Polybrominated Diphenyl Ether-Associated Alterations in Cell

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Environ. Sci. Technol. 2009, 43, 3356–3364

Polybrominated Diphenyl Ether-Associated Alterations in Cell Biochemistry as Determined by Attenuated Total Reflection Fourier-Transform Infrared Spectroscopy: a Comparison with DNA-Reactive and/or Endocrine-Disrupting Agents V A L O N L L A B J A N I , †,‡ K E V I N C . J O N E S , ‡ GARETH O. THOMAS,‡ LEE A. WALKER,‡ RICHARD F. SHORE,‡ AND F R A N C I S L . M A R T I N * ,† Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster LA1 4YQ, U.K., and NERC Centre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP, U.K.

Received December 19, 2008. Revised manuscript received February 28, 2009. Accepted March 9, 2009.

Whether polybrominated diphenyl ethers (PBDEs) induce effects in target cells is increasingly important given that their environmental burdens are rising. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy can be used to biochemically signature cells based on the notion that a detailed “biochemical-cell fingerprint” in the form of an infrared (IR) spectrum is derived. By employing subsequent computational approaches such as principal component analysis (PCA) and/ or linear discriminant analysis (LDA), data reduction is achieved to allow for the identification of wavenumber-related biomarkers of effect. Clustering of similar spectra (or scores) away from dissimilar ones highlights the variance responsible for discriminating classes. Discriminating biomarkers might include protein conformational changes, structural alterations to DNA/RNA, glycogen content, or protein phosphorylation. Employing this approach, we investigated in MCF-7 cells the doserelated effects of PBDEs (congeners 47, 153, 183, and 209), benzo[a]pyrene (B[a]P), 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PHIP), 17β-Oestradiol (E2), or lindane (γ-hexachlorocyclohexane). Cultures concentrated in G0/G1- or S-phases were treated for 24 h. Following treatment, MCF-7 cells were fixed and applied to IR reflective Low-E windows for interrogation using ATR-FTIR spectroscopy. At concentrations as low as 10-12 M in culture, significant separation (P e 0.05) of PBDEtreated and vehicle control cell populations was noted. In some cases this was associated with alterations in lipid or the secondary structure of proteins; with DNA-reactive compounds (e.g., B[a]P), variance was primarily noted in the DNA/RNA * Corresponding author tel.: +44 1524 594505; fax: +55 1524 593192; e-mail: [email protected]. † Centre for Biophotonics. ‡ NERC Centre for Ecology and Hydrology. 3356

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region. This study points to a novel nondestructive approach capable of identifying contaminant effects at environmental concentrations in target cells.

