Mutagenesis, Vol. 15, No. 2, 149-154,
March 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Pathways of heterocyclic amine activation in the breast: DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) formed by peroxidases and in human mammary epithelial cells and fibroblasts
Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey SM2 5NG, UK
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Most human mammary carcinomas originate in the epithelial cells of the breast ducts. A potential role of heterocyclic amines (HAs) in the aetiology of this disease has led us to investigate peroxidase-catalysed and stromal (non-epithelial) activation of the HA 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), which may subsequently lead to DNA damage in the adjacent human mammary epithelial cells (HMECs). HAs are formed when proteinaceous foods are cooked at high temperature and some, but not all, can cause mammary tumours in rats. Myeloperoxidase (MPO) and lactoperoxidase (LPO) are peroxidase enzymes present in breast secretions. 32P-post-labelling analysis showed that IQ–DNA adducts were formed after co-incubation of IQ (500 µM) with calf thymus DNA, hydrogen peroxide and either bovine LPO or horseradish peroxidase (HRP). The major HRP-mediated IQ–DNA adduct co-migrated on TLC and HPLC with the major adduct formed in HMECs, suggesting a common reactive intermediate (nitrenium ion). IQ–DNA adducts were also formed in extracellular DNA when phorbol myristate acetate-stimulated neutrophils (which activate IQ via MPO) were co-incubated with IQ (500 µM) and extracellular plasmid (4 ± 1 adducts/108 nucleotides) or calf thymus DNA (6 ± 2). Mean adduct formation was five to seven times greater in neutrophil DNA (31 ± 20). Primary cultures of human mammary fibroblasts or epithelial cells isolated from reduction mammoplasty tissues (n = 4 individuals) were incubated with IQ (500 µM) and formed 2.5 and 14.8 adducts/108 nucleotides (mean values), respectively. Our results indicate the possible contribution of stromal cells and breast peroxidases to the metabolic activation of carcinogens in the mammary gland.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Known risk factors for breast cancer include lifetime oestrogen exposure and inheritance of high penetrance cancer susceptibility genes, but can account for only one third of cases. Environmental carcinogens may contribute to the aetiology of human mammary tumours (Martin et al., 1996
, 1999; Zheng et al., 1998). A recent estimate suggests that between 20 and 42% of all cancer deaths could be avoided by changes in the diet (Willett, 1995) and for breast cancer a potential reduction of 50% has been proposed (Doll and Peto, 1981). One study has reported a positive association between higher intake of `well done' meat (compared to medium done or rare meat) and breast cancer risk (Zheng et al., 1998). Another study found no association between red meat consumption and breast cancer risk, although the degree of cooking was not taken into account (Ambrosone et al., 1998). High dietary fat intake during middle life may not necessarily be a risk factor for breast cancer (reviewed by Hunter and Willett, 1996).
Heterocyclic amines (HAs) have been implicated in human breast carcinogenesis (Zheng et al., 1998) and some are rat mammary carcinogens when administered at high doses (Nagao et al., 1994). These compounds are formed during the cooking of proteinaceous foods such as meat and fish at high temperatures (Nagao et al., 1994). Aromatic amines (AAs) are also putative breast carcinogens (Ambrosone et al., 1996; DeBruin et al., 1999) in humans and include aminobiphenyls which are present in cigarette smoke (Skipper and Tannenbaum, 1994). HAs demonstrate genotoxicity only after undergoing metabolic activation and considerable variation exists between individuals in the ability of human mammary duct epithelial cells (HMECs) to mediate this activation (Stone et al., 1998). The major pathway of activation in these cells is thought to be a two-step process mediated by cytochrome P450 (CYP)1A1- and CYP1B1-catalysed hydroxylation (Williams et al., 1998), followed by O-esterification catalysed by N-acetyltransferases (NATs) (Sadrieh et al., 1996) or by sulfotransferases (Lewis et al., 1998; Stone et al., 1999) to form the acetoxy or sulphate esters. The major site of DNA damage by activated HAs is the C8 atom of guanine, although the N2 atom of guanine is also modified (Turesky et al., 1992), and the mutagenic consequences of HA–DNA adduct formation are mainly transversions and frameshift mutations (for a review of HA–DNA adducts see Schut and Snyderwine, 1999).
Lactoperoxidase (LPO) is present in human milk (Ueda et al., 1997; Goldman et al., 1998) and is secreted by HMECs into the breast ducts (Anderson et al., 1979). We have investigated peroxidase-catalysed IQ activation by a mammalian (bovine) peroxidase (LPO) and a plant peroxidase (horseradish peroxidase, HRP), to investigate the possible existence of a general mechanism of peroxidative activation of HAs. A novel method of digestion of 32P-post-labelled adducts which recovers 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)–DNA adducts as mononucleotides (5'-phosphates) (Ochiai et al., 1999) has allowed us to compare the adducts formed by peroxidases with those formed by the activating enzymes present in HMECs and overcomes the problems of incomplete digestion of IQ–DNA oligonucleotides resulting from 32P-post-labelling using the ATP-deficient method (Hall et al., 1990).
