Cytotoxicity, DNA adduct formation and DNA repair induced by 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline and 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in cultured human mammary epithelial cells.
2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are heterocyclic amines (HAs) found in cooked meats. Both compounds are mammary gland carcinogens in rats. The initiation of carcinogenesis by the HAs is believed to be associated with their DNA adduct formation that occurs after metabolic activation of the parent amines via cytochrome P-450-mediated N-hydroxylation and esterification. To assess the capacity of the human mammary epithelium to metabolically activate the HAs, we used the 32P-postlabeling method to measure the levels of DNA adducts in a culture of human mammary epithelial cells exposed to IQ, PhIP or their N-hydroxylamine metabolites. Whereas 50 microM parent amines did not form detectable levels of DNA adducts in cultured human mammary epithelial cells after 24 h incubations, concentrations as low as 1 microM N-hydroxylamines produced detectable levels of adducts after 2 h incubations. N-Hydroxy-PhIP formed higher adduct levels than N-hydroxy-IQ at all concentrations tested. For example, following a 2 h incubation at 50 microM, adduct levels (per 10(7) nucleotides) were 674 and 16 for N-hydroxy-PhIP and N-hydroxy-IQ, respectively. At similar initial adduct levels (10-11/10(7) nucleotides), 60-80% of IQ- and PhIP-DNA adducts were removed after 24 h, indicating that the mammary epithelial cell culture showed efficient repair of HA adducts. Whereas neither IQ nor PhIP was cytotoxic, both N-hydroxy-IQ and N-hydroxy-PhIP were cytotoxic as assessed by a dose-dependent inhibition of colony formation. After exposure to 0.1, 1, 10 or 50 microM N-hydroxy-PhIP (or N-hydroxy-IQ), colony formation was 103 (94), 84 (74), 37 (29) and 3 (2)% of the control values, respectively. Only N-hydroxy-PhIP (at 10 and 50 microM), however, was cytotoxic as assessed by the MTT cell survival assay (which measures the capacity of mitochondria to metabolize a tetrazolium salt). The ability of the N-hydroxylamines to form DNA adducts and to be cytotoxic in a culture of human mammary epithelial cells may implicate these metabolites as proximate carcinogenic forms of IQ and PhIP in the human mammary gland. However, whether there are inter-individual differences in N-hydroxylamine metabolism, adduct formation and repair in human mammary epithelial cells requires further study. The results from this study support the usefulness of cultured human mammary epithelial cells for studies on the genotoxicity and metabolism of the N-hydroxylamines of HA food mutagens.
Fan
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Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD 20892-4255, USA.
Schut
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Snyderwine
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D001940:Breast / Q000187:drug effects* / Q000378:metabolism*; D002273:Carcinogens / Q000378:metabolism* / Q000493:pharmacokinetics / Q000633:toxicity*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D018736:DNA Adducts / Q000096:biosynthesis*; D004260:DNA Repair; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005562:Formazans; D006801:Humans; D007093:Imidazoles / Q000378:metabolism* / Q000493:pharmacokinetics / Q000633:toxicity*; D009153:Mutagens / Q000378:metabolism* / Q000493:pharmacokinetics / Q000633:toxicity*; D011725:Pyridines / Q000378:metabolism* / Q000633:toxicity*; D011804:Quinolines / Q000378:metabolism* / Q000493:pharmacokinetics / Q000633:toxicity*; D013778:Tetrazolium Salts
D002273:Carcinogens; D018736:DNA Adducts; D005562:Formazans; D007093:Imidazoles; D009153:Mutagens; D011725:Pyridines; D011804:Quinolines; D013778:Tetrazolium Salts; C060533:2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine; C079782:MTT formazan; C029216:2-amino-3-methylimidazo(4,5-f)quinoline; C032012:2-hydroxyamino-3-methylimidazolo(4,5-f)quinoline; D004247:DNA; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine