Transactivation Potencies of the Baikal Seal (Pusa sibirica

Mar 7, 2011 - The present study assessed the transactivation potencies of the Baikal seal (Pusa sibirica) peroxisome proliferator-activated receptor Î...
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Transactivation Potencies of the Baikal Seal (Pusa sibirica) Peroxisome Proliferator-Activated Receptor r by Perfluoroalkyl Carboxylates and Sulfonates: Estimation of PFOA Induction Equivalency Factors Hiroshi Ishibashi,† Eun-Young Kim,‡ and Hisato Iwata*,† † ‡

Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan Department of Life and Nanopharmaceutical Science and Department of Biology, Kyung Hee University, Hoegi-Dong, Dongdaemun-Gu, Seoul 130-701, Korea

bS Supporting Information ABSTRACT: The present study assessed the transactivation potencies of the Baikal seal (Pusa sibirica) peroxisome proliferator-activated receptor R (BS PPARR) by perfluorochemicals (PFCs) having various carbon chain lengths (C4-C12) using an in vitro reporter gene assay. Among the twelve PFCs treated with a range of 7.8-250 μM concentration, eight perfluoroalkyl carboxylates (PFCAs) and two perfluoroalkyl sulfonates (PFSAs) induced BS PPARR-mediated transcriptional activities in a dose-dependent manner. To compare the BS PPARR transactivation potencies of PFCs, the present study estimated the PFOA induction equivalency factors (IEFs), a ratio of the 50% effective concentration of PFOA to the concentration of each compound that can induce the response corresponding to 50% of the maximal response of PFOA. The order of IEFs for the PFCs was as follows: PFOA (IEF: 1) > PFHpA (0.89) > PFNA (0.61) > PFPeA (0.50) > PFHxS (0.41) > PFHxA (0.38) ≈ PFDA (0.37) > PFBA (0.26) = PFOS (0.26) > PFUnDA (0.15) . PFDoDA and PFBuS (not activated). The structure-activity relationship analysis showed that PFCAs having more than seven perfluorinated carbons had a negative correlation (r = -1.0, p = 0.017) between the number of perfluorinated carbons and the IEF of PFCAs, indicating that the number of perfluorinated carbon of PFCAs is one of the factors determining the transactivation potencies of the BS PPARR. The analysis also indicated that PFCAs were more potent than PFSAs with the same number of perfluorinated carbons. Treatment with a mixture of ten PFCs showed an additive action on the BS PPARR activation. Using IEFs of individual PFCs and hepatic concentrations of PFCs in the liver of wild Baikal seals, the PFOA induction equivalents (IEQs, 5.3-58 ng IEQ/g wet weight) were calculated. The correlation analysis revealed that the hepatic total IEQs showed a significant positive correlation with the hepatic expression levels of cytochrome P450 4A-like protein (r = 0.53, p = 0.036). This suggests that our approach may be useful for assessing the potential PPARR-mediated biological effects of complex mixtures of PFCs in wild Baikal seal population.

’ INTRODUCTION Perfluorochemicals (PFCs) such as perfluoroalkyl carboxylates (PFCAs) and perfluoroalkyl sulfonates (PFSAs) are persistent contaminants that have been ubiquitously detected in human and wildlife.1-4 Contaminants in this group of PFCs are not only limited to perfluorooctanoic acid (PFOA, C8) and perfluorooctane sulfonate (PFOS, C8) but also include commercially available short/long-chain PFCs (C2-C18).5-7 However, studies on the toxicological properties of PFCs have been mostly focused on PFOA and PFOS. Previous reviews have shown that PFOA and PFOS can cause increase in liver weight, peroxisome proliferation, and hepatocellular adenomas in experimental animals.8,9 It is also well-known that chronic peroxisome proliferation induced by chemical exposure is associated with the promotion of tumors in the liver of rats and mice.10 These results r 2011 American Chemical Society

suggest that these adverse effects may be mediated by the peroxisome proliferator-activated receptor R (PPARR) signaling pathway.8,9 PPARR is a member of the ligand-activated nuclear hormone receptor superfamily, in which three different isoforms, PPARR, PPARβ/δ, and PPARγ, have been identified so far.11-13 In response to specific ligands, PPARR heterodimerizes with retinoid X receptor R (RXRR), interacts with the peroxisome proliferator-responsive element (PPRE) in the promoter region of target genes, and consequently regulates the transcription. Received: November 7, 2010 Accepted: February 23, 2011 Revised: February 15, 2011 Published: March 07, 2011 3123

