Detection of deoxyadenosine-4-aminobiphenyl adduct in DNA of human uroepithelial cells treated with N-hydroxy-4-aminobiphenyl following nuclease P1 enrichment and 32P-postlabeling analysis.
To characterize the DNA adducts in human uroepithelial cells (HUC) exposed to 4-aminobiphenyl and its proximate N-hydroxy metabolites, we used 32P-postlabeling analyses following butanol extraction of the DNA hydrolysates. Using this method, we identified N-(deoxyguanosin-3',5'-bisphospho-8-yl)-4-aminobiphenyl (pdGp-ABP) as a major adduct and N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4-aminobiphenyl (pdAp-ABP) as a minor adduct in an immortalized non-tumorigenic cell line of HUC following exposure to N-hydroxy-4-aminobiphenyl (N-OH-ABP). Towards characterization of pdAp-ABP, we postlabeled the synthetic N-(deoxyadenosin-3'-phospho-8-yl)-4-aminobiphenyl (dAp-ABP) adduct to generate pdAp-ABP and determined its chromatographic (TLC and HPLC) properties and sensitivity to nuclease P1 digestion. In contrast to pdGp-ABP, which was cleaved to the corresponding 5'-monophosphate by nuclease P1, the pdAp-ABP adduct was unaffected when incubated with nuclease P1 under similar conditions. To test whether nuclease P1 digestion could be adopted for enrichment of the dAp-ABP adduct in HUC samples, postlabeling analyses were carried out after butanol extraction following nuclease P1 digestion of the DNA hydrolysate. Under these conditions, the pdAp-ABP adduct was detected in DNA from HUC E7 cells treated with N-OH-ABP and in calf thymus DNA reacted with N-OH-ABP under acidic (pH 5.0) conditions. These data indicate that pdGp-ABP and pdAp-ABP adducts are generated in HUC E7 on treatment with N-OH-ABP and that nuclease P1 enrichment may provide a method for qualitative and quantitative analyses of the pdAp-ABP adduct in DNA.
University of Wisconsin, Comprehensive Cancer Center, Madison 53792.
J F
JF
Hatcher
S
S
Swaminathan
D000611:Aminobiphenyl Compounds / Q000378:metabolism* / Q000633:toxicity*; D002273:Carcinogens / Q000378:metabolism* / Q000633:toxicity*; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D004247:DNA / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism*; D018736:DNA Adducts / Q000032:analysis* / Q000096:biosynthesis*; D003839:Deoxyadenosines / Q000378:metabolism*; D004847:Epithelial Cells; D004848:Epithelium / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D010761:Phosphorus Radioisotopes; D012680:Sensitivity and Specificity; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism* / Q000494:pharmacology*; D014551:Urinary Tract / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*
D000611:Aminobiphenyl Compounds; D002273:Carcinogens; D018736:DNA Adducts; D003839:Deoxyadenosines; D010761:Phosphorus Radioisotopes; C006757:4-biphenylamine; C014593:N-hydroxy-4-aminobiphenyl; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases