Four Forms of Cytochrome P-450 in Human Fetal Liver: Purification and Their Capacity to Activate Promutagens

Four forms of cytochrome P-450 were separated and purified to electrophoretic homogeneity from human fetal livers. These forms of cytochrome P-450, termed P-450HFLa, P-450HFLb, P-450HFLc and P-450HFLd, were distinguishable from each other in their molecular weights, spectral properties, immunochemical properties and mutagen-producing activities from promntagens. The molecular weights of P-450HFLa, b, c and d were estimated to be 51,500, 49,000, 51,500 and 50,000, respectively. Antibodies to P-450HFLa recognized P-450HFLc but not P-450HFLb or d, and antibodies to rat P-448-H (P-450IA2) cross-reacted with P-450HFLb but not with other forms of cytochrome P-450. The N-terminal amino acid sequence of P-450HFLc was highly homologous, but not identical, to that of P-450HFLa. Each form of cytochrome P-450 catalyzed mutagenic activation of aflatoxin Bl (AFB1), 2-amino-3-methylimidazo[4,5- f ]quinoline (IQ) and 2-amino-6-methyldipyrido-[l,2-a:3',2'- d ]imidazole (Glu-P-1) at different rates. P-450 HFLa showed activities to produce mutagen(s) from AFB1, IQ and to a lesser extent from Glu-P-1. P-450 HFLb activated IQ at a faster rate than did the other forms. P-450 HFLc produced a mutagen from AFB1 and Glu-P-1 but not from IQ. P-450 HFLd did not activate these promutagens at significant rates.     

Metabolic activation of aflatoxin B1 and 2-amino-3-methylimidazo[4,5-f]-quinoline by human adult and fetal livers

The mutagenic activation of promutagens by human adult and fetal livers was investigated using the umu test system. Among the promutagens studied, aflatoxin B1 (AFB1) and 2-amino-3-methyl-imidazo[4,5-f] quinoline (IQ) were efficiently activated to mutagens by both adult and fetal livers. 7,8-Benzoflavone inhibited the activation of IQ by fetal livers, but the inhibition observed in fetal livers was much less than that observed in adult livers. Antibodies to P450HM1 (P450111A4) and P450HFLa markedly inhibited the activation of AFB1 by adult and fetal livers, respectively. The formation of genotoxic product(s) from IQ in human adult livers was almost completely inhibited by anti-P448H (P4501A2) antibodies but not by anti-P450HM1 antibodies, whereas that in fetal livers was inhibited by both anti-P450HFLa and anti-P450IA2 antibodies. P450HFLa catalyzed the mutagenic activation of both AFB1 and IQ in a reconstituted system. On the contrary, P450HM1 catalyzed the mutagenic activation of AFB1 but not IQ. A preparation of cytochrome P450 partially purified from human fetal livers and cross-reactive with anti-P450IA2 antibodies was found to be active for mutagenic activation of IQ in a reconstituted system. These results indicate that P450HFLa and P450HM1 are mainly involved in the genotoxic product formation from AFB1 in fetal and adult livers, respectively, and that the metabolic activation of IQ in fetal livers is catalyzed by two forms of cytochrome P450, P450HFLa, and cytochrome P450 immunochemically related to P450IA2 but that in adult livers it is mainly catalyzed by cytochrome P450 related to P450IA2.     

A new form of cytochrome P-450 responsible for mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline in human livers.

Antibodies to P-450IA2 strongly inhibited the mutagenic activation of 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate but not aflatoxin B1 in human liver microsomes. The anti-rat P-450IA2 antibodies were capable of recognizing two proteins which show different mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of human liver microsomes. A new form of cytochrome P-450 (designated P-450-HM4) cross-reactive with anti-rat P-450IA2 antibodies showing that the smaller molecular weight was purified from human liver microsomes by means of the fast-performance liquid chromatography system. The molecular weight of P-450-HM4 was estimated to be 49,000, which was apparently different from that of P-450PA (human P-450IA2). The antibodies to P-450-HM4 did not cross-react with P-450PA (human P-450IA2) but inhibited to various extents the mutagenic activation of IQ in microsomes from human livers. In addition, P-450-HM4 showed significant mutagen-producing activity from IQ in a reconstituted system. Together with these and other results reported previously, it is concluded that at least two forms of cytochrome P-450 [P-450-HM4 and P-450PA (human P-450IA2)] are involved in the mutagenic activation of IQ in human liver.     

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.     

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.     

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.     

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.     

Mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline by complementary DNA-expressed human liver P-450

Eight forms of human liver microsomal P-450 were individually expressed in human hepatoma Hep G2 cells with a vaccinia virus cDNA expression system. Using the Ames test, each expressed P-450 was examined for its ability to activate to mutagenic products the compounds, 2-amino-3-methylimidazo[4,5-f]quinoline, 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, respectively. Three forms of human P-450 significantly activated 2-amino-3-methylimidazo[4,5-f]quinoline when the latter was at high substrate concentrations, but only a single form, P-450IA2, showed very high activation of all promutagens at lower substrate concentrations. Human IA2 had extraordinarily high affinity towards four promutagens tested and is likely the predominant P-450 enzyme responsible for their mutagenic activation in human liver.     

Human cDNA-expressed cytochrome P450 IA2: mutagen activation and substrate specificity

The vaccinia virus cDNA expression system was used to produce human cytochrome P450 IA2 in a hepatoma cell line that is devoid of significant basal levels of P450. The expressed enzyme yielded a reduced carbon monoxide-bound difference spectrum with a lambda max of 449 nm. Catalytic activities and mutagen activation ability of the human enzyme were assessed and directly compared with results obtained with the orthologous mouse IA2, which was also expressed using vaccinia virus. Both the human and mouse enzymes were able to catalyze efficiently the p-hydroxylation of aniline. Mouse IA2 also catalyzed ethoxyresorufin O-deethylation, and its activity was sevenfold greater than expressed human IA2. The mouse and human enzymes also activated several promutagens and procarcinogens. Mouse IA2 was five- to sevenfold more active than the human enzyme for activation of the procarcinogens 2-acetylaminofluorene and benzo[a]pyrene-trans-7,8-dihydrodiol and the promutagens Glu-P-2 and Trp-P-1. Comparable activities were observed with 2-aminoanthracene, 2-aminofluorene, and Glu-P-1. These data demonstrate the utility of cDNA expression for examining the activities of human P450s and further suggest potentially important differences in catalytic activities of orthologous P450s found in different species.     

Activation of the food-derived mutagen 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine by rabbit and human liver microsomes and purified forms of cytochrome P-450

The specificity of rabbit cytochrome P-450 involved in the mutagenicactivation of 2-amino-1-methyl-6-phenylimid-azo[4, 5-b]pyridine(PhIP) was assessed using control and induced rabbit liver andlung microsomes, and six purified forms of cytochrome P-450.The number of revertants produced/2.5µg PhIP by controlrabbit liver was 260 ± 196/10 µg of microsomalprotein (mean ± SD; n = 3), and this increased to 1265± 248 when 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD)-inducedliver microsomes were used as the activation source in the Amestest. Microsomes form phenobarbital-, rifampicin-and acetone-pretreatedrabbits showed no increase in activity over controls. Controllung microsomes did not activate PhIP to a mutagen, whereasTCDD-induced lung microsomes produced 1443 ± 136 (mean± SD; n=4) Ames/Salmonella revertants/100 µg protein.In reconstitution experiments cytochrome P450 forms 4 and 6were found to be efficient activators of PhIP to a mutagen.Form 6 was 3.1-fold more active than form 4 and produced 4577revertants/10 pmol with a 20-min preincubation step in the Amestest. Cytochrome form 5 produced 17 revertants/10 pmol and forms2, 3b and 3c were not active in metabolizing PhIP to a mutagen.A highly significant statistical correlation existed betweenthe capacity of control and induced liver microsomes to activatePhIP to a mutagen and their cytochrome P-450 form 4 (r = 0.97,r² = 0.94) and form 6 (r = 0.95, r² = 0.90) content. These datastrongly support the involvement of polycyclic hydrocarbon-inducibleforms of cytochrome P450 in the activation of PhIP in the rabbit.Anti-rabbit forms 4 and 6 IgGs recognized proteins in sevenhuman liver microsomes of comparable mol. wt to rabbit cytochromeP-450 forms 4 and 6. However, no correlation existed betweenthe content of these proteins and the capacity of human livermicrosomes to activate PhIP.     

Differential effects on the metabolism of dimethylnitrosamine and aflatoxin B1 by hepatic microsomes from senescent rats

The ability of hepatic microsomes from senescent rats to metabolize the two potent hepatocarcinogens dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) was investigated. Seven and 24-month-old male Sprague-Dawley rats were used. Liver weights, and microsomal protein per gram tissue weight were higher, whereas cytochrome P-450 and cytochrome b5 were significantly lower in older rats. Glutathione S-transferases and NADPH cytochrome c reductase activities were dramatically reduced in senescent rats. There was no difference in the formation of formaldehyde from DMN in vitro (31 vs. 34 pmol/nmol P-450) between the young and old rats. In contrast, increased microsome mediated binding of AFB1 to DNA was observed in older rats (116 vs. 228 pmol/nmol P-450) suggesting the possibility of either quantitative or qualitative changes in P-450 species. Additionally the cytoplasmic GSH S-transferases from older rats affected lower inhibition of binding of AFB1 to DNA. These results indicated differential abilities in the hepatic microsomal metabolism of these two carcinogens which may cause differential effects of these carcinogens in senescent rats.     

