phosphorus-radioisotopes and chloroacetaldehyde

32P-Postlabelling procedures coupled with HPLC have been developed to detect and measure a range of cyclic DNA adducts formed by bifunctional genotoxic agents. The methods are based on reverse-phase HPLC, particularly column-switching HPLC, to enrich adduct 3'-monophosphates before labelling. Following 3'-dephosphorylation of the 3'5'-[5'-32P]bisphosphates with nuclease P1, the resulting 5'-[32P]monophosphate adducts are resolved, identified and characterized by co-chromatography with synthetic reference standards. The procedures have been applied to a number of cyclic adducts including those formed by chloroacetaldehyde, glycidaldehyde and malonaldehyde. In general, labelling efficiencies measured as chromatographed 5'-[32P]monophosphates were in the range 30-40%. However, the values for the malonaldehyde deoxyguanosine adduct were much lower. The techniques have been applied to studies on the formation of DNA adducts in the skin of male C3H mice treated cutaneously with glycidaldehyde. The HPLC-32P-postlabelling analysis of epidermal DNA hydrolysates indicated that a single major cyclic adduct was formed by reaction with deoxyadenosine residues in mouse skin DNA. The adduct was identified as a hydroxymethyl ethenodeoxyadenosine adduct by comparison with a synthetic standard. This adduct was highly fluorescent and it was possible to make quantitative comparisons of the amounts of adduct determined by either HPLC-32P-postlabelling or HPLC-fluorescence detection.

Topics: Acetaldehyde; Animals; Carcinogens; Chromatography, High Pressure Liquid; Deoxyguanosine; DNA; DNA Damage; Male; Mice; Mice, Inbred C3H; Nucleotides, Cyclic; Phosphorus Radioisotopes; Skin

Traces of damaged bases in DNA can be detected without the use of radioactive chemical agent or radiolabelled DNA substrate by incorporation of radiolabel into DNA after exposure to the chemical. This postlabelling approach to carcinogen adduct analysis is potentially useful for adduct detection in the DNA of humans exposed to carcinogens. We describe here the postlabelling analysis of DNA by chromatography of deoxynucleoside monophosphates. Separation and characterization of modified residues in nucleotide digests of DNA treated in vitro with chloroacetaldehyde is demonstrated.

Topics: Acetaldehyde; Chromatography, Thin Layer; Deoxyribonucleotides; DNA Damage; Phosphorus Radioisotopes