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.     

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.     

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.     

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.     

Metabolism of nitrosamines by purified rabbit liver cytochrome P-450 isozymes

The metabolism of nitrosamines by microsomal cytochrome P-450 (P-450) isozymes was studied in a reconstituted monooxygenase system. P-450 LM2, LM3a, LM3b and LM3c, LM4, and LM6 were purified, respectively, from the livers of phenobarbital-treated, ethanol-treated, untreated, isosafrole-treated, and imidazole-treated rabbits. Of these isozymes, LM3a had the highest N-nitrosodimethylamine demethylase (NDMAd) activity with a Km of 2.9 mM and Vmax of 9.3 nmol/min/nmol. LM2, LM4, and LM6 exhibited NDMAd activity only at high N-nitrosodimethylamine concentrations, and isozymes LM3b and LM3c had poor activity even at the highest substrate concentrations examined. LM2, however, was more active than LM3a in the metabolism of N-nitrosomethylaniline. With each isozyme (LM3a or LM4), only one Km for NDMAd was observed, whereas with rabbit liver microsomes, multiple Km of 0.07, 0.27, and 36.8 mM were obtained. P-450 isozymes also catalyzed the denitrosation of nitrosamines at rates comparable to or lower than the demethylation, and the ratio of these two reactions was different with different nitrosamines. 2-Phenylethylamine and 3-amino-1,2,4-triazole, which were believed previously to affect NDMAd by mechanisms independent of P-450, were shown to be potent inhibitors of P-450-dependent NDMAd. These results further establish the role of P-450 isozymes in the metabolism of nitrosamines and indicate that LM3a is apparently responsible for the increased N-nitrosodimethylamine metabolism associated with ethanol treatment.     

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.     

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 promutagens. 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 B1 (AFB1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-6-methyldipyrido-[1,2-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.     

Species Difference among Experimental Rodents in the Activity and Induction of Cytochrome P-450 Isozymes for Mutagenic Activation of Carcinogenic Aromatic Amines

The expressions of hepatic microsomal cytochrome P-450 isozymes in male rats, mice, hamsters and guinea pigs were studied comparatively with or without an ip injection of a cytochrome P-450 inducer. The activity and quantity of microsomal cytochrome P-450 isozymes were determined respectively by a bacterial mutation assay with Salmonella typhimurium TA98 and immunochemical assays using monoclonal antibodies against rat cytochrome P-450 isozymes. 3-Methoxy-4-aminoazobenzene (3-MeO-AAB), 2-amino-3-methyl-9 H -pyrido[2,3- b ]indole acetate (MeAαC) and 3-methylcholanthrene were used as cytochrome P-450 inducers, and 7 carcinogenic aromatic amines including 3-MeO-AAB and MeAαC were used as substrates for the mutation assay. By means of these assays, we examined the species differences among rodents in the activity and induction rate of hepatic cytochrome P-450 isozymes responsible for the mutagenic activation of carcinogenic aromatic amines.     

Purification and characterization of cytochrome P-450 induced by benz(a)anthracene in mouse skin microsomes

Topical application of benz(a)anthracene to mouse skin elicited a 2-fold increase in cytochrome P-450 content, with accompanying increases in monooxygenase activities such as benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and acetanilide 4-hydroxylation, in the microsomes. A major form of cytochrome P-450 was purified from skin microsomes of mice treated with polycyclic aromatic hydrocarbon. A specific content of 1.95 nmol/mg of protein, which corresponded to 48-fold purification from the microsomes, was observed. The purified protein produced a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a molecular weight of 55,000. Using Western blotting, the band immunochemically cross-reacted with antibody which had been raised against rat liver cytochrome P-450MC-1. The purified preparation efficiently catalyzed benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation when reconstituted with NADPH-cytochrome P-450 reductase. These activities were inhibited by 7,8-benzoflavone as well as anti-cytochrome P-450MC-1 antibody, but not by P-450PB-1 antibody. The results indicate that, in mouse skin microsomes, a cytochrome P-450 induced by benz(a)anthracene is enzymatically and immunochemically similar to rat liver cytochrome P-450MC-1. It is suggested that this enzyme plays an important role in the activation of carcinogenic polycyclic aromatic hydrocarbons.     

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.     

