Detection of 1,N6-etheno-2'-deoxyadenosine and 3,N4-etheno-2'-deoxycytidine occurring endogenously in DNA.

1,N6-Etheno-2'-deoxyadenosine (epsilon dA) and 3,N4-etheno-2'-deoxycytidine (epsilon dC) are DNA adducts formed by a number of genotoxic chemicals, including vinyl chloride. They are also formed endogenously in tissue DNA, probably from a reactive metabolite of lipid peroxidation. Both the qualitative and quantitative detection of endogenous adducts is important in order to place adduct formation by chemicals such as vinyl chloride in the context of this natural background level. Methods with sufficient sensitivity are therefore being developed to measure the natural background of epsilon dA and epsilon dC adducts. We have developed a high-performance liquid chromatography (HPLC)-32P-postlabelling method to measure epsilon dA and epsilon dC at alkylation frequencies of 1 adduct in 10(7)-10(8) nucleotides in 10-microgram samples of DNA. In HPLC-32P-postlabelling analysis of liver DNA from control Wistar rats, epsilon dA and epsilon dC were determined at levels of 1 adduct in 8.1 x 10(7) and 1 adduct in 1.8 x 10(7) nucleotides, respectively. The levels of epsilon dA and epsilon dC measured in liver DNA of animals exposed orally to five daily doses of 50 mg/kg body weight vinyl chloride were found by this method to be 1 adduct in 2.9 x 10(7) and 1 adduct in 1.4 x 10(7) nucleotides, respectively. In contrast, in a direct labelling study, radiolabelled epsilon dA and epsilon dC were not detected in liver DNA of rats exposed for 6 h by nose-only inhalation to [1,2-14C]vinyl chloride at up to 45 ppm v/v. Immunochemical procedures are also being developed for recognizing etheno adducts. Thus, a monoclonal antibody raised to protein conjugates of epsilon dC showed high selectivity in the recognition of this DNA adduct. When the antibody was immobilized on a solid support and used in an immunoenrichment procedure to purify epsilon dC from a large excess of normal nucleotides, one epsilon dC adduct from about 10(8) normal nucleotides could be resolved. Coupling the immunoaffinity enrichment procedure with capillary zone electrophoresis permitted the detection of approximately one epsilon dC adduct in 3 x 10(6) nucleotides.

32P-postlabelling methods for cyclic DNA adducts.

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.

Formation of ribonucleotides in DNA modified by oxidative damage in vitro and in vivo. Characterization by 32P-postlabeling.

Oxygen free radicals generated by the interaction of Fe2+ and H2O2 (Fenton reaction) are capable of reacting with DNA bases, which may induce premutagenic and precarcinogenic lesions. Products formed in DNA by such reactions have been characterized as hydroxylated derivatives of cytosine, thymine, adenine, and guanine and imidazole ring-opened derivatives of adenine and guanine. As shown here by 32P-postlabeling, incubation of DNA under Fenton reaction conditions gave rise to additional oxidation products in DNA that were characterized as putative ribonucleosides by enzymatic hydrolysis of the oxidized DNA, 32P-postlabeling, and co-chromatography in multiple systems with authentic markers. Formation of these products in DNA was enhanced by the presence of L-ascorbic acid in the reaction mixtures and their total amounts were similar to those of the major DNA oxidation product, 8-hydroxy-2'-deoxyguanosine. The ribonucleoside guanosine was also formed in kidney DNA of male rats treated with ferric nitrilotriacetate, a renal carcinogen. It is postulated that ribonucleotides alter conformation and function of DNA and thus their presence in DNA may lead to adverse health effects.

Molecular dosimetry of DNA adducts in C3H mice treated with glycidaldehyde.

The formation of DNA adducts in the skin of male C3H mice treated cutaneously with glycidaldehyde (2 or 10 mg/animal) in acetone has been investigated by HPLC coupled with fluorescence detection and 32P-postlabelling analysis. Following a 24 h exposure period, epidermal DNA was isolated from treated dorsal skin and enzymically digested to nucleoside-3'-monophosphates. HPLC-32P-postlabelling analysis of the DNA hydrolysate indicated that a single major cyclic adduct was formed from the reaction of glycidaldehyde with deoxyadenosine residues in mouse skin DNA. This adduct was identified as 3-beta-D-deoxyribofuranosyl-7-(hydroxymethyl)-3H- imidazo[2,1-i]purine-3'-monophosphate by comparison with a synthetic standard. This adduct was stable, strongly fluorescent and readily detected by HPLC with fluorescence detection. There was no evidence for the formation of deoxyguanosine adducts in epidermal DNA of treated animals. Glycidaldehyde also reacted with calf thymus DNA in vitro at pH 7.0 to give the same deoxyadenosine adduct observed in vivo. At pH 10, however, this was a relatively minor product and the major adduct was 5,9-dihydro-7-(hydroxymethyl)-9- oxo-3-beta-D-deoxyribofuranosyl-3H-imidazo[1,2-a]purine-3'- monophosphate formed by the initial reaction of glycidaldehyde with deoxyguanosine residues.

Quantitative and kinetic examination of 32P-postlabeling of etheno-substituted nucleotides.

1,N6-ethenodeoxyadenosine-, 1,N2-ethenodeoxyguanosine- and 3,N4-ethenodeoxycytidine-3'-monophosphates were labeled by [gamma-32P] ATP using T4 polynucleotide kinase in conditions commonly used for the 32P-postlabeling assay. Kinetic studies showed that the reaction is fast reaching a plateau after 15-30 min. The efficiency of phosphorylation, as studied by substrate-product concentration dependency, was between 50-100% at the lower substrate concentrations. The adducts are labeled efficiently at sub-femtomole levels. All the adducts were sensitive to the 3'-dephosphorylation by P1 nuclease although the guanine derivative appeared to be more resistant than the two other adducts.

HPLC-32P-postlabelling analysis of 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine.

A 32P-postlabelling procedure coupled with HPLC has been developed to detect and measure the cyclic nucleic acid adducts 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine in DNA. Chloroacetaldehyde-modified DNA containing these adducts was enzymatically digested to 3'-monophosphates and adducts were separated by ion-pair reverse-phase HPLC on PL RP-S prior to 32P-postlabelling with carrier free [gamma-32P]ATP. Following 3'-dephosphorylation with nuclease P1 the resulting [5'-32P]monophosphate adducts were finally resolved by HPLC on PL RP-S and assayed by liquid scintillation counting.