DNA repair in human cells: quantitative assessment of bulky anti-BPDE-DNA adducts by non-competitive immunoassays.
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 N(2)-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 < 0.1 fmol adducts in immobilized DNA. Non-competitive immuno-slot blot assay could detect 0.2 adducts/10(6) nucleotides induced by < 1 nM ( +/- )-anti-BPDE. The high sensitivity and mono-adduct specificity of non-competitive immunoassays allowed the detailed study of ( +/- )-anti-BPDE-DNA adduct processing human cells exposed to very low levels of the genotoxin. Analysis of polyclonal antiserum binding sites in DNA from repair-proficient human fibroblasts revealed adduct removal rates directly proportional to the initial genotoxic insult. Despite efficient repair, substantial damage persisted in repair-proficient cells exposed to high doses of carcinogen. At low levels of initial damage (0.882 and 3.44 +/- 0.17 adducts/ 10(6) nucleotides) approximately 50% repair was observed after 4 and 8 h respectively. Cells removed approximately 40% of the lesions in 8 h at an intermediate level of damage (20.7 +/- 1.5 adducts/10(6) nucleotides). At higher DNA damage levels (105 +/- 8 and 177 +/- 1 adduct/10(6) 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.
Venkatachalam
S
S
Department of Radiology, Ohio State University, Columbus 43210, USA.
Denissenko
M
M
Wani
A A
AA
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000032:analysis* / Q000378:metabolism / Q000633:toxicity; D000327:Adsorption; D000906:Antibodies / Q000378:metabolism; D002273:Carcinogens / Q000378:metabolism / Q000633:toxicity; D002478:Cells, Cultured; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis* / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D012015:Reference Standards; D012680:Sensitivity and Specificity
D000906:Antibodies; D002273:Carcinogens; D018736:DNA Adducts; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA