Activation of genotoxins to DNA-damaging species in exfoliated breast milk cells.
ABSTRACT Exfoliated cells, isolated from breast milk samples donated by UK-resident women (n=15), were incubated, either immediately or after culture for 7 days, with one of a series of genotoxins, either in the presence or absence of the DNA-repair inhibitors, hydroxyurea (HU), and cytosine arabinoside (ara-C). The numbers of DNA single-strand breaks induced were then assessed as comet tail length (CTL) (microm) using the alkaline single cell-gel electrophoresis ('Comet') assay; cell viability was measured by trypan blue exclusion. The heterocyclic aromatic amines (HAAs) (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (0.4 mM), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) (1.67 mM), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) (1.77 mM)), a polycyclic aromatic hydrocarbon (benzo[a]pyrene (B[a]P) (0.36 mM)), a nitro-polycyclic aromatic hydrocarbon (1-nitropyrene (1-NP) (1.84 mM)) and aromatic amines (o-toluidine (0.85 mM), p-chloroaniline (0. 71 mM)) each induced statistically significant (P<0.0001, Mann-Whitney test) increases in median CTLs in breast milk cells from all the donors examined when incubated (30 min, 37 degrees C) in the presence of HU/ara-C. In some cases, these compounds were also active in the absence of the repair inhibitors. There were marked variations in comet formation between donors and between genotoxins. Cell culture appeared to increase the epithelial cell proportion and cultured cells retained their ability to activate genotoxins. The results suggest that breast milk is a valuable source of human mammary cells for the study of the metabolic activation of possible carcinogens.
- Carcinogenesis 01/1992; 13(5):907-909. · 5.64 Impact Factor
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ABSTRACT: The heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a potent mutagen and is a mammary carcinogen in rodents. In man, hepatic activation is carried out by cytochrome P450 (CYP) 1A2 and the ultimate DNA-reactive species is thought to be a nitrenium ion formed via an acetoxy ester of an exocyclic amino group. Because most human breast tumours are ductal in origin, we investigated the ability of cell types present in the mammary gland (breast epithelial cells and neutrophils present in milk) to activate IQ to DNA-binding species using 32P-postlabelling. Phorbol myristate acetate-stimulated neutrophils produced a similar pattern of IQ-DNA adducts to that produced by human mammary epithelial cells. Adduct formation in stimulated neutrophils was inhibited 80% by the myeloperoxidase inhibitor sodium azide (1 mM) but was not affected by proadifen (100 microM), indomethacin (100 microM), or eicosatetraynoic acid (100 microM), inhibitors of cytochrome P450, prostaglandin synthetase, and lipoxygenase, respectively. Similar experiments in human mammary epithelial cells showed no azide inhibition of IQ-DNA adduct formation. Analysis of gene expression by reverse transcription-polymerase chain reaction showed that CYP1A1 and CYP1B1, but not CYP1A2, were expressed at detectable levels in untreated mammary epithelial cells, whereas in neutrophils cytochrome P450 expression was confined to low levels of CYP1A1. In cultured epithelial cells, IQ-DNA adduct formation and CYP1A1, but not CYP1B1 expression were induced threefold by benz[a]anthracene treatment; IQ-DNA adduct formation was inhibited by alpha-naphthoflavone. Our results indicate possible mechanisms for the metabolic activation of dietary carcinogens in the human breast.Pharmacogenetics 01/1999; 8(6):519-28.
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ABSTRACT: Cultures of human mammary epithelial cells were treated with one of seven heterocyclic amine food mutagens [2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-di-methylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx), 2-amino-3,4,7,8-tetramethylimidazo[4,5-f]quinoxaline (4,7,8-TriMeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP)], four nitropyrenes (1-nitropyrene (1-NP), 1,3-dinitropyrene (1,3-DNP), 1,6-dinitropyrene (1,6-DNP) or 1,8-dinitropyrene (1,8-DNP)] or the polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P). DNA isolated from the cultures was analysed by 32P-post-labelling and in each case the presence of carcinogen-DNA adducts was detected. The patterns and numbers of adducts obtained when human mammary cell DNA digests were separated on polyethyleneimine-cellulose TLC were found to closely resemble those previously demonstrated to be present in the DNA of tissues from rodents and other primates treated with the same agents. Up to six DNA adducts were detected in human breast cells treated with IQ and MeIQ. Fewer adducts (1-3) were detected following treatment with MeIQx or its methylated derivatives, whilst PhIP gave rise to at least four distinct adduct spots. Five adduct spots were detected in breast cells treated with DB[a,l]P or with 1-NP, but fewer adduct spots were formed by 1,3-, 1,6- and 1,8-DNP. These data demonstrate the ability of human breast epithelial cells to activate to DNA binding species a range of carcinogenic compounds known to be present in the human diet or to which humans are known to be exposed environmentally.Carcinogenesis 09/1996; 17(8):1769-72. · 5.64 Impact Factor