Introduction Polybrominated diphenyl ethers (PBDEs) have been used in large volumes worldwide for incorporation as flame retardants into commercial products; they consist of pentabromodiphenyl ether (pentaBDE), octabromodiphenyl ether (octaBDE), and decabromodiphenyl ether (decaBDE) (1). These leach out into the environment where lower-brominated congeners (tetra to hexa) seem to persist in different environmental compartments and bioaccumulate (2, 3). Higher-brominated congeners (e.g., decaBDEs) have a lower tendency to bioaccumulate whereas BDE47 is typically the most abundant congener detected in the environment (2). PBDEs may be transferred directly into the food chain (4). Tissue concentrations in humans progressively increased through the 1980s and 1990s (5-7); little information exists regarding possible health effects. Depending on the test agent and dosage, exposure to PBDEs has been observed to induce in vitro (8, 9) and in vivo (10) effects. In vivo effects might include neurotoxicity and endocrine (thyroid) disruption (e.g., reduced plasma thyroxine (T4) levels), as well as liver and kidney damage (10-13). Exposure to PBDEs during development has been associated with neurotoxicity (14-16). In vitro studies suggest that µM PBDE concentrations induce neurological effects by altering calcium signaling pathways (17, 18). Exposure to BDE47 induces oxidative stress, apoptosis, and DNA damage in rat hippocampal neurons (19). Low-concentration exposures resulted in detectable formation of reactive oxygen species in human neurophil granulocytes cells (20). In addition, BDE99 exposure in the developing rat uterus was associated with an increase in estrogen receptor (ER)R and ERβ mRNA transcripts (21). They may also have weak ER activity in vitro (22); treatment of ER-positive MCF-7 cells with BDE-71 increased cell proliferation, an effect prevented by an antiestrogen (23). PBDEs might also modulate phase I and phase II metabolizing enzymes in rodents (24) including aromatic hydrocarbon receptor-mediated induction of cytochrome P450 (CYP)1A1 and CYP1A2 (12, 25-27). Predicting biological responses to low-level exposures based on adverse effects induced by high concentrations is difficult (2). Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy has been used to examine for test-agent effects (9, 28, 29) and might be used to biochemically signature different target cells (30). This approach is based on the notion that depending on the interrogated cells, a detailed “biochemical-cell fingerprint” in the form of an infrared (IR) spectrum relating to chemical structure is obtained (31). Subsequent application of computational approaches such as principal component analysis (PCA) and/or linear discriminant analysis (LDA) allows for data reduction to facilitate identification of wavenumberrelated biomarkers ((32); Figure 1). PCA allows for the reduction of data with a high level of intrinsic dimensionality (e.g., IR spectra) into scores in which variance between different classes may be visualized along coordinates known as principal components (PCs). By clustering similar spectra and segregating dissimilar ones, one might readily identify the molecular alterations contributing to variance. By inputting the PCA output into LDA, one reduces the influence of intraclass variation and maximize interclass variance (30, 32). A loadings plot identifies the wavenumbers responsible for segregating the different classes. 10.1021/es8036127 CCC: $40.75

 2009 American Chemical Society

Published on Web 04/01/2009

FIGURE 1. Overview of principles underlying the application of mid-IR spectroscopy for the analysis of biomolecular structures such as biological cells. In this study, we investigated whether the application of ATR-FTIR spectroscopy to interrogate variously treated MCF-7 cells and with subsequent PCA-LDA of derived spectra might allow for the identification of molecular alterations. Dose-related effects of PBDEs (congeners 47, 153, 183, and 209), benzo[a]pyrene (B[a]P), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PHIP), 17β-Oestradiol (E2), or lindane (γ-hexachlorocyclohexane) were studied. Test concentrations ranged from 10-12 to 10-3 M, with lower levels (pM to nM) being expected to be consistent with environmental exposures (7). Our aim was to determine whether this platform might facilitate the identification of low-dose effects consistent with relevant environmental exposure levels, and allow contaminant-specific identification based on induced molecular alterations in target cells.

Experimental Section Culture. MCF-7 cells were grown in Dulbecco’s modified essential medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/mL), and streptomycin (100 µg/mL) in a humidified atmosphere with 5% CO2 in air at 37 °C. Cells were disaggregated with trypsin (0.05%)/EDTA (0.02%) solution before incorporation into experiments. Culture consumables were obtained from Invitrogen Life Technologies (Paisley, UK), unless otherwise stated. Test Agents. Test agents were added as solutions in dimethylsulfoxide (DMSO); the maximum concentration of DMSO/culture mix was 1% (v/v). Chemicals were obtained from Sigma Chemical Co. (Poole, Dorset, UK), unless otherwise stated. On two separate occasions, once at the beginning of the experimental period and again at the end, all stock solutions were analytically tested (8, 9); treatment concentrations remained accurate throughout (data not shown). Treatments and ATR-FTIR Spectroscopy. Routinely cultured MCF-7 cells were disaggregated, resuspended in complete medium, and seeded in T25 flasks. Cultures were concentrated in either Go/G1-phase or S-phase (33) prior to treatment. Following treatment, cells were again disaggregated into cell suspensions and were immediately fixed with 70% ethanol. Resultant cell mixes were then applied to 1 cm × 1 cm Low E-reflective glass slides and allowed to air-dry prior to storage in a desiccated environment until analysis. IR spectra were obtained using a Bruker Vector 22 FTIR spectrometer with Helios ATR attachment containing a

