Mutagenesis, Vol. 16, No. 2, 155-161,
March 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Genotoxins and the initiation of sporadic breast cancer
Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey SM2 5NG, UK
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Abstract |
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 Top Abstract Scale of the problem...Why the breast could...Tissue acquisitionExtractionIn vitro simulation of...Bacterial mutagenicity and...Single cell gel electrophoresis...Morphological transformation in...DiscussionConclusions and future workReferences |
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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.
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Scale of the problem and risk factors |
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TopAbstractScale of the problem...Why the breast could...Tissue acquisitionExtractionIn vitro simulation of...Bacterial mutagenicity and...Single cell gel electrophoresis...Morphological transformation in...DiscussionConclusions and future workReferences |
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Breast cancer is the most frequent malignancy occurring in women world wide, accounting for 20% of all female cancers (Higginson et al., 1992
). Currently, one in every 8–9 women in Western Europe and North America will develop the disease by the age of 85. Aetiological risk factors that could account for the majority of cases, which are post-menopausal and sporadic, remain obscure and genetic predisposition probably accounts for only some 5–10% of breast cancer cases (Higginson et al., 1992). The observation that the mutational spectrum in the p53 gene in human mammary tumours differs from that attributable solely to background endogenous processes points to environmental factors playing a role in the aetiology of breast cancer (Biggs et al., 1993).
Breast cancer incidence varies widely across the globe, with a much higher incidence in Western countries than in, for example, the Far East (Higginson et al., 1992). Migration studies have shown that breast cancer incidence in Japanese-American women increases to match the resident incidence by the second or third generation (Ziegler et al., 1993). There is evidence linking alcohol intake and breast cancer incidence amongst individuals lacking both the GSTM1 and GSTT1 genes (Park et al., 2000). Intensity of alcohol intake rather than duration may also be a risk factor (Bowlin et al., 1997). Such epidemiological investigations into the relationship of breast cancer risk to gene–environment interactions are still in their infancy (Williams and Phillips, 2000). For instance, observation of an increased risk of breast cancer from smoking in slow NAT2 acetylators (Ambrosone et al., 1996) has not been verified (Hunter et al., 1997; Delfino et al., 2000). The only environmental exposure proven to induce breast cancer is ionizing radiation (Tokunaga et al., 1987). Total cumulative exposure to oestrogen is also seen as a risk factor: thus nulliparity, late age at first pregnancy, early menarche and late menopause may play a role in breast cancer risk (Feigelson and Henderson, 1996). However, oestrogens probably act as tumour and growth promoters rather than as complete carcinogens (Higginson et al., 1992).
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Why the breast could be peculiarly susceptible to fat-soluble genotoxins |
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TopAbstractScale of the problem...Why the breast could...Tissue acquisitionExtractionIn vitro simulation of...Bacterial mutagenicity and...Single cell gel electrophoresis...Morphological transformation in...DiscussionConclusions and future workReferences |
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In simple terms, the breast consists of 70–90% adipose tissue, in which are dispersed the functional elements that are lined with epithelial cells from which breast cancers commonly arise. Carcinogen–DNA adducts in many tissues, including the breast, have been observed in vivo in different human populations (Perera et al., 1995
; Perera, 2000). Human exposures to carcinogens via the diet and the environment include exposure to polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs and heterocyclic aromatic amines (HAAs, `food mutagens'), many of which are fat soluble and capable of inducing mammary tumours in rodents (El-Bayoumy, 1992). This lends credence to the proposition (Beer and Billingham, 1978) that human mammary lipid could act as a reservoir or depot for mutagenic/genotoxic agents capable of inducing DNA damage in adjacent epithelial cells in vivo. These epithelial cells express CYP1B1 and CYP1A1, which are involved in PAH and HAA activation (Larsen et al., 1998; Williams et al., 1998), as well as N-acetyltransferases and sulphotransferases, that further metabolically activate hydroxylated derivatives of these compounds (Sadrieh et al., 1996; Williams et al., 2000a). human mammary epithelial cells (HMECs) can thus activate HAAs and PAHs to DNA-binding species (Grover et al., 1980; Pfau et al., 1992; Carmichael et al., 1996). It has also been hypothesized that peroxidases, including lactoperoxidase, synthesized in HMECs, and myeloperoxidase, present in neutrophils, may play a key role in the initial activation of aromatic amines to DNA-binding species (Josephy, 1996). |
Tissue acquisition |
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TopAbstractScale of the problem...Why the breast could...Tissue acquisitionExtractionIn vitro simulation of...Bacterial mutagenicity and...Single cell gel electrophoresis...Morphological transformation in...DiscussionConclusions and future workReferences |
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A large amount of lipid can be obtained from essentially healthy women following collagenase digestion of tissues removed at elective reduction mammoplasty (Easty et al., 1980
). Such tissues can be obtained from young women compared with the usual age of subjects from whom tumour-adjacent breast tissue is obtained (Li et al., 1996; Perera et al., 1995). An advantage of using reduction mammoplasty tissue is that it can be examined for DNA damage and for the presence of agents capable of inducing genotoxic events many years before the age-related peak in sporadic breast cancer incidence.