Effect of beta-naphthoflavone on the metabolism of aflatoxin B1 in hamsters

The effect of beta-naphthoflavone (BNF) pretreatment of hamsters on the hepatic metabolism of aflatoxin B1 (AFB1) has been examined in studies in vitro and in vivo. Pretreatment with BNF not only increased microsomal cytochrome P-450 by 50-80% but also increased microsome-mediated AFB1 epoxidation as measured by AFB1-DNA binding 2.6 fold without significantly affecting other hydroxylations. Neither cytosolic GSH S-transferases' activities nor AFB1-GSH (AFB1-SG) conjugation were affected. In vivo, hepatic AFB1-DNA binding was also increased about 3-4-fold. These results in contrast to those observed in the rat indicate that induced species of cytochrome P-450 are primarily responsible for higher epoxidation of AFB1 in the hamster.     

Evidence of direct generation of oxygen free radicals from heterocyclic amines by NADPH/cytochrome P-450 reductase in vitro.

Highly purified cytochrome P-450 reductase (also called cytochrome c reductase; EC 1.6.2.4.) and NADPH were used to generate superoxide radical (O2.-) from 11 different heterocyclic amines (HCAs) as identified by electron spin resonance spectroscopy using the spin trapping method with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The signal intensity of DMPO-OOH(-O2-) (i.e., the DMPO spin adduct of O2.-) was strongest for 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). The O2.- generation with HCAs decreased in the following order: 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) = 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) > 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (diMeIQx) > or = other HCAs; O2.- generation was lowest with 2-amino-3-methyl-9H-pyrido[2,3-b]indole .CH3COOH (MeA alpha C). By using Lineweaver-Burk plots, Km values of cytochrome P-450 reductase for mitomycin C, IQ, and MeIQ were determined to be 1.60 x 10(-6) M, 1.97 x 10(-5) M, and 2.83 x 10(-6) M, respectively. The present findings have important implications for carcinogenesis because of the known effect of oxygen radicals on cell proliferation.     

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.     

Structure-activity relationships of chlorinated benzenes as inducers of hepatic cytochrome P-450 isozymes in the rat

This study compared the ability of hexachlorobenzene (HCB) and of other chlorinated benzenes to induce cytochrome P-450 isozymes in rat liver. HCB (greater than 99% pure) induced both the phenobarbital-inducible forms (cytochrome P-450b and P-450e) and the 3-methylcholanthrene (3-MC)-inducible forms (P-450c and P-450d) of cytochrome P-450. However, HCB differed from many 3-MC-type inducers by inducing P-450d preferentially over P-450c. In contrast to HCB, the lower chlorinated benzenes did not induce significant amounts of P-450c or P-450d in the rat, but were phenobarbital-type inducers, inducing P-450b and P-450e. These data indicate that the hepatic effects of HCB differ markedly from those of other chlorinated benzenes. However, chlorinated dibenzodioxins also induce P-450c and P-450d in the rat, and although chlorinated dibenzodioxins and dibenzofurans contaminate certain commercial products, none were detected by gas chromatography/mass spectrometry (detection limit 0.5 ppm) in the HCB used in this study. The evidence that HCB interacted with the receptor for 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) was equivocal. At a concentration of 10(-6) M, HCB produced a slight decrease (18%) in the binding of 3H-TCDD to this protein in vitro, but had no effect at lower concentrations. However, as an inducer of two 3-MC-inducible isozymes of P-450, HCB was clearly more effective in aromatic-hydrocarbon-responsive mice (C57Bl/6J) than in non-responsive mice (DBA/2J), suggesting that HCB may act through the Ah receptor.     

