A comparison of two ultrasensitive methods for measuring 1,N6-etheno-2'- deoxyadenosine and 3,N4-etheno-2'-deoxycytidine in cellular DNA

1,N⁶-Ethenodeoxyadenosine (edA) and 3,N⁴-ethenodeoxycytidine(edC) are two mutagenic adducts associated with exposure toethyl carbamate (urethane) and vinyl chloride. We have recentlydeveloped two ultrasensitive methods for determining the moleculardose of these adducts in cellular DNA. In both methods, purifiedDNA was first enzymatically digested to 2c-deoxyribonucleotide3c-monophosphates. Etheno-modified nucleotides were then separatedfrom normal nucleotides in one of two ways: either by reversephase, ion-pair HPLC coupled with 260 nm UV detection, or byimmunoaffinity chromatography using reusable microcolumns containingspecific monoclonal antibodies coupled to Protein A–Sepharose.Fractions enriched for the adducted nucleotides were labeledusing T4 polynucleotide kinase and [³²P]ATP, and individualnucleotides were subsequently resolved by two-dimensional TLC,visualized by autoradiography, and quantified by liquid scintillationcounting. When used to analyze the same sample of etheno-modifiedcalf thymus DNA, both assays produced similar results. However,when both methods were used to analyze rat liver DNA ‘spiked’with known amounts of etheno nucleotide standards, the immunoaffinity/³²PTLC procedure proved to be more sensitive and more reproduciblethan the HPLC/³²P TLC method: while the detection limit of theimmunoaffinity/³²P TLC technique was < 4 etheno adducts/10⁹parent deoxynucleotides, the HPLC/³²P TLC method often failedto detect adducts at concentrations <2/108. In other experiments,the immunoaffinity/³²P TLC method was used to demonstrate formationof edA and edC in cells treated with vinyl chloride monomer.Because of its exquisite sensitivity, the immunoaffinity/³²PTLC method promises to be extremely useful for measuring bothbackground and induced levels of etheno adducts, making it possibleto examine the role of these adducts in inducing mutations and/orcarcinogenesis.