Formation of hemoglobin-benzo[a]pyrene adducts in human erythrocytes incubated with benzo[a]pyrene and hamster embryo cells.
Evidence is accumulating that the levels of covalent carcinogen-macromolecule adducts, including adducts with hemoglobin, reflect biologically effective levels of carcinogen exposure. The purposes of the present study were (a) to establish a cellular system for obtaining adducts between intracellular human hemoglobin and metabolites of polycyclic aromatic hydrocarbons (PAH), and (b) to evaluate techniques for chromatographic characterization of the adducts. We showed that hemoglobin-benzo[a]pyrene adducts were formed when human erythrocytes were treated with [3H]benzo[a]pyrene (BP) in the presence of hamster embryo fibroblasts, which are known to be effective for BP metabolism. After lysis of the erythrocytes, noncovalently bound BP and its metabolites were effectively removed from hemoglobin under mild conditions by using hydrophobic interaction and size-exclusion liquid chromatography. Three to five distinct adducts were resolved by reversed-phase and ion-exchange liquid chromatography. As determined by a two-step, reversed-phase liquid chromatographic procedure, trypsin treatment of globin from the cellular system yielded at least three of the four 7,8,9,10-tetrahydro-7,8,9,10-tetrahydroxy BP tetrols known to arise from mammalian metabolism of BP. This observation is consistent with both (a) the recently described formation of labile carboxyl esters via reaction of BP-7,8-dihydrodiol-9,10-epoxide (BPDE) with hemoglobin and (b) the known formation of both anti- and syn-BPDE in hamster embryo fibroblasts. In addition, high-performance liquid chromatographic analysis demonstrated the presence of other products presumed to be BP-peptide adducts because of their susceptibility to thermolysin treatment.