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Table 1. Transactivation Potencies of Baikal Seal PPARr by Perfluoroalkyl Carboxylates (PFCAs) and Perfluoroalkyl Sulfonates (PFSAs) chemical PFCAs PFBA

full name

perfluorobutyric acid

perfluorinated

purity

REC50a

Efficacy b

REP20-80 rangec

molecular formula

carbon number

(%)

(μM)

(% of PFOA)

(REP20/REP80)

IEFd

CF3(CF2)2COOH

3

0.42-0.16

0.26

98g

273

53 ( 6

(2.66) PFPeA

perfluoropentanoic acid

CF3(CF2)3COOH

>98h

4

142

78 ( 25

0.56-0.44

0.50

(1.27) PFHxA

perfluorohexanoic acid

CF3(CF2)4COOH

>98h

5

189

64 ( 9

0.54-0.26

0.38

(2.08) PFHpA

perfluoroheptanoic acid

CF3(CF2)5COOH

99g

6

132 ( 17

0.87-0.92

71e

1 0.61

79

100

(0.95) 1

PFNA

perfluorononanoic acid

CF3(CF2)7COOH

8

>98

117

106 ( 26

0.53-0.69

PFDA

perfluorodecanoic acid

CF3(CF2)8COOH

9

98g

190

75 ( 11

PFOA

perfluorooctanoic acid

CF3(CF2)6COOH

7

>98h h

0.89

(0.78) 0.31-0.45

0.37

(0.69) PFUnDA

perfluoroundecanoic acid

CF3(CF2)9COOH

10

95g

488

32 ( 10

0.33-0.06

PFDoDA PFSAs

perfluorododecanoic acid

CF3(CF2)10COOH

11

97g

NAj

18 ( 15

—

—

PFBuS

perfluorobutane

CF3(CF2)3SO3K

4

98g

NAj

10 ( 1

—

—

CF3(CF2)5SO3K

6

>98g

173

74 ( 18

0.40-0.41

0.41

0.15f

(5.22)

sulfonate PFHxS

perfluorohexane sulfonate

PFOS

perfluorooctane sulfonate

(0.98) CF3(CF2)7SO3K

98i

8

271

45 ( 5

0.27-0.25

0.26

(1.08)

50% relative effective concentration (REC50); concentration of the test compounds showing 50% of the relative luciferase activity of 250 μM PFOA. Percent of luciferase activity maximally induced by each PFC relative to the maximum luciferase activity of PFOA (250 μM). c PFOA X % relative potency (REPx) = PFOA-ECX/a given chemical-RECX. d PFOA induction equivalency factor (IEF) = PFOA-EC50/a given chemical-REC50. e 50% effective concentration. f Predicted value with uncertainly. g Data from Sigma-Aldrich, St. Louis, MO, USA. h Data from Tokyo Chemical Industry Ltd., Tokyo, Japan. i Data from Wako Pure Chemical Industries, Ltd., Tokyo, Japan. j NA: not activated. a b

Studies on PPARR gene knockout mice demonstrated PPARRmediated regulation of cytochrome P450 (CYP) 4A that can catalyze the oxidation of fatty acids including arachidonic acid and other eicosanoids.14,15 The expression of rat CYP4A mRNA/protein is induced by exposure to PFOA.16 Several cellbased in vitro reporter assay systems also revealed the activation of the human, mouse, and rat PPARR by PFCs including PFOA and PFOS.17,18 However, information on such effects is available mostly for PFOA and PFOS. Only a few studies on the PPARR activation by other PFCs have been done.18 The Baikal seal (Pusa sibirica), a top predator in Lake Baikal, Russia, accumulates high levels of dioxins and related compounds.19,20 In addition to these contaminants, our recent study has shown that this seal species is contaminated with perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA), suggesting the induction of hepatic expression levels of PPARR mRNA and CYP4A protein by these chemicals.21 We also constructed an in vitro reporter gene assay system where the Baikal seal PPARR (BS PPARR) expression vector and a PPRE containing reporter vector were transiently transfected in CV-1 cells. Results showed PPARR-mediated transactivation potencies by selected PFCs, such as PFOS, PFOA, PFNA, PFDA, and perfluoroundecanoic acid (PFUnDA).22 These results indicate that BS PPARR-mediated response may be a useful biomarker to

evaluate potential biological effects of PFCs in this species. However, there is no comprehensive study on the BS PPARR activation by other shorter/longer-chain analogues. In this study, we investigated the transactivation potencies of BS PPARR by PFCAs and PFSAs with various carbon chain lengths (C4-C12) using the in vitro reporter gene assay. The structure-activity relationship was assessed for the BS PPARR activation by PFCs. This study also examined the effects of a complex mixture of PFCs on the BS PPARR transactivation. In addition, the present study estimated the PFOA induction equivalency factors (IEFs) for the BS PPARR transactivation potencies by selected PFCs. To validate the utility of the IEFs, the relationship of hepatic total PFOA induction equivalent (IEQ) levels with CYP4A-like protein expression levels was examined in wild Baikal seals.

’ MATERIALS AND METHODS Chemicals. A total of 12 PFCs including 9 PFCAs and 3 PFSAs and bezafibrate (2-[4-[2-[(4-chlorobenzoyl)amino]ethyl] phenoxy]-2-methylpropanoic acid, a potent agonist of the PPARR) were treated as ligands in this study (Table 1). The distributors and purity of test compounds are shown in Table 1. For all the test compounds, 250 mM stock solutions (bezafibrate: 3124

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Environmental Science & Technology 40 mM stock solution) were prepared by dissolving each chemical in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA), and the final DMSO concentration was adjusted to 0.1% w/v. For in vitro reporter gene assays, test solutions of PFCs (final concentration: 7.8-250 μM) and bezafibrate (0.6340 μM) were prepared by serially diluting the stock solutions with the medium for cell culture. To evaluate the effects of a mixture of PFCs, a PFC solution including equal molar concentrations of PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFHxS, and PFOS (final concentration of each PFC: 1.56-50 μM) was also prepared. Luciferase Reporter Gene Assay. The luciferase reporter gene assay for the BS PPARR transactivation was performed as previously described22 with some modifications. In this study, we used CV-1 cells, which express RXR endogenously.23 Detailed procedures can be found in the Supporting Information (SI). Relative Effective Concentration, Relative Potency, and Induction Equivalency Factor. For BS PPARR transactivation, X % effective concentration (ECX) of PFOA as a standard chemical was calculated from a dose-response curve using GraphPad Prism version 5.0b (GraphPad Software, Inc., San Diego, CA). To estimate the relative potencies of PPARR-dependent transactivation of the test compounds, the concentration of each compound that can induce the response corresponding to X % of the maximal response of PFOA (X% PFOA max) was defined as X % relative effective concentration (RECX). The X % potency of each test compound relative to the maximal response of PFOA (PFOA X % relative potency, REPx) was calculated as the ratio of PFOA-ECX to RECX of the compound. As the dose-response curves may not be parallel between the test compounds and PFOA, ranges of concentrations at which linearity of responses based on log-doses was observed both for PFOA and a given congener were only considered. For making a linear regression model fit, the linear portion of each dose response was defined by dropping points from the tails until an R2 > 0.95 was obtained by least-squares regression. Following the recommendation by Villeneuve et al.,24 the standard range of response over which REPs are calculated was defined as 20 to 80% (REP20-80 range) of the maximum response achieved for the reference compound, PFOA (20-80% PFOA max). For compounds whose observed maximum responses were more than 20%, but less than 50% and 80% PFOA max., the REP50 and REP80 values were extrapolated from the linear regression model of PFC concentration and % PFOA max. Compounds which had less than 20% PFOA max. were considered as inactive for BS PPARR transactivation. Since the use of a single REP estimate may result in misleading and/or inaccurate interpretations, the multiple point estimates (MPE), over the range of responses from REP20 to REP80 (REP20-80 range), can be used for evaluating the parallelism; the REP20-80 range is directly proportional to the degree of deviation from the parallelism between test chemical and standard dose-response curves.24 Thus, in this study we calculated the REP20/REP80 ratio of each test compound. When the parallelism of the dose response for the test compound was confirmed (REP20/REP80 ratio 3.0), REP50 was regarded as a predicted IEF value with uncertainty due to nonparallel slopes. Induction Equivalents and CYP4A Protein Levels. Data on hepatic concentrations of six PFCs (PFOA, PFNA, PFDA, PFUnDA, PFDoDA, and PFOS) (SI Table S1) and CYP4A-like