Breast milk is a natural, lipid-containing secretion of the breast that is more readily available than elective reduction mammoplasty tissues and that can be obtained non-invasively from a larger cohort of individuals. There is also evidence that body lipid, as opposed to dietary lipid, turns over to provide lipid for secretion in breast milk (Villalprando and del Prado, 1999), bringing with it, one could reasonably surmise, any contaminating genotoxins. The detection of low (p.p.b.) concentrations of aromatic amines, one of which is known to be a rodent mammary carcinogen, has been reported in breast milk (DeBruin et al., 1999). The presence of organochlorines such as lindane has also been documented, both in breast tissue and in breast milk (Lunden and Noren, 1998; Aronson et al., 2000).
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Extraction |
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TopAbstractScale of the problem...Why the breast could...Tissue acquisitionExtractionIn vitro simulation of...Bacterial mutagenicity and...Single cell gel electrophoresis...Morphological transformation in...DiscussionConclusions and future workReferences |
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Prior to their incorporation into assays for genotoxicity, a reliable clean-up procedure for breast lipid or breast milk is required in order to remove the growth-inhibitory and potentially cytotoxic effects of lipids and also to concentrate any putative genotoxins that are present. Without such a procedure only minute lipid samples can be tested in vitro (Johnson and Smith, 1984
). The method employed to generate extracts of both lipid and breast milk was a solid phase tandem extraction procedure originally developed for the extraction of HAAs from lipid-rich food matrices (Gross, 1990). Extracts for genotoxicity testing were prepared exactly as has been described for lipid analyses (Martin et al., 1996, 1997, 1998) and for breast milk analyses (Martin et al., 1999a, 2000a). |
In vitro simulation of the in vivo situation |
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TopAbstractScale of the problem...Why the breast could...Tissue acquisitionExtractionIn vitro simulation of...Bacterial mutagenicity and...Single cell gel electrophoresis...Morphological transformation in...DiscussionConclusions and future workReferences |
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The biological activities of extracts obtained from human mammary lipid or breast milk prepared from different donors were compared using a metabolically competent human cell line, MCL-5 cells (Martin et al., 1997
, 1999a). The use of fresh materials (adipose tissues from elective reduction mammoplasties or breast milk) allows the recovery of viable HMECs, which can be analysed for their ability to activate genotoxins and for the presence of pre-existing DNA single-strand breaks (SSBs) in otherwise untreated cells. In addition, they can be placed in primary cultures and used for further analyses, i.e. of the repair of pre-existing DNA damage and/or the maintenance of metabolic capacity (Martin et al., 1997, 1999a, 2000a). Such primary cultures were obtained following collagenase digestion of tissues (O'Hare et al., 1991) or centrifugation of breast milk (Taylor-Papadimitriou and Stampfer, 1992). Breast milk is known to contain a mixture of cell types, including macrophages, lymphocytes and neutrophils, as well as HMECs (Thompson et al., 1998), and some non-epithelial cells may contribute to the activation of genotoxins (Eltom et al., 1998; Josephy and Coomber, 1998; Williams et al., 1998, 2000b). |
Bacterial mutagenicity and clastogenicity in human cells in vitro |
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TopAbstractScale of the problem...Why the breast could...Tissue acquisitionExtractionIn vitro simulation of...Bacterial mutagenicity and...Single cell gel electrophoresis...Morphological transformation in...DiscussionConclusions and future workReferences |
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Plate incorporation assays for bacterial mutagenicity (Venitt et al., 1984
), with or without Aroclor 1254-induced rat liver S9, were performed and of 40 mammary lipid extracts examined, 16 (40%) tested positive (Martin et al., 1996). Fifteen were positive in Salmonella typhimurium TA1538 and 11 were positive in TA98, with samples from 10 individuals being positive in both strains. Micronucleus formation induced by extracts in MCL-5 cells blocked at cytokinesis by cytochalasin B was used as an indicator of chromosomal damage (Crofton-Sleigh et al., 1993). Extracts from 17 of 40 lipid samples exhibited significant micronucleus-forming activity as judged by a 
2 test for trend of dose–response data (Martin et al., 1996). There was a significant positive correlation between mutagenicity of lipid samples in S.typhimurium TA98 and TA1538 and micronucleus formation in MCL-5 cells as judged by Spearman rank order correlation tests (TA98 versus micronucleus formation, r = 0.817, P <0.0001; TA1538 versus micronucleus formation, r = 0.793, P <0.0001).