Speculation on the contribution of enzymes present in the breast to the activation of carcinogens (Josephy, 1996; Josephy and Coomber, 1998; Martin et al., 1999) in the breast epithelium has led us to investigate the IQ-activating ability of peroxidase enzymes. Neutrophils stimulated to respiratory burst by phorbol myristate acetate (PMA) activate IQ, and inhibitor studies have identified myeloperoxidase (MPO) as the enzyme responsible (Williams et al., 1998). In this study we have investigated the ability of neutrophils to form IQ–DNA adducts in extracellular DNA. The results from these experiments could indicate whether the cellular components of breast fluid, in contact with environmental carcinogens present in breast tissue (Martin et al., 1996), including possible `cooked meat' carcinogens in the diet (Zheng et al., 1998), could pose a genotoxic risk to ductal epithelial cells, which are the most common site for the development of breast carcinomas.
Human mammary fibroblasts (HMFs) have recently been shown to express CYP1B1 mRNA and immunoreactive protein (Eltom et al., 1998), which indicates that these cells, adjacent to HMECs in the breast, may express enzymes capable of metabolically activating potential breast carcinogens such as HAs and polycyclic aromatic hydrocarbons (PAHs). In this paper, we have also compared the metabolic activation of IQ by HMFs and HMECs.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Chemicals
IQ was purchased from Toronto Research Chemicals (Ontario, Canada). pBlueScript plasmid was obtained from Stratagene (Cambridge, UK). T4 polynucleotide kinase was obtained from Kramel Biotech (Cramlington, UK). [
-32P]ATP was purchased from ICN (Basingstoke, UK). Unless stated otherwise all other materials were obtained from Sigma Chemical Co. (Poole, UK).
HRP- and LPO-catalysed IQ activation
Calf thymus DNA was dissolved in Dulbecco's phosphate-buffered saline A (PBS, 172 mM NaCl, 0.33 mM KCl, 1.05 mM KH2PO4, 0.17 mM Na2HPO4, pH 7.4). A mammalian peroxidase (bovine LPO) and a plant peroxidase (HRP) were chosen for the study. Complete incubations (1 ml volume, 37°C, 1 h) contained IQ (500 µM) and DNA (1 mg), H2O2 (0.2 mM) and enzyme (500 U). Controls omitted enzyme, H2O2 or enzyme and H2O2. Reactions were terminated by addition of ice-cold phenol and vortexing and DNA was isolated and purified according to the method of Gupta et al. (1985). Each IQ/peroxidase incubation experiment was performed twice and in each experiment single and duplicate incubations, respectively, were carried out for the LPO and HRP experiments.
Exposure of cultured human mammary epithelial cells and fibroblasts to IQ
Human female breast tissue was obtained from healthy reduction mammoplasty patients (aged between 19 and 28 years). Cells were prepared, as previously described in detail (Stone et al., 1998; Williams et al., 1998), by collagenase digestion. HMFs were separated from HMEC organoids by sequential filtration through gauze of 140 and 53 µm gauge. HMFs were present in the filtrate. HMECs (as `organoids') were seeded in RPMI 1640 medium plus 10% foetal calf serum (FCS) and supplements in culture flasks, and were gassed with 5% CO2 in air for 5 min before incubation at 37°C for 48 h. HMFs were cultured (24 h at 37°C) in DMEM (with 10% FCS), before washing to remove unwanted red blood cells with sterile PBS. HMF cells were cultured for another 48 h or until confluent. Fluorescence microscopy was used to examine the morphological characteristics of the HMECs (organoids) or HMF cell preparations (see below). Single cell suspensions were prepared by trypsin/EDTA treatment and HMECs or HMFs in suspension were then exposed to IQ at 500 µM for 22 h before DNA extraction and 32P-post-labelling analysis. Comparisons between HMF and HMEC cell activation of IQ were performed on cell preparations from four individuals.
Establishment of epithelial cell phenotype by immunohistochemical staining
The purity of the HMEC populations was investigated by immunohistochemical staining of cell-specific cytokeratins [intermediate size (10 nm) filaments composed of keratin complexes]. Mammary myoepithelial cells express cytokeratin 14 (CK14) whereas mammary luminal epithelial cells express cytokeratin 18 (CK 18). A double antibody stain for both cytokeratins was used to establish the purity of the epithelial cells used for IQ activation experiments (Clarke et al., 1994). Primary HMEC cultures were seeded onto sterile coverslips in 4-well dishes, left to attach overnight at 37°C, then fixed in cold methanol for 1 h (–20°C). The fixed myoepithelial or luminal cells were probed with primary antibody first, then with subclass-specific mouse anti-keratin antibodies conjugated, respectively, with the fluorophores tetramethylrhodamine isothiocyanate (TRITC, red fluorescence) or fluorescein isothiocyanate (FITC, green fluorescence). Epifluorescence microscopy using narrow band fluorescein and rhodamine emission filters showed cells under view either to fluoresce red, confirming myoepithelial cell identity, or to fluoresce green, confirming the identity of luminal cells. Controls (cells not treated with primary antibody) did not fluoresce.
For confirmation of fibroblast identity, cells were trypsinized from the base of culture flasks and, after washing with sterile PBS for 15 min at 4°C, were exposed to mouse anti-human fibroblast antibody (MCA 1399; Serotec, Oxford, UK). The cells were then washed and exposed to FITC-conjugated rabbit anti-mouse immunoglobulin for 10 min at 4°C. Fluorescence microscopy was carried out after further washing. Cells which had no primary antibody were used as a control.