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protein expression levels in 16 Baikal seals (8 males and 8 females) have been reported elsewhere.21,22 The PFOA induction equivalents (IEQs) in the liver of the seals were obtained by summing up the hepatic concentrations of the six PFCs multiplied by the respective IEFs. Statistical Analysis. Statistical analyses were performed using SPSS 16.0J (SPSS Japan, Tokyo, Japan). For the in vitro reporter gene assay, the experimental data were initially analyzed by Kolmogorov-Smirnov and Levene’s test to evaluate the normality and the homogeneity of variance. Given the result of initial analyses, differences in luciferase activities between groups treated with vehicle or test chemicals were analyzed by a oneway analysis of variance (ANOVA) followed by Dunnett’s post hoc test. Spearman’s rank correlation test was performed to assess the relationships between the IEFs of PFCs and the respective perfluorinated carbon numbers. The relationship between hepatic IEQ levels and CYP4A-like protein levels in the liver of Baikal seals was also examined by Spearman’s rank correlation test. Statistical significance was regarded as p < 0.05.

’ RESULTS AND DISCUSSION Reporter Gene Assay System. Our earlier study constructed an in vitro reporter gene assay where pcDNA3.2TOPO plasmid with BS PPARR cDNA and pGL3-based reporter plasmid were transiently transfected in CV-1 cells. Treatment with each PFOA, PFNA, PFDA, PFUnDA, and PFOS has demonstrated induction of the BS PPARR-dependent transcriptional activity.22 However, a weak but steady induction by these compounds was also observed in CV-1 cells transfected with an empty plasmid where no PPARR cDNA was inserted. This implied that endogenous transcription factors other than PPARR in CV-1 cells may be involved in the PFC-dependent transactivation.22 It was recently shown that newly developed pGL4 vector has reduced numbers of transcription factor binding sites in its backbone compared with pGL3 vector.25 Hence, we constructed pGL4.23-(PPRE)3Luc as a reporter plasmid in this study. No significant induction of the luciferase activity was observed by using this pGL4-based construct, in non-PPARR transfected cells treated with individual PFCs (Figure 1A-1K). The luciferase activity in non-PPARR transfected cells treated with individual PFOA, PFNA, PFDA, and PFUnDA isomers increased only up to a maximum of 1.4-, 1.2-, 1.7-, and 1.8-fold, respectively (Figure 1E, 1F, 1G, and 1H). The maximum fold induction values under the pGL4-based construct were lower than those under the pGL3-based construct (2.0 for PFOA, 2.0 for PFNA, 3.0 for PFDA, and 4.0 for PFUnDA).22 The only exception was seen for PFOS. Exposure to 250 μM PFOS resulted in a 3.5-fold increase in no PPARRexpressed cells, although the fold induction was lower than that in PPARR-expressed cells (Figure 1L). Overall, the reduction of the PPARR-independent responses indicates that the pGL4.23(PPRE)3-Luc reporter plasmid is more suitable for evaluating the PPARR-dependent transcriptional activity than the pGL3-based reporter plasmid. This study also investigated transactivation potency of BS PPARR by bezafibrate, a fibrate drug used for the treatment of hyperlipidaemia, which is also a potent agonist of human and rodent PPARRs.26 Treatment with bezafibrate induced PPARRmediated transcriptional activity in a dose-dependent manner (SI Figure S1). The lowest-observed-effect concentration (LOEC) and EC50 value of bezafibrate were found to be 2.5 and 4.4 μM, respectively. In several published reports,26,27 EC50 values of 3125

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Figure 1. Transcriptional activities of Baikal seal PPARR by each PFBA (A), PFPeA (B), PFHxA (C), PFHpA (D), PFOA (E), PFNA (F), PFDA (G), PFUnDA (H), PFDoDA (I), PFBuS (J), PFHxS (K), and PFOS (L) using in vitro reporter gene assay. Bars indicate the fold induction of transcriptional activities in CV-1 cells into which non-PPARR (white bars) or Baikal seal PPARR (black bars) expression vector was transfected. As a positive control, 20 μM of bezafibrate (BF) was used. Data are presented as the mean and standard deviation of triplicate assays. Asterisk denotes statistical difference (p < 0.05) from the transcriptional activities in control cells treated with DMSO (vehicle).

