Mutagenesis Advance Access originally published online on September 16, 2006
Mutagenesis 2006 21(5):351-360; doi:10.1093/mutage/gel038
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Low-dose treatment with polybrominated diphenyl ethers (PBDEs) induce altered characteristics in MCF-7 cells
1 Department of Environmental Science, Lancaster University Lancaster LA1 4YQ, UK 2 Department of Biological Sciences, Lancaster University Lancaster LA1 4YQ, UK
Polybrominated diphenyl ethers (PBDEs) are hydrophobic and persistent additive flame retardants that seemingly transfer into environmental compartments where they bioaccumulate i.e. in human biota. We examined the micronucleus-forming activities of low-dose PBDEs (congeners 47, 99, 153, 183 or 209) in MCF-7 cells along with their ability to modulate growth, cell biochemistry [by infrared (IR) microspectroscopy], clonogenic survival or quantitative expression of cytochrome P450 isoenzymes (CYP1A1, CYP1A2 and CYP1B1), cyclin-dependent kinase inhibitor 1A [CDKN1A (P21WAF1/CIP1)], B-cell leukaemia/lymphoma-2 (BCL-2) and Bcl-2-associated X (BAX). Elevations in micronucleus formation were observed following treatment with 10–12 to 10–9 M PBDE concentrations despite the fact that less than one-fourth of the concentration of each test agent administered partitioned out of the media and into the incubating cells. However, low-dose treatment levels remained within the range of reported concentrations measured in UK serum samples collected in 2003. Clonogenic survival and gene expression was unaltered following 10–12 to 10–9 M PBDE treatment but significant (P < 0.05) elevations in growth kinetics were observed. Significant alterations in IR cell spectra were associated with treatments, and plotted clusters following principal component analysis highlighted these changes. Whether such in vitro effects point to an underlying ability of PBDEs to initiate and drive target-cell alterations in vivo now needs to be addressed.
Polybrominated diphenyl ethers (PBDEs) are commercially available as pentabromodiphenyl ether (pentaBDE), octabromodiphenyl ether (octaBDE) or decabromodiphenyl ether (decaBDE) technical mixtures (1
). These brominated flame retardants (BFRs) are dissolved in a wide range of household and workplace products e.g. polyurethane foam, computers. More than 200 000 metric tons of BFRs are manufactured per year (2) and in the UK alone, some 3000 metric tons of pentaBDE have been produced since 1970 (3). There are 209 possible PBDE congeners with pentaBDE containing PBDE47, PBDE99 and PBDE153 among others, octaBDE consisting of several hexa- to nona-brominated congeners (dominated by PBDE183), and decaBDE being almost entirely composed of PBDE209 (1). Over time, PBDEs may leach out into the environment and there are now concerns regarding their ubiquity and persistence along with year-on-year concentration increases in humans because they possess a chemical structure not dissimilar to that of polychlorinated biphenyls (PCBs) (4–6). The question of whether PBDEs possess a toxicological profile (neurodegenerative, endocrine disrupting and possibly carcinogenic) similar to PCBs remains unanswered (4,7). PBDEs have been shown to affect thyroid hormone status in rats, mice and seals (8–11).
Low-dose effects are associated with the apparent induction of biological alterations at concentrations that fall within the range of typical human exposures. It has been suggested that hormones and persistent organochlorines (OCs) are agents that induce low-dose effects in the form of elevated levels of micronuclei, altered growth kinetics or modulated gene-expression profiles (12,13). For the purposes of regulatory toxicology, agents are often tested at concentrations that far exceed those that organisms, including humans, would be exposed to (14). However, genome-damaging agents possessing hormone-like characteristics may act as ‘clever carcinogens’ capable, at much lower concentrations, of facilitating clonal expansion of an initiated phenotype (15). To clarify actual risk posed by environmental exposures, a re-evaluation of agents employing an integrated platform of biological endpoints is required in order to determine whether threshold concentrations are achievable below which no-risk predictions are feasible (16).
