Inter-individual Differences in the Ability of Human Milk-Fat Extracts

MCF-7 cells were treated for 24 h with or without BP (0.001−1.0 μM) in the presence .... Full-scan GC−MS analysis was performed on a HP5890 GC eq...
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Environ. Sci. Technol. 2004, 38, 3614-3622

Inter-individual Differences in the Ability of Human Milk-Fat Extracts To Enhance the Genotoxic Potential of the Procarcinogen Benzo[a]pyrene in MCF-7 Breast Cells OLGA I. KALANTZI,† REBECCA HEWITT,‡ KIRSTIE J. FORD,§ RUTH E. ALCOCK,| GARETH O. THOMAS,† JAMES A. MORRIS,§ ALAN HEWER,⊥ DAVID H. PHILLIPS,⊥ KEVIN C. JONES,† AND F R A N C I S L . M A R T I N * ,‡ Department of Environmental Science and Department of Biological Sciences, IENS, Lancaster University, Lancaster LA1 4YQ, U.K., Department of Histopathology, Royal Lancaster Infirmary, Ashton Road, Lancaster LA1 4RP, U.K., Environmental Research Solutions, Ghyll Cottage, Mill Side, Witherslack, Nr Grange-over-Sands, Cumbria LA11 6SG, U.K., and Section of Molecular Carcinogenesis, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG, U.K.

Environmental factors are believed to play an important role in cancer aetiology. Whether environmental pollutants act in isolation or in combination within mixtures remains unclear. Four human milk-fat extracts (from resident U.K. women) were screened for levels of organochlorinated and brominated compounds prior to being tested (1-50 mgequiv) for micronucleus (MN)-forming activity in MCF-7 cells. Using the cytokinesis-block micronucleus assay, micronuclei (MNi) were scored in 1000 binucleate cells per treatment. Cell viability (% plating efficiency) and immunohistochemical detection of p53 induction were also measured. The effects of treatment with 1 mg-equiv of extract in combination with benzo[a]pyrene (BP) were also examined. BP-DNA adducts were detected and quantified by 32P-postlabeling analysis. Dose-related increases in MNi independent of pollutant concentrations were induced by milk-fat extracts. All four extracts elevated the percentage of p53 positive cells, although not always in a doserelated fashion. Some combinations resulted in profound lowdose-induced increases in MNi and significant elevations in the percentage of p53 positive cells, which occurred without further reduction in cell viability or mitotic rate. When one particular extract was combined with BP, a 100-fold increase in BP-DNA adducts was detected as compared with the levels induced by BP alone; an effect not induced by other extracts. This adduct-enhancing extract * Corresponding author telephone: +44 1524 594505; fax: +44 1524 843854; e-mail: [email protected]. † Department of Environmental Science, IENS, Lancaster University. ‡ Department of Biological Sciences, IENS, Lancaster University. § Royal Lancaster Infirmary. | Environmental Research Solutions. ⊥ Institute of Cancer Research. 3614

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was fractionated into 14 fractions that were subsequently tested (1 mg-equiv of original extract) in combination with 0.01 µM BP. Fraction 1, into which nonpolar pollutants mostly eluted, enhanced MN-forming activity with BP. Surprisingly, the more polar and less likely to contain fatsoluble pollutants fractions 5 and 8 also enhanced MNforming activity. No identifiable pollutants were present in these fractions. The results suggest that different environmental pollutants present in human tissue may influence the susceptibility of target cells to initiating events.

Introduction Breast cancer is the commonest-occurring malignancy in women while also being responsible for the highest female cancer death rate worldwide (1, 2). Currently, 1 in every 8-9 women in the Western world will develop breast cancer by the age of 85, and genetic predisposition accounts for only a minority (5%) of cases (3). A large number of different risk factors for this disease point to a complex but as-yetundefined aetiology (4). Identifying causative agents has proved difficult even though epidemiological evidence points to environmental and/or dietary factors being responsible for most “sporadic” breast cancer cases (5). Over several decades, halogenated hydrocarbon chemicals have entered the ecosystem and the food chain as environmental pollutants (6). Such organochlorinated or, more recently, brominated chemicals are persistent in the environment and are fat-soluble. The primary route of organochlorine (OC) exposure in the general population is through dietary intake (7), particularly via meat and dairy products (8), while lactation can be an important means of clearance (9). Human exposure, particularly to mixtures of such agents, is of concern since many of these chemicals possess oestrogenic activity, can suppress immune function, and may induce hepatic microsomal enzymes (10-12). The majority of sporadic breast cancers contain point mutations clustered within exons 5-8 of the TP53 gene, and evidence suggests that a significant proportion of such mutations may be due to exposure to mutagenic environmental agents rather than to endogenous/background processes (13, 14). In addition, epidemiological evidence points to total cumulative exposure to oestrogen as an important determinant of breast cancer risk (e.g., early age at menarche and late onset of menopause) (15). Thus, hormonal exposures are considered important modulating factors in the epidemiology of this disease (16). Human mammary lipid extracts from a sizable proportion of individuals exhibit genotoxic activity, thus supporting the proposition that human mammary lipid may act as a reservoir for fat-soluble genotoxic agents (17, 18). A similar profile of genotoxicity was observed in extracts of individual breast milk samples (19, 20). While the genotoxic component(s) present in these extracts remain to be identified, it is not inconceivable that there is a mixture of agents present at low concentrations that interact additively or synergistically. How individual genotoxins act within the complex mixtures to which humans are continuously exposed remains to be ascertained. In this study, four human milk samples were extracted using a methodology developed to isolate persistent OCs. Subsequently, the effects of these extracts either alone or in combination with the procarcinogen benzo[a]pyrene (BP) were determined in the oestrogen receptor-positive breast carcinoma MCF-7 cell line. Levels of genotoxicity were 10.1021/es035422y CCC: $27.50