When 20 breast milk extracts were tested in S.typhimurium TA1538 and YG1019 in the presence of Aroclor 1254-induced rat liver S9, six were adjudged to produce a positive mutagenic response in one or both bacterial strains (Martin et al., 1999a). Six samples (four of which were positive for bacterial mutagenicity) also induced significant micronucleus formation in MCL-5 cells. The activity of the milk extracts in the micronucleus assay correlated significantly with bacterial mutagenicity (versus S.typhimurium TA1538, r = 0.74, P <0.0002; versus S.typhimurium YG1019, r = 0.75, P <0.0001; Spearman rank order correlation test) (Martin et al., 1999a).
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Single cell gel electrophoresis `Comet' assay |
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TopAbstractScale of the problem...Why the breast could...Tissue acquisitionExtractionIn vitro simulation of...Bacterial mutagenicity and...Single cell gel electrophoresis...Morphological transformation in...DiscussionConclusions and future workReferences |
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The alkaline version of the Comet assay is a sensitive, simple and rapid method for visualizing and measuring DNA SSBs in single cell preparations (Singh et al., 1988
). In the assay a small number of treated cells suspended in a thin agarose sandwich are lysed, subjected to electrophoresis at high pH and stained with a fluorescent DNA-binding dye (Fairbairn et al., 1995; Tice, 1995). As a result of SSBs, DNA migrates at different, size-related rates according to the extent of damage and forms a `comet tail' (although the tail precedes the head, i.e. a comet in reverse), whose length is measured. The results obtained using the Comet assay are presented as frequency distributions of comet tail lengths (CTLs) (in µm) for each treatment condition, derived from the measurement of 50 nuclei (Martin et al., 1999b). A shift to the right in distribution away from 0 indicates an increase in tail length that is interpreted as an increase in DNA damage. Cell viability, as assessed by trypan blue exclusion, is measured in parallel.
Examples of the fluorescence microscopy images obtained with HMECs which were either untreated or treated with an active human mammary lipid extract, both in the presence of HU/ara-C, prior to incorporation into the Comet assay are shown in Figure 1A and B, respectively. When the assay is carried out under alkaline conditions, the extent of DNA SSB formation can be quantified by measuring the extent of DNA migration (CTL); the greater the CTL, the more numerous the strand breaks. By incorporating two known inhibitors of DNA repair, hydroxyurea (HU) and cytosine arabinoside (ara-C), the sensitivity of the Comet assay is greatly enhanced (Martin et al., 1997, 1999b; Pfau et al., 1999). These DNA repair inhibitors allow the recognition and incision stages of nucleotide excision repair to occur but inhibit subsequent DNA resynthesis: this results in an accumulation of SSBs (Collins and Johnson, 1984; Thompson et al., 1989; Kinley et al., 1995).
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With MCL-5 cells, extracts of human mammary lipid induced comet formation only in the presence of HU/ara-C, which may indicate that these cells are repair efficient (Martin et al., 1997
). The induction of comet formation in the presence of HU/ara-C was also observed after treatment of MCL-5 cells with extracts of breast milk (Martin et al., 1999a). With HMECs, however, comets were observed both in the absence and presence of the repair inhibitors, but CTLs increased when the inhibitors were added; this may indicate that HMECs have a capacity to repair some DNA damage (Martin et al., 1997). In addition, it was found that several samples of HMECs, obtained either following collagenase digestion of elective reduction mammoplasty tissues or recovered as exfoliated breast milk cells, contained pre-existing DNA damage. It was also noticed that the most active extracts, either of lipid or of milk, tended to come from those donors whose cells also contained the most pre-existing DNA SSBs (Martin et al., 1997, 2000a). Genotoxicity, whether detected in the form of pre-existing or as induced SSBs, was observed in the absence of an appreciable loss of cell viability (Martin et al., 2000a,b).
