GLOBIN ADDUCTS OF BENZO[A]PYRENE - MARKERS OF INHALATION EXPOSURE AS MEASURED IN F344/N RATS

We have explored methods for determining benzo[a]pyrene (BaP) dosimetry by measuring adduction levels to F344/N rat blood hemoglobin, and have refined and validated an assay that measures the in vivo binding of the 7, 8-diol 9, 10-epoxide metabolite of BaP to globin. The assay for BaP-globin adducts was based on the release of tetrahydroxy-BaP (BaP-tetrols) from globin by mild acid hydrolysis. After extensive isolation, BaP-tetrols were quantitated by high-pressure liquid chromatography using fluorescence detection. BaP-tetrol levels were measured in rats dosed intraperitoneally with 242, 71 and 24-mu-mol BaP kg-1 body weight in corn oil. The formation of BaP-tetrols was not linear with dose. The lowest dose yielded adduct levels that represented the limits of sensitivity for the method, as performed in this laboratory. Once this limit of sensitivity was established, the potential use of the assay was assessed by measuring the radiochemical binding of inhaled [C-14]BaP or its metabolites to the globin of F344/N rats. Rats were exposed for 4 h per day, 1 day per week, for 12 weeks to pure aerosols of [C-14]BaP at a level of 2 mg m-3. At the conclusion of exposure, rats were sacrificed and globin was isolated. The extent of [C-14]BaP binding to the globin was determined by liquid scintillation spectrometry. Rats exposed to aerosols of [C-14]BaP had statistically increased levels of binding to globin, and the levels were comparable to those observed previously after intragastric administration. Based on the spectroscopic assay data, the sensitivity of the proposed analytical method was projected to be insufficient to measure BaP-tetrol levels after inhalation exposures similar to those described above. The use of the globin-BaP adduct approach may be limited by the small fraction of BaP-tetrols that can be released by acid hydrolysis.