In this study, we investigated the effects of a panel of PBDEs (congeners 47, 99, 153, 183 and 209) in the oestrogen-receptor-positive breast carcinoma MCF-7 cell line (7). Levels of chromosomal damage were assessed as micronucleus-forming activity in the cytokinesis-block micronucleus (CBMN) assay. Cell growth (assessed as cell number following 72-h incubation), cell biochemistry (monitored through attenuated total reflection-Fourier transform infrared (ATR) microspectroscopy) and colony-forming ability along with quantitative gene-expression analyses of cytochrome P450 isoforms (CYP1A1, CYP1A2, and CYP1B1), cyclin-dependent kinase inhibitor 1A [CDKN1A (P21WAF1/CIP1)], B-cell leukaemia/lymphoma-2 (BCL-2) and Bcl-2-associated X (BAX) were also assessed. Cells were exposed to low-dose concentrations (10–12 to 10–9 M) to determine whether environmental exposures (7) to PBDEs might be associated with genotoxic, cytotoxic and/or altered cell characteristics.
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Materials and methods |
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Chemicals
Chemicals were obtained from Sigma Chemical Co. (Poole, Dorset, UK) and cell culture consumables were obtained from Invitrogen Life Technologies (Paisley, UK), unless otherwise stated.
Cell culture and standard preparation
The human mammary carcinoma MCF-7 cell line was grown in Dulbecco's modified essential medium supplemented with 10% heat-inactivated foetal calf serum (FCS), penicillin (100 U/ml) and streptomycin (100 µg/ml). Cells were grown in 5% CO2 in air at 37°C in a humidified atmosphere, and disaggregated using a trypsin (0.05%)/EDTA (0.02%) solution, to form single cell suspensions prior to sub-culture or incorporation into experiments.
PBDE standards (purity >99% except PBDE209, purity >95%) were purchased from Cambridge Isotope Laboratories, Inc (Andover, Massachusetts, USA) and were pre-dissolved in nonane at 50 µg/ml (50 p.p.m.), except for analytical standard PBDEs 51, 69 and 128 which were purchased from AccuStandard (New Haven, Connecticut, USA). Test solutions were formulated by diluting PBDE standards (in nonane) with required amounts of dimethyl sulphoxide (DMSO) to give a 2 µM stock solution. This required dilutions of 1 in 50 for PBDE47 (2,2',4,4'-tetraBDE; Mol. Wt. = 485.82), 1 in 45 for PBDE99 (2,2',4,4',5-pentaBDE; Mol. Wt. = 564.8), 1 in 40 for PBDE153 (2,2',4,4',5,5'-hexaBDE; Mol. Wt. = 643.59), 1 in 35 for PBDE183 (2,2',3,4,4',5',6'-heptaBDE; Mol. Wt. = 722.5) and, 1 in 25 for PBDE209 (2,2',3,3',4,4',5,5',6,6'-decaBDE; Mol. Wt. = 959.22). From these stock solutions, serial dilutions were then set up so that upon addition to culture mixtures (at 10–9 to 10–12 M PBDE), DMSO concentrations would not exceed 1% v/v. In all experiments, an appropriate negative control (NC) containing nonane (purity >95%) (Promochem, Welwyn Garden City, Hertfordshire, UK) dissolved in DMSO at the highest-PBDE-concentration equivalent for that particular experiment was incorporated, as indicated. Standards were stored in 2-ml screw-capped amber glass vials with Teflon-lined lids (Aldrich, Poole, Dorset, UK).
The cytokinesis-block micronucleus (CBMN) assay
Routinely-cultured cells were disaggregated and re-suspended in complete medium prior to seeding aliquots (3 ml; 
1 x 104 cells) into 30-mm petri dishes containing 20-mm coverslips (12). They were then cultured for 24 h to allow them to attach. Attached cells were then treated with or without PBDE congener for a further 24 h, as indicated. Medium was then replaced with fresh medium, without test agent but containing cytochalasin B (2 µg/ml). After another 24 h in culture, cells were fixed with 70% ethanol (EtOH), stained with 5% Giemsa and mounted on microscope slides (7). Prior to analysis, slides were coded by an individual uninvolved with the experiment. Employing established criteria (17), micronuclei in 1000 binucleate cells were then scored.
Cell growth and clonogenic survival
To assess growth, cells were re-suspended in complete medium prior to seeding aliquots (5 ml; 0.5–1 x 105 cells) into 25-cm2 flasks. Cells were allowed to attach for 24 h prior to addition of test agent, as indicated; designated T0, an initial cell count was taken. Following 24-h treatment, medium was replaced with fresh medium without test agent (13). At 72-h incubation after T0, the cells were disaggregated, re-suspended in phosphate-buffered saline (PBS) and applied to a haemocytometer with a coverslip after which cell number (and trypan blue exclusion) was estimated.
For the clonogenic assay, disaggregated cells were re-suspended in complete medium (0.5 x 103 cells in a 5-ml aliquot) and seeded into 25-cm2 flasks in the presence or absence of test agent for 24 h, as indicated. The medium was then replaced with fresh medium without test agent. Cells were cultured undisturbed for a further 7 days prior to removal of medium and fixation with 70% EtOH. Colonies were then stained with 5% Giemsa after which they were counted and percentage plating efficiencies calculated (12,13).
Quantitative real-time reverse transcription (RT)–PCR
Routinely-cultured cells were disaggregated and re-suspended in complete medium prior to seeding aliquots (5 ml; 1 x 105 cells) into 60-mm petri dishes. After 24 h, attached cells were then treated with or without test agent, as indicated, for a further 24 h. Cells were then washed twice with PBS prior to lysis and total RNA extraction using the Qiagen RNeasy® Kit in combination with the Qiagen RNase free DNase kit (Qiagen Ltd, Crawley, West Sussex, UK). DNase was incorporated into the extraction procedure in order to remove any residual DNA e.g. pseudogene. RNA quality was routinely assessed in a 1.2% formaldehyde agarose gel; yield and purity were checked using a spectrophotometer. RNA (0.4 µg) was reverse transcribed in a final volume of 20 µl containing Taqman® reverse transcription reagents (Applied Biosystems, Warrington, Cheshire, UK): 1 x Taqman RT buffer; MgCl2 (5.5 mM); oligo d(T)16 (2.5 µM); dNTP mix (dGTP, dCTP, dATP and dTTP; each at a concentration of 500 µM); RNase inhibitor (0.4 U/µl); reverse transcriptase (MultiScribeTM) (1.25 U/µl) and RNase-free water. Reaction mixtures were then incubated at 25°C (10 min), 48°C (30 min) and 95°C (5 min).
cDNA samples were stored at –20°C prior to use. Primers (Table I) for CYP1A1 (GenBank accession no. BC023019 [GenBank] ), CYP1A2 (GenBank accession no. NM_000761 [GenBank] ), CYP1B1 (GenBank accession no. NM_000104 [GenBank] ), P21WAF1/CIP1 (GenBank accession no. NM_078467 [GenBank] ), BCL-2 (GenBank accession no. NM_000633 [GenBank] ), BAX (GenBank accession no. AF007826 [GenBank] ), and endogenous control ß-ACTIN (GenBank accession no. AK222925 [GenBank] ) were chosen using Primer Express software 2.0 (Applied Biosystems, Warrington, UK) and designed so that one primer spanned an exon boundary. Specificity was confirmed using the NCBI BLAST search tool. Quantitative real-time PCR was performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Warrington, UK). Reaction mixtures contained 1 x SYBR® Green PCR master mix (Applied Biosystems, Warrington, UK); forward and reverse primers (Invitrogen, Paisley, UK) at a concentration of 300 nM (CYP1A1, CYP1A2, CYP1B1, P21WAF1/CIP1, BCL-2, BAX, or ß-ACTIN); for CYP1A1, CYP1A2, CYP1B1, P21WAF1/CIP1, BCL-2 or BAX amplification 20 ng cDNA template or for ß-ACTIN amplification 5 ng cDNA template; made to a total volume of 25 µl with sterile H2O. Thermal cycling parameters included activation at 95°C (10 min) followed by 40 cycles each of denaturation at 95°C (15 s) and annealing/extending at 60°C (1 min). Each reaction was performed in triplicate and ‘no-template’ controls were included in each experiment. Dissociation curves were run to eliminate non-specific amplification, including primer-dimers.
