Quantitation of 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline DNA adducts in specific sequences using alkali or uvrABC excinuclease.
2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) are carcinogens found in cooked meats that form DNA adducts upon metabolic activation. Purified DNA from Chinese hamster ovary (CHO) cells was reacted in vitro with the active metabolites N-acetoxy-IQ or N-acetoxy-MelQx, and the adduct levels in the 5' dihydrofolate reductase (DHFR) gene and downstream region were quantitated by Southern hybridization. Adducted and restricted DNA was treated with Escherichia coli uvrABC excinuclease or alkali (0.1 N NaOH, 37 degrees C, 60 min) to incise DNA at IQ and MelQx adduct sites. The DNA was then denatured with formamide, electrophoresed on a neutral agarose gel, transferred to a support membrane, and hybridized with sequence-specific DNA probes. Both uvrABC and alkali reduced the intensity of Southern hybridization in proportion to the number of IQ or MelQx adducts in DNA, indicating that these adducts are substrates for uvrABC and that they form alkali-labile lesions in DNA. IQ and MelQx adduct levels were the same in the 5' DHFR gene and in the downstream region. Southern hybridization analysis of pBR322 containing known levels of IQ or MelQx adducts showed that the efficiency of cutting IQ or MelQx adducts by uvrABC excinuclease and alkali was approximately 30% and 15%, respectively. 32P-postlabeling studies examining adduct level in bulk DNA further showed that the adduct profiles were identical in pBR322, CHO DNA, and cultured CHO cells exposed to the reactive metabolites of IQ or MelQx. The results indicate that IQ and MelQx adducts can be quantitated in specific genomic sequences and that this method should be directly applicable to studies of gene-specific repair of these adducts in cultured cells.
Nouso
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Laboratory of Experimental Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Bohr
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Schut
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Snyderwine
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D000468:Alkalies; D000818:Animals; D001345:Autoradiography; D015139:Blotting, Southern; D016466:CHO Cells; D003296:Cooking; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000032:analysis / Q000187:drug effects / Q000378:metabolism; D004586:Electrophoresis; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli; D005506:Food Contamination; D007364:Intercalating Agents; D009153:Mutagens / Q000633:toxicity*; D010761:Phosphorus Radioisotopes; D010957:Plasmids; D011804:Quinolines / Q000032:analysis / Q000633:toxicity*; D011810:Quinoxalines / Q000032:analysis / Q000633:toxicity*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014466:Ultraviolet Rays
D000468:Alkalies; D004269:DNA, Bacterial; D007364:Intercalating Agents; D009153:Mutagens; D010761:Phosphorus Radioisotopes; D011804:Quinolines; D011810:Quinoxalines; C036990:2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D004706:Endodeoxyribonucleases; C036989:2-amino-3,4-dimethylimidazo(4,5-f)quinoline