IQ–DNA adduct formation in neutrophils and extracellular DNA
Neutrophils (>90% purity) were prepared from whole blood by centrifugation as previously described (Williams et al., 1998). Aliquots (107 cells) were stimulated to respiratory burst (confirmed by flow-assisted cell sorting analysis; Williamset al., 1998) by the addition of PMA (2.5 ng/ml) and were then incubated (1 h, 37°C) in a final volume of 2 ml with IQ (50 µl of a 20 mM solution in dimethylsulphoxide, final concentration 500 µM) in the presence of calf thymus DNA (1 mg/ml) or pBluescript K+ plasmid DNA (1 mg/ml) in PBS for 1 h at 37°C. Neutrophils were separated from extracellular DNA by centrifugation (1500 r.p.m., 10 min). After purification (Gupta et al., 1985), DNA (4 µg) from neutrophils or plasmid was electrophoresed (100 V for 30 min) in a 1% (w/v) agarose gel (containing ethidium bromide) and was viewed under UV light. Genomic neutrophil and calf thymus DNA displayed a distinct `smear' appearance, in contrast to supercoiled plasmid DNA, which displayed a discrete band on the gel.
Analysis of IQ–DNA adducts by 32P-post-labelling
For detection of the IQ–DNA adducts formed by the peroxidase enzymes in vitro and in IQ-treated HMECs, the modified method II of Ochiai et al. (1999) was employed. This method overcomes the problem of incompletely digested IQ-modified DNA by successive enzymatic incubations of the 32P-labelled adducts with polynucleotide kinase, nuclease P1 and phosphodiesterase I. For quantitative comparison of IQ–DNA adduct formation between HMECs and HMFs and between PMA-stimulated neutrophils and in co-incubated extracellular DNA, the ATP-deficient method described by Hall et al. (1990) was used, as for previous quantitative analyses of IQ–DNA neutrophil-mediated (Williams et al., 1998) and HMEC-mediated (Stone et al., 1998; Williams et al., 1998) IQ activation. 32P-post-labelled IQ–DNA adducts were chromatographed on polyethyleneimine (PEI)–cellulose TLC plates under the following solvent conditions: D1, 1.0 M sodium dihydrogen orthophosphate, pH 6.0; D2, 2.3 M lithium formate, 5.5 M urea, pH 3.5; D3, 0.8 M lithium chloride, 0.5 M Tris–HCl, 8.5 M urea, pH 8.0. DNA adducts were detected and quantified with a two-dimensional scanner (Instantimager; Canberra Packard, Pangbourne, UK). The level of DNA adducts was determined by relating levels of radioactivity in adducts to the specific activity of the [-32P]ATP and expressed as relative adduct labelling (RAL, adducts/108 nucleotides). The results from all experiments are the means of data obtained from duplicate labellings. In some subsequent analyses of groups of means (from separate preparations of mammary-derived cells), values are expressed as mean adducts/108 nucleotides ± SD.
For HPLC analysis, IQ–DNA adduct spots, cut out from the PEI–cellulose plates after two-dimensional chromatography, were eluted with 4 M pyridinium formate (0.5 ml, pH 4.5). After centrifugation at 10 000 g for 10 min to pellet contaminating solids the supernatant was evaporated and the residue was dissolved in water (200 µl). HPLC analysis was carried out using a Waters 2690 Separations Module linked to a Packard 150TR Flow Scintillation Analyser. Aliquots (60 µl) were separated on a Phenomenex 250x4.6 mm 5µ C18 Jupiter Column using a two-solvent system. Solvent A was 2 M ammonium formate (pH 4.0) and solvent B was a 6:1 (v/v) mixture of acetonitrile and methanol. The gradient was 10–30% B for 70 min, followed by 30–50% B for 10 min at a flow rate of 1 ml/min.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Using the 32P-post-labelling and DNA digestion method described by Ochiai et al. (1999), DNA adducts were observed after incubation of IQ with either LPO (Figure 1a
) or HRP (Figure 1b) in the presence of H2O2 (0.2 mM) and DNA. The formation of IQ–DNA adducts in these experiments strongly indicates that the metabolic activation of IQ proceeds in this system via a H2O2-dependent peroxidative reaction, as no IQ–DNA adducts were observed when H2O2 was absent from the incubations or when IQ was incubated with H2O2 alone (not shown).
|
The C-8 position of guanine in DNA is the major site of reaction of the potent electrophile acetoxy-IQ (Endo et al., 1994
), which forms a nitrenium ion, by heterolytic loss of the acetoxy group, as the ultimate DNA-reactive species. This is also the major target site of the other HAs (for a review see Schut and Snyderwine, 1999). Formation of adduct spot 1 (marked by an arrow) was observed after co-incubation of calf thymus DNA with LPO and H2O2 (Figure 1a, 
24% of the total radioactivity), with HRP and H2O2 (Figure 1b, 47% of the total radioactivity) and in genomic DNA from IQ-treated HMECs (22 h at 37°C, Figure 1c, 69% of the total). We have tentatively identified this adduct as pdG-C8-IQ. A number of pieces of evidence support this identification (Boteju and Hanna, 1994; Ochiai et al., 1999; Schut and Snyderwine, 1999). The major IQ–DNA adduct formed in rat liver and confirmed as the C-8 adduct of guanine was found to constitute 62% of the total adducts formed (Ochiai et al., 1999).