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Attenuated total reflection-Fourier transform infrared (ATR) microspectroscopy
Spectra were acquired using a Bruker Vector 22 FTIR spectrometer with Helios ATR attachment that contained a diamond crystal (Bruker Optics Ltd, Coventry, UK). Routinely-cultured cells were disaggregated and re-suspended in complete medium prior to seeding aliquots (5 ml;
1 x 105 cells) into 60-mm petri dishes containing 1 x 1 cm Low-E reflective glass slides (Kevley Technologies, Chesterland, Ohio, USA). After 24 h, attached cells were then treated with or without test agent, as indicated, for a further 24 h prior to fixation with 70% EtOH; these were allowed to air dry prior to being placed in a dessicator until analysis (18,19). Data was collected in ATR mode and spectra (8 cm–1 spectral resolution, co-added for 32 scans) were converted into absorbance using Bruker OPUS software (20). From each experimental treatment, 10 spectra were acquired. Sodium dodecyl sulphate (SDS; Sigma Chemical Co., Poole, Dorset, UK) was used to clean the ATR crystal prior to analysis of a new sample. Each time the crystal was cleaned a new background reading was also taken prior to recommencing spectral analysis. Spectra were baseline corrected using OPUS software and normalised to the amide I (1650 cm–1) absorbance band. Principal component analysis (PCA) was carried out on the spectra using the Pirouette software package (Infometrix Inc., Woodinville, USA). In PCA, each spectrum becomes a single point, or score, in n-dimensional space and using selected principal components (PCs) as coordinates, the data was analysed for clustering when viewed in different directions. A resultant 3D scores plot on PCs selected to demonstrate best segregation of cell spectra from different treatment groups was obtained.
PBDE cell/media partitioning
Routinely-cultured cells were disaggregated and re-suspended in complete medium prior to seeding aliquots (5 ml; 1 x 104 cells) into 25-cm2 flasks. After 24 h, attached cell incubates, in triplicate, were then treated for a further 24 h with individual PBDE congeners at 10–9 M concentration. Following treatment, media was collected into sterile Teflon tubes for subsequent chemical analysis. Cells were then disaggregated using trypsin/EDTA solution; the cell suspension was also collected into sterile Teflon tubes for subsequent chemical analysis. Sample analysis was performed based on previously-reported methods for blood analysis (21). Briefly, samples were denatured with hydrochloric acid and propan-2-ol (BDH Laboratory Supplies, Poole, Dorset, UK; purity >99.7%). This was followed by extraction with a hexane:methyl tert-butyl ether (MTBE; Aldrich, Stenheim, Germany; purity >99.8%) mixture. Samples were then cleaned using concentrated sulphuric acid, followed by gel permeation chromatography (Biobeads S-X3), before the addition of internal standards [PBDE69 (2,3',4,6-tetraBDE; Mol. Wt. = 485.82) and PBDE181 (2,2',3,4,4',5,6-heptaBDE; Mol. Wt. = 722.5)], final volume reduction, and analysis by gas chromatography (GC)-mass spectrometry (MS). All samples were spiked with recovery standards [PBDEs 51 (2,2',4,6'-tetraBDE; Mol. Wt. = 485.82), 128 (2,2',3,3',4,4'-hexaBDE; Mol. Wt. = 643.59) and 190 (2,3,3,4,4',5,6-heptaBDE; Mol. Wt. = 722.5) at 50 pg/µl], in 25 µl acetone, before extraction. Concentrations were corrected for the recoveries of these standards, which averaged 65–119% (Table II). Samples were analysed for PBDEs 47, 99, 153 and 183 using a Fisons MD800 GC-MS. The GC used splitless injection and was fitted with a 30 m DB5 capillary column. The MS used a negative ion chemical ionization (NICI) source in SIM mode, and used ammonia as the reagent gas, monitoring m/z ratios 79 and 81 for the PBDEs (21). PBDE209 was analysed by GC-electron capture detection (ECD) using the method described below.
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Chemical stability
Breakdown of brominated diphenyl ethers (BDEs) can occur by debromination, resulting in the formation of lower BDEs (22
). Any possible bromine-containing breakdown products will be detectable by ECD. The purity of PBDE standards and the chemical stability of test solutions over the course of the experimental period was examined using GC–ECD. Analysis was performed using a Hewlett-Packard 5890 Series II GC (Avondale, Pennsylvania, USA) in splitless injector mode with a 260°C injector temperature. Separation was achieved using a 0.1 µm film thickness, 0.25 mm i.d., 15 m DB5-MS column (J & W Scientific, Folsom, California, USA), fitted with a 2 m 0.53 mm i.d. retention gap, and operated with helium carrier gas at a 1 ml/min flow rate. Two GC oven temperature programs were used: 110°C, 2 min, 25°C/min to 200°C, then 4°C/min to 300°C hold for 10 min (for PBDE209), and: 110°C, 2 min, 25°C/min to 160°C, then 4°C/min to 300°C hold for 2 min (for the other PBDEs). The ECD was maintained at 350°C with a nitrogen make-up gas. Aliquots of PBDE standard stock solutions were diluted in dodecane (1 µl; 500 ng/µl solutions) and were injected directly into the GC–ECD. At the end of the experimental period, test solutions were transferred from DMSO to dodecane for analysis by GC–ECD. Firstly, 10 µl 2 mM solutions (in DMSO) were dissolved in 1 ml de-ionized water (Millipore Milli-Ro 30, Beford, Massachusetts, USA). This solution was then sequentially extracted three times with n-hexane (BDH Laboratory Supplies, Poole, Dorset, UK; purity >97%), after which the three hexane extracts were combined. The extracts had 100 µl dodecane (Aldrich, Stenheim, Germany; purity >99%; cleaned with concentrated sulphuric acid) added to them, and were then reduced under nitrogen to 100 µl volume. Aliquots (1 µl; 500 ng/ml) of these PBDE solutions were injected into the GC–ECD.
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Results |
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Micronucleus-forming activity
Employing established criteria (17
), micronuclei were scored in populations of 1000 binucleate MCF-7 cells either as micronucleated binucleate cells or total number of micronuclei (12,13,23). Under x1260 magnification, scoring of binucleate MCF-7 cells that were identifiable as free of micronuclei (Figure 1A) or, for instance, containing one micronucleus (Figure 1B), three (Figure 1C) or four micronuclei (Figure 1D) was conducted. In instances where there was a mixed population of closely-associated cells (Figure 1E), great care was taken to appropriately score binucleate MCF-7 cells.
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Following 24-h treatment with 10–9 M PBDE congeners 47, 153, 183 or 209 (mean ± SD derived from five independent experiments), marked increases in micronuclei were apparent following exposure to each of these four congeners (Figure 2). Significant elevations (P < 0.05) in PBDE-induced micronucleus formation compared to NC were noted following treatment with PBDE congeners 47 or 183. When the dose-related micronucleus-forming activities of PBDE congeners 47, 99, 153, 183 or 209 (mean ± SD derived from triplicate measurements) were investigated, no elevations compared to NC were apparent following 24-h treatment with 10–12 M but there were marked increases after 10–10 or 10–9 M exposure (Figure 3). Following exposure to these latter two concentrations, significant elevations (P < 0.05) in micronucleated binucleate MCF-7 cells and/or total micronuclei were observed with PBDE47, PBDE99, PBDE153, PBDE183 and PBDE209 with one or both treatments; in instances where significance was not observed, elevations in micronucleus-forming activity compared with the corresponding NC were still noted (Figure 3). Benzo[a]pyrene (B[a]P) (10–6 M) was employed as a positive control and 24-h treatment resulted in 408 ± 98 micronucleated binucleate MCF-7 cells or 638 ± 121 total micronuclei in 1000 binucleate cells (mean ± SD, n = 3), an
8-fold increase compared to NC.
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Cell growth and clonogenic survival
Cell viability, as ascertained by trypan blue exclusion, was consistently in excess of 90%. In comparison with corresponding NC, none of the PBDE congeners (in the 10–12 to 10–9 M concentration range) tested in this study altered clonogenic survival (measured as % plating efficiency) 7 days following 24-h treatment (Figure 4). However, a feature associated with 24-h treatment with 10–9 M PBDE congener was a marked elevation in MCF-7 cell number at 72 h after T0 (Figure 5). This was a consistent finding independent of inter-experimental variability i.e. higher cell numbers were scored in PBDE-treated cultures compared to corresponding NCs (data not shown). Compared to corresponding NCs, this was noted to be a significant (P < 0.05) increase following exposure to PBDE153 or PBDE183 (Figure 5).
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ATR microspectroscopy
Figure 6A–F shows average infrared (IR) spectra (n = 10 in each group) derived from 10–9 M PBDE-treated MCF-7 cells compared to corresponding NCs and obtained using the
250 x 250 µm octagon-shaped sampling area of ATR microspectroscopy. A vibrational IR spectrum of tissue biochemistry was obtainable from such EtOH-fixed cells adhered to Low-E reflective glass slides. Throughout the spectral region interrogated (900–1800 cm–1), clear differences in the ‘biochemical-cell fingerprint’ of PBDE-treated cells compared to corresponding NCs were noted (Figure 6). Indicating a protein-conformation alteration, a marked shift (5 cm–1) in the centroid of the amide I peak of PBDE99-treated and PBDE153-treated cells (1640 cm–1) compared to NCs (1645 cm–1) occurred. There were also marked differences in the spectral region containing DNA/RNA (1490–1000 cm–1) with marked intensity alterations in glycoproteins (1380 cm–1), 
antisymmetric stretch (as
, 1350 cm–1 to 1260 cm–1), C–O ring vibrations of nucleic acid ‘sugars’ (1185 cm–1 to 1120 cm–1), carbohydrates (1155 cm–1) and, symmetric stretching vibrations of nucleic acids and phospholipids (1084 cm–1).
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PCA (3D) was performed to determine if segregation of IR spectra derived from different cell incubates might be achieved along chosen PCs (1, 2 and 3, identified to account >97% variance) and whether rotated data might point to similarities or differences (Figure 7). Using these three PCs it proved possible to obtain segregated clustering for the different treatment groups i.e. spectra derived from each cell incubate (NC or PBDE-treated) exhibited significant clustering in this scores plot (Figure 7). Nearness in multivariate distance implies spectral similarity and separation in the 3D plots signifies spectral differences. There was good separation between IR spectra derived from NC and PBDE-treated cell incubates pointing to the potential of multivariate analysis to segregate cells following such exposures; of interest was the observation that cell-incubate IR spectral clusters for PBDE183 and PBDE209 were closely aligned with that of B[a]P whereas clusters for PBDE47 and PBDE183 were more distally segregated (Figure 7). Loadings curves for each PC were plotted (data not shown) and these allowed the influence of specific spectral features on each PC to be identified. On PC1 the spectral region between
1000 cm–1 to 1200 cm–1 accounted for the majority of the variance, on PC2 a shift in amide I was the most prominent variable and on PC3, the spectral region between 1700 cm–1 to 1750 cm–1 was picked out as contributing to variation between the different treatment groups.
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Cell/media partitioning and chemical stability
Under tissue culture conditions PBDEs may rapidly partition out of media solution and into cells (a rodent cell line) (24
). When we investigated this in our cell culture system, partitioning of PBDEs from the media into the MCF-7 cells was less significant. Of the combined amounts measured in the cells and media, 94, 98, 96, 83 and 71% remained in the media for PBDEs 47, 99, 153, 183 and 209, respectively (Table II). There was, however, a large amount of added chemical unaccounted for following extraction of the cells and the media; of the total amount initially administered to each cell incubate, some 73, 74, 84, 81 and 52% of PBDE congeners 47, 99, 153, 183 and 209, respectively, appeared to have been lost e.g. absorbed into plastic. This was despite the fact that the recovery efficiencies of the extraction-spiked PBDEs (congeners 51, 128, 190) were good (Table II), showing that the extraction method performed well, and suggesting that these losses occurred during cell incubation. Given the low volatility of PBDEs, losses by volatilization are not expected, and one would reasonably expect that degradation or metabolism of these BFRs would be negligible over such a short treatment period. If absorption into plastic was responsible, such rapid partitioning would probably have resulted in the cells being exposed to a lower (by a factor of 4) dose than that intended. The results of the GC–ECD analysis of some standards used in our experimental studies are shown in Figure 8A–H. The nonane stock standards for PBDE47, PBDE153 and PBDE183 contained no significant brominated impurities, and none were observed in the test solutions at the end of the experimental period either (Figure 8A–F). The nonane standard and test solutions (at the end of the experimental period) for PBDE209 were found to show evidence of the presence of nonaBDEs. Area ratios of decaBDE:nonaBDEs were 11.5:1 in the nonane PBDE209 standard and 10.5:1 in the test solution, indicating that the nonaBDE proportion of the solution increased by <1% over the entire experimental period (Figure 8G–H).
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Gene-expression analysis
Exposure of MCF-7 cells for 24 h to 10–12 M or 10–9 M PBDE congeners 47, 99, 153 or 183 was not associated with marked up-regulation and/or down-regulation in the expression of CYP isoforms or P21WAF1/CIP1, BCL-2 or BAX (Table III). A rigorous examination of intra-experimental variation for the expression of genes analysed in this study revealed that fluctuations in excess of 60% down-regulation and 70% up-regulation of expression are outside the normal variability (data not shown). In some cases marked inter-experimental variation was associated with apparent elevations in gene expression but whether these were outlier observations or a consequence of genuine low-dose effects remains to be ascertained.
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Discussion |
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Environmental contaminants such as PCBs and OCs are potential carcinogens (25
,26). Currently-used BFRs (e.g. PBDEs) are structurally similar, possess the same chemical properties of environmental persistence and lipophilicity, and may be capable of inducing a matching profile of intra-cellular and organism toxicity (27,28). Despite their widespread production and use, investigations into the toxicity of PBDEs remains limited (28). This is now a concern as these agents have been shown to migrate from the products in which they are used to accumulate in the environment (29,30) and human biota (31).
The CBMN assay facilitates the measurement of chromosomal aberrations that are microscopically visualised as micronuclei expressed in cells that have completed a nuclear division (17). Micronuclei may arise as a result of exposure to agents that induce either chromosomal breaks (i.e. clastogens) or a numerical chromosomal change (i.e. aneugens) (32). Marked low-dose PBDE-induced elevations in micronucleus-forming activity were apparent (Figures 2 and 3). This was despite the losses of PBDEs from the cell-incubate mixture (probably through absorption into plastic) and the low level of partitioning into cells (Table II).
Median concentrations of UK serum PBDE levels (collected 2003) were reported to be 0.82 ng/g lipid for PBDE47 (range 0.30–180 ng/g), <0.16 ng/g lipid for PBDE99 (range <0.16–150 ng/g), 1.7 ng/g lipid for PBDE153 (range 0.36–87 ng/g), 0.30 ng/g lipid for PBDE183 (range 0.14–1.8 ng/g) and <15 ng/g lipid for PBDE209 (<15–240 ng/g) (21). Based on a blood-lipid content of 5 mg/ml, this is equivalent to 4.1 pg/ml for PBDE47 (range 1.5–900 pg/ml), 8.5 pg/ml for PBDE153 (range 1.8–435 pg/ml), <0.8 pg/ml for PBDE99 (range <0.8–750 pg/ml), 1.5 pg/ml for PBDE183 (range 0.7–9 pg/ml), and <75 pg/ml for PBDE209 (range <75–1200 pg/ml). The treatment concentrations employed in this study equated to 0.5 pg/ml (10–12 M) to 500 pg/ml medium (10–9 M); even if losses were factored in (Table II), levels of 0.13–130 pg/ml, 0.13–130 pg/ml, 0.08–80 pg/ml, 0.09–90 pg/ml, and 0.17–170 pg/ml for 10–12 to 10–9 M PBDE47, PBDE99, PBDE153, PBDE183 and PBDE209, respectively, would have been present. Thus we would suggest that our low-dose effects occurred in response to environmentally-relevant exposures.