 2004 American Chemical Society Published on Web 05/28/2004

assessed using the cytokinesis-block micronucleus (CBMN) assay, and DNA adducts were detected and quantified by 32 P-postlabeling analysis. Cell viability (% plating efficiency) and mitotic rate (% binucleate cells in the CBMN assay) were measured, and immunocytochemical analyses for p53 were carried out. Our experimental objective was to determine whether environmentally relevant mixture effects associated with enhanced genotoxicity would be observed.

Experimental Section All chemicals, including test chemicals, were obtained from Sigma Chemical Co. (Poole, Dorset, U.K.) unless otherwise stated. Cell culture consumables were obtained from Life Technologies (Paisley, U.K.) unless otherwise stated. Antibodies were obtained from DakoCytomation (Ely, Cambridgeshire, U.K.). For polybrominated diphenyl ether (PBDE) and polychlorinated biphenyl (PCB)/OC analysis, solvents were of HPLC or glass-distilled grade, and standards were purchased from Promochem (Welwyn Garden City, Hertfordshire, U.K.) or QMx (Thaxted, Essex, U.K.). Human Milk Samples. Individual human milk samples were donated anonymously, after appropriate ethical approval. Four samples were collected from the maternity units of hospitals in the Lancaster (northwest of England) and London (Hammersmith, West London) areas between late 2001 and early 2003. Lancaster samples were designated NW1 and NW2 while London samples were designated SE1 and SE2. Donated milk samples were immediately frozen and stored at -20 °C prior to extraction and analysis. Cell Culture. The human mammary carcinoma (MCF-7) cell line was grown in Dulbecco’s modified essential medium supplemented with 10% heat-inactivated foetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were cultured routinely in 75 cm2 flasks at 5% CO2 in air and 37 °C in a humidified atmosphere and subcultured (1:10, v/v) twice weekly. Prior to subculture or incorporation into experiments, cells were disaggregated using a 0.05% trypsin/0.02% EDTA solution to form single-cell suspensions. Dimethyl sulfoxide (DMSO) was used as the solvent vehicle for additions of test agents or of suspensions of milk-fat extracts to culture medium. DMSO concentrations in culture medium did not exceed 1% v/v. MCF-7 cells were treated for 24 h with or without BP (0.001-1.0 µM) in the presence or absence of milk-fat extracts, as indicated. An appropriate vehicle control was always incorporated into each experiment. CBMN Assay. Routinely cultured MCF-7 cells were disaggregated and resuspended in complete medium prior to seeding 3-mL aliquots (≈ 1 × 104 cells) into 30-mm Petri dishes containing 20-mm coverslips (21). After being left for 24 h to attach, cells were treated with either vehicle control, BP in DMSO or milk-fat extract in DMSO for 24 h, as indicated. Medium was then replaced with fresh medium, without test agent but containing 2 µg/mL cytochalasin-B. Following a further 24-h incubation, medium was removed, and the cells were washed with phosphate-buffered saline (PBS) and fixed with 70% ethanol (EtOH) prior to being stained with 5% Giemsa. For each treatment, micronuclei (MNi) in 1000 binucleate cells from a minimum of three pooled experiments were scored. Mitotic rate was assessed as percent of binucleate cells (mean ( SD, n ) 3). Clonogenic Assay. Following disaggregation, MCF-7 cells were resuspended in complete medium (1 × 103 cells/5 mL aliquots) and seeded into 25 cm2 flasks in the presence or absence of a 24-h treatment with either vehicle control, BP in DMSO or milk-fat extract in DMSO, as indicated. The medium was then replaced with fresh medium. Cells were cultured undisturbed for a further 7 d prior to removal of medium, washing with PBS, and fixation with 70% EtOH.

