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Biomedical subjects

S Venkatachalam

Publications and source records attributed to S Venkatachalam.

23 records · Page 2Linked to original sources
PubMed1995

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.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1994

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. We have developed a novel quantitative assay combining the selective recovery of DNA damage containing genomic fragments with the PCR amplification. DNA isolated from 7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) treated human skin fibroblasts was immunoprecipitated with polyclonal antibody BP-1. Recovered target sequences were amplified by PCR using primers encompassing a 149 bp target region around codon 12 of the H-ras proto-oncogene. Quantitative DNA damage specific response was observed with nanogram amounts of genomic DNA. This approach allowed analysis of the initial DNA damage at a level less than 1 anti-BPDE adduct per 6.4 kbp ras gene fragment. Repair proficient GM637 cells exposed to 2 microM anti-BPDE showed a faster removal of the adducts from the H-ras gene segment than from the genome overall. Gene-specific repair was not apparent in GM4429 xeroderma pigmentosum (complementation group A) cells. The established technique could be extended to the quantitative measurement of the repair of diverse DNA base lesions in any genomic region of known sequence.

Animals↗
PubMed1994

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. The polyclonal antibody (PAb BP1) distinctly recognized the highly carcinogenic lesion (+)-anti-BPDE-N2-dG with a 40-fold preference over the (-)-anti-BPDE-N2-dG adduct. In contrast, the monoclonal antibody (MAb 5D2) bound avidly to (-)-anti-BPDE-N2-dG and exhibited very little affinity for the (+)-anti-BPDE-N2-dG adduct. The overall sensitivity of detection of polyclonal antibodies for adducts in (+/-)-anti-BPDE-modified DNA was about 90-fold higher than monoclonal antibodies. Neither antibody showed any detectable reactivity with (+)-or (-)-anti-BPDE-N6-dA and with unmodified DNA antigens. The distinct preference of antibodies for particular enantiomeric adducts was observed in both single and duplexed oligomeric conformations. The demonstrated differential interaction of antibodies with the established conformations of (+)- and (-)-enantiomer anti-BPDE-DNA adducts (de los Santos et al., Biochemistry, 31, 5245-5252, 1992), has significant implications for in vitro and in vivo adduct processing and risk assessment biomonitoring studies.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

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 1.5 h following treatment with varying concentrations of (+/-)-anti-BPDE. The fraction of apoptotic cell population and corresponding DNA fragmentation indicated a minimum threshold damage (approximately 1-2 x 10(-5) adduct/base or about 1 putative lesion/human gene) necessary to elicit apoptosis. Suppression of apoptosis (60-90% inhibition of DNA fragmentation) by 3-aminobenzamide and lack of an effect by aphidicolin indicated the role of poly(ADP-ribosylation) but not of blocked DNA replication. Our data suggest that DNA damage triggers an acute response that sets the responsive cells on path of an "immediate apoptotic" mode of cell death.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