A distribution of CTLs from a preparation of HMECs that showed some pre-existing DNA damage is provided in Figure 2. This preparation had median CTLs that were not significantly different when incubations were carried out either in the presence (19.5 µm) or absence (26.0 µm) of HU/ara-C. When an aliquot of these cells was treated with an extract of the donor's own lipid, increases in median CTLs were observed, both in the absence (39.0 µm) and in the presence (38.0 µm) of HU/ara-C. These HMECs were also capable of activating a known rodent mammary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), at a concentration of 0.2 mM, which induced median CTLs of 29.0 µm in the absence of HU/ara-C and of 43.5 µm in their presence. Treatment of these HMECs with the N-hydroxy metabolite of PhIP (0.001 mM, a 200-fold lower concentration) induced median CTLs of 25.0 µm in the absence of HU/ara-C and of 56.5 µm in their presence.
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Figure 3
shows a distribution of CTLs from a preparation of breast milk cells after culture for 1 week. This preparation had a median CTL of 16.0 in the absence and 16.0 in the presence of HU/ara-C after 30 min incubation at 37°C. When an aliquot of these cells was treated with an extract of the donor's own breast milk, an increase in median CTL was observed in the presence (72.5 µm) of HU/ara-C, but not in their absence (18.5 µm). Again, these cells were capable of activating PhIP at a concentration of 0.4 mM, which induced median CTLs of 20.0 µm in the absence of HU/ara-C and of 82.0 µm in their presence. Treatment with the N-hydroxy metabolite (0.001 mM) induced median CTLs of 64.5 µm in the absence of HU/ara-C and of 105.5 µm in their presence. The ability of these preparations of exfoliated breast milk cells to activate a range of carcinogens to DNA-damaging species has also been demonstrated (Martin et al., 2000b).
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Morphological transformation in vitro |
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TopAbstractScale of the problem...Why the breast could...Tissue acquisitionExtractionIn vitro simulation of...Bacterial mutagenicity and...Single cell gel electrophoresis...Morphological transformation in...DiscussionConclusions and future workReferences |
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Mammalian cell transformation assays have been developed as in vitro test systems in order to predict the carcinogenic potential of chemical, biological and physical agents (Huberman et al., 1972
; Isfort et al., 1996). Morphologically transformed foci of C3H/M2 mouse fibroblasts form tumours that metastasize in nude mice (Marquardt et al., 1976).
Interindividual variations were observed in the ability of mammary lipid extracts to induce morphological transformation. Transformation rates in C3H/M2 mouse fibroblasts of 0.27 (70 mg), 0.33 (70 mg), 0.07 (30 mg), 0.29 (80 mg), 0.21 (30 mg), 0.00 (110 mg), 0.07 (70 mg) and 0.13 (50 mg) transformed foci/treated dish (mg lipid equivalent), respectively, were obtained with extracts from eight donors (transformation rate of 0 with vehicle control). In comparison, results with the positive controls N-methyl-N'-nitro-N-nitrosoguanidine and 3-methylcholanthrene were 0.33–0.78 (0.5 µg/ml) and 0.27–0.46 (10 µg/ml), respectively (Martin et al., 1998). The ability of mammary lipid extracts to induce morphological transformation of C3H/M2 mouse fibroblasts certainly heightens the suspicion that genotoxins detected in breast lipid may be involved in mammary tumour initiation, but does not prove it (Martin et al., 1998).
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Discussion |
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TopAbstractScale of the problem...Why the breast could...Tissue acquisitionExtractionIn vitro simulation of...Bacterial mutagenicity and...Single cell gel electrophoresis...Morphological transformation in...DiscussionConclusions and future workReferences |
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The basis of the work described in this paper is the hypothesis that the initiation of breast cancer occurs following exposure of HMECs to lipophilic dietary and/or environmental carcinogens. The proposition that human mammary lipid can act as a reservoir for such genotoxic compounds has been examined in several ways. A significant proportion (~40%) of extracts of human mammary lipid, obtained from different donors, have been found to be mutagenic in bacterial cells and clastogenic towards human cells (Martin et al., 1996
). Such extracts also induced increases in CTLs in MCL-5 cells and in HMECs (Martin et al., 1997). DNA SSBs were observed in some otherwise untreated preparations of HMECs. In addition, it was found that extracts of mammary lipid obtained from those donors whose cells exhibited higher levels of pre-existing DNA SSBs were more active in inducing comet formation. Mammary lipid extracts were also found to be capable of inducing morphological transformation of C3H/M2 mouse fibroblasts (Martin et al., 1998).
Since breast milk can be obtained non-invasively from a much larger cohort of women and since its composition, in terms of genotoxins, may reflect that of breast lipid because body fat turnover occurs during lactation (Villalprando and del Prado, 1999), we have started to examine it. In a preliminary study using different donors ~25% of human milk extracts were positive in bacterial mutagenicity tests and more than half the milk extracts were active in increasing CTLs in MCL-5 cells (Martin et al., 1999a). Previous studies have reported the mutagenicity of extracts of human aortal lipid (Johnson and Smith, 1984), breast milk (McClure et al., 1999), nipple aspirates (Petrakis et al., 1980; Scott and Miller, 1990) and cyst fluid (Scott and Miller, 1991).