Participation of cytochrome P-450 in reductive metabolism of 1-nitropyrene by rat liver microsomes

Reductive metabolism of carcinogenic 1-nitropyrene by rat liver microsomes and reconstituted cytochrome P-450 systems was investigated. Under the nitrogen atmosphere, 1-aminopyrene was the only detected metabolite of 1-nitropyrene. The reductase activity in liver 105,000 X g supernatant fraction was ascribed to DT-diaphorase, aldehyde oxidase, and other unknown enzyme(s) from the results of cofactor requirements and inhibition experiments. The microsomal reductase activity was inhibited by oxygen, carbon monoxide, 2,4-dichloro-6-phenylphenoxyethylamine, and n-octylamine. Flavin mononucleotide markedly enhanced the activity, and 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride also enhanced it, but slightly. The microsomal activity was induced by the pretreatment of rats with 3-methylcholanthrene, sodium phenobarbital, or polychlorinated biphenyl, and the increments of the activity correlated well with those of the specific contents of cytochrome P-450 in microsomes. The reductase activity could be reconstituted by NADPH-cytochrome P-450 reductase and forms of cytochrome P-450 purified from liver microsomes of polychlorinated biphenyl-induced rats. Among four forms of cytochrome P-450 examined, an isozyme P-448-IId which showed high activity in hydroxylation of benzo(a)pyrene catalyzed most efficiently the reduction of 1-nitropyrene. The results of this study indicate the central role of cytochrome P-450 in the reductive metabolism of 1-nitropyrene in liver microsomes.     

Absence of p53 Mutations in Rat Colon Tumors Induced by 2-Amino-6-methyldipyrido[1,2-a:3', 2'-d]imidazole, 2-Amino-3-methylimidazo[4,5-f]quinoline, or 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

Colon tumors were induced in F344 rats by three heterocyclic amines (HCAs), 2-amino-6-methyl-dipyrido[1,2- a :3', 2'- d ]imidazole (Glu-P-1), 2-amino-3-methylimidazo[4,5- f ]quinoline (IQ) or 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP), and examined for p53 mutations. Seven carcinomas induced by Glu-P-1, and nine carcinomas and two adenomas induced by IQ were examined by cDNA-polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis from codon 103 to 391 of p53, which encompasses the conserved regions II to IV. Nine carcinomas induced by PhIP were examined by genomic PCR-SSCP analysis of exons 5 to 7 (from codon 124 to 304), which encompasses the 3'half of the conserved region II and all the conserved regions III-V. No band shifts were found in any of these tumors under at least two conditions of SSCP analysis. Our previous study had shown a Ki- ras mutation in only one Glu-P-1-induced adenocarcinoma among the same 27 colon tumors, and no other mutation of ras family genes had been found. HCA-induced rat colon tumors appear to represent a group of human colon tumors in which neither Ki- ras nor p53 is involved.     

Hepatocarcinogenic heterocyclic aromatic amines that induce cytochrome P-448 isozymes, mainly cytochrome P-448H (P-450IA2), responsible for mutagenic activation of the carcinogens in rat liver

Male F344 rats were treated with hepatocarcinogenic heterocydicaromatic amines such as amino acid-and protein-pyrolysate components(Trp P-1, Trp P-2, Glu P-1, Glu P-2, AC, MeAC, IQ and MeIQx)and changes in microsomal cytochrome P-450 isozymes in the liverswere examined by means of immuno-Western blotting using anti-ratcytochrome P-450 monoclonal antibodies. The results suggestedthat all chemicals tested induce cytochrome P-448 isozymes,particularly cytochrome P-448H (P-450IA2), which efficientlymediate mutagenic activation of the carcinogens. This was substantiatedby the enzymatic analyses with the substrates showing differentcharacters to rat cytochrome P-450 isozyme-mediated mutagenesis.     

Comparative carcinogenicity of 4-aminobiphenyl and the food pyrolysates, Glu-P-1, IQ, PhIP, and MeIQx in the neonatal B6C3F1 male mouse.

The tumorigenic activities of four representative heterocyclic amine food pyrolysates, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), were assessed in the neonatal male B6C3F1 mouse and were compared with that of the potent human carcinogen, 4-amino-biphenyl (4-ABP). These aromatic amines were administered by i.p. injection at two dose levels on days 1, 8, and 15 after birth; and the incidence of tumors was examined at 8 and 12 months. Glu-P-1, IQ, PhIP, MeIQx, and 4-ABP each induced a significant incidence of hepatic adenomas, as compared to the solvent-treated (DMSO) control. Hepatocellular carcinomas were also observed with 4-ABP, SO, and MeIQx. Overall tumorigenicity was in the order: 4-ABP greater than Glu-P-1 greater than IQ approximately PhIP greater than MeIQx greater than DMSO. In the neonatal B6C3F1 mouse, these heterocyclic aromatic amines showed potent tumorigenicity after 8 and 12 months at total doses that were 5-10,000-fold less than those employed in standard chronic bioassays.