diamond crystal (Bruker Optics Ltd., Coventry, UK); 10 spectra were acquired per slide. The ATR crystal was cleaned using sodium dodecyl sulfate (Sigma Chemical Co.) and a new background was collected prior to analysis of each new sample. IR spectra were individually baseline corrected using OPUS software and normalized to the Amide I (1650 cm-1) absorbance band. Each experimental regimen (2 months to complete) was conducted on three separate occasions over a 12-month period, once by one individual and twice by a second individual. PCA-LDA. PCA was carried out using the Pirouette software package (Infometrix Inc., Woodinville, WA). In PCA, each spectrum becomes a single point, or score, in ndimensional space and using selected PCs as coordinates, the data were analyzed for clustering when viewed in different directions. For each experimental condition, 10 IR spectra were acquired and following baseline correction/normalization, an average spectrum was acquired for subsequent computational analyses. PCA was used for preliminary data reduction and the output processed using LDA (30, 32, 34). PCA-LDA scores and loadings plots were derived for the biochemical-cell fingerprint region (1800 cm-1 to 900 cm-1; Figure 1). Ellipses of Confidence. The mean and standard deviation (SD) of each class used (x and y values) were calculated from the PCA-LDA scores plots. The values for mean and SD were then imported into MatLab where ellipses of confidence with a P-value of 0.95 around the averaged scores/individual treatment were estimated. The contours are ellipses of constant Mahalanobis distance from the group center. Points lying outside a contour drawn at a particular value of P have a probability of e(1 - P) of belonging to that cluster. At the same time however, if a point lies within an ellipse, we are told nothing about its probability of it belonging to that cluster as it could be an outlier from a different class. In practice, in such a case a point may be assigned to the nearest cluster.

Results For a typical IR spectrum (average of n ) 10/treatment) of MCF-7 cells derived using the ≈250 µm × 250 µm octagonshaped crystal used for this ATR-FTIR spectroscopic application, see Supporting Information. Throughout the spectral region (1800 cm-1 to 900 cm-1), no clear difference(s) between different treatment groups can be readily observed VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. PCA-LDA scores plots following PBDE treatment, as indicated. Panels in the left column represent MCF-7 populations concentrated in G0/G1-phase; those in the right column were concentrated in S-phase. Each symbol (score) represents one independent experiment and is the average of 10 separate IR spectra acquired per slide. Confidence ellipses show probability contours assuming normal distribution for each of the clusters. Each ellipsis has a probability value of P ) 0.95. and the data sets are multidimensional. Given the large numbers of spectra generated, computational analysis is required to discriminate classes and identify associated biomarkers contributing to variance. This approach might include PCA-LDA (34); each spectrum becomes a point, or 3358

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score, in which nearness implies spectral similarity and segregation points to spectrally dissimilar classes (Figure 1). Dose-related effects of four PBDEs (congeners 47, 153, 183, and 209) in MCF-7 cells that were concentrated in G0/ G1-phase or S-phase were examined (Figure 2). BDE47-

