Phosphatase activity in commercial spleen exonuclease decreases the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling.
Spleen exonuclease, which degrades nucleic acids into single 3'-nucleotides, is used in the detection of DNA adducts by 32P-postlabeling. Contamination of the exonuclease with phosphatase activity can reduce the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-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 mumol of benzo[a]pyrene adducts per mole of DNA. Recovery of benzo[a]pyrene adducts was reduced to 0.56 mumol 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 P1 than when using 1-butanol extraction for adduct enrichment, since arylamine DNA adducts have previously been reported to be poorly detected by 32P-postlabeling after nuclease P1 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 32P-postlabeling.
Adams
S P
SP
Merck Research Laboratories, West Point, Pennsylvania 19486.
Laws
G M
GM
Selden
J R
JR
Nichols
W W
WW
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000032:analysis; D000135:Acid Phosphatase / Q000032:analysis*; D000243:Adenosine Deaminase / Q000032:analysis*; D000818:Animals; D001564:Benzo(a)pyrene / Q000032:analysis*; D002417:Cattle; D018736:DNA Adducts / Q000032:analysis*; D004340:Drug Contamination; D005092:Exonucleases / Q000032:analysis*; D007202:Indicators and Reagents; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis*; D010761:Phosphorus Radioisotopes; D011865:Radioisotope Dilution Technique; D013154:Spleen / Q000201:enzymology
D018736:DNA Adducts; D007202:Indicators and Reagents; C088856:N-hydroxy-2-naphthylamine DNA adduct; D010761:Phosphorus Radioisotopes; C056612:benzo(a)pyrene-DNA adduct; D001564:Benzo(a)pyrene; D015081:2-Naphthylamine; D005092:Exonucleases; C022650:spleen exonuclease; D000135:Acid Phosphatase; D010744:Phosphoric Monoester Hydrolases; D000243:Adenosine Deaminase