Site-specific induction and repair of benzo[a]pyrene diol epoxide DNA damage in human H-ras protooncogene as revealed by restriction cleavage inhibition.

Most genotoxic DNA base modifications localized at key genomic sequences constitute the molecular alterations crucial or mutagenesis and tumorigenesis. We have utilized lesion-rendered inhibition of restriction endonuclease cleavage for the analysis of site-specific DNA damage induced by (+/-)-7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene diol epoxide, anti-BPDE) in human genes. The H-ras protooncogene and insulin gene sequences were used as targets for modification in vitro and in vivo.

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).

Assessment of DNA damage and repair in specific genomic regions by quantitative immuno-coupled PCR.

Fine analysis of DNA damage and repair at the subgenomic level has indicated a microheterogeneity of DNA repair in mammalian cells, including human. In addition to the well established Southern hybridization-based approach to investigate gene-specific DNA damage and repair, alternative methods utilizing the sensitivity of PCR have been evaluated. The latter technique has relied on decreased PCR amplification due to damage in template DNA.

Differential recognition of stereochemically defined base adducts by antibodies against anti-benzo[a]pyrene diol-epoxide-modified DNA.

The configurational isomers of benzo[a]pyrene diol-epoxide exhibit a range of reactivity, adduct profiles, genotoxic, mutagenic and tumorigenic responses. Whilst the (+)-enantiomer of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) is the most potent genotoxic species, studies dealing with many critical aspects of BPDE genotoxicity have predominantly been done with the racemic mixture of anti-BPDE. By utilizing highly sensitive non-competitive immunoassays, we have shown that both polyclonal and monoclonal antibodies developed against anti-BPDE-modified DNA exhibit a high degree of stereospecific adduct selectivity with 11mer oligodeoxynucleotides containing a single well-defined base adduct.

Rapid activation of apoptosis in human promyelocytic leukemic cells by (+/-)-anti-benzo[a]pyrene diol epoxide induced DNA damage.

Genotoxic damage in responsive mammalian cells is implicated as a critical event in the induction of apoptosis. We have evaluated the time course of activation of apoptosis in HL-60 cells following treatment with (+/-)-anti-BPDE metabolite, a well established DNA damaging carcinogen. Programmed cell death, determined by typical cellular and molecular markers of apoptosis, was apparent with 1.