DNA repair in human cells: quantitative assessment of bulky anti-BPDE-DNA adducts by non-competitive immunoassays

Abstract

Mutagenicity and carcinogenicity of the ubiquitous environmental pollutant benzo[a]pyrene is mediated via its reactive diol epoxide metabolite, anti-BPDE, with the predominant formation of N²-deoxyguanine adducts in genomic DNA. Polyclonal and monoclonal antibodies specific for (±)-anti-BPDE DNA adducts were used for the quantitative detection of genotoxic damage in DNA treated in vitro and in vivo with (±)-anti-BPDE. In non-competitive enzyme-linked immunosorbent assay the polyclonal antiserum (BP1) exhibited higher affinity, avidity and sensitivity than the monoclonal antibody (5D2). A linear antibody binding response was observed over a wide carcinogen dose range with a detection limit of 6 nucleotides induced by anti-BPDE. The high sensitivity and mono-adduct specificity of non-competitive immunoassays allowed the detailed study of (±)-anti-BPDE-DNA adduct processing in human cells exposed to very low levels of the genotoxin. Analysis of polyclonal antiserum binding sites in DNA from repairproficient human fibroblasts revealed adduct removal rates directly proportional to the initial genotoxic insult. Despite efficient repair, substantial damage persisted in repairproficient cells exposed to high doses of the carcinogen. At low levels of initial damage (0.882 and 3.44 ± 0.17 adducts/ 10⁶ nucleotides) ∼50% repair was observed after 4 and 8 h respectively. Cells removed ∼40% of the lesions in 8 h at an intermediate level of damage (20.7 ± 1.5 adducts/10⁶ nucleotides). At higher DNA damage levels (105 ± 8 and 177 ± 1 adduct/10⁶ nucleotides) 33 and 19% of the lesions respectively were repaired in 24 h. Repair-deficient xeroderma pigmentosum group A fibroblast cells did not show any significant loss of antibody binding sites at high or low initial modification levels. These data suggest that the level of initial DNA damage has a significant impact on the overall efficiency of cellular repair, with potential implications for the biological consequences of deleterious DNA lesions in mammalian cells.