Further co-chromatographic analysis of this major adduct spot by HPLC and TLC required greater (20 µg) than the usual (4 µg) amount of DNA. This resulted in sufficient radioactivity in adduct spot 1 for co-migration analysis by TLC and co-elution analysis by HPLC (Figure 2). IQ–DNA adduct 1 formed in IQ-treated epithelial cells (Figure 2a, 61% of the total radioactivity) was observed to co-migrate on TLC and co-elute on HPLC with IQ–DNA adduct 1 formed by HRP (Figure 2b) when they were chromatographed together (Figure 2c). This observation confirms that a one- or two-electron oxidation of IQ forms the same reactive species, a nitrenium ion, as that produced by the two-step metabolic pathway (hydroxylation and O-esterification) occurring in HMECs, as suggested by our previous report on MPO-mediated IQ activation (Williams et al., 1998).
|
In order to distinguish between extracellular DNA and genomic DNA in neutrophils, supercoiled plasmid DNA was used in some experiments. The pattern of IQ–DNA adducts observed in DNA from neutrophils (Figure 3a
) was very similar to patterns of adducts seen in the extracellular calf thymus DNA (shown in Figure 3b) or plasmid DNA (Figure 3c). Mean adduct formation was five to seven times greater in neutrophil DNA (31 ± 20 adducts/108 nucleotides) than in the co-incubated extracellular DNA from calf thymus (6 ± 2 adducts/108 nucleotides) or from plasmid (4 ± 1 adducts/108 nucleotides). These results suggest that reactive intermediates of IQ produced inside the granulocytic cells can pass out of the cell and can bind covalently to extracellular DNA. It is also possible that peroxidases may be released into the extracellular compartment by neutrophil degranulation.
|
Fluorescence microsocopy using monoclonal antibodies confirmed the identity of typical cultures of HMECs and HMFs. The results of the comparison of IQ activation [using the ATP-deficient method of Hall et al. (1990), which results in incomplete DNA digestion] between HMECs and HMFs from four individuals are shown in Table I
. IQ–DNA adduct formation varied 4-fold in HMFs and 6-fold in HMECs. The pattern of adducts observed from IQ-treated HMECs was similar to the pattern of adducts observed in DNA from HMFs. The mean level of IQ–DNA adducts formed in HMECs (14.8 ± 15.2 adducts/108 nucleotides) was six times greater than in HMFs (2.5 ± 1.2 adducts/108 nucleotides), although this difference was not statistically significant as judged by non-parametric paired analysis (P = 0.125). Comparison between adduct levels in HMECs and HMFs from each individual (r2 = 0.71, n = 4) showed that there was some association between the two cell types in terms of their abilities to metabolically activate IQ to DNA-binding products.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Peroxidases such as MPO and LPO are present in the breast, and peroxidative activation of IQ forms an IQ–DNA adduct of identical chromatographic mobility to that formed by the two-step (N-hydroxylation and O-esterification) activation occurring in HMECs (Figures 1 and 2
). This confirms the identity of the nitrenium ion of IQ as the reactive DNA-binding intermediate and suggests that peroxidative activation of IQ can cause mutagenesis (for a review of IQ–DNA adducts see Schut and Snyderwine, 1999). The formation of IQ–DNA adducts in extracellular plasmid or calf thymus DNA after co-incubation with PMA-stimulated neutrophils indicates that neutrophils may export activated IQ intermediates from the cell and/or release peroxidase-containing granules into the extracellular compartment. In the human breast in vivo, epithelial cells may therefore be targets for HA intermediates activated at adjacent sites such as stromal cells, neutrophils or extracellular peroxidases. Our results show that non-epithelial cell-mediated metabolic activation occurs in primary cell cultures derived from reduction mammoplasty tissues. Stromal cells such as HMFs have IQ-activating ability, but the levels of IQ–DNA adducts formed were lower than in primary cultures of HMECs.
Levels of cytochrome P450 enzymes may be up to 500 times lower in the breast than in the liver (Hellmold et al., 1998), and it has been suggested that peroxidases may be responsible for a greater proportion of the total amount of carcinogen activation in the mammary gland than in the liver (Josephy, 1996). The contribution of peroxidases to the total level of carcinogen activation in other extra-hepatic tissues may also be significant, since individuals with mutations in the promoter sequence of the MPO gene, resulting in decreased transcription, are apparently less at risk of developing lung cancer than those with the wild-type protein (London et al., 1997).
