Metabolic activation of alpha-naphthoflavone by 2,3,7,8-tetrachlorodibenzodioxin-induced rat liver microsomes

Previous studies in our laboratory had demonstrated that addition of alpha-naphthoflavone (ANF) to lymphocytes from smokers or polychlorinated biphenyls (PCB)s-exposed individuals caused an increase in sister chromatid exchange (SCE) frequency whereas lymphocytes from controls were relatively unaffected. In order to investigate the mechanism responsible, metabolism of ANF by uninduced and 2,3,7,8-tetrachlorodibenzodioxin (TCDD)-induced microsomes was studied as a function of microsomal protein concentration and incubation time. Nonpolar metabolites were analyzed and the amount of conjugated (polar) and protein-bound metabolites determined. The initial ANF-metabolism rate was 10-fold higher in TCDD-induced microsomes (4.9 +/- 0.6 nmol/min per mg TCDD-induced microsomal protein vs. 0.5 +/- 0.2 nmol/min per mg uninduced microsomal protein) than in uninduced microsomes. Moreover, uninduced microsomes no longer metabolize ANF after 30-40 min while TCDD-induced microsomes metabolize ANF for longer than 2 h or until all the ANF is gone. In addition to the metabolites formed by uninduced microsomes 7,8-dihydro-7,8-dihydroxy-ANF (7,8-dihydrodiol); 5,6-dihydro-5,6-dihydroxy-ANF (5,6-dihydrodiol); 5,6-oxide-ANF and 6-hydroxy-ANF], TCDD-induced microsomes from unidentified metabolites. When TCDD-induced microsomes and 40 microM ANF were added to Chinese hamster ovary (CHO) cells, we found a correlation between the concentration of 5,6-oxide-ANF and clastogenicity to CHO cells. However, purified 5,6-oxide-ANF did not induce SCEs in CHO cells in the absence or presence of TCDD-induced microsomes. However, a minor metabolite (identified as the 9,10-dihydro-9,10-dihydroxy-ANF by acid dehydration) formed with TCDD-induced microsomes produces clastogenicity in CHO cells. These data indicate that a minor metabolite of ANF is a potent clastogen which suggests that this metabolite may be responsible for the ANF-mediated increases in SCE frequency in lymphocytes from smokers or PCB-exposed individuals.