Activation of genotoxins to DNA-damaging species in exfoliated breast milk cells

Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, SM2 5NG, Surrey, UK.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis (Impact Factor: 3.68). 11/2000; 470(2):115-24. DOI: 10.1016/S1383-5742(00)00046-6
Source: PubMed


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.

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    • " . , 2000b ; Gorlewska - Roberts et al . , 2002 ; Phillips et al . , 2002 ) . Thus , adducts of environmental chemicals in milk indicate exposure of the ductal epithelial cells to compounds that may act as mammary carcinogens . In addition , exfoliated cells in human milk can be used for the study of metabolic activation of suspected carcinogens ( Martin et al . , 2000b ) . Practical information for future breast cancer investiga - tions using ductal epithelial cells is available ( Thompson et al . , 1998 ) . For example , DNA yield is significantly associated with number of weeks post - partum that a milk sample is collected ( optimal yield 6 – 8 weeks post - partum ) ( Thompson et al . , 1998 ) . In "
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    • "Cells were then seeded into 12-well multi-well dishes with each well containing 1 ml RPMI 1640 medium supplemented with 10% fetal calf serum and 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml hydrocortisone (Sigma Chemical Co., UK), 5 µg/ml insulin (Sigma Chemical Co.) and 5 µg/ml cholera toxin (Sigma Chemical Co.) and were incubated at 37°C in 5% CO 2 in air. The medium was changed after 3 days in order to concentrate the proportion of HMECs (Martin et al., 2000). Prior to subculture or incorporation into experiments, cultured cells were disaggregated, using a 0.05% trypsin/0.02% "

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