Incubation in the presence of PMA-stimulated neutrophils results in IQ becoming bound covalently to extracellular DNA, which supports our hypothesis that genotoxic compounds which bind to HMEC DNA could conceivably have undergone metabolic activation in other cells in the mammary gland. Local bioactivation in the breast may be important in the initiation of breast carcinomas. We have shown previously that neutrophils, which are present in breast milk as well as being abundant in blood and inflamed tissue, can activate IQ via MPO when stimulated to respiratory burst (Williams et al., 1998). Neutrophils also mediate metabolic activation of PAHs (Trush et al., 1985), present in the diet (Phillips, 1999), in cigarette smoke and vehicle exhausts, as well as catalysing the activation of AAs (Tsuruta et al., 1985) that humans are exposed to in the environment. Two prospective epidemiological studies (Mills et al., 1992; Eriksson et al., 1995) have reported that women with atopic diseases (associated with inflammation) are at increased risk of developing breast cancer. Another peroxidase, LPO, is excreted by breast epithelial cells into breast, and is thought to play a protective role in neonates by killing harmful bacteria, but may also have a damaging effect through activation of carcinogens. The presence of hydrogen peroxide would be necessary for these reactions to occur, and suggestions for its origin include milk xanthine oxidase and breast milk neutrophils (Josephy, 1996). The contribution of peroxidative carcinogen activation in breast duct secretions could therefore be biologically significant in terms of damaging DNA in HMECs.
CYP1A1 and CYP1B1 mRNAs (Larsen et al., 1998; Williams et al., 1998), protein (Larsen et al., 1998) and active enzymes (Larsen et al., 1998) are expressed in HMECs. Pretreatment of HMECs with the aryl hydrocarbon receptor ligand benz[a]anthracene (a CYP1 inducer) increased IQ–DNA adduct formation (Williams et al., 1998). This effect, combined with results from experiments using selective CYP and peroxidase inhibitors (Williams et al., 1998) and the work of others (for example Crofts et al., 1998) provide very strong evidence that CYP1A1 and CYP1B1 are the major enzymes in HMECs catalysing the N-hydroxylation of HAs. NATs (Sadrieh et al., 1996) and sulfotransferase (Lewis et al., 1998) can catalyse the O-esterification step. This two-step activation results in formation of a nitrenium ion as the putative reactive species that binds to DNA. HMFs have a lower IQ-activating ability than HMECs, and this difference may be due in part to selective expression of the CYP1B1 enzyme catalysing N-hydroxylation of IQ in HMFs (Eltom et al., 1998), since neither immunodetectable CYP1A1 protein nor CYP1A1-catalysed enzyme activity is expressed in these cells (Eltom et al., 1998). The catalytic efficiency (Vmax/Km) of CYP1A1 in catalysing N-hydroxylation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is greater than that of CYP1A2, and is far greater than that of CYP1B1 (Crofts et al., 1998): the lower levels of IQ activation in HMFs compared with HMECs support these studies. The ability of HMFs to form adducts in extracellular DNA has not yet been investigated.
The role of hepatic enzymes in controlling systemic levels of ingested HAs or their metabolites has been described (Boobis et al., 1994), but the overall levels of HA bioactivation in the breast do not appear to have been measured. The stability of N-hydroxy-HAs is inherently low. Malfatti et al. (1999) were unable to detect the N-hydroxy derivative of the HA PhIP in human plasma 1 h after oral administration, whereas the parent compound was still systemically available at high levels (17–56% of the recovered dose) for metabolic activation at extrahepatic sites. These results support a role for mammary activation of HAs, for which evidence is provided in this paper, although insufficient evidence is available to exclude a hepatic role for HA-induced DNA damage in mammary cells.
The human mammary gland is uniquely structured in terms of morphology, where the ductal elements are suspended in adipose tissue. HMECs lining the mammary ducts are the most common site of origin of breast carcinomas. HMECs could be exposed to exogenous compounds present in breast milk and/or in circulating blood, as well as to lipophilic compounds accumulating in adipose tissue (Martin et al., 1996).
We suggest at least six factors which may contribute to the levels of locally activated mammary carcinogen (e.g. HAs, AAs and PAHs) that reach this target tissue: (i) the levels of activating enzymes in the epithelial cells, which for the carcinogenic amines are likely to be the N-hydroxylating CYP1A1 and CYP1B1 enzymes (Williams et al., 1998) and (ii) the enzymes responsible for O-esterification of the N-hydroxylated parent HAs, the NATs (Sadrieh et al., 1996) and/or sulfotransferase (Lewis et al., 1998; Stone et al., 1999); (iii) the levels of carcinogen activation enzymes in the fibroblasts surrounding the mammary epithelium (Eltom et al., 1998); (iv) breast fluid/milk neutrophil number and intracellular MPO activity (Williams et al., unpublished observations); (v) LPO levels in the breast milk/fluid itself; (vi) diffusion of carcinogen from the site of activation to the ductal cells of the epithelium. Of the above, quantitative risks have only been established for those with gene polymorphisms effecting the catalytic activity of carcinogen-metabolizing enzymes. For example, for women commencing smoking before the age of 18 who possess homozygous MspI or exon 7 polymorphisms in the CYP1A1 gene (resulting in increased enzyme activity) (Ishibe et al., 1998), the relative risk (odds ratio, OR) of breast cancer was found to be as high as 5.65 [95% confidence interval (CI) 1.50–21.3] compared with non-smokers possessing the MspI genotype. Also, a possible increase in breast cancer incidence was suggested (Zheng et al., 1999) for those with the NAT1*11 allele who consistently consumed their red meat well done (OR = 5.6, 95% CI = 0.5–62.7).
