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.     

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.     

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.     

Strain Differences in Susceptibility to 2-Acetylaminofluorene and Phenobarbital Promotion of Hepatocarcinogenesis: Immunohistocliemical Analysis of Cytochrome P-450 Isozyme Induction by 2-Acetylaminofluorene and Phenobarbital

Strain differences in the expression of cytochrome P-450 isoenzymes (P-450s) during enhancement of hepatocarcinogenesis by 2-acetylaminofluorene (2-AAF) and phenobarbital (PB) were investigated immunohistochemically using monoclonal antibodies against phenobarbital (PB) (APF3) or 3-methylcholanthrene (3-MC) (APH8) inducible P-450s. LEW, SD, WBN, F344, SHR, NAR, Wistar and ODS rats were studied, the first five strains proving to be less susceptible to 2-AAF induction of APH8 while responding strongly to the promoting influence of this chemical, as reported previously. The other three strains, NAR, Wistar and ODS, demonstrated greater inducibility, this correlating with an observed resistance to promotion by 2-AAF. PB administration was not associated with any strain difference in APF3 cytochrome P-4SO inducibility except in the ODS rat, in which its effects were minimal. The results provide direct evidence that differential expression of cytochrome P-450 species plays a major role in determining responsiveness to hepatocarcinogenesis-promoting agents such as 2-AAF.     

Saperconazole: a selective inhibitor of the cytochrome P-450-dependent ergosterol synthesis in Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes

The N-1-substituted triazole antifungal, saperconazole, is a potent inhibitor of ergosterol synthesis in Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes. Fifty % inhibition is already achieved at nanomolar concentrations. The saperconazole-induced inhibition of ergosterol synthesis coincides with an accumulation of 14-methylated sterols, such as 24-methylenedihydrolanosterol, lanosterol, obtusifoliol, 14 alpha-methylfecosterol, 14 alpha-methylergosta-8,24(28)-dien-3 beta-6 alpha-diol and 14 alpha-methylergosta-5,7,22,24(28)-tetraenol. This indicates that saperconazole interferes with the cytochrome P-450 (P-450)-dependent 14 alpha-demethylation of lanosterol and/or 24-methylenedihydrolanosterol. Saperconazole forms stable drug-P-450-complexes by binding via its free triazole nitrogen to the heme iron and via its N-1 substituent to the apoprotein moiety. The triazole derivative is a highly selective inhibitor of the 14 alpha-demethylase in fungal cells. It is a poor inhibitor of the 14 alpha-demethylation of lanosterol in rat and human liver cells. Saperconazole is, at concentrations as high as 10 microM, devoid of effects on the P-450-dependent cholesterol side-chain cleavage and 11 beta-hydroxylase, 17,20-lyase,21-hydroxylase and aromatase. Saperconazole does not interfere with the 2 alpha, 6 alpha-, 6 beta- and 7 alpha-hydroxylations of testosterone in microsomes from male rat liver. At high concentrations (greater than 5 microM) an inhibition of the 16 beta-hydroxylations is seen.     

Human hepatic and renal microsomes, cytochromes P450 1A1/2, NADPH:cytochrome P450 reductase and prostaglandin H synthase mediate the formation of aristolochic acid-DNA adducts found in patients with urothelial cancer

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Understanding which human enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual's susceptibility to this plant carcinogen. Using the (32)P postlabeling assay, we examined the ability of microsomal samples from 8 human livers and from 1 human kidney to activate AAI, the major component of the plant extract AA, to metabolites forming adducts in DNA. Microsomes of both organs generated DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed from AAI by all human hepatic and renal microsomes. To define the role of human microsomal enzymes in the activation of AAI, we investigated the modulation of AAI-DNA adduct formation by cofactors and selective inhibitors of microsomal reductases, cytochrome P450 (CYP) enzymes, NADPH:CYP reductase and NADH:cytochrome b(5) reductase. We also determined whether the activities of CYP and NADPH:CYP reductase in different human hepatic microsomal samples correlated with the levels of AAI-DNA adducts formed by the same microsomal samples. On the basis of these studies, we attribute most of the activation of AAI in human hepatic microsomes to CYP1A2. In contrast to human hepatic microsomes, in human renal microsomes NADPH:CYP reductase is more effective in AAI activation. In addition, prostaglandin H synthase is another enzyme activating AAI in renal microsomes. The results demonstrate for the first time the potential of microsomal enzymes in human liver and kidney to activate AAI by nitroreduction.