Characterization of epoxide adducts of polycyclic aromatic hydrocarbons with hemoglobin.
Authors: Steven R. Myers, Harrell E. Hurst, Terry Wright, Christopher Cunningham
Journal: Cancer Research (2004)

Proc Amer Assoc Cancer Res, Volume 45, 2004

3191 

In the following experiments we investigated the binding of selected PAH epoxides (benzo(a)pyrene, benzo(b)fluoranthene, dibenz(a,hanthracene, fluoranthene, benzo(ghi)perylene, and benz(a)anthracene) to hemoglobin in order to determine the kinetics of binding of the PAH epoxide to hemoglobin. The following experiments were carried out using mouse (C57BL/6 male mice) hemoglobin in vitro . Through a series of binding studies as well as known carcinogenicity of the parent PAHs, we correlated extent of reaction of various epoxides with known carcinogenicity of the parent PAH. PAH epoxides arise via metabolism of the parent PAH to epoxides, followed by the addition of water to generate a diol. These reactions are catalyzd by microsomal enzymes as well as epoxide hydrolase. A second epoxidation reaction occurs at an adjacent double bond to yield a diol-epoxide of the parent PAH. Packed red cells (mouse) were resuspended in isotonic saline (2 mls). 100 μl aliquots of RBC suspension were used in reactions (volume brought to 500 μl with saline). PAH epoxide stock solutions were dissolved in tetrahydrofuran (1 mg/ml). 10 μl epoxide PAH stock was added to reactions. Reactions were carried out at 37° for 0,1,2,4,8,10,15,20,45,and 60 minutes. Reactions were stopped by addition of 500 μl water to hydrolyze unreacted PAH epoxide. Red cells were precipitated by centrifugation at 3,000 rpm for 10 minutes. The packed red cells were lyzed using 1 volume cold water and globin precipitated by addition of hemoglobin to acidified (0.1% HCl) acetone. Globin was collected, rinsed with acetone (100%), dried under nitrogen and stored at -20° until analysis. Control reactions were performed by preincubation of the PAH epoxides in saline for 15 minutes prior to addition of the red cell suspension. PAH epoxides were released from the globin by incubation (1 ml) with pronase for 24 hours at 37°. After reaction, released PAH tetrols were extracted using combinations of liquid and solid phase extraction and the tetrols taken to dryness under vacuum. The resulting PAH tetrols were analyzed spectrophotometrically by HPLC using both UV and fluorescence detection. Results from the study indicated that PAH epoxides bound covalently to the protein hemoglobin. The extent of PAH binding to the protein was similar with all epoxides, however there appears to be a correlation between the extent of binding and the overall carcinogenicity of the PAH with the stronger carcinogens having a faster rate of protein adduction than weaker carcinogens. These results suggest that the formation of hemoglobin adducts to PAH carcinogens may serve as reliable biomarkers of exposure as well as carcinogenicity. The limit of sensitivity of the assay by fluorometric detection of the released PAH tetrols is <10 pmols PAH epoxide/ mg protein. This limit of sensitivity suggests that the methods developed may be useful in routine monitoring of PAHs in population studies.