Full term pregnancy has a protective effect against breast carcinogenesis (Kelsey and Bernstein, 1996; Guzman et al., 1999). A higher overall lifetime exposure to oestrogen increases breast cancer risk, which may be due to the higher number of cyclical growth changes occurring during more menstrual cycles, when secretory material enters the mammary duct lumen. Breast tissue responds to the hormone prolactin by producing the secreted constituents of milk. Levels of prolactin are 35% higher in nulliparous women (Yu et al., 1981) and this may increase risk by raising the levels of LPO secreted into the ducts. According to King et al. (1975) the normal cellular components of nipple aspirate fluid include ductal epithelial cells, colostral cells, lymphocytes, neutrophils and necrotic cells. Studies on the nature of nipple aspirate fluid (Petrakis, 1993) have suggested that there is retention of weak carcinogens and promoters within the breast ductal system and contact with peroxidase enzymes could therefore be a mechanism for carcinogen activation in the non-lactating mammary gland.
In summary, the present study shows that partial or complete metabolic activation of putative mammary carcinogens can occur in a number of cellular or extracellular compartments of the organ and may contribute to genetic damage in the target cells for breast cancer, the luminal epithelial cells.
|
Acknowledgments |
|---|
We thank Frances Neville for phlebotomy, Danny Lloyd for preparation of the plasmid and Professor Barry Gusterson for the supply of reduction mammoplasty material. This work has been funded by The Cancer Research Campaign and the Association for International Cancer Research.
|
Notes |
|---|
1 To whom correspondence should be addressed. Tel: +1 181 643 8901; Fax: +1 181 770 7290; Email: andyw{at}icr.ac.uk
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Ambrosone,C.B., Freudenheim,J.L., Graham,S., Marshall,J.R., Vena,J.E., Brasure,J.R., Michalek,A.M., Laughlin,R., Nemoto,T., Gillenwater,K.A. and Shields,P.G. (1996) Cigarette smoking, N-acetyltransferase 2 genetic polymorphisms and breast cancer risk. J. Am. Med. Assoc., 276, 1494–1501.[Abstract]
Ambrosone,C.B., Freudenheim,J.L., Sinha,R., Graham,S., Marshall,J.R., Vena,J.E., Laughlin,R., Nemoto,T. and Shields,P.G. (1998) Breast cancer risk, meat consumption and N-acetyltransferase (NAT2) genetic polymorphisms. Int. J. Cancer, 75, 825–830.[ISI][Medline]
Anderson,W.A., Kang,Y.H. and Mohla,S. (1979) Mammalian endogenous peroxidases as cellular markers and as biosynthetic endpoints of hormone-mediated activity: viewpoint from cytochemistry. Prog. Histochem. Cytochem., 11, 1–27.[ISI][Medline]
Boobis,A.R., Lynch,A.M., Murray,S., de la Torre,R., Solans,A., Farre,M., Segura,J., Gooderham,N.J. and Davies,D.S. (1994) CYP1A2-catalyzed conversion of dietary heterocyclic amines to their proximate carcinogens is their major route of metabolism in humans. Cancer Res., 54, 89–94.
Boteju,L.W. and Hanna,P.E. (1994) Arylamine-nucleoside adduct formation: evidence for arylnitrene involvement in the reactions of an N-acetoxyarylamine. Chem. Res. Toxicol., 7, 684–689.[ISI][Medline]
Clarke,C., Titley,J., Davies,S. and O'Hare,M.J. (1994) An immunomagnetic separation method using superparamagnetic (MACS) beads for large-scale purification of human mammary luminal and myoepithelial cells. Epithelial Cell Biol., 3, 38–46.[ISI][Medline]
Crofts,F.G., Sutter,T.R. and Strickland,P.T. (1998) Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by human cytochrome P4501A1, P4501A2 and P4501B1. Carcinogenesis, 19, 1969–1973.
DeBruin,L.S., Pawliszyn,J.B. and Josephy,P.D. (1999) Detection of monocyclic aromatic amines, possible mammary carcinogens, in human milk. Chem. Res. Toxicol., 12, 78–82.[ISI][Medline]
Doll,R. and Peto,R. (1981) The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J. Natl Cancer Inst., 66, 1191–1308.
Eltom,S.E., Larsen,M.C. and Jefcoate,C.R. (1998) Expression of CYP1B1 but not CYP1A1 by primary cultured human mammary stromal fibroblasts constitutively and in response to dioxin exposure: role of the Ah receptor. Carcinogenesis, 19, 1437–1444.
Endo,H., Schut,H.A. and Snyderwine,E.G. (1994) Mutagenic specificity of 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the supF shuttle vector system. Cancer Res., 54, 3745–3751.
Eriksson,N.E., Holmen,A., Hogstedt,B., Mikoczy,Z. and Hagmar,L. (1995) A prospective study of cancer incidence in a cohort examined for allergy. Allergy, 50, 718–722.[ISI][Medline]
Goldman,A.S., Chheda,S. and Garofalo,R. (1998) Evolution of immunologic functions of the mammary gland and the postnatal development of immunity. Pediatr. Res., 43, 155–162.[ISI][Medline]
Gupta,R.C., Reddy,M.V. and Randerath,K. (1985) 32P-Postlabelling analysis of non-radioactive aromatic carcinogen DNA adducts. Cancer Res., 45, 5656–5662.[ISI][Medline]
Guzman,R.C., Yang,J., Rajkumar,L., Thordarson,G., Chen,X. and Nandi,S. (1999) Hormonal prevention of breast cancer: mimicking the protective effect of pregnancy. Proc. Natl Acad. Sci. USA, 96, 2520–2525.
