Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey SM2 5NG and
1 Department of Paediatrics, Queen Charlotte's and Chelsea Hospital, Goldhawk Road, London W6 0XG, UK
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Abstract |
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Abbreviations: ara-C, cytosine arabinoside; CTL, comet tail length; HMECs, human mammary epithelial cells; HU, hydroxyurea; PAH, polycyclic aromatic hydrocarbon; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.
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Introduction |
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However, the exact nature of these factors remains obscure. High penetrance breast cancer susceptibility genes are likely to account for only 5% of cases. The spectrum of p53 mutations associated with breast cancer implies that exogenous environmental influences are responsible for a proportion of breast cancers (3) but the only environmental agent that has been clearly shown to induce breast cancer in women is ionizing radiation (4). It has been postulated that lipophilic carcinogens could be sequestered in the adipose tissue of the human breast (5), thus exposing mammary epithelial cells, from which breast carcinomas arise, to DNA damage and mutation (68). Candidate environmental carcinogens include polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs and heterocyclic aromatic amines (food mutagens), many of which are fat-soluble rodent mammary carcinogens (9).
When mammary lipid extracts, obtained from UK women undergoing elective reduction mammoplasties, were examined for the presence of genotoxic components, using a range of short-term tests, some 40% were found to be positive (6). In addition, otherwise untreated human mammary epithelial cells (HMECs), prepared from the same tissue samples, were found to contain DNA damage in the form of single-strand breaks that could be increased in number by incubation with mammary lipid extracts (7). However, the use of such tissues and extracts is limited to the small cohorts of women that undergo reduction mammoplasty. Other studies have attempted to assess such genotoxic exposures by examining related biological fluids, such as nipple aspirates (1012) and cyst fluid (13), in short-term genotoxicity assays, but with mixed results.
As a surrogate breast tissue, breast milk can be obtained from a much larger and more representative cohort of, albeit lactating, women. The recovery of viable human breast cells from freshly expressed milk provides a ready source, obtained non-invasively, of HMECs in which to study the presence of, and their susceptibility to, DNA damage (14,15). Analysis, in tandem, of the milk from which the cells were obtained provides a means of detecting possible carcinogens excreted in breast milk during lactation (16). Recently, two independent pilot studies have shown that a significant percentage of human breast milk extracts are positive in tests for genotoxic activity (15,17).
In the present study we have used the Comet assay to examine fresh and 7 day cultures of breast milk cells for the presence of DNA damage and to determine if treatment of such cells with the donors' own breast milk extracts can induce DNA strand breaks. The study included a comparison of early milk samples (obtained ~4 weeks post-partum) with late samples (~4 months post-partum). Experiments were carried out in the presence or absence of the DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (ara-C), which can enhance the sensitivity of the Comet assay by blocking DNA repair synthesis (18).
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Materials and methods |
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Human milk
Samples of human milk were donated by 16 breast-feeding volunteers after ethical approval had been obtained for the study. Where possible, each volunteer provided two early (~4 weeks post-partum) and two late (~4 months post-partum) milk samples and each volunteer completed a brief questionnaire on dietary, drinking and smoking habits (see Table I for details). Each pair of samples comprised one breast milk sample expressed during the evening and stored at 4°C and another sample expressed the following morning immediately prior to the transportation of both samples, at 4°C, to the laboratory. Samples were processed within 2 h of expression of the second sample: milk extracts were prepared from the first of each pair of samples and cells from the second.
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Extraction of milk
Milk was saponified and extracted using a solid phase tandem extraction procedure originally described by Gross (19) and used in previous studies on human mammary lipid (68). Extracts in methanolic ammonia (9:1 v/v, 800 µl) were evaporated to dryness and the residues were resuspended in DMSO (5 µl) for incorporation into assays.
Isolation of cells from milk
Fresh samples of milk (80100 ml, a single expression containing both fore- and hind-milk) were diluted 1:1 with RPMI 1640 medium in order to facilitate separation (20) and centrifuged at 1000 g for 20 min. The supernatant was removed without disturbing the pellet and the pelleted cells were washed twice more by resuspension in medium and centrifugation. Cells were then either examined immediately using the Comet assay or were seeded into 25 cm2 flasks containing 10 ml RPMI 1640 medium supplemented with 10% fetal calf serum and 2 mM L-glutamine, 100 µg/ml penicillin/streptomycin mix, 5 µg/ml hydrocortisone, 5 µg/ml insulin and 5 µg/ml cholera toxin and were incubated at 37°C in 5% CO2 in air with a change of medium after 4 days (20). Prior to examination in the Comet assay, cultured cells were disaggregated, using a 0.25% trypsin/EDTA solution, to form a single cell suspension (~1x106 cells/ml) in Dulbecco's minimal essential medium with 5% fetal calf serum.
