Binding of 3‐hydroxybenzo[ a ]pyrene to bovine hemoglobin and albumin

Previous studies examined the bioavailability and first‐pass biotransformation of 3‐hydroxy[ 3 H]benzo[ a ]pyrene ([ 3 H]‐3‐OHBaP) in an isolated perfused catfish intestinal model. This work showed that 3‐OHBaP, or a metabolite formed in intestine, bound covalently to blood protein. In this study, the blood adducts were characterized in vitro by incubating bovine ferric hemoglobin or albumin with [ 3 H]‐3OHBaP under various conditions. Incubation of 2 μM [ 3 H]‐3‐OHBaP with hemoglobin for 1 h resulted in 7.49 pmol bound/mg protein, while albumin binding was 1.37 pmol/mg protein. Mild acid hydrolysis released only 5% of the radioactivity from 3‐OHBaP–hemoglobin adducts. After gel filtration, the 3‐OHBaP–hemoglobin adducts were examined by HPLC analysis. A single peak of radioactivity was detected at the same retention time as the heme component of hemoglobin. Unbound 3‐OHBaP was oxidized to BaP‐3,6‐dione during incubation with ferric hemoglobin. Treatment of hemoglobin with ascorbic acid decreased the formation of hemoglobin adducts by 33%, while hydrogen peroxide treatment increased adduct formation by 44%. Incubation of [ 3 H]‐BaP‐3‐β‐ D ‐glucuronide (BaP‐3G) with hemoglobin and β‐glucuronidase resulted in greater binding to hemoglobin than incubation with [ 3 H]‐3‐OHBaP alone. The hemoglobin adduct obtained from [ 3 H]‐BaP‐3G also co‐migrated with heme. These results indicate that an oxidative process is involved in formation of the heme adduct and that 3‐OHBaP or BaP‐3G might be a precursor of the bound metabolite. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:239–247, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10084