Immunological quantitation of O4-ethylthymidine in alkylated DNA: repair of minor miscoding base in human cells

Rabbit antisera specific for O4-ethyldeoxythymidine (O4-EtdThd) were developed and used in competitive and non-competitive immunoassays for the quantitation of the miscoding promutagenic adduct O4-EtdThd in DNA. High titer antiserum TB3 (affinity constant, 8.1 X 10(8)) gave 50% inhibition of the tracer antigen-antibody binding with 0.26 pmol of O4-EtdThd in radioimmunoassay and negligible inhibition with unmodified DNA components. In competitive enzyme-linked immunosorbent assay (ELISA), using ENU-poly(dT) instead of hapten-protein conjugate as antigen, inhibition of antigen-antibody binding was proportional to both, the concentration of ethylnitrosourea (ENU) and alkylated DNA. The sensitivity of detection of O4-EtdThd in DNA was enhanced to fmol of O4-EtdThd and ng amounts of alkylated DNA by using avidin-biotin linked immunoreagents in non-competitive ELISA and immunoslot blot assays. An O4-EtdThd concentration-dependent binding of specific antibody, detecting 0.12 fmol of the modified base, was observed with DNA alkylated in vitro with ENU at an O4-EtdThd/Thd molar ratio of 2.6 X 10(-6). The immunological quantitation of O4-EtdThd in ENU-treated human skin fibroblast and kidney epithelial cells indicates a gradual removal of the modified base as a function of post-treatment time in culture. In both cell types about 50% of the initial damage was repaired during a 72-h period. Earlier studies using human organ extracts ruled out the participation of an alkyltransferase or glycosylase enzyme for the repair of O4-alkylthymine. None the less, the kinetics data presented indicate that normal human cells in culture are proficient for the repair of critical pyrimidine alkylation adduct that may be associated with cellular oncogenic transformation of susceptible mammalian cells.