Hall,M., Ni She,M., Wild,D., Fasshauer,I., Hewer,A. and Phillips,D.H. (1990) Tissue distribution of DNA adducts in CDF1 mice fed 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). Carcinogenesis, 11, 1005–1011.
Hellmold,H., Rylander,T., Magnusson,M., Reihner,E., Warner,M. and Gustafsson,J.A. (1998) Characterization of cytochrome P450 enzymes in human breast tissue from reduction mammaplasties. J. Clin. Endocrinol. Metab., 83, 886–895.
Hunter,D.J. and Willett,W.C. (1996) Nutrition and breast cancer. Cancer Causes Control, 7, 56–68.[ISI][Medline]
Ishibe,N., Hankinson,S.E., Colditz,G.A.,Spiegelman,D., Willett,W.C., Speizer,F.E., Kelsey,K.T. and Hunter,D.J. (1998) Cigarette smoking, cytochrome P450 1A1 polymorphisms and breast cancer risk in the Nurses' Health Study. Cancer Res., 58, 667–671.
Josephy,P.D. (1996) The role of peroxidase-catalyzed activation of aromatic amines in breast cancer. Mutagenesis, 11, 3–7.
Josephy,P.D. and Coomber,B.L. (1998) The 1996 Veylien Henderson Award of the Society of Toxicology of Canada. Current concepts: neutrophils and the activation of carcinogens in the breast and other organs. Can. J. Physiol. Pharmacol., 76, 693–700.[ISI][Medline]
Kelsey,J.L. and Bernstein,L. (1996) Epidemiology and prevention of breast cancer. Annu. Rev. Public Health, 17, 47–67.[ISI][Medline]
King,E.B., Barrett,D., King,M.C. and Petrakis,N.L. (1975) Cellular composition of the nipple aspirate specimen of breast fluid. I. The benign cells. Am. J. Clin. Pathol., 64, 728–738.[ISI][Medline]
Larsen,M.C., Angus,W.G., Brake,P.B., Eltom,S.E., Sukow,K.A. and Jefcoate,C.R. (1998) Characterization of CYP1B1 and CYP1A1 expression in human mammary epithelial cells: role of the aryl hydrocarbon receptor in polycyclic aromatic hydrocarbon metabolism. Cancer Res., 58, 2366–2374.
Lewis,A.J., Walle,U.K., King,R.S., Kadlubar,F.F., Falany,C.N. and Walle,T. (1998) Bioactivation of the cooked food mutagen N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by oestrogen sulfotransferase in cultured human mammary epithelial cells. Carcinogenesis, 19, 2049–2053.
London,S.J., Lehman,T.A. and Taylor,J.A. (1997) Myeloperoxidase genetic polymorphism and lung cancer risk. Cancer Res., 57, 5001–5003.
Malfatti,M.A., Kulp,S.K., Knize,M.G., Davis,C., Massengill,J.P., Williams,S., Nowell,S., MacLeod,S., Dingley,K.H., Turteltaub,K.W., Lang,N.P. and Felton,J.S. (1999) The identification of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans. Carcinogenesis, 20, 705–713.
Martin,F.L., Carmichael,P.L., Crofton-Sleigh,C., Venitt,S., Phillips,D.H. and Grover,P.L. (1996) Genotoxicity of human mammary lipid. Cancer Res., 56, 5342–5346.
Martin,F.L., Cole,K.J., Weaver,G., Williams,J.A., Millar,B.C., Grover,P.L. and Phillips,D.H. (1999) Genotoxicity of human milk extracts and detection of DNA damage in exfoliated cells recovered from breast milk. Biochem. Biophys. Res. Commun., 259, 319–326.[Medline]
Mills,P.K., Beeson,W.L., Fraser,G.E. and Phillips,R.L. (1992) Allergy and cancer: organ site-specific results from the Adventist Health Study. Am. J. Epidemiol., 136, 287–295.
Nagao,M., Ushijima,T., Wakabayashi,K., Ochiai,M., Kushida,H., Sugimura,T., Hasegowa,R., Shirai,T. and Ito,N. (1994) Dietary carcinogens and mammary carcinogenesis. Induction of rat mammary carcinomas by administration of heterocyclic amines. Cancer, 74, 1063–1069.[ISI][Medline]
Ochiai,M., Nakagama,H., Turesky,R.J., Sugimara,T. and Nagao,M. (1999) A new modification of the 32P-post-labelling method to recover IQ–DNA adducts as mononucleotides. Mutagenesis, 14, 239–242.
