Some quantitative considerations about DNA adduct enrichment procedures for 32P-postlabelling

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Abstract
The concentrations of 2′-deoxyribonucleoside-3′-mono-phosphates remaining in calf-thymus DNA digests after nuclease P1 digestion or extraction into 1-butanol, the most commonly used adduct enrichment procedures prior to the application of the 32P-postlabelling assay, were measured using HPLC and 32P-postlabelling methods. When 10 μg of DNA digested to mononudeotides was used, the total amount of nucleotides remaining in the samples were ∼4 and ∼ 14 pmol after nudease P1 treatment or 1-butanol extraction respectively. The influence of various concentrations of normal nucleotides on the labelling efficiency of a 2′-deoxy-guanosine-3′-monophosphate adduct of benzo[a]pyrene diol-epoxide was also studied and found to depend upon the ratio of normal nucleotides/adducted nucleotides present in the sample. Also, the ATP/normal nucleotides ratio in the phosphorylation reaction may affect the quantitation of the adducts and thus deserves due consideration.

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