bezafibrate for the mouse and human PPARR were reported to be 17-90 μM. The difference in the EC50 values may be explained by species differences in response to bezafibrate via PPARR, although direct comparison of the data obtained in the previous and present studies was difficult due to different experimental conditions such as cell type, reporter gene, and incubation time. These results indicate that, as a typical PPARR agonist, bezafibrate activates PPARR of diverse species of mammals

including the Baikal seal, and also that our constructed reporter gene system works well as well as those constructed for human and rodent PPARRs.28 Transactivation of PPARr by Individual PFCs. We investigated the transactivation potencies of the BS PPARR by PFCAs and PFSAs having various carbon chain lengths (C4-C12) using the in vitro reporter gene assay. Among the 12 PFCs tested, eight PFCAs (PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, 3126

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Environmental Science & Technology and PFUnDA) and two PFSAs (PFHxS and PFOS) induced BS PPARR-mediated transcriptional activity in a dose-dependent manner (in a range of 7.8-250 μM) (Figure 1). PFDoDA and PFBuS did not increase the response significantly (Figure 1I and 1J), showing less than 20% efficacy (Table 1). To estimate the relative potencies of BS PPARR-dependent transactivation of the test compounds, the concentration of each compound that can induce the response corresponding to 50% PFOA max was defined as REC50 values (Table 1). The 50% potency of each test compound relative to the maximal response of PFOA (PFOA REP50) was calculated as the ratio of PFOAEC50 to REC50 of the compound (Table 1). This approach has been applied to estimate the relative potency of a wide variety of chemicals, such as dioxin-like and estrogenic compounds, by in vitro bioassays.24,29 However, the single point estimate, such as REP50, can lead to misleading and/or inaccurate interpretations, when dose-response curves between standard and test compounds are not parallel.24 In contrast, the multiple point estimates (MPE), over the range of responses from REP20 to REP80 (REP20-80 range), can be used for evaluating the nonparallel relationship.24 Thus, in this study we initially calculated the REP20/REP80 ratio of each test compound to verify the parallelism of the dose-response. PFCs including PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFHxS, and PFOS indicated low (0.69-2.66) REP20/REP80 ratios, and only PFUnDA showed relatively high value (5.22) (Table 1). In addition, efficacies of PFCs except PFDoDA and PFBuS were more than 20% PFOA max (Table 1, SI Figure S2 and S3). A previous study24 has recommended that MPE methods can be used to calculate an approximate REP20-80 range, if the observed maximum response for the sample is greater than 20% PFOA max. Evaluation of the parallelism and efficacy indicates that the uncertainties of IEFs from the single point estimate (REP50) are substantially low for PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFHxS, and PFOS, and in the case of PFUnDA the uncertainty was up to five. IEFs of PFDoDA and PFBuS were not estimated, because the efficacies of these compounds were less than 20% PFOA max. The order of IEF estimated from REC50 values for the transactivation of BS PPARR for the twelve PFCs was as follows: PFOA (IEF: 1) > PFHpA (0.89) > PFNA (0.61) > PFPeA (0.50) > PFHxS (0.41) > PFHxA (0.38) ≈ PFDA (0.37) > PFBA (0.26) = PFOS (0.26) > PFUnDA (0.15) . PFDoDA and PFBuS (not activated) (Table 1). It is known that PFOA and PFNA are potent activators of the mouse/human PPARRs.18 However, to our knowledge, no study is available on the potential effects of PFHpA and PFPeA in animals. Our previous study showed the presence of PFHpA in the serum of wild Baikal seals, although the PFHpA concentration was much lower than the most dominant compound, PFNA.21 For PFHpA and PFPeA, more attention should be paid to investigate the contamination levels in animal bodies. Structure-Activity Relationship. The molecular models based on the crystal structure analysis of the ligand-binding domain of PPARs have demonstrated binding affinity of human PPARR with short/long-chain fatty acids.30,31 Nonetheless, limited information is available on the structure-activity relationship of short/long-chain PFCs for PPARR activation.18 To evaluate the structure-activity relationship of PFCs and BS PPARR activation, we investigated the relationship between the activation potencies of PFCs and the respective perfluorinated carbon numbers. Spearman’s rank correlation test showed