Colonies were then stained with 5% Giemsa and counted, and percent of plating efficiency was calculated. Immunohistochemical Staining. Routinely cultured cells were disaggregated with trypsin/EDTA and resuspended in complete medium prior to seeding 5-mL aliquots (≈1 × 105 cells) into 60-mm Petri dishes containing 24-mm glass coverslips. After being allowed 24 h to attach, cells were treated for 24 h with either vehicle control, BP in DMSO or milk-fat extract in DMSO, as indicated. Posttreatment, medium was aspirated, and the cells were washed with PBS and fixed with CytoFixx fixative (CellPath plc, Newtown, Powys, U.K.). The p53 mouse monoclonal (DO-7, Isotype: IgG2b) antiserum was diluted 1:20 in 0.2% bovine serum albumin in Tris-buffered saline (pH 7.6) (BSAT). Fixative was removed by soaking the coverslips in 95% industrial methylated spirits (IMS) for 30 min. Following a 5-min wash with tap water, coverslips were incubated in 1:5 normal goat sera in 0.05 M Tris-buffered saline (pH 7.6) (TBS) for 15 min in a humidified environment. Excess sera were removed, and the coverslips were incubated with primary antibody (see above) for 1 h at room temperature. Coverslips were washed with TBS for 5 min and then, using the StreptABComplex duet kit (DakoCytomation, U.K.), incubated for 30 min with secondary anti-sera (goat anti-mouse/rabbit) in BSAT, washed with TBS for 5 min, incubated with tertiary anti-sera (avidin-biotin complex) in BSAT for 30 min, and washed again with TBS for 5 min following the manufacturer’s instructions. 3,3′-Diaminobenzidine (DAB) chromogen in 0.05 M Tris-HCl buffer (pH 7.6) with 0.1% H2O2 was applied to preparations for 15 min after which they were washed for 5 min with tap water. Finally, slides were transferred to a rack and stained with haemotoxylin (50%) and eosin (0.1%) as previously described (22). Expression levels of proteins were determined as the mean ( SD of positive cells following five separate counts. Extraction of Milk for PBDE and PCB/OC Analyses. Silica gel (Merck, 0.063-0.200 mm) and/or Na2SO4 was heated at 450 °C overnight and stored in sealed containers. After thawing, milk samples were centrifuged at 3000 rpm for 15 min. A mixture of milk fat (0.5 g), Na2SO4 (5 g), and hexane (50 mL) was boiled for 10 min and allowed to cool prior to lipid determination. Evaporated to 5 mL, these mixtures were applied to 25 mm i.d. columns containing 15 g of acidified silica gel (2:1 silica gel:acid by weight) and eluted with hexane. Eluted samples were evaporated to 1 mL and applied to gel permeation chromatography (GPC) columns packed with Biobeads S-X3 and eluted with hexane: dichloromethane (DCM) (1:1 by volume). 13C-labeled C12PCB recovery (added at the beginning of the procedure) and internal standards (added at the end of the procedure) were incorporated when subsequent GC-MS analysis was carried out on whole milk-fat extracts (23) but were excluded when extracts were incorporated into biological experiments. To check the possibility of artifactual contamination giving rise to positive results in biological assays a “run-blank” extract (extraction run without sample incorporation) and “negativeblank” extract (hexane wash of a previously unused sample collection vessel) were also prepared. Fractionation of Extracts. Following GPC cleanup, one extract (SE1) was applied to a silica gel fractionation column, packed with 3 g of silica gel (previously activated at 450 °C). This was eluted with 20 mL of hexane (fraction 1), followed by 20 mL of hexane:DCM (0.92:0.08) (fraction 2). Eleven further fractions (fractions 3-13) were collected, at 8% DCM increments; the final fraction (fraction 14) collected was 100% DCM. The samples were evaporated to 25 µL for subsequent GC-MS analysis. GC-MS Analysis. PBDE analysis was performed on a Finnigan Trace GC-MS. Separation was achieved on a 30-m DB-5MS capillary column (J&W Scientific, Stockport, U.K.) VOL. 38, NO. 13, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Relative Micronucleus-Forming Effects of Milk-Fat Extracts in MCF-7 Cellsa mg-equiv of milk-fat extract control 1 5 25 50

micronuclei

SE1

treatment SE2 NW1

MN TMN MN TMN MN TMN MN TMN MN TMN

17 18 27 27 28 33 44 54 47 58

17 18 24 27 22 30 37 48 47 63

17 18 39 52 37 50 42 52 39 52

NW2 17 18 19 22 39 50 45 57 34 38

a

MCF-7 cells were treated for 24 h with or without graded concentrations of human milk-fat extracts prior to cytokinesis block with 2 µg/mL cytochalasin-B. After a further 24-h incubation, cells were stained with 5% Giemsa as described in the Experimental Section. Control refers to treatment performed in the presence of the vehicle solvent, DMSO. MN, micronucleated cells/1000 binucleate MCF-7 cells; TMN, total number of micronuclei/1000 binucleate MCF-7 cells.

