Chromatographic characterization of hemoglobin benzo[a]pyrene-7,8-diol-9,10-epoxide adducts

The formation of hemoglobin-carcinogen adducts has been detected in carcinogen-treated animals and in human populations. Although polynuclear aromatic hydrocarbons are ubiquitous in the human environment and DNA-aromatic hydrocarbon adducts have been detected in human tissue, the occurrence of hemoglobin-polynuclear aromatic hydrocarbon adducts in humans has not been thoroughly described. In this study we examined the effects of reaction conditions on the extent of in vitro reaction of human hemoglobin and (+) [3H]benzo[a]pyrene-7,8-diol-9,10-epoxide (anti)(BPDE), a metabolite thought to be largely responsible for the carcinogenic effect of benzo[a]pyrene. The chromatographic properties of the resulting hemoglobin-BPDE adducts were examined by conventional DEAE-cellulose ion exchange liquid chromatography and by reversed phase high performance liquid chromatography. Several adducts were formed which were chromatographically resolved from hemoglobin and from the individual globins. Some adducts were basic and some acidic relative to unaltered hemoglobin, suggesting adduct formation by reaction at carboxyl and basic nitrogen groups, respectively. Alteration of the ion-chromatographic properties of the adducts by an ionic sulfhydryl reagent, together with only a moderate effect of pH on the extent of adduct formation, indicated that the adducts were not formed via reaction with the beta 93 cysteine sulfhydryl group. The chromatographic techniques employed may be applicable for the characterization and analysis of other hemoglobin-carcinogen adducts.