induced segregation was observable at lower concentrations (10-11 and 10-10 M) in cells concentrated in G0/G1-phase. In S-phase cells, there appeared to be an inverse correlation between concentration and effect with BDE47 exposure, with scores for the lowest concentration (10-12 M) segregating most clearly away from those of the vehicle control whereas those for the highest concentration were the least segregated. For the remaining three PBDE congeners, the pattern of segregation of treatment scores from those of the vehicle control was less marked. Following exposure to BDE153 in cultures concentrated in S-phase, a dose-related increase in segregation of spectral scores compared to vehicle control was observed up to a concentration of 10-9 M, above which higher concentrations were not significantly separated. However, 24-h treatment of MCF-7 cells concentrated in G0/ G1-phase resulted in segregation of scores for the vehicle control compared to exposure to lower concentrations (10-12, 10-11, 10-10, or 10-9 M) of BDE183 or BDE209 (Figure 2). This inverse relationship of concentration with effect was more pronounced in cells concentrated in G0/G1-phase following exposure to higher-brominated congeners whereas lowerbrominated congeners induced such effects in cell populations concentrated in S-phase. B[a]P, PhIP, E2, and lindane were examined for their doserelated effects in MCF-7 cells concentrated in either G0/G1phase or S-phase (Figure 3). Such agents might be expected to mediate their intracellular effects via different mechanisms. Over the concentration range of B[a]P exposure, spectral scores for treated cells in S-phase were more segregated from the corresponding vehicle control compared to those in G0/ G1-phase; in the latter, only the higher concentration (10-5 M) significantly discriminated. In contrast, in PhIP-treated cells concentrated in either G0/G1-phase or S-phase, segregation of spectral scores was induced over the entire concentration range, surprisingly most pronounced in the former. Different E2-induced dose-related responses were observed depending on whether MCF-7 cells were in either G0/G1-phase or S-phase. In the former, spectral scores for 10-10 M-treated cells only segregated away from the corresponding vehicle control. In contrast, following 24-h exposure of cells in S-phase, a marked shift away from the corresponding vehicle control was observed following exposure to 10-12 or 10-11 M; higher concentrations induced a doserelated realignment (Figure 3). A distinction between lowdose and high-dose effects was most observable following exposure to lindane, independent of whether cells were in G0/G1-phase or S-phase. Segregation of spectral scores from the corresponding vehicle control progressively occurred following exposure to lower concentrations (10-12, 10-11, or 10-10 M), but was increasingly more pronounced in the presence of higher concentrations (10-5, 10-4, or 10-3 M) (Figure 3). Loadings plots identified the wavenumbers responsible for segregation of scores of spectral clusters following exposure at low-dose (10-12 M) or high-dose (10-7 M) along with the vehicle control (Figure 4). A PCA-LDA loadings plot displays the coefficients by which each of the original wavenumber variables must be multiplied to obtain the hyperspace vector passing through the median of a chosen cluster (32). It thereby picks out those spectral bands that are primarily responsible for the discrimination of those spectra from all the spectra taken as a whole. Categorydistinguishing wavenumbers and PBDE-induced effects based on cell cycle (i.e., G0/G1-phase or S-phase) were noted. To assist with the identification of such distinguishing wavenumbers, only the effects of a single low-dose (10-12 M) and high-dose (10-7 M) are shown; for others, see Supporting Information. In cell populations in G0/G1-phase, BDE47induced spectral alterations (consistently opposite to corresponding vehicle control) at 1750 cm-1 (CdO stretching

