Nuclease P1-mediated enhancement of sensitivity of 32P-Postlabeling test for structurally diverse DNA adducts

Abstract

Exceedingly sensitive procedures are required to detect the presence of covalent DNA adducts in humans exposed to environmental genotoxicants because of low levels of such derivatives (1 adduct in 10⁸-10¹⁰ DNA nucleotides). A ³²P-postlabeling assay for detection and quantitation of DNA adducts with a sensitivity limit of 1 adduct in 10⁷-10⁸ nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are quantitatively ³²P-labeled at their 5'-hydroxyl groups by T4 polynudeotide kinase-catalyzed [³²P]phosphate transfer from [γ^-32P]ATP. ³²P-labeled derivatives are resolved by t.l.c, detected by autoradiography and quantitated by counting. We now report that a minor modification of this procedure, entailing the postincubation of DNA digests with Penicillium citrinum nuclease P1 before ³²P-labeling, enhanced the technique's sensitivity to 1 adduct in ∼10¹⁰ nucleotides for a 10-μg DNA sample. Nuclease PI cleaves deoxyribonucleoside 3'-monophosphates of normal nucleotides to deoxyribonudeo-sides which do not serve as substrates for polynudeotide kin-ase, while most adducted nucleotides were found to be totally or partially resistant to the 3'-dephosphorylating action of nuclease PI. The additional enzymatic step enabled specific labeling of adducts in up to 20 μg of DNA with excess carrier-free [γ^-32P]ATP. The enzymatic digestion conditions were standardized to afford optimal adduct recovery. The new procedure was found to be simple, highly reproducible, and applicable to the detection and measurement of aromatic or bulky non-aromatic DNA adducts formed with such structurally diverse carcinogens as benzo[a]pyrene, 7, 12-dimethyl-benz[a]anthracene, dibenzo[c, g]carbazole, 4-aminobiphenyl, safrole and mitomycin C; most adducts were recovered quantitatively with a 500- to 1000-fold increase in ³²P-count rates as compared with the standard procedure.