Formation of 1,N6-ethenoadenine and 3,N4-ethenocytosine by lipid peroxidation products and nucleic acid bases.

Lipid peroxidation (LPO) products are known to interact with DNA, yielding several types of adduct with nucleobases. In this study, we demonstrate the formation of two ethenobase adducts, 1,N6-ethenoadenine and 3,N4-ethenocytosine, by reaction of LPO products with nucleic acid bases. Rat liver microsomes were incubated at 37 degrees C for 30 min in the presence of inducers of LPO [Fe(II) or cumene hydroperoxide] and adenine or cytosine nucleotides or nucleosides, followed by further heating at 80 degrees C for 30 min to complete the reactions. The etheno adducts detected after immunoaffinity chromatography were 1,N6-etheno-cAMP and 1,N6-etheno-2'-deoxyadenosine (HPLC/fluorimetry), 3,N4-etheno-2'-deoxycytidine (competitive radioimmunoassay), and 1,N6-etheno-2'-deoxyadenosine 3'-monophosphate and 3,N4-etheno-2'-deoxycytidine 3'-monophosphate (32P-postlabeling). Incubation of arachidonic acid supplemented with Fe(II) also led to the formation of the 1,N6-etheno adduct from cAMP. LPO intermediates that may be involved are discussed. These data suggest that etheno adducts may be markers of DNA damage associated with LPO.

Immunoaffinity clean-up combined with 32P-postlabelling analysis of 1,N6-ethenoadenine and 3,N4-ethenocytosine in DNA.

Immunoaffinity gels able to bind 1,N6-ethenodeoxyadenosine 3'-monophosphate (3'-epsilon dAMP) and 3,N4-ethenodeoxycytidine 3'-monophosphate (3'-epsilon dCMP) were prepared. These gels retained their specific binding capacity for 3'-epsilon dAMP or 3'-epsilon dCMP in the presence of a large excess (> 2 mg per column) of unmodified 3'-deoxynucleotide monophosphates. 3'-epsilon dAMP and 3'-epsilon dCMP were 32P-postlabelled in the presence of [gamma-32P]ATP and T4 polynucleotide kinase to give either 3',5'-[5'-32P]-bisphosphates or 5'-[32P]monophosphates. 3',5'-[5'-32P]Bisphosphates were recovered from the labelling mixture by immunoprecipitation and quantitated by Cerenkov counting (method A). The detection limit of this procedure was 1 fmol with an efficiency of 80% for both ethenonucleotides. Alternatively, 5'-[32P]epsilon dAMP and 5'-[32P]epsilon dCMP were analysed by two-dimensional TLC on PEI cellulose and autoradiography (method B). The detection limit of method B was 50 amol of ethenonucleotide. Methods A and B are complementary and can quantify the formation and repair of 3'-epsilon dAMP and 3'-epsilon dCMP in DNA from rats exposed to a low level of vinyl chloride.