Quantitation of Benzo(a)pyrene-deoxyguanosine Adducts by Radioimmunoassay

Calf thymus DNA was modified with the benzo(a)pyrene (BP) derivative, (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+/-) BPDE I], under conditions which yielded greater than 99% of the binding product in the form of trans-(7R)-N2-(10[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl) deoxyguanosine. Rabbits were immunized with modified DNA coupled to methylated bovine serum albumin, and the resulting antiserum was utilized in a competition radioimmunoassay for the quantitation of products of BP covalently bound to DNA. The antiserum was specific for both native and denatured immunogen DNA's as well as for the major isolated BP binding product, but it did not recognize BP, the tetrol of (+/-)BPDE I, or unmodified deoxyguanosine. The modified DNA was assayed in quantities as low as 2 pmol of adduct, a sensitivity sufficient to quantitate the extent of modification of cellular DNA when epidermal cell cultures were exposed either to BP or to (+/-)BPDE I. High-pressure liquid chromatographic analysis of DNA hydrolsates, obtained from epidermal cells exposed to BP or to (+/-)BPDE I, indicated that the major adduct was the same as than on the immunogen DNA. This approach should prove valuable for further studies on the mechanism of carcinogenesis and for monitoring human exposure to this ubiquitous carcinogen.