fitted with a retention gap, using helium as the carrier gas. The injection port was kept at 270 °C, in splitless mode with pressure surge. The mass spectrometer was used in selected ion recording mode; negative chemical ionization was performed using ammonia as the reagent gas and a source temperature of 200 °C. The following PBDE congeners, chosen because of their reported occurrence in environmental samples, were screened for: PBDE 17, 28, 32, 35, 37, 47, 49, 71, 75, 85, 99, 100, 119, 153, and 154 (23). PCB and OC pesticide analysis was performed on a Fisons 8000 GC equipped with a 50-m CPSil8 capillary column (Chrompak, Walton-on-Thames, U.K.) fitted with a retention gap, using helium as the carrier gas. The injection port was kept at 250 °C in splitless mode. The Fisons MD800 MS (EI+) was used in selected ion recording mode. Full-scan GC-MS analysis was performed on a HP5890 GC equipped with a 30-m HP5 MS capillary column (Agilent Technologies, Stockport, U.K.), using helium as the carrier gas. The injection port was kept at 280 °C in splitless mode. The HP5972 MSD (EI+) was used to scan for masses 50-600 amu throughout each run. The following PCB congeners and OC pesticides were screened: PCB 18, 22, 28, 31, 41/64, 44, 49, 52, 54, 60/56, 70, 74, 87, 90/101, 95, 99, 104, 105, 110, 114, 118, 123, 132, 138, 141, 149, 151, 153, 155, 156, 157, 158, 167, 170, 174, 180, 183, 187, 188, 189, 194, 199, and 203; R-, β-, γ-, and δ-HCH; HCB; R- and γ-chlordane; and o,p′-DDD, p,p′DDD, o,p′-DDE, p,p′-DDE, o,p′-DDT, and p,p′-DDT (23). 32P-Postlabeling Analysis. DNA isolated from MCF-7 cells was subjected to 32P-postlabeling analysis (4 µg per sample) using the nuclease P1 digestion method of sensitivity enhancement (24). Solvents for chromatography of the labeled digests on polyethyleneimine-cellulose TLC plates were D1, 1.0 M phosphate, pH 6.0; D2, 2.5 M ammonium formate, pH 3.5; D3, 3.5 M lithium formate, 8.5 M urea, pH 3.5; D4, 0.8 M lithium chloride, 0.5 M Tris-HCl, 8.5 M urea, pH 8.0. Chromatograms were scanned for radioactivity using an InstantImager (Canberra Packard, Pangbourne, U.K.). Relative levels of DNA modification were calculated from the levels of radioactivity in the DNA adduct spots detected on the postlabeling chromatograms and from the specific activity of the [γ-32P]ATP used in the labeling procedure.

Results Micronucleus (MN)-forming activity of milk-fat extracts in MCF-7 cells and pollutant levels in the milk fat are shown in Tables 1 and 2, respectively. Extracts (SE1, SE2, NW1, and NW2), tested at up to 50.0 mg-equiv of milk fat, induced 3616

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TABLE 2. Screened Levels of Selected Pollutants in Human Milk-Fat Extractsa human milk-fat extract

total PCBs (ng/g)

ΣDDT (ng/g)

total PBDE (ng/g)

HCB (ng/g)

total HCHs (ng/g)

SE1 SE2 NW1 NW2

47 410 29 210

2300 710 55 150

7.9 11 1.9 5.7

8.0 34 8.0 19

15,000 300 14 220

a Individual human milk samples were donated anonymously. PBDE, PCB, and OC pesticide analysis was performed as described in the Experimental Section. Values are reported to two significant figures.