Activation of 3,3'-dichlorobenzidine in rat liver microsomes to mutagens: involvement of cytochrome P-450d

Studies were carried out, using antibodies to specific cytochromeP-450 isozymes, to identify the isozymes involved in the NADPH-dependentactivation of 3,3'-dichlorobenzidine (DCB) by rat hepatic microsomesto mutagens in the Ames test. DCB activation was not affectedby a monoclonal antibody specific for P-450c or by a monoclonalantibody specific for P-450b, but was inhibited 69% by a polyclonalantibody made against P-450d. DCB activation was also inhibited46% by antibody specific for NADPH-cytochrome P-450 reductase.Further, addition of methimazole, a high affinity substratefor the flavin-containing monooxygenase, reduced the residualmutagenicity in the systems containing antibody to P-450d andcytochrome P-450 reductase to 9% and 19%, respectively, of theappropriate control values. It is concluded that P-450d contributesto a majority of the P-450-dependent activation of DCB in hepaticmicrosomes. The results also suggest that the flavin-containingmonooxygenase may contribute to the microsomal activation ofDCB.     

Immunological identification and effects of 3-methylcholanthrene and phenobarbital on rat pulmonary cytochrome P-450

Rabbit antibodies to the phenobarbital (PB) inducible rat liver microsomal cytochrome P-450s b and e and to 3-methylcholanthrene (MC) inducible P-450c were used to examine the expression of these isozymes in rat lungs. Western blots of total lung microsomes demonstrated that about 40 pmol P-450b/mg protein (and no detectable P-450e) were present in lungs from control or MC treated rats and that pretreatment with PB caused a small but significant (P less than 0.05) increase in the expression of P-450b. Microsomes from control and PB treated lung contained minimal levels of P-450c, and MC induced this isozyme to 185 pmol/mg. Immunocytochemistry was used to demonstrate immunoreactivity to these isozymes in specific cell types. Neither P-450b nor P-450c was detectable in endothelial cells from control or PB treated lungs, but MC increased immunoreactivity to P-450c in pulmonary endothelial cells. Type II alveolar cells showed distinct immunoreactivity to P-450b and weak immunoreactivity to P-450c in control or PB treated rats. Individual Clara cells stained for either P-450c or P-450b in control, MC treated, and PB treated rats, and colocalization was observed in some cells. An increase in type II alveolar cell and Clara cell immunoreactivity to P-450c was observed after MC induction. Mast cells, identified by metachromatic Giemsa staining, appeared to react nonspecifically with both antisera. In conclusion, P-450c is highly inducible by MC in rat lung (detected in microsomes by Western blot), specifically in endothelial cells, Clara cells, and alveolar type II cells (as visualized by immunocytochemistry); and P-450b is present in rat lung microsomes, and immunoreactivity to this isozyme is localized in alveolar type II and Clara cells.     

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(6 beta-1) (testosterone 6 beta-hydroxylase) and P-450e (a phenobarbital-inducible form). In accordance with the reduction in P-450-male, capacities for microsomal 16 alpha- and 2 alpha-hydroxylations, but not 6 beta-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.     

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)     

Reconstitution of rabbit liver microsomal N-nitrosopyrrolidine alpha-hydroxylase activity

The in vitro alpha-hydroxylation of N-nitrosopyrrolidine (NPYR) by both isolated rabbit liver microsomes and purified cytochrome P-450 isozymes was investigated. Microsomes from untreated rabbits catalyzed the alpha-hydroxylation of NPYR at rates similar to those reported previously for rats, mice, and hamsters. The effect of established inducers of microsomal P-450 caused complex changes in apparent rates of alpha-hydroxylation of NPYR which made interpretation of responses to inducer pretreatment difficult and suggested the participation of multiple cytochrome P-450 isozymes in the metabolism of NPYR. Partial inhibition of alpha-hydroxylase activity by antibodies against rabbit isozymes 2, 3a, and 5 indicated the participation of at least these three isozymes in microsomal catalysis. Reconstitution studies using purified rabbit isozymes 2, 3a, 3b, 3c, 4, and 6 indicated that isozymes 2, 3a, 4, and 6 possessed significant alpha-hydroxylase activity with isozymes 3a and 6 exhibiting the highest activity when assayed at 20 mM NPYR. As NPYR concentrations were decreased, the rates of catalysis for the reconstituted systems were differentially decreased such that isozyme 3a exhibited the highest activity at low NPYR concentrations. These data indicate that isozyme 3a is the preferred catalyst for the alpha-hydroxylation of NPYR at low substrate concentrations and suggest that conditions such as chronic ethanol consumption which lead to the induction of isozyme 3a in rabbits or its orthologue in other species can account for enhanced rates of alpha-hydroxylation and metabolic activation of NPYR.     

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.     

