S. Venkatachalam et al., DNA-REPAIR IN HUMAN-CELLS - QUANTITATIVE ASSESSMENT OF BULKY ANTI-BPDE-DNA ADDUCTS BY NONCOMPETITIVE IMMUNOASSAYS, Carcinogenesis, 16(9), 1995, pp. 2029-2036
Mutagenicity and carcinogenicity of the ubiquitous environmental pollu
tant benzo[a]pyrene is mediated via its reactive diol epoxide metaboli
te, anti-BPDE, with the predominant formation of N-2-deoxyguanine addu
cts 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 irt vivo with (+/-)-anti
-BPDE. In non-competitive enzyme-linked immunosorbent assay the polycl
onal antiserum (BP1) exhibited higher affinity, avidity and sensitivit
y than the monoclonal antibody (5D2). A linear antibody binding respon
se was observed over a wide carcinogen dose range with a detection lim
it of <0.1 fmol adducts in immobilized DNA. Non-competitive immune-slo
t blot assay could detect 0.2 adducts/10(6) nucleotides induced by <1
nM (+/-)-anti-BPDE. The high sensitivity and mono-adduct specificity o
f non-competitive immunoassays allowed the detailed study of (+/-)-ant
i-BPDE-DNA adduct processing in human cells exposed to very low levels
of the genotoxin. Analysis of polyclonal antiserum binding sites in D
NA from repair-proficient human fibroblasts revealed adduct removal ra
tes directly proportional to the initial genotoxic insult. Despite eff
icient repair, substantial damage persisted in repair-proficient cells
exposed to high doses of the carcinogen. At low levels of initial dam
age (0.882 and 3.44 +/- 0.17 adducts/ 10(6) nucleotides) similar to 50
% repair was observed after 4 and 8 h respectively. Cells removed simi
lar to 40% of the lesions in 8 h at an intermediate level of damage (2
0.7 +/- 1.5 adducts/10(6) nucleotides). At higher DNA damage levels (1
05 +/- 8 and 177 +/- 1 adduct/10(6) nucleotides) 33 and 19% of the les
ions respectively were repaired in 24 h. Repair-deficient xeroderma pi
gmentosum group A fibroblast cells did not show any significant loss o
f antibody binding sites at high or low initial modification levels. T
hese data suggest that the level of initial DNA damage has a significa
nt impact on the overall efficiency of cellular repair, with potential
implications for the biological consequences of deleterious DNA lesio
ns in mammalian cells.