PHOSPHATASE-ACTIVITY IN COMMERCIAL SPLEEN EXONUCLEASE DECREASES THE RECOVERY OF BENZO[A]PYRENE AND N-HYDROXY-2-NAPHTHYLAMINE DNA-ADDUCTS BY P-32 POSTLABELING

Spleen exonuclease, which degrades nucleic acids into single 3'-nucleotides, is used in the detection of DNA adducts by P-32-postlabeling. Contamination of the exonuclease with phosphatase activity can reduce the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by P-32-postlabeling. Four preparations of spleen exonuclease containing varying levels of phosphatase activity (<1-62% of the unmodified 3'-nucleotides being dephosphorylated) were used to hydrolyze the DNA. The exonuclease with the lowest phosphatase activity produced a recovery of up to 9.60 mu mol of benzo[a]pyrene adducts per mole of DNA. Recovery of benzo[a]pyrene adducts was reduced to 0.56 mu mol of adduct per mole of DNA using the exonuclease with the highest phosphatase activity. Phosphatase in the exonucleases also dephosphorylated N-hydroxy-2-naphthylamine DNA adducts. Surprisingly, recovery of these DNA adducts was nearly 10 times greater using nuclease P-1 than when using 1-butanol extraction for adduct enrichment, since arylamine DNA adducts have previously been reported to be poorly detected by P-32-postlabeling after nuclease P-1 treatment. Our data indicate that the hydrolysis of DNA by spleen exonuclease may be an important source of variability in both qualitative and quantitative analysis of adducts by P-32-postlabeling. (C) 1994 Academic Press, Inc.