Epithelial cell proportion
The total number of cells present in milk samples was determined in resuspended cell pellets using a Coulter counter. In five determinations the cell number ranged from 3.2x104/ml breast milk to 1.08x106/ml, in line with published values (14). The numbers of epithelial cells present were measured by flow cytometry using an anti-epithelial cell antibody (15,21). Briefly, approximately 105106 cells were exposed to anti-human epithelial cell monoclonal antibody (20 µl) (HEA 125; Serotec, UK) for 10 min at 4°C, before washing with saline (Dulbecco's phosphate-buffered saline A). Rabbit and mouse immunoglobulins conjugated to fluorescein isothiocyanate were added and incubation was continued for a further 10 min at 4°C. The cells were washed, resuspended in 0.5 ml ortholysis solution (Ortho Diagnostics, UK) and incubated for 10 min at room temperature before examination in an Orthocytoron (Ortho Diagnostics) with gating for epithelial cells. A control culture was included in each analysis, which had not been treated with primary antibody. Such cell preparations were found to consist of 17 ± 9% epithelial cells (n = 3) and under the culture conditions used enrichment of epithelial cells to 47% (n = 1) could be achieved.
Cell viability
Trypan blue exclusion was used. Individual cell suspensions were gently mixed 1:1 with trypan blue (0.4% solution in 0.85% saline; Flow Laboratories, UK), allowed to stand for 510 min and placed in a haemocytometer. The percentage of cells that excluded trypan blue was used as an indicator of cell viability and was estimated both before and after treatment of cells with milk extracts or test chemicals.
Single-cell gel electrophoresis (Comet) assay
In order to detect DNA single-strand breaks in breast milk cells, alkaline lysis followed by alkaline gel electrophoresis was employed (7,18). Aliquots of otherwise untreated breast milk cell suspensions (~1x105 cells/75 µl) were incubated in the presence or absence of HU and ara-C (10 and 1.8 mM final concentrations, respectively) at 37°C for 30 min (7). Cultured cells were also treated with 8 g equivalents of milk extract (15) or with a test chemical (18) in DMSO (maximum concentration of 5% v/v) in the presence or absence of HU/ara-C. Following incubation, cells were embedded in an agarose sandwich on fully frosted microscope slides (Curtin Matheson Scientific, Houston, TX) on a cold surface. Slides were submerged in cold lysis solution (2.5 M NaCl, 100 mM EDTA disodium salt, 10 mM Tris, 1% Triton X-100 and 10% DMSO), protected from light and stored at 4°C for at least 1 h. Under red light, slides were transferred to a horizontal electrophoresis tank, covered in electrophoresis solution (0.3 M NaOH, 1 mM EDTA, freshly prepared and assumed to be pH > 13), and stored in a chilled incubator at 10°C for 40 min to allow unwinding of the DNA before electrophoresis at 0.8 V/cm and 300 mA for 36 min. After electrophoresis, slides were neutralized (0.5 M Tris, pH 7.5) and stained with ethidium bromide (20 ng/ml), after which comet tail lengths (CTLs) were visualized by epifluorescence using a Leitz Laborlux S microscope. A total of 50 digitized images/data point, 25 from each of two duplicate slides, was measured. CTLs were compared using a MannWhitney test.
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Results |
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The ability of extracts of breast milk to induce DNA damage in exfoliated cells, obtained from the same donor and cultured for 1 week, and the effects of these extracts on cell viability were also investigated. Extracts of milk from donors whose untreated, exfoliated cells contained only low levels of pre-existing DNA damage (donors 1, 2, 4, 5, 12, 13 and 15) were relatively inactive in inducing further damage and had median CTLs ranging from 4.0 to 15.5 µm with and from 2.5 to 14.0 µm without HU/ara-C (Table II). However, extracts prepared from milk of individuals whose exfoliated cells exhibited higher levels of pre-existing DNA damage (donors 3, 611, 14 and 16) were found to be actively comet forming, with median CTLs ranging from 16.5 to 75.5 µm in the presence of HU/ara-C and from 5.5 to 27.5 µm in the absence of HU/ara-C. The enhanced genotoxicity detected after treatment with milk extracts was accompanied by some cytotoxicity; cell viability ranged from 81 to 94% and from 88 to 97% following incubations carried out in the presence or absence of HU/ara-C, respectively.
