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Activation of Amino--carboline, 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and a Copper Phthalocyanine Cellulose Extract of Cigarette Smoke Condensate by Cytochrome P-450 Enzymes in Rat and Human Liver Microsomes¹

Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

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--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--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--carboline and PhIP. In rat liver microsomes PhIP may also be activated by P-450 3A enzymes; activity was induced in rats treated with pregenenolone 16-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. The following results collectively indicate that P-450 1A2 may be the major catalyst for the activation of the components in this blue cotton extract: (a) isosafrole and Aroclor 1254 induced the activities seen in rat liver microsomes; (b) in different human liver preparations the activation of the extract was correlated with activities catalyzed by P-450 1A2 and was inhibited by 7,8-benzoflavone or a human autoimmune antiserum that strongly and specifically recognizes human P-450 1A2; (c) reconstituted systems containing purified human, rat, or rabbit P-450 1A2 catalyzed the activation of the extract. The participation of the human P-450 2D6 enzyme in the activation of these chemicals does not seem as likely because the activities were not inhibited by quinidine or by antibodies raised against rat P-450 2D1. Thus, the work does not provide evidence for the activation of genotoxic components of tobacco smoke by P-450 2D6 as being the basis for the reported association between debrisoquine 4-hydroxylation and incidence of lung cancer. The present results show for the first time that cigarette smoke condensate contains both inhibitors of P-450 enzymes and procarcinogens capable of being activated by P-450 enzymes and that the P-450 1A2 enzymes appear to be the most important catalysts for the (hepatic) activation reaction of a copper phthalocyanine cellulose extract of cigarette smoke condensate.

¹ Supported in part by USPHS Grants CA 44353 and ES 00267 (F. P. G.) and by a Grant in Aid for Cancer Research from the Ministry of Education, Science, and Culture of Japan (T. S.).

² Present address: Osaka Prefectural Institute of Public Health, Nakamichi, Higashinari-ku, Osaka 537, Japan.

³ To whom requests for reprints should be addressed.

Received 4/ 4/91. Accepted 7/19/91.

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