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Figure 2. Relationship between the perfluorinated carbon number of PFCs and the PFOA induction equivalency factor (IEF) based on the Baikal seal PPARR transactivation. PFCAs with more than seven perfluorinated carbons (indicated in the circle) had a negative correlation with IEFs (r = -1.0, p = 0.017). Asterisk denotes the predicted IEFs of the test chemicals for which the dose-responses were not paralleled with that of PFOA.

that PFCAs with more than seven perfluorinated carbons had a negative correlation (r = -1.0, p = 0.017) with IEFs (Figure 2). On the other hand, PFCAs with less than seven perfluorinated carbons had a tendency for increase with IEFs (Figure 2). A previous study has demonstrated that transactivation potencies of mouse/human PPARRs increased with the number of perfluorinated carbons of PFCAs (PFNA: 8 > PFOA: 7 > PFDA: 9 > PFHxA: 5 > PFBA: 3).18 These findings together with our results suggest that the perfluorinated carbon number of PFCAs is one of factors determining the transactivation potencies of the BS PPARR. In addition, the comparison of the PPARR transactivation potencies of PFCAs and PFSAs with same number of perfluorinated carbons (PFPeA vs PFBuS, PFHpA vs PFHxS, and PFNA vs PFOS) revealed that the potency of the PFCA was higher than that of the PFSA (Figure 2, Table 1). Similar findings were also reported in mouse or human PPARR-expressed cells treated with PFNA and PFOS.18 It has been documented that the hydrogen bond between the carboxylate group of GW409544 (human PPARR agonist) and the human PPARR protein acts as a molecular switch to activate the transcriptional activity of the receptor.31 Our results support this model. Effects of a Mixture of PFCs. Our previous studies have demonstrated that the hepatic concentrations of individual PFCs in wild Baikal seal population were orders of magnitude lower than the respective LOEC levels for BS PPARR.21,22 However, assuming that a mixture of PFCs may additively or synergistically contribute to the transcriptional activation of BS PPARR, even at lower concentrations of each compound, total PFC concentrations may reach the level in which BS PPARR is activated. Hence, this study investigated the effects of a mixture of PFCs on the BS PPARR transcativation. A mixture containing equimolar concentrations of ten PFCs induced PPARR-mediated transcriptional activity in a dose-dependent manner (Figure 3A). Estimated LOEC values (6.25 μM of each concentration) (Figure 3A) of the ten PFC mixtures were 10-40 times lower than those (62.5-250 μM) of each PFC (Figure 1). To further evaluate whether the transactivation potencies of BS PPARR by a mixture of PFCs are additive or not, we compared the dose-response curves of PFOA and total PFOA IEQs. The comparison showed similar dose-response curves between total PFOA IEQs and 3127

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Figure 4. Relationship between total PFOA induction equivalents (IEQs) and CYP4A-like protein expression levels in the liver of wild Baikal seals. Total PFOA IEQs were obtained by summing up hepatic concentrations of six PFCs (PFOA, PFNA, PFDA, PFUnDA, PFDoDA, and PFOS) in wild Baikal seals21 multiplied by the respective IEFs. Data on relative CYP4A-like protein expression levels were cited from our previous study.22 The expression level of CYP4A-like protein in each sample was expressed as a relative value of staining intensity from the antibody cross-reactive protein in a given sample.

Figure 3. (A) Transcriptional activity of Baikal seal PPARR by a mixture of ten PFCs (PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFHxS, and PFOS) using in vitro reporter gene assay and (B) comparison of the dose-response curve of a mixture of ten PFCs (black circles) with that of PFOA (white circles). Bars indicate the fold induction of transcriptional activities in CV-1 cells into which nonPPARR (white bars) or Baikal seal PPARR (black bars) expression vector was transfected. Data are presented as the mean and standard deviation of triplicate assays. Asterisk denotes the detection of a statistical difference in DMSO (vehicle) control cells at the level of p < 0.05. Total PFOA induction equivalents (IEQs) were obtained by summing up the test concentrations of the ten PFCs (PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFHxS, and PFOS) multiplied by the respective IEFs.

