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.
"Microscope slides were cleaned with 70% EtOH and pre-coated on one side with a 1% solution of normal melting point (NMP) agarose in H2O . MCF-7 cells grown to confluence in 12-well multi-well dishes were exposed in 1 ml of complete medium to various concentrations (0.01 μg/ml, 0.1 μg/ml and 1.0 μg/ml final concentrations) of test compound added as a solution in DMSO; DMSO was employed as the vehicle control and 1.0 μM benzo[a]pyrene (B[a]P) as positive control [21,22]. "
[Show abstract][Hide abstract] ABSTRACT: The environmental impact of metal complexes such as organotin(IV) compounds is of increasing concern. Genotoxic effects of organotin(IV) compounds (0.01 μg/ml, 0.1 μg/ml or 1.0 μg/ml) were measured using the alkaline single-cell gel electrophoresis (comet) assay to measure DNA single-strand breaks (SSBs) and the cytokinesis-block micronucleus (CBMN) assay to determine micronucleus formation. Biochemical-cell signatures were also ascertained using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. In the comet assay, organotin(IV) carboxylates induced significantly-elevated levels of DNA SSBs. Elevated micronucleus-forming activities were also observed. Following interrogation using ATR-FTIR spectroscopy, infrared spectra in the biomolecular range (900 cm-1 – 1800 cm-1) derived from organotin-treated MCF-7 cells exhibited clear alterations in their biochemical-cell fingerprint compared to control-cell populations following exposures as low as 0.0001 μg/ml. Mono-, di- or tri-organotin(IV) carboxylates (0.1 μg/ml, 1.0 μg/ml or 10.0 μg/ml) were markedly cytotoxic as determined by the clonogenic assay following treatment of MCF-7 cells with ≥ 1.0 μg/ml. Our results demonstrate that ATR-FTIR spectroscopy can be applied to detect molecular alterations induced by organotin(IV) compounds at sub-cytotoxic and sub-genotoxic concentrations. This biophysical approach points to a novel means of assessing risk associated with environmental contaminants.
PACS codes: 87.15.-v, 87.17.-d, 87.18.-h
" . , 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 "
[Show abstract][Hide abstract] ABSTRACT: Extensive research indicates that the etiology of breast cancer is complex and multifactorial and may include environmental risk factors. Breast cancer etiology and exposure to xenobiotic compounds, diet, electromagnetic fields, and lifestyle have been the subject of numerous scientific inquiries, but research has yielded inconsistent results. Biomonitoring has been used to explore associations between breast cancer and levels of environmental chemicals in the breast. Research using breast tissues and fluids to cast light on the etiology of breast cancer is, for the most part, predicated on the assumption that the tissue or fluid samples either contain measurable traces of the environmental agent(s) associated with the cancer or that they retain biological changes that are biomarkers of such exposure or precursors of carcinogenic effect. In this paper, we review breast cancer etiology research utilizing breast biomonitoring. We first provide a brief synopsis of the current state of understanding of associations between exposure to environmental chemicals and breast cancer etiology. We then describe the published breast cancer research on tissues and fluids, which have been used for biomonitoring, specifically human milk and its components, malignant and benign breast tissue, nipple aspirate fluid (NAF) and breast cyst fluid. We conclude with a discussion on recommendations for biomonitoring of breast tissues and fluids in future breast cancer etiology research. Both human milk and NAF fluids, and the cells contained therein, hold promise for future biomonitoring research into breast cancer etiology, but must be conducted with carefully delineated hypotheses and a scientifically supportable epidemiological approach.
Journal of Exposure Science and Environmental Epidemiology 10/2007; 17(6):525-40. DOI:10.1038/sj.jes.7500548 · 3.19 Impact Factor
"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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