Based on our results, low-dose PBDE concentrations appear to be capable of damaging cell genomes. Through a modulation of Bcl-2:Bax, potential carcinogens have been shown to delay the involution of the rat mammary gland (33); dysregulation effects that may not be dissimilar to those induced by 17ß-oestradiol (34). In this study, PBDEs (10–12 or 10–9 M) tested were not found to markedly modulate the expression levels of BCL-2 and/or BAX (Table III). Exposure to PBDEs has also been associated with induced reductions in CYP expression (35); at the concentrations used in this study, no such observations were noted (Table III).
Cellular biomolecules absorb the mid-IR (
= 2–20 µm) via vibrational transitions that are derived from individual chemical bonds yielding richly-informative ‘fingerprint’ spectra relating to structure and conformation (18). A received sample (i.e. cells) absorbs IR energy and this permits the detection and measurement of cellular biomarkers including DNA, RNA, lipids, phosphate and carbohydrates (19). IR microspectroscopy often generates large datasets, which are readily interrogated using advanced computational techniques (19,20). PCA is a multivariate technique that allows cluster analysis by plotting each spectrum as a point in n-dimensional space; using selected PCs as coordinates, the data may then be viewed in particular directions in order to identify the best segregation (Figure 7). Using this approach, clear variance between control and PBDE-treated cells was noted (Figure 7) consistent with the spectral profiles derived from the different cell incubates (Figure 6). The most marked changes in the pattern of IR absorbance occurred between 1000 cm–1 and 1200 cm–1 especially following treatment with PBDE99 (Figure 6B), PBDE153 (Figure 6C) or PBDE209 (Figure 6E); this was also associated with B[a]P treatment (Figure 6F). This particular region is associated with structural alterations in nucleic acids and these changes translated into strong cluster separation (Figure 7). Structural damage reflected in differences in base functional groups and conformational properties in response to environmental contaminants are also detectable in vivo using IR microspectroscopy (36). Whether these observations point to the presence of PBDE-DNA adducts or to epigenetic events remains to be determined.
The extrapolation of observed effects from high-dose rodent cancer tests to predict risk(s) following human low-dose exposures remains controversial (16). However, despite suggestions that environmental exogenous agents play a negligible role in the aetiology of human diseases such as cancer (37), epidemiological studies strongly suggest that they must (38). The question then arises as to whether typical environmental exposures, be they by-products of combustion (38) or synthetic chemicals (7,13), are sufficient to modulate normal cellular functioning [8,9] to give rise to a pathogenic state; it is also conceivable that unidentified factors may be responsible (39). With regards to the PBDE congeners 47, 153 and 183, GC-ECD analysis excluded the potential role for impurities from debromination breakdown products contributing towards the cellular effects observed in this study (40). The presence of nonaBDEs in the PBDE209 analytical standard mirrors their presence in the technical decaBDE mix where they are typically found at <3% (41). No other brominated impurities were detectable in the PBDE209 standard solutions, indicating that the chemical stability of these agents over the experimental period was good.
PBDEs may possess endocrine properties and the MCF-7 cell line is hormone-responsive. Our results point to a hitherto unrecognised potential for a range of PBDEs at low-dose concentrations (
10–9 M) to induce altered characteristics in such cells. The implications of our findings raise important questions for the development of future strategies to assess risk associated with these persistent agents.
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Acknowledgments |
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The study was supported by a research grant (GR/S75918/01) from the Engineering and Physical Sciences Research Council (EPSRC), UK.
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*To whom correspondence should be addressed. Tel: +44 1524 594505; Fax: +44 1524 593192; Email: f.martin{at}lancaster.ac.uk
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Received on October 9, 2005; revised on July 28, 2006; accepted on July 31, 2006.
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