Petrakis,N.L. (1993) ASO Distinguished Achievement Award Lecture. Studies on the epidemiology and natural history of benign breast disease and breast cancer using nipple aspirate fluid. Cancer Epidemiol. Biomarkers Prev., 2, 3–10.[Abstract]
Phillips,D.H. (1999) Polycyclic aromatic hydrocarbons in the diet. Mutat. Res., 443, 139–147.[ISI][Medline]
Sadrieh,N., Davis,C.D. and Snyderwine,E.G. (1996) N-acetyltransferase expression and metabolic activation of the food-derived heterocyclic amines in the human mammary gland. Cancer Res., 56, 2683–2687.
Schut,H.A. and Snyderwine,E.G. (1999) DNA adducts of heterocyclic amine food mutagens: implications for mutagenesis and carcinogenesis. Carcinogenesis, 20, 353–368.
Skipper,P.L. and Tannenbaum,S.R. (1994) Molecular dosimetry of aromatic amines in human populations. Environ. Health Perspect., 102 (suppl. 6), 17–21.
Stone,E.M., Williams,J.A., Grover,P.L., Gusterson,B.A. and Phillips,D.H. (1998) Interindividual variation in the metabolic activation of heterocyclic amines and their N-hydroxy derivatives in primary cultures of human mammary epithelial cells. Carcinogenesis, 19, 873–879.
Stone,E.M., Williams,J.A. and Phillips,D.H. (1999) N-acetyltransferases (NAT) and sulphotransferases activate heterocyclic amines in the human breast, but NAT mRNA levels are not influenced by NAT genotype (abstract). Mutagenesis, 14, 652.
Trush,M.A., Seed,J.L. and Kensler,T.W. (1985) Oxidation-dependent metabolic activation of polycyclic aromatic hydrocarbons by phorbol ester-stimulated human polymorphonuclear leukocytes: possible link between inflammation and cancer. Proc. Natl Acad. Sci. USA, 82, 5194–5198.
Tsuruta,Y., Josephy,P.D., Rahimtula,A.D. and O'Brien,P.J. (1985) Peroxidase-catalyzed benzidine binding to DNA and other macromolecules. Chem. Biol. Interact., 54, 143–158.[ISI][Medline]
Turesky,R.J., Rossi,S.C., Welti,D.H., Lay,J.O.Jr and Kadlubar,F.F. (1992) Characterization of DNA adducts formed in vitro by reaction of N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline at the C-8 and N2 atoms of guanine. Chem. Res. Toxicol., 5, 479–490.[ISI][Medline]
Ueda,T., Sakamaki,K., Kuroki,T., Yano,I. and Nagata,S. (1997) Molecular cloning and characterization of the chromosomal gene for human lactoperoxidase. Eur. J. Biochem., 243, 32–41.[ISI][Medline]
Willett,W.C. (1995) Diet, nutrition and avoidable cancer. Environ. Health Perspect., 103 (suppl. 8), 165–170.
Williams,J.A., Stone,E.M., Millar,B.C., Gusterson,B.A., Grover,P.L. and Phillips,D.H. (1998) Determination of the enzymes responsible for activation of the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline in the human breast. Pharmacogenetics, 8, 519–528.[ISI][Medline]
Yu,M.C., Gerkins,V.R., Henderson,B.E., Brown,J.B. and Pike,M.C. (1981) Elevated levels of prolactin in nulliparous women. Br. J. Cancer, 43, 826–831.[ISI][Medline]
Zheng,W., Gustafson,D.R., Sinha,R., Cerhan,J.R., Moore,D., Hong,C.P., Anderson,K.E., Kushi,L.H., Sellers,T.A. and Folsom,A.R. (1998) Well-done meat intake and the risk of breast cancer. J. Natl Cancer Inst., 90, 1724–1729.
Zheng,W., Deitz,A.C., Campbell,D.R., Wen,W.Q., Cerhan,J.R., Sellers,T.A., Folsom,A.R. and Hein,D.W. (1999) N-acetyltransferase 1 genetic polymorphism, cigarette smoking, well-done meat intake and breast cancer risk. Cancer Epidemiol. Biomarkers Prev., 8, 233–239.
Received on August 16, 1999; accepted on October 14, 1999.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. M. Knaapen, N. Gungor, R. P. F. Schins, P. J. A. Borm, and F. J. Van Schooten Neutrophils and respiratory tract DNA damage and mutagenesis: a review Mutagenesis, July 1, 2006; 21(4): 225 - 236. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Meisel Cancer, genes and gender Carcinogenesis, June 1, 2002; 23(6): 1087 - 1088. [Full Text] [PDF]
|
|
|
|
|
|
F. L. Martin Genotoxins and the initiation of sporadic breast cancer Mutagenesis, March 1, 2001; 16(2): 155 - 161. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.A. Williams Single nucleotide polymorphisms, metabolic activation and environmental carcinogenesis: why molecular epidemiologists should think about enzyme expression Carcinogenesis, February 1, 2001; 22(2): 209 - 214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.A. Williams, F.L. Martin, G.H. Muir, A. Hewer, P.L. Grover, and D.H. Phillips Metabolic activation of carcinogens and expression of various cytochromes P450 in human prostate tissue Carcinogenesis, September 1, 2000; 21(9): 1683 - 1689. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Williams and D. H. Phillips Mammary Expression of Xenobiotic Metabolizing Enzymes and Their Potential Role in Breast Cancer Cancer Res., September 1, 2000; 60(17): 4667 - 4677. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||