Using the Comet assay, the presence and induction of DNA damage in single cells can be determined. Several studies have shown that the incorporation of DNA repair inhibitors, HU and ara-C, permits accumulation of SSBs (Gedik and Collins, 1991; Kinley et al., 1995). This greatly enhances the sensitivity of the Comet assay (Lynn et al., 1997; Martin et al., 1997, 1999b, 2000a). It is believed that HU/ara-C allows the identification of repair-induced strand breaks in the Comet assay that would normally have disappeared well within the time course of treatment (Martin et al., 1999b). On the basis of this premise, Figure 2
illustrates two different kinds of inducible damage. One form of damage, that induced by PhIP or by N-hydroxy PhIP (Stone et al., 1998), is readily and efficiently repaired. Hence, incorporation of HU/ara-C is required in order to detect DNA damage induced by these agents. A second form of DNA damage, that observed either as pre-existing DNA damage or that induced by extracts, appears to be less readily repaired. On the other hand, Figure 3 shows an example where HU/ara-C is required to detect the DNA damage induced by a breast milk extract in a different donor's own breast milk cells. This suggests either different genotoxins in extracts obtained from different donors or different repair capabilities. Hence, the incorporation of HU/ara-C is not always required in order to detect the DNA damage induced by the as yet unidentified genotoxins present in extracts of lipid or milk. The DNA strand-breaking activity of such extracts may be important because of the correlations found between SSB induction and morphological transformation induced both by such extracts and by HAAs (Martin et al., 1998; Pfau et al., 1999). Concomitant measurement of cellular status, other than by trypan blue exclusion, may be necessary for future validation of the Comet assay and such determinations could include energy status (Martin and McLean, 1995, 1998).
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Conclusions and future work |
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TopAbstractScale of the problem...Why the breast could...Tissue acquisitionExtractionIn vitro simulation of...Bacterial mutagenicity and...Single cell gel electrophoresis...Morphological transformation in...DiscussionConclusions and future workReferences |
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The identities of the biologically active components present in mammary lipid or breast milk extracts remain to be established. Although the extraction procedure we used is known to isolate a variety of HAAs (Gross, 1990
) and bacterial mutagenicity was detected only in frameshift-detecting strains (Martin et al., 1996, 1999a), none of the common HAAs were detected when a small number of extracts were examined using selective ion monitoring (Martin et al., 1998). Human mammary DNA has been found to contain as yet unidentified hydrophobic adducts (Perera et al., 1995). Our studies show that mammary lipid and breast milk, as a surrogate for breast tissue, contain genotoxic constituents and lend support to the hypothesis that genetic damage leads to the initiation of human mammary carcinogenesis. However, it must be emphasized that the benefits of breast feeding, in terms of childhood health and development, outweigh any hypothetical risks to infants as a result of the presence of genotoxins in breast milk (Wilson et al., 1998; Martin et al., 1999a). It is now important to determine the identities of the biologically active genotoxins that have been detected and to test them for carcinogenicity. The origin of these genotoxins, either endogenous or exogenous, also needs to be established and efforts made to determine what factors affect their occurrence in mammary fat. Should these substances be identified as carcinogens, strategies designed to reduce or to eliminate human exposure to them could then be devised.
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Acknowledgments |
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This paper is based on the UKEMS Young Scientist Award Lecture delivered by the author at the University of Wales, Swansea, in July 2000. The work was supported by grants from the Association for International Cancer Research, the Cancer Research Campaign and the Department of Health. The assistance of my colleagues, without whom many of these experiments would not have been possible, is also gratefully acknowledged: David H.Phillips, Philip L.Grover, Stan Venitt, Christopher Crofton-Sleigh, Kathleen J.Cole, Paul L.Carmichael, Wolfgang Pfau, Elaine M.Stone, Gillian Weaver, David Harvey, J.Andrew Williams and Barbara C.Millar.
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Notes |
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Tel: +44 20 8643 8901; Fax: +44 20 8770 7290; Email:flmartin{at}icr.ac.uk
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TopAbstractScale of the problem...Why the breast could...Tissue acquisitionExtractionIn vitro simulation of...Bacterial mutagenicity and...Single cell gel electrophoresis...Morphological transformation in...DiscussionConclusions and future workReferences |
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Received on September 11, 2000; accepted on November 14, 2000.
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