dose-related increases in MN formation. All extracts appeared to possess roughly comparable levels of MN-forming activities although NW1 was the most active at the lowest concentrations (Table 1). Residue analysis of these human milk-fat extracts showed that the most marked fluctuations occurred in levels (ng/g) of total PCBs, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p′-DDT) and its metabolites (ΣDDT), and total 1,2,3,4,5,6-hexachlorocyclohexane (HCHs) (Table 2). NW1 extracts contained the lowest pollutant levels. The MN-forming effects of BP in combination with 1 mgequiv of milk-fat extract in MCF-7 cells are shown in Figure 1. Marked enhancements in the induction of MNi were observed following certain treatments. BP treatment alone at concentrations of 0.001, 0.01, or 0.1 µM resulted in 2-, 4-, and 5-fold increases in total MNi in 1000 binucleate MCF-7 cells (Figure 1A). In combination with 1 mg-equiv of SE1 or NW2, a more than additive effect on the levels of MN induction was observed (Figure 1B,E). The effect was most evident at a BP concentration of 0.01 µM. Figure 2A demonstrates that the MN-forming effects hitherto described could not be attributable to either the extraction procedure (run blank) or the sample collection vessels (negative blank). While all four milk-fat extracts induced increases in MN-forming activity in the 1-50 mgequiv concentration range, similar treatment did not result in marked decreases in colony-forming activity (Figure 2B). Figure 3A shows that 24-h treatment of MCF-7 cells with 0.01 µM BP resulted in a decrease in percent of plating efficiency from control levels of 53.7 ( 7.6 to 38.8 ( 2.4. Decreases in plating efficiency, in comparison with untreated control levels, were also observed following 24-h treatment with 1 mg-equiv of SE1, SE2, and NW2 but not with NW1. However, 24-h treatment with 1 mg-equiv of any of the four milk-fat extracts plus 0.01 µM BP did not further enhance BP-induced reductions in percent of plating efficiency (Figure 3A). Treatment with milk-fat extracts SE1, SE2, or NW1 alone induced significant (p < 0.05) reductions in percent of binucleate cells (Figure 3B). However, only 0.01, 0.1, or 1.0 µM BP in the presence of NW2 (15.1 ( 0.4, 13.9 ( 0.3, and 10.6 ( 0.8% binucleate MCF-7 cells, respectively) induced a significant (p < 0.05) reduction in percent of binucleate MCF-7 cells in comparison with the corresponding treatment control (16.71 ( 0.5, 14.9 ( 0.4, and 14.5 ( 0.4% binucleate MCF-7 cells, respectively) (Figure 3B). BP-DNA adduct levels detected following treatment with or without 1 mg-equiv of SE1 are shown in Table 3. Treatment for 24 h with 0.001-1.0 µM BP alone resulted in dose-related increases of 2.1-128.2 BP-DNA adducts per 108 nucleotides, respectively. In combination with 1 mg-equiv of SE1, the same concentrations of BP resulted in adduct levels between 48.1 and 1237.6 per 108 nucleotides (Table 3). This enhancement in BP-DNA adduct formation was only observed in the presence of SE1 and not with other extracts (SE2 or NW2); in the presence of these extracts, slight reductions in the levels of BP-DNA adduct formation were observed (data not shown).

FIGURE 1. Micronucleus (MN)-forming activity of benzo[a]pyrene (BP) in the presence or absence of 1 mg-equiv of human milk-fat extract following 24-h treatment in MCF-7 cells. Control refers to treatment performed in the presence of the vehicle solvent, DMSO. MCF-7 cells were treated with (A) BP alone; (B) BP in combination with 1 mg-equiv of SE1 extract; (C) BP in combination with 1 mg-equiv of SE2 extract; (D) BP in combination with 1 mg-equiv of NW1 extract; (E) BP in combination with 1 mg-equiv of NW2 extract. MN formation was scored in 1000 binucleate cells. MCF-7 cells express wild-type p53 (25), and the percentage of cells staining positive for p53 following 24-h treatment with up to 50.0 mg-equiv of human milk-fat extract are shown in Figure 4. SE1 and NW2 appear to induce dose-related increases in the percentage of MCF-7 cells staining positive for p53. However, SE2 and NW1 induced elevations in the percentage of p53 positive cells in the absence of a clear dose-related response (Figure 4). Treatment (24 h) with BP resulted in dose-related increases in the percentage of MCF-7 cells staining positive for p53 (Figure 5). These BP-induced dose-related increases in the percentage of p53 positive cells were not markedly elevated in the presence of SE2, NW1, or NW2. However, 1 mg-equiv of SE1 plus 0.01, 0.1, or 1.0 µM BP gave rise to increases in the percentage p53 positive cells of 62.2 ( 4.6 (p < 0.0005), 87.6 ( 4.6 (p < 0.0005), and 94.4 ( 2.1 (p < 0.05), respectively, as compared to a background levels of 27.4 ( 2.5, 62.2 ( 2.3, and 88.2 ( 3.7 (Figure 5). In an attempt to isolate the milk-fat extract components responsible for SE1-induced genotoxicity-enhancing effects,

