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.
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ABSTRACT: Phortress is a novel, potent, and selective experimental antitumor agent. Its mechanism of action involves induction of CYP1A1-catalyzed biotransformation of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) to generate electrophilic species, which covalently bind to DNA, exacting lethal damage to sensitive tumor cells, in vitro and in vivo. Herein, we investigate the effects of DNA adduct formation on cellular DNA integrity and progression through cell cycle and examine whether a relevant pharmacodynamic end point may be exploited to probe the clinical mechanism of action of Phortress and predict tumor response. Single cell gel electrophoresis (SCGE) was applied to quantify DNA damage and cell cycle analyses conducted upon 5F 203 treatment of benzothiazole-sensitive MCF-7 and inherently resistant MDA-MB-435 breast carcinoma cells. Following treatment of xenograft-bearing mice and mice possessing hollow fiber implants containing MCF-7 or MDA-MB-435 cells with Phortress (20 mg/kg, i.p., 24 hours), tumor cells and xenografts were recovered for analyses by SCGE. Dose- and time-dependent DNA single and double strand breaks occurred exclusively in sensitive cells following treatment with 5F 203 in vitro (10 nmol/L-10 micromol/L; 24-72 hours). In vivo, Phortress-sensitive and Phortress-resistant tumor cells were distinct; moreover, DNA damage in xenografts, following treatment of mice with Phortress, could be determined. Interrogation of the mechanism of action of 5F 203 in silico by self-organizing map-based cluster analyses revealed modulation of phosphatases and kinases associated with cell cycle regulation, corroborating observations of selective cell cycle perturbation by 5F 203 in sensitive cells. By conducting SCGE, tumor sensitivity to Phortress, an agent currently undergoing clinical evaluation, may be determined.Molecular Cancer Therapeutics 01/2005; 3(12):1565-75. · 5.60 Impact Factor
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ABSTRACT: Breast cancer is the most frequently diagnosed female malignancy world-wide. The aetiology of the majority of cases remains obscure and the only genotoxin as yet known to initiate breast cancer is ionizing radiation. High penetrance susceptibility genes probably account for no more than 5-10% of cases. The breast, which consists of 70-90% adipose tissue, has a unique morphological structure. Dispersed within it are the functional elements that are lined with cancer-susceptible epithelial cells. Numerous dietary and/or environmental fat-soluble compounds are known to be rodent mammary carcinogens. Extracts of lipid obtained following collagenase digestion of elective reduction mammoplasty tissues from UK resident women showed activity in short-term genotoxicity assays in 40% of cases. More active lipid extracts tended to come from donors whose human mammary epithelial cells (HMECs) exhibited pre-existing DNA single-strand breaks (SSBs). Lipid extracts also induced morphological transformation of mammalian cells in vitro. To increase cohort size, extracts of UK breast milk were examined for genotoxicity and similar activity profiles were observed. Viable cells, a large percentage of which were HMECs, were recovered from breast milk and examined for pre-existing SSBs and for the ability of the donor's own milk extract to induce SSBs. Again, donors whose untreated cells contained the most SSBs tended to yield genotoxic breast milk extracts. Breast milk cells were also able to activate rodent mammary carcinogens to DNA-damaging species. These studies provide good evidence for in vivo exposure of HMECs to genotoxic agents years before the peak in occurrence of sporadic breast cancer. Work is in progress to characterize these agents and to determine their possible role in breast cancer aetiology.Mutagenesis 04/2001; 16(2):155-61. · 3.50 Impact Factor
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ABSTRACT: Epidemiological studies indicate the involvement of environmental factors in the etiology of breast cancer, but have not provided clear indications of the nature of the agents responsible. Several environmental carcinogens are known to induce mammary tumors in rodents, and the abundance of adipose tissue in the human breast suggests that the epithelial cells, from which breast tumors commonly arise, could be exposed to lipid-soluble carcinogens sequestered by the adipose tissue. In this report we review our studies in which we have examined human mammary lipid, obtained from elective reduction mammoplasties from healthy donors, and human milk from healthy mothers, for the presence of components with genotoxic activity in several in vitro assays. A significant proportion of lipid extracts induced mutations in bacteria and micronuclei in mammalian cells. They also caused DNA damage, detected as single-strand breaks in the alkaline single-cell gel electrophoresis (comet) assay, in both the MCL-5 cell line and in primary cultures of human mammary epithelial cells. Genotoxic activity was also found in a significant proportion of extracts of human breast milk. Viable cells recovered from milk samples showed evidence of DNA damage and were susceptible to comet formation by genotoxic agents in vitro. Genotoxic activity was found to be less prevalent in milk samples from countries of lower breast cancer incidence (the Far East) compared with that in samples from the UK. The agents responsible for the activity in milk appear to be moderately polar lipophilic compounds and of low molecular weight. Identification of these agents and their sources may hold clues to the origins of breast cancer. Environ. Mol. Mutagen. 39:143–149, 2002. © 2002 Wiley-Liss, Inc.Environmental and Molecular Mutagenesis 03/2002; 39(2‐3):143 - 149. · 3.71 Impact Factor