isosafrole and alpha-naphthoflavone

1. In vitro studies were conducted to identify the hepatic cytochrome P450 isoform involved in the metabolism of YM992, ((S)-2-[[(fluoro-4-indanyl)oxy]methyl]morpholine monohydrochloride), a novel serotonin re-uptake inhibitor, in human liver microsomes. 2. Microsomes prepared from yeast expressing CYP1A1, CYP1A2 and CYP2D6 effectively metabolized YM992. A significant correlation was observed between the rate of YM992 metabolism and 7-ethoxyresorufin O-deethylation, CYP1A1/2 specific activity, in liver microsomes from 16 individual donors (r2 = 0.628, p < 0.001). Alpha-naphtoflavone and isosafrole, CYP1A1/2 inhibitors, suppressed the metabolism of YM992 in human liver microsomes in a concentration-dependent manner. 3. The metabolism of YM992 in human liver microsomes was inhibited by approximately 95% by antibodies which recognize both CYP1A1 and CYP1A2 whereas antibodies specific for CYP1A1 did not show inhibitory effects. 4. The same major metabolites, M6 and M7, were generated from YM992 after incubation with human liver microsomes and recombinant human CYP1A2. 5. These results suggest that the metabolism of YM992 in human liver microsomes is mainly catalysed by CYP1A2, and that YM992 might increase plasma concentration of concomitant drugs metabolized by CYP1A2 due to competitive inhibition.

Topics: Benzoflavones; Chromatography; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP2D6; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; DNA, Complementary; Dose-Response Relationship, Drug; Humans; Kinetics; Microsomes, Liver; Models, Biological; Morpholines; Protein Binding; Protein Isoforms; Recombinant Proteins; Safrole; Selective Serotonin Reuptake Inhibitors; Serotonin Antagonists

The ability of cigarette smoke condensate to induce a genotoxic response has been measured in liver microsomal and reconstituted monooxygenase systems containing rat and human cytochrome P-450 (P-450) enzymes, as determined by umu gene expression in Salmonella typhimurium TA1535/pSK1002. The reactivities of amino-alpha-carboline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), two compounds known to be present at considerable levels in cigarette smoke condensate, were also determined and compared with regard to genotoxicity. Amino-alpha-carboline and PhIP are activated principally by P-450 1A2 enzymes in human and rat liver microsomes: (a) activation of both compounds was catalyzed efficiently by liver microsomes prepared from rats treated with 5,6-benzoflavone, isosafrole, or the commercial polychlorinated biphenyl mixture Aroclor 1254, and the activities could be considerably inhibited by antibodies raised against P-450 1A1 or 1A2; (b) the rates of activation of these compounds were correlated with the amount of human P-450 1A2 and of phenacetin O-deethylation activity in different human liver microsomal preparations, and these activities were inhibited by anti-P-450 1A2; (c) reconstituted enzyme systems containing P-450 1A enzymes isolated from rats and humans showed the highest rates of activation of amino-alpha-carboline and PhIP. In rat liver microsomes PhIP may also be activated by P-450 3A enzymes; activity was induced in rats treated with pregnenolone 16 alpha-carbonitrile and was inhibited by anti-human P-450 3A4. However, in humans the contribution of P-450 3A enzymes could be excluded as judged by the very low effects of anti-P-450 3A4 on the microsomal activities and poor correlation with P-450 3A4-catalyzed activities in various liver samples. Cigarette smoke condensate strongly inhibited the activation of several potent procarcinogens by human liver microsomes, particularly the reactions catalyzed by P-450 1A2, but was not so inhibitory of the activation reactions catalyzed by P-450 3A4 and of P-450 2D6-catalyzed bufuralol 1'-hydroxylation. Genotoxic components of the cigarette smoke condensate were extracted by using copper phthalocyanine cellulose (blue cotton). Genotoxicity of this extract was observed only after activation by P-450, and the inhibition of P-450 1A2 activities by these extracts was slight.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Aflatoxin B1; Animals; Aroclors; Benzoflavones; beta-Galactosidase; Carbolines; Chlorodiphenyl (54% Chlorine); Cytochrome P-450 Enzyme System; DNA; Dose-Response Relationship, Drug; Humans; Imidazoles; In Vitro Techniques; Male; Microsomes, Liver; Mutagens; Nicotiana; Plants, Toxic; Quinidine; Rats; Rats, Inbred Strains; Safrole; Smoke; SOS Response, Genetics

The present study compared the induction and inhibition of the metabolism of the prototype polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in rat and hamster liver microsomes. The production of total polar metabolites was quantitated by separating 3H-metabolites from [3H]-BaP using reverse-phase thin-layer chromatography. The rate of hepatic microsomal BaP metabolism was similar in the rat and hamster (0.81 vs 0.72 nmol/min/nmol cytochrome P-450 respectively). In the rat, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 5 micrograms/kg, i.p.) and 3-methylcholanthrene (3-MC; 50 mg/kg, i.p., X 3 days) pretreatments doubled the rate of BaP metabolism, whereas phenobarbital pretreatment (PB; 80 mg/kg, i.p., X 3 days) had no effect. In contrast, hamster hepatic microsomal BaP metabolism was elevated 2.3-fold by PB pretreatment, whereas TCDD and 3-MC pretreatments had no effect. Isosafrole pretreatment (ISO; 150 mg/kg, i.p., X 3 days) elevated the rate by almost 2-fold in each species. Another cytochrome P-448-mediated activity, 7-ethoxyresorufin O-deethylase (EROD), was induced by the same compounds that induced BaP metabolism in the rat. In hamster liver microsomes, in contrast to BaP metabolism, EROD was induced by TCDD and 3-MC but not PB or ISO pretreatments. The results suggest differences in the substrate specificity of the cytochromes P-448-450 induced by TCDD, 3-MC and PB in these species. This was supported by the different selectivity of the in vitro inhibitors, metyrapone and 7,8-benzoflavone, towards BaP metabolism and EROD in hepatic microsomes from TCDD- or PB-pretreated rats and hamsters. Reverse-phase HPLC analysis indicated that, while 3-hydroxy-BaP was the major metabolite formed by the untreated rat, untreated hamster liver microsomes formed predominantly BaP-4,5-diol. Microsomes from TCDD-treated rats generated elevated levels of all BaP-diols, diones and 3-hydroxy-BaP, with the major metabolites being BaP-9,10- and BaP-7,8-diols. In contrast, the metabolite profile from TCDD-pretreated hamsters was unchanged from the control. PB-treated hamster microsomes produced elevated levels of BaP-diones and 3-hydroxy-BaP. However, the major hepatic metabolite formed by PB-pretreated hamsters was BaP-4,5-diol, while BaP-9,10- and BaP-7,8-diols were not detected. The results of the study indicate differences in the induced cytochrome P-450s and the generation of toxic BaP metabolites in the liver of the rat and hamster.

Topics: Animals; Benzo(a)pyrene; Benzoflavones; Cricetinae; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Induction; Male; Mesocricetus; Methylcholanthrene; Metyrapone; Microsomes, Liver; Oxidoreductases; Oxidoreductases, N-Demethylating; Phenobarbital; Polychlorinated Dibenzodioxins; Rats; Rats, Inbred Strains; Safrole; Species Specificity