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

TLDR

Detection of covalent DNA adducts in humans exposed to environmental genotoxicants requires extremely sensitive methods because adducts occur at levels as low as one per 10^8–10^10 nucleotides, and a 32P‑postlabeling assay with a limit of 1 per 10^7–10^8 nucleotides has been previously described. The study reports that adding a post‑incubation step with Penicillium citrinum nuclease P1 before 32P labeling increases the assay’s sensitivity to one adduct per ~10^10 nucleotides in a 10‑µg DNA sample. The method digests DNA to 3′‑phosphorylated nucleotides, labels them with 32P via T4 pol.

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 10 8 -10 10 DNA nucleotides). A 32 P-postlabeling assay for detection and quantitation of DNA adducts with a sensitivity limit of 1 adduct in 10 7 -10 8 nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are quantitatively 32 P-labeled at their 5'-hydroxyl groups by T4 polynudeotide kinase-catalyzed [ 32 P]phosphate transfer from [γ -32 P]ATP. 32 P-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 32 P-labeling, enhanced the technique's sensitivity to 1 adduct in ∼10 10 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 -32 P]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 32 P-count rates as compared with the standard procedure.