the extract was fractionated into 14 fractions. Figure 6 compares the MN-forming effects of 1 mg-equiv of whole extract in the presence or absence of 0.01 µM BP (Figure 6A) with the MN-forming effects of individual fractions (1-14, at a concentration of 1 mg-equiv of original extract) in the presence of 0.01 µM BP (Figure 6B). Fractions 1, 5, and 8 appeared to enhance MN-forming activity. Fractions 1 and 8, in the presence of 0.01 µM BP (Figure 6B), resulted in the induction of MN levels comparable to combination treatment with whole extract (Figure 6A). Fraction 5 appeared to be less active. GC-MS analysis of the fractions, and interpretation of these results in line with the known elution order for a silica gel column showed that most OCs [including PCBs, polychlorinated naphthalenes (PCNs), HCB, chlordane, 1,1dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p′-DDE), 1,1dichloro-2,2-bis(p-chlorophenyl)ethane (p,p′-DDD), most p,p′-DDT, PBDEs, and polychlorinated dibenzo-p-dioxins/ dibenzofurans (PCDDs/Fs)] would be found in fraction 1 (Table 4). In fraction 2, some p,p′-DDT was found along with R-, β-, and γ-HCHs. Approximately 25% of the HCHs were VOL. 38, NO. 13, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Exclusion of artifactual contamination and the effects of human milk-fat extract on percent plating efficiency in MCF-7 cells. A run blank extract (extraction run without sample incorporation) and negative blank (hexane wash of a previously unused sample collection vessel) were tested. Cells were fixed prior to being stained with 5% Giemsa. Control refers to treatment performed in the presence of the vehicle solvent, DMSO. (A) Micronucleus formation was scored in 1000 binucleate cells. (B) Plating efficiency was calculated by estimating the percentage of colonies counted over the number of cells initially seeded. found in fraction 3, and some of the more polar dioxins/ furans (PCDD/Fs, PBDD/Fs, and mixed Cl/Br dioxins/furans) could also be present in this fraction and fraction 2. However, no OC chemicals were detectable from fraction 4 onward (Table 4). No other organohalogen chemicals were found at concentrations detectable by GC-MS in full-scan mode.

Discussion Environmental and/or dietary factors appear to play an important role in the aetiology of breast cancer (4). The breast consists primarily of adipose tissue (70-90%, by weight); suspended in this unique morphological structure are the functional elements that are lined with epithelial cells from which most breast cancers may arise. Humans are exposed, via their environment and diet, to fat-soluble carcinogens, among them agents that induce mammary tumors in rodents (4). These may be chemicals requiring metabolic activation to DNA-reactive species (4). Other pollutants may be labeled under the enigmatic umbrella of endocrine disrupters (i.e., exogenous chemical agents that interfere with the synthesis, secretion, transport, binding, action, or elimination of natural hormones) (26). Because the potency of endocrine-disrupting pollutants is so much lower than endogenous hormones, it has been suggested that potentially harmful hormonal effects of the former are negligible (27). It is simple logic to suggest that human mammary lipid may sequester fat-soluble pollutants, thus exposing adjacent epithelial cell populations (17, 18). Persistent pollutants such as OCs can accumulate in human fat to levels in excess of 3618

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FIGURE 3. Effects of benzo[a]pyrene (BP) in the presence or absence of 1 mg-equiv of human milk-fat extract on percent plating efficiency and mitotic rate (percent binucleate cells) following 24-h treatment in MCF-7 cells. Cells were fixed prior to being stained with 5% Giemsa. Control refers to treatment performed in the presence of the vehicle solvent, DMSO. (A) Plating efficiency (%) was calculated by estimating the percentage of colonies counted over the number of cells initially seeded. (B) Mitotic rate was estimated by calculating the percent of binucleate MCF-7 cells following treatment (mean ( SD). *, p < 0.05 (extract-treated cells vs background control) as determined by an unpaired t-test with Welch’s correction.

TABLE 3. BP-DNA Adduct Formation in MCF-7 Cellsa BP-DNA adducts per 108 nucleotides BP (µM)

no addition

+1 mg-equiv of SE1

0.001 0.01 0.1 1.0

2.1 2.2 12.3 128.2

48.1 282.6 1090.2 1237.6

a MCF-7 cells were treated for 24 h in the presence of graded concentrations of BP, with or without 1 mg-equiv of SE1. Isolated DNA was subjected to 32P-postlabeling analysis (4 µg per sample) using the nuclease P1 digestion method of sensitivity enhancement (24). Relative levels of DNA modification were calculated from the levels of radioactivity in the DNA adduct spots detected on the postlabeling chromatograms and from the specific activity of the [γ-32P]ATP used in the labeling procedure.

