1, specific for the MC-inducible forms of cytochrome P-450 (P-450IA1 and A2), to the incubation mixtures resulted in a 55-70% inhibition of AHH activity in both lung and liver assays, regardless of the inducing agent used, while having no effect on AHH activity from oil-treated mice. RNA blot analysis carried out in parallel with enzyme assays demonstrated that the levels of enzyme activity correlated very well with the levels of steady-state RNAs. MC caused maximal induction of P-450IA1 RNA levels 4 h after injection in both organs and a biphasic secondary increase was observed in the lung. Maximal levels of P-450IA1 RNA were seen at 12-16 h following injection of beta NF. However, the ratio of P-450IA1 RNAs present at 16 versus 2 h in the beta NF-treated liver appeared greater than that in the lung. P-450IA2 was also induced in fetal liver and lung, but at low levels relative to P-450IA1. The results indicate that the increase in functional AHH activity was primarily due to induction of cytochrome P-450IA1. The differences in induction kinetics observed for cytochromes P-450IA1 and A2 suggest that these enzymes exhibit both tissue- and inducer-dependent specificity.     

Effects of phenobarbital and 3-methylcolanthrene treatment on microsomes of Morris hepatoma 3924-A, tumour-bearing and normal rat liver.

Microsomal cytochromes and some oxidative activities were determined in normal rat liver, tumour-bearing rat liver and Morris hepatoma 3924-A. Except for a moderate lowering of cytochromes and enzymes in host livers, the relation between TPNH-cytochrome c reductase activity and cytochrome P-450 TPNH reduction, both increased by phenobarbital (PB) and decreased by 3-methylcolanthrene (3-MC) treatment, is noteworthy. In tumour cytochromes b5 and P-450 are absent and TPNH-cytochrome c reductase is unmeasurable and not induced by PB or 3-MC treatment. Aminopyrine demethylase activity, instead, is comparable with normal or host liver and it is modified by PB or 3-MC treatment in the same way, despite the microsomal enzymes pathway disorganization. Microsomal enzymatic defect selectivity in tumours may be due to a deranged microsome-linked growth control.