Of the 10 donors who provided both early and late samples, five showed activity in both samples (donors 79, 11 and 16), one showed activity in the early sample only (donor 1) and four showed no activity in either sample (donors 2, 4, 13 and 15). Curiously, the five positive pairs of samples were all from women who reported exposure to passive smoking at home. Two out of three samples from early-only donors were positive and two out of three from late-only donors were positive. Of those who drank <3 units of alcohol/week, six out of eight gave a positive sample, while of those who drank more, only four out of eight were positive.
The results obtained, either in the absence or presence of HU/ara-C, with cells isolated from breast milk provided by donor 2, both before and after they had been maintained in culture for 1 week, are shown in Figure 1. Median CTLs did not exceed 11.0 µm and the decreases in CTLs that were detected following cell culture were not reversed by incubation with an extract of this donor's milk.
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In addition, when cultured breast milk cells from donor 4 were treated, in the presence of HU/ara-C, with an extract of the early milk sample provided by donor 8, median CTLs increased from 6.0 µm in the untreated cells to 33.0 µm in those incubated with the extract (data not shown).
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Discussion |
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Although relatively large amounts of lipid and large numbers of HMECs can be obtained from reduction mammoplasties, only small numbers of women undergo such surgery, which limits the usefulness of this approach to the investigation of breast cancer aetiology. Breast milk, however, can be obtained non-invasively from a much larger cohort of women and its composition, in terms of genotoxins, may reflect that of breast lipid, since breast lipid turnover occurs during lactation (24).
In a preliminary study, using different donors, ~25% of human milk extracts were positive in bacterial mutagenicity tests when they were prepared by the same method that was used to extract mammary lipid (15). In addition, more than half the milk extracts were active in increasing CTLs in MCL-5 cells. As previously stated (15), it must be emphasized that breast feeding exerts a moderate protective effect against some childhood cancers and lactation itself confers a weak protective effect against pre-menopausal breast cancer.
In the present study, the possibility that the genotoxicity found in breast milk samples resulted from a progressive clearance of genotoxins that had accumulated in breast lipid was examined by comparing breast milk samples supplied by donors earlier and later in lactation. The results obtained (Table II) do not support this proposed mechanism. The fluctuations in activity seen here in extracts of different milk samples provided by the same donors, when taken together with the fluctuations in the levels of DNA damage seen in untreated breast cells from those donors, probably point to variations in diet and/or lifestyle as the source of genotoxicity. Intervention studies could be designed that would enable the source, or sources, to be identified but the small number of donors who participated in the present study precludes conclusions being made on this point at present.
The decrease in the amount of DNA damage that was detected by the Comet assay when breast milk cells were kept in culture for 7 days (Table II) may be due either to a selective loss of the most damaged cells through death or to DNA repair; more detailed studies will be required to distinguish between these alternative mechanisms. If DNA repair is involved, then this will have to be considered alongside the finding that increases in DNA damage induced by breast milk extracts were only detected when incubations were carried out in the presence of the repair inhibitors HU and ara-C. Thus the damage present in untreated breast milk cells may be different in type from the damage that can be induced in those cells by incubation with milk extracts.
Freshly expressed breast milk is known to contain a mixture of cell types, including macrophages, lymphocytes and neutrophils as well as luminal epithelial cells (14), and some of the non-epithelial cells may contribute to the activation of genotoxins (21,25). In the present work, cultures of breast cells that included epithelial cells were found to be capable of activating the rodent mammary carcinogens o-toluidine (22) and PhIP (23) to DNA-damaging species. Cultures of breast cells also activated the genotoxic components of breast milk extracts to metabolites that caused DNA single-strand breaks, even if the donor's untreated cells did not contain damaged DNA. This shows that the capability to activate genotoxins was present in cells, even though no damage appeared to have been induced in these cells in vivo. The enzymes involved in the activation steps have not been identified so far but may, under normal conditions, function as steroid metabolizing enzymes since the breast is a hormone-dependent tissue.
In conclusion, this study has shown that genotoxic activity can be detected, using the Comet assay, in both early and late breast milk extracts and that such extracts can induce DNA single-strand breaks in the donor's own exfoliated breast cells. The relevance of these genotoxic effects to the initiation of breast cancer, and the nature of the genotoxins involved, are currently under investigation.
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Acknowledgments |
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Notes |
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References |
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