Species difference among experimental rodents in the activity and induction of cytochrome P-450 isozymes for mutagenic activation of carcinogenic aromatic amines

The expressions of hepatic microsomal cytochrome P-450 isozymes in male rats, mice, hamsters and guinea pigs were studied comparatively with or without an ip injection of a cytochrome P-450 inducer. The activity and quantity of microsomal cytochrome P-450 isozymes were determined respectively by a bacterial mutation assay with Salmonella typhimurium TA98 and immunochemical assays using monoclonal antibodies against rat cytochrome P-450 isozymes. 3-Methoxy-4-aminoazobenzene (3-MeO-AAB), 2-amino-3-methyl-9H-pyrido[2,3-b]indole acetate (MeA alpha C) and 3-methylcholanthrene were used as cytochrome P-450 inducers, and 7 carcinogenic aromatic amines including 3-MeO-AAB and MeA alpha C were used as substrates for the mutation assay. By means of these assays, we examined the species differences among rodents in the activity and induction rate of hepatic cytochrome P-450 isozymes responsible for the mutagenic activation of carcinogenic aromatic amines.     

Role of xanthine oxidase in the interferon-mediated depression of the hepatic cytochrome P-450 system in mice

Interferon, interferon inducers, and a variety of other immunomodulators are known to depress the hepatic cytochrome P-450 drug-metabolizing system. Two concepts have been proposed to explain this phenomenon. (a) The steady-state of cytochrome P-450 is altered through decreased synthesis and increased degradation of cytochrome P-450 apoprotein. (b) Interferon induces xanthine oxidase; superoxide generated by interferon-induced xanthine oxidase destroys cytochrome P-450. The current study investigated the second concept. Administered polyribonucleotides [polyriboinosinic acid.polyribocytidylic acid (poly IC), polyriboinosinic acid.polycytidylic acid, polylysine and carboxymethylcellulose, mismatched poly IC], recombinant murine gamma-interferon, and a natural murine alpha/beta-interferon were shown to depress hepatic cytochrome P-450 and selected microsomal cytochrome P-450-dependent monooxygenase reactions and to induce hepatic xanthine oxidase activity. The feeding of tungstate in the drinking water largely depleted xanthine oxidase in mice; cytochrome P-450 levels and monooxygenase activities were not affected by tungstate treatment. Tungstate rendered the level of xanthine oxidase much below that in mice that had not received tungstate regardless of whether or not they had received poly IC or interferon; nevertheless, poly IC and interferon produced losses of cytochrome P-450 and monooxygenase activities in these tungstate-treated mice equivalent to those observed in mice that had not received tungstate. The administration of N-acetylcysteine did not prevent the loss of cytochrome P-450 induced by poly IC, as has been reported, nor did the incubation of microsomal cytochrome P-450 with buttermilk xanthine oxidase and hypoxanthine cause a loss of cytochrome P-450, which has also been reported. It is concluded from these studies that the induction of xanthine oxidase and the loss of cytochrome P-450 generated by interferon are coincidental rather than causally related phenomena.     

Oxidative metabolism of 1-nitropyrene by rabbit liver microsomes and purified microsomal cytochrome P-450 isozymes

Rabbit liver (male) microsomal metabolism of 10 microM [4,5,9,10-3H]-1-nitropyrene (1NP) was investigated. The total metabolism was not appreciably different with rates of 4.44 +/- 0.45, 3.98 +/- 0.19, 3.90 +/- 0.16, and 3.75 +/- 0.27 nmol/min/mg protein, respectively, for microsomes from phenobarbital, Aroclor-1254, ethanol-treated, and untreated rabbits. However, a more noticeable difference was found in the formation of specific metabolites. Phenobarbital treatment induced changes which favored 1-nitropyrene-3-ol formation, and Aroclor-1254 and ethanol-induced changes which favored 1-nitropyren-6-ol and 1-nitropyren-8-ol formation. 1NP was incubated with untreated microsomes and alpha-naphthoflavone, an inhibitor of rabbit cytochrome P-450 form 6 at low concentrations (less than 1 microM), and an activator of form 3c at high concentrations. The presence of alpha-naphthoflavone changed the profile of metabolites while not affecting the total metabolism. Using purified isozymes of rabbit P-450, we found the constitutive form 3b metabolized 1NP at the highest rate with a catalytic activity of 26.8 nmol/min/nmol P-450. Forms 2 and 6 exhibited rates of 2 and 2.2 nmol/min/nmol P-450. Forms 3a, 3c, and 4 had rates about 50- to 300-fold lower than form 3b. High performance liquid chromatography was used to identify the metabolites when the incubations were carried out in the presence of purified rabbit epoxide hydrolase. With form 6, 54% of the metabolites were accounted for as 1-nitropyren-3-ol, while with form 3b, 73% of the metabolites were 1-nitropyren-6-ol and 1-nitropyren-8-ol. The K-region dihydrodiols were formed by forms 2 and 3b, but not by forms 3c or 6. These results demonstrate that 1NP is a preferential substrate for form 3b, and that a preponderance of the metabolism with untreated rabbit liver microsomes can be attributed to this isozyme.     

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.