1 ppm (µg/g) (28) whereas adipose concentrations of morereadily metabolized procarcinogens (e.g., BP) probably never exceed 1 ppb (ng/g) levels (29). A single causative factor in the aetiology of gender-associated cancers is unlikely (4), and humans are exposed, continuously and variously, to mixtures of different agents. It is also highly probable that exposures to as-yet-unidentified pollutants occur and their effects remain to be ascertained; one would reasonably expect milk-fat extracts to contain pollutants other than those specifically screened. Within such mixtures, the levels of

TABLE 4. Fractionation of Human Milk-Fat Extract on a Silica Fractionation Columna fraction

chemicals elutedb

solvent

F1

hexane

F2

8% DCM in hexane

F3

16% DCM in hexane

F4 24% DCM in hexane F5-F14 32% DCM in hexane-100% DCM (8% DCM increment per fraction)

PCBs*, PCNs, HCB, chlordane, p,p′-DDE*, p,p′-DDD, p,p′-DDT, PBDEs and PCDD/Fs, polybrominated dioxins/furans, and mixed chlorinated/brominated dioxins/furans R-, β*-, and γ-HCH, p,p′-DDT*, p,p′-DDD, PBDEs and PCDD/Fs, polybrominated dioxins/furans, and mixed chlorinated/brominated dioxins/furans approximately 25% of R-, β*-, and γ-HCH in F2, more polar dioxins/furans (PCDD/Fs, PBDD/Fs, and mixed Cl/Br dioxins/furans) no OCs detected no OCs detected

a Fractionation of SE1 on a silica fractionation column and subsequent PBDE, PCB, and OC pesticide analysis as described in the Experimental Section was carried out. b Chemicals we would expect based on an interpolation from knowledge of their elution order on an activated silica column. An asterisk (*) indicates chemicals detected.

FIGURE 4. Immunohistochemical analysis of p53 protein expression in MCF-7 cells following treatment with human milk-fat extracts. The antibody employed was p53 mouse monoclonal (DO-7, Isotype: IgG2b) antiserum. Control refers to treatment performed in the presence of the vehicle solvent, DMSO. Expression levels of p53 protein were determined as the mean ( SD of positive cells following five separate counts. *, p < 0.05; **, p < 0.005; ***, p < 0.0005 (treatment vs control) as determined by an unpaired t-test with Welch’s correction.

FIGURE 5. Immunohistochemical analysis of p53 protein expression in MCF-7 cells following treatment with benzo[a]pyrene (BP) in the presence or absence of 1 mg-equiv of human-milk-fat extract. The antibody employed was p53 mouse monoclonal (DO-7, Isotype: IgG2b) antiserum. Control refers to treatment performed in the presence of the vehicle solvent, DMSO. Expression levels of p53 protein were determined as the mean ( SD of positive cells following five separate counts. *, p < 0.05; **, p < 0.005; ***, p < 0.0005 (extract-treated cells vs background control) as determined by an unpaired t-test with Welch’s correction. individual components will undoubtedly fluctuate with age, lifestyle changes, and a plethora of other factors. A multicomponent mixture of exogenous endocrine-disrupting chemicals was shown to produce observable effects, pre-

