ACTIVATION OF TRANS-1,2-DIHYDRO-1,2-DIHYDROXY-6-AMINOCHRYSENE TO GENOTOXIC METABOLITES BY RAT AND HUMAN CYTOCHROMES P450

In order to address the hypothesis that 6-aminochrysene (6-AC) is conv erted to genotoxic products by cytochrome P450 enzymes via two activat ion pathways (N-hydroxylation and epoxidation), the activation of 6-AC and trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysen (6-AC-diol) to gen otoxic metabolites was examined in rat and human liver microsomal cyto chrome P450 enzymes using Salmonella typhimurium TA1535/pSK1002 and TA 1535/pSK1002/pNM12 (NM2009) as tester strains. The latter bacteria, an O-acetyl-transferase-overexpressing strain, was highly sensitive to m etabolites derived from activation of 6-AC, but not those from 6-AC-di ol, using liver microsomes from phenobarbital-treated rats or a recons tituted monooxygenase system containing P4502B1 or -2B2, thus suggesti ng the roles of P450 and acetyltransferase systems in the activation p rocess. 6-AC-diol, on the other hand, was activated very efficiently b y liver microsomes prepared from beta-naphthoflavone-treated rats or a reconstituted system containing P4501A1 or -1A2; the activation react ion is considered to proceed through diolepoxide formation. The contri bution of rat P4501A enzymes towards activation of 6-AC-diol was confi rmed by the inhibitory effects on the activation process of alpha-naph thoflavone, a specific inhibitor of P4501A-related activities, and ant ibodies raised against purified P4501A1 and -1A2. In humans, P4501A2 w as found to be the major enzyme involved in the activation of 6-AC-dio l to genotoxic metabolites while the parent compound 6-AC was activate d mainly by P4503A4. Experiments using recombinant P450. proteins expr essed in human lymphoblastoid cell lines showed that human P4501A1 cou ld also activate 6-AC-diol to reactive metabolites at almost the same rate measured,vith P4501A2. In addition, P4502B6 was found to efficien tly catalyze the activation of 6-AC to genotoxic metabolites, and P450 3A4 was active in the activation of 6-AC-diol as well as 6-AC. Additio n of purified rat epoxide hydrolase to the incubation mixture containi ng purified rat P4501A1 or microsomes expressing human P4501A1 caused inhibition of activation of 6-AC-diol. These results suggest the exist ence of different enzymatic activation pathways for 6-AC and 6-AC-diol . The former carcinogen may be N-hydroxylated principally by P4502B en zymes in rats and P4503A4 and -2B6 in humans and activation to its ult imate metabolites may proceed through esterification of the N-hydroxy metabolites by an N-acetyltransferase. The 6-AC-diol is metabolized to its ultimate diolepoxide product by P4501A enzymes in rat and human l iver microsomes. P4503A4 (humans) and P4503A2 (rats) may also contribu te to some extent in the activation of Q-AC-diol, albeit at lower rate s than those of P4501A enzymes.