vibrations of lipids), 1650 cm-1 (Amide I), 1600 cm-1, 1550 cm-1 (Amide II), 1400 cm-1, 1350 cm-1, 1250 cm-1, 1225 cm-1 [asymmetric phosphate stretching vibration (νasPO2-)], 1150 cm-1, 1080 cm-1 [symmetric phosphate stretching vibration (νsPO2-)], 1050 cm-1, 1030 cm-1 (glycogen), and 970 cm-1 (protein phosphorylation) were noted (Figure 4). Surprisingly, low-dose BDE47 induced such profound effects, over the entire biochemical-cell fingerprint region, in cells in S-phase that no apparent distinguishing wavenumbers were associated with either the corresponding vehicle control or the 10-7 M treatment. Such cell cycle-associated susceptibility was not as apparent following BDE153 exposure (Figure 4). Following 24-h exposure, low-dose and high-dose treatments gave rise to discriminating loadings throughout the biochemical-cell fingerprint region. These effects were associated with alterations to lipids (around 1700 cm-1), the secondary structure of proteins (Amide I and Amide II), DNA conformational alterations (1225 and 1080 cm-1), RNA levels and conformation (1100 to 1150 cm-1), glycogen (1030 cm-1), and protein phosphorylation (970 cm-1) (Figure 4). Independent of cell cycle phase, high-dose BDE183 induced the most marked effects in cells in either G0/G1-phase or S-phase; distinguishing wavenumbers included 1650 cm-1 (Amide I), 1600 cm-1, 1550 cm-1 (Amide II), 1500 cm-1, 1400 cm-1 (only in cells in S-phase, but low-dose or high-dose; COOsymmetric stretching of fatty acids and amino acids), 1225 cm-1 (νasPO2-), 1080 cm-1 (νsPO2-), 1050 cm-1, 1030 cm-1 (glycogen), and 970 cm-1 (protein phosphorylation) (Figure 4). In contrast to the lower-brominated congeners, low-dose exposure to BDE209 induced the more marked effects on distinguishing wavenumbers in cells in G0/G1-phase whereas high-dose effects were more marked in cycling cells i.e., S-phase (Figure 4). Of interest is the observation that there seem to be two clusters of effect: approximately 1700 to 1500 cm-1, and 1300 to 1000 cm-1. In comparison with the corresponding vehicle control, the DNA-reactive pro-carcinogen B[a]P-induced spectral alterations at both test exposures in cell populations in G0/ G1-phase were associated with Amide I, Amide II, νasPO2-, and νsPO2-. In fact the lower exposure (10-7 M or 0.1 µM) was clearly associated with opposing effects compared to the higher level (100 × 10-7 M or 10.0 µM); the corresponding vehicle control exhibited an intermediate biochemical-cell fingerprint (Figure 5). In S-phase cells, the higher exposure appeared to be associated with the majority of the distinguishing wavenumbers. Following exposure to PhIP, very different loadings plots were generated dependent on whether cell populations were in G0/G1-phase or S-phase. In more quiescent cells, the corresponding vehicle control appeared to give rise to the most pronounced distinguishing wavenumbers whereas in S-phase cultures, the higher (10 µM) exposure gave rise to marked loadings associated with Amide I (≈1650 cm-1), 1500 cm-1, 1450 cm-1, 1400 cm-1, 1225 cm-1, 1080 cm-1, glycogen (1030 cm-1), and protein phosphorylation (970 cm-1) (Figure 5). This might point to markedly altered metabolic rates in target cells in addition to alterations in RNA/DNA conformation and the secondary structure of proteins. In contrast, E2 exposure resulted in loadings associated with vehicle control cell populations in either G0/G1-phase or S-phase being the most segregating; however, cell cycle-dependent distinguishing wavenumbers were noted at 1300 and 1100 cm-1 in cells in G0/G1-phase, while 1750 and 1700 cm-1 were most pronounced in S-phase cells (Figure 5). Following lindane exposure of cells in G0/ G1-phase, category-distinguishing wavenumbers resulted for the corresponding vehicle control and the 10-3 M group associated with Amide I (≈1650 cm-1), Amide II (1550 cm-1), 1500 cm-1, νasPO2- (1225 cm-1), νsPO2- (1080 cm-1), glycogen (1030 cm-1), and protein phosphorylation (970 cm-1). These distinguishing features were less pronounced in cells in VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. PCA-LDA scores plots following treatment with test agent, as indicated. Panels in the left column represent MCF-7 populations concentrated in G0/G1-phase; those in the right column were concentrated in S-phase. Each symbol (score) represents one independent experiment and is the average of 10 separate IR spectra acquired per slide. Confidence ellipses show probability contours assuming normal distribution for each of the clusters. Each ellipsis has a probability value of P ) 0.95. S-phase, where the lower lindane exposure (10-12 M) gave rise to marked loadings at 1750 and 1700 cm-1 (Figure 5). The ability of low-dose exposures to induce segregating characteristics in the biochemical-cell fingerprint of MCF-7 cells in either G0/G1-phase or S-phase was further examined 3360

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(see Supporting Information). In quiescent cells, three PBDEs (congeners 47, 153, and 209) gave rise to spectral scores that clustered very distinctly away from the corresponding vehicle control; those for BDE183 were less segregated. However, only spectral scores for BDE47-treated MCF-7 cells segregated

FIGURE 4. PCA-LDA loadings plots indicating wavenumber variations responsible for segregation of different class clusters following treatment of MCF-7 cells with PBDE congener, as indicated. away from the corresponding vehicle control in exponentially growing cells. In addition, B[a]P, PhIP, E2, and lindane all gave rise to spectral scores that clearly segregated away from the corresponding vehicle control in cells in G0/G1-phase, whereas the segregation was less apparent for three of these test agents (B[a]P, PhIP, and lindane), albeit clustering was still significant, in S-phase cultures.

Discussion In vitro testing is an important part of risk assessment and in short-term test systems typical concentration ranges are often high (µM to mM); consequently, extrapolating to predict cell responses to environmental exposures in vivo is difficult (35). Recently, there has been interest regarding thresholds VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. PCA-LDA loadings plots indicating wavenumber variations responsible for segregation of different class clusters following treatment of MCF-7 cells with test agent, as indicated. of effect, and the possibility that a particular concentration needs to be reached before toxicity might be induced (36, 37). A number of factors might modulate concentration-driven susceptibility to a particular test agent in vitro (35). These might include intercellular differences in susceptibility to the agent (38) or cell cycle (33, 39). In addition, different agents, sometimes despite structural similarities, may medi3362

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ate their effects via very different mechanisms that are either linearly dose-related or threshold-driven. Distinguishing such a range of different end points within a single test system is difficult. Our rationale was to examine the use of mid-IR spectroscopy to detect measurable phenotypical alterations in a concentration range defined as low-dose (e10-9 M) and high-dose (g10-7 M).

Environmental contaminants might mediate their effects via DNA reactivity, generation of reactive oxygen species, or endocrine activity (35). In general, persistent pollutants (e.g., lindane) tend to be endocrine active whereas more readily bioactivated pro-carcinogens (e.g., B[a]P or PhIP) are metabolized to electrophiles that covalently bind to nucleophilic sites on macromolecules (33, 40). A class of compounds currently generating concern regarding their possible effects are PBDEs; these brominated flame retardants are now appearing as environmental contaminants and measurable levels are detectable in human tissues (7, 8, 41). FTIR spectroscopy can identify environmental-contaminant effects in cellular macromolecules such as DNA pre- and postremediation (42). Our findings suggest that four different PBDEs (congeners 47, 153, 183, and 209) are capable of inducing phenotypical changes as determined by mid-IR spectroscopy, especially at lower exposure levels (pM to nM); it is unlikely that such levels have been rigorously tested. BDE47 induced the most marked alterations in MCF-7 cells, and such effects were associated with alterations to lipids, the secondary structure of proteins (Amide I and Amide II), DNA conformational alterations, RNA levels and conformation, glycogen, and protein phosphorylation (Figure 4). PBDE-induced alterations were often induced at concentrations lower than those for other test agents (B[a]P, PhIP, E2, or lindane) (Figure 5). Whether PBDEs mediate their effects via receptor-mediated mechanisms or as DNA reactivity remains to be ascertained. This study examined the applicability of mid-IR spectroscopy to identify the dose-related effects of a panel of test agents over a large concentration range. The approach appears capable of identifying low-dose (pM to nM) effects, much lower than those required to induce cytotoxicity. Employing PCA-LDA in order to determine segregation of clusters associated with different exposure categories further allows for the derivation of loadings plots that then facilitate the identification of distinguishing wavenumbers (43). Such profiles of distinguishing wavenumbers might allow for the identification of a spectral fingerprint allowing for risk prediction associated with unknown or new agents. The correlation of such alterations with effect (i.e., damage or stimulated proliferation) remains to be conducted to validate this experimental approach.

Acknowledgments V.L. is a NERC-CEH algorithm student (NE/F008643/1).

Supporting Information Available Additional data plots. This information is available free of charge via the Internet at http://pubs.acs.org.

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