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-methylimidazo4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo4,5-b]pyridine(PhIP) are heterocyclic amines (HAs) found in cooked meats.Both compounds are mammary gland carcinogens in rats. The initiationof carcinogenesis by the HAs is believed to be associated withtheir DNA adduct formation that occurs after metabolic activationof the parent amines via cytochrome P-450-mediated N-hydroxylationand esterification. To assess the capacity of the human mammaryepithelium to metabolically activate the HAs, we used the ³²P-postlabelingmethod to measure the levels of DNA adducts in a culture ofhuman mammary epithelial cells exposed to IQ, PhIP or theirN-hydroxylamine metabolites. Whereas 50 µM parent aminesdid not form detectable levels of DNA adducts in cultured humanmammary epithelial cells after 24 h incubations, concentrationsas low as 1 µM N-hydroxylamines produced detectable levelsof adducts after 2 h incubations. N-Hydroxy-PhIP formed higheradduct levels than N-hydroxy-IQ at all concentrations tested.For example, following a 2 h incubation at 50 µM, adductlevels (per 10⁷ nucleotides) were 674 and 16 for N-hydroxy-PhIPand N-hydroxy-IQ, respectively. At similar initial adduct levels(10–11/10⁷ nucleotides), 60–80% of IQ- and PhIP—DNAadducts were removed after 24 h, indicating that the mammaryepithelial cell culture showed efficient repair of HA adducts.Whereas neither IQ nor PhIP was cytotoxic, both N-hydroxy-IQand N-hydroxy-PhIP were cytotoxic as assessed by a dose-dependentinhibition of colony formation. After exposure to 0.1, 1, 10or 50 µM N-hydroxy-PhIP (or N-hydroxy-IQ), colony formationwas 103 (94), 84 (74), 37 (29) and 3 (2)% of the control values,respectively. Only N-hydroxy-PhIP (at 10 and 50 µM), however,was cytotoxic as assessed by the MTT cell survival assay (whichmeasures the capacity of mitochondria to metabolize a tetrazoliumsalt). The ability of the N-hydroxylamines to form DNA adductsand to be cytotoxic in a culture of human mammary epithelialcells may implicate these metabolites as proximate carcinogenicforms of IQ and PhIP in the human mammary gland. However, whetherthere are inter-individual differences in N-hydroxylamine metabolism,adduct formation and repair in human mammary epithelial cellsrequires further study. The results from this study support the usefulness of culturedhuman mammary epithelial cells for studies on the genotoxicityand metabolism of the N-hydroxylamines of HA food mutagens.