sumably additive, in a recombinant yeast oestrogen screen even when each individual component was present below its individual threshold effect level (30). Mixture effects on target epithelial cells remain to be ascertained. We examined the effects of environmentally relevant mixtures obtained following the extraction of human milk fat using a methodology designed to isolate persistent pollutants. Extracts were found to induce marked increases in MNi following 24-h treatment of MCF-7 cells with 1-50 mg-equiv of milk fat. The MN-forming activity of milk-fat extracts was similar to the activity of human mammary lipid extracts obtained following an extraction procedure originally developed for the extraction of heterocyclic aromatic amines (17, 31) and were markedly more active than whole-milk extracts (19, 32). The MN-forming activity of milk-fat extracts did not correlate with the levels of any of the identified pollutants present (Tables 1 and 2). When MCF-7 cells were treated with 1 mg-equiv of milkfat extract in combination with BP, more than additive increases in MN formation were observed (Figure 1). This was most marked following treatment with SE1 plus 0.01 µM BP. Two possible mechanisms may underlie such observations: first, OCs may induce metabolising enzymes (12, 33), thus resulting in more efficient BP activation to toxic metabolites; second, OCs may inhibit detoxification mechanisms (34, 35). Uncertainties in predicting the outcome of a maze of modulating factors (11, 36, 37) could explain the difficulties in implicating environmental exposures in the aetiology of breast cancer (10). Exposures to persistent pollutants have been linked to the aetiology of other cancers. Several epidemiological studies have suggested elevated cancer risks among farmers (38), and positive associations have been identified between lindane exposure and incidence of prostate cancer (39) and non-Hodgkin’s lymphoma (40). Low-dose concentrations of lindane can induce MNi in MCF-7 and PC-3 cells (22). However, the role of OCs in cancer causation still remains controversial (41), and whether hormonal factors may influence susceptibility of target cells to genotoxins such as BP remains to be ascertained (42). Because low-dose effects were being examined, the possibility of artifactual contamination was excluded (Figure 2A). Milk-fat extracts induced or enhanced MN formation (Table 1, Figure 1) without further reducing cell survival (Figures 2B and 3A) and mitotic rate (Figure 3B). This supports observations that oestrogenic agents facilitate cell survival even in the presence of enhanced genotoxicity (42). Genotoxic effects may result in alterations in cell cycle kinetics and apoptosis activation mechanisms. The TP53 gene product, p53, is an important mediator of such processes (43). The ability of the SE1 and NW2 extracts to induce dose-related increases in the percentage of p53 positive MCF-7 cells was suggestive of the presence of genotoxic components in these VOL. 38, NO. 13, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Micronucleus (MN)-forming activity, following 24-h treatment in MCF-7 cells, of 0.01 µM benzo[a]pyrene (BP) in the presence of (A) 1 mg-equiv of human milk-fat extract or (B) 14 extract fractions (1-14, at a concentration of 1 mg-equiv of original extract) isolated on a GPC column. Control refers to treatment performed in the presence of the vehicle solvent, DMSO. MN formation was scored in 1000 binucleate cells. extracts (Figure 4). In line with the ability of 1 mg-equiv of SE1 plus 0.01 µM BP to result in enhanced MN formation, a similar treatment also resulted in an elevated percentage of p53 positive MCF-7 cells in comparison with levels observed following treatment with BP alone (Figure 5). Although this points to the presence of other genotoxic components, Table 3 suggests the presence of modulating components in this particular extract that enhance BP-DNA adduct formation. In an attempt to isolate the genotoxic principles of SE1, whole extract was fractionated on a GPC column into 14 fractions prior to testing individual fractions in the CBMN assay in the presence of 0.01 µM BP (Figure 6A,B). A GC-MS screen for identifiable pollutants in each fraction was also run (Table 4). Fraction 1, into which most nonpolar (fatsoluble) agents would elute, resulted in an elevated level of 3620

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MN formation in the presence of 0.01 µM BP (Figure 6B) comparable to levels induced in the presence of whole extract (Figure 6A). Surprisingly, it appears that significantly more polar components of this extract that eluted in fractions 5 and 8 resulted in comparable levels of MN formation following treatment in the presence of 0.01 µM BP (Figure 6B). Due to the cleanup method employed, any activity shown by fraction 4 onward must come from pollutants that fulfill the following criteria: they must be resistant to concentrated sulfuric acidsthis excludes most unsaturated aliphatic chemicals and most unhalogenated aromatic chemicals; they are likely to have less than 15 carbon atoms (due to a size exclusion effect); and they must be relatively polar as compared to pollutants such as DDE and HCH. This suggests the presence of unidentified components that possess the ability to modulate genotoxicity in MCF-7 cells.

MCF-7 cells are considered to represent an “early” hormone-responsive phenotype with low metastatic potential (44), but it is a cell line known to have subpopulations (45). They are also known to differ widely in their proliferative response to 17β-oestradiol and show extensive variation in copy number changes affecting specific chromosomal regions (46). This may explain the variable responses to treatment with 1 mg-equiv of milk-fat extracts; a much higher susceptibility is observed in Figure 1 as opposed to Table 1. Albeit, MCF-7 cells remain a robust cell model for genotoxic analyses (21, 22, 25, 42). Despite these observations, it must be emphasized that breast feeding is protective to the neonate (47, 48) and also appears to confer a protective effect against breast cancer in the mother (4). The role that complex mixtures of pollutants may play in breast cancer aetiology remains obscure. The present work highlights the difficulties in characterizing the toxicological relevance of individual pollutants. Hydrophobic DNA adducts have been detected in normal breast tissues (49); the question remains as to whether carcinogenic pollutants reach target epithelial cells at sufficient concentrations to induce initiating events or whether other modulating effects influence the susceptibility of such cells to toxic insults. Future work is required to build up a database of effects by testing milk-fat extracts from a much larger cohort of individuals and to characterize the entities responsible for genotoxic effects or enhanced susceptibility. Isolating and identifying such modulating factors may improve the toxicological assessment of environmental exposures.

Acknowledgments The authors gratefully acknowledge grants from the Lancaster University Research Committee under the Small Grants Scheme (O.I.K.), the North West Cancer Research Fund, UK (R.H.), Cancer Research UK (D.H.P. and A.H.), and the Morecambe Bay Area NHS Trust (K.J.F.). We also thank the neonatal staff at the Royal Lancaster Infirmary and Gillian Weaver at the Queen Charlotte’s and Chelsea Hospital for the provision of breast milk samples.

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Received for review December 18, 2003. Revised manuscript received April 9, 2004. Accepted April 21, 2004. ES035422Y