PFOA (Figure 3B). This result indicates that a mixture of 10 PFCs acts on the BS PPARR in an additive manner. Meanwhile, during treatment with the highest dose tested (50 μM of each PFC), the fold induction slightly declined (Figure 3A), implying that some PFCs at high dosages may have antagonistic potential for BS PPARR. PFOA Induction Equivalent Approach. Studies on experimental animals have demonstrated that clofibrate and PFOA induced CYP4A expression level in the liver of rodents, whereas no such an effect was observed in PPARR knockout mice.15,16 Our earlier studies also indicated that PFNA and PFDA, which are predominantly detected in wild Baikal seals, induced hepatic CYP4A-like proteins via PPARR signaling. On the other hand, no significant relationship was observed between total concentrations of ten PFCs and expression levels of hepatic CYP4A-like protein.22 This implies that the transactivation potency might be different for individual PFCs. The toxic equivalency factor (TEF) approach developed by the World Health Organization has been

widely applied to quantify 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalents for complex mixtures of aryl hydrocarbon receptor ligands like dioxins and related compounds.32,33 However, this approach is yet to be applied for evaluating the exposure of complex mixtures of PPARR ligands like PFCs. Thus, the present study attempted to estimate PFOA IEFs for transactivation potencies of BS PPARR by selected PFCs. We then calculated PFOA IEQ using the estimated PFOA IEFs and the respective PFC concentrations in the liver of wild Baikal seals.21 For validating the IEF approach, we further examined the relationship between hepatic IEQ levels and CYP4A-like protein expression levels in the seals. Total PFOA IEQs were found to be in the range of 5.3-58 ng IEQ/g wet weight in the liver of Baikal seals (SI Table S1). Comparison of the IEQ of individual PFCs showed that the contribution of PFNA (54%) to the total IEQs was the most dominant, followed by PFOS (16%), PFDA (14%), PFOA (11%), and PFUnDA (5.4%). Spearman’s rank correlation analysis showed that the total IEQ levels had a significant positive correlation (r = 0.53, p = 0.036) with the hepatic CYP4A-like protein levels (Figure 4). This result was in contrast with our previous findings which showed no such significant relationship when we used total PFC concentrations for calculation.22 On the other hand, the total PFOA IEQ levels in the wild Baikal seal population were much lower than the PFOA LOEC (62.5 μM = 26 μg/g) for BS PPARR-mediated transactivation, although the cause(s) that fill the gap remains unknown. These results suggest that certain PFC mixtures induce hepatic CYP4A-like proteins via PPARR signaling in Baikal seals, and also this IEF approach may be useful for assessing the potential PPARR-mediated effects of complex mixtures of PFCs on wild population. In conclusion, this is the first study in which the transactivation potencies of PPARR by multiple PFCs in aquatic mammals were clarified. The in vitro reporter gene assay driven by BS PPARR reveals high activation potencies by PFOA and PFHpA and 3128

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Environmental Science & Technology additive action by a mixture of PFCs. Structure-activity relationship analysis clearly indicated that the BS PPARR transactivation mostly depends on the number of perfluorinated carbon of PFCAs. The PFOA IEQ was able to predict the induction of CYP4A-like protein by multiple PFCs in the liver of wild Baikal seals. The PFOA IEF approach derived from in vitro PPARR transactivation may be applicable not only for this species but also to other wildlife for assessing environmental risk associated with exposure to PFCs.

’ ASSOCIATED CONTENT

bS

Supporting Information. Information on additional descriptions of experimental procedures and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone/Fax: þ81-89-927-8172. E-mail: [email protected].

’ ACKNOWLEDGMENT The authors thank Prof. An. Subramanian, Ehime University, for critical reading of this manuscript. This study was supported by Grant-in-Aid for Scientific Research (S) (no. 21221004) from Japan Society for the Promotion of Science, and “Global COE Program” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and Japan Society for the Promotion of Science (JSPS). Financial assistance was also provided in part by Basic Research in ExTEND2005 (Enhanced Tack on Endocrine Disruption) from the Ministry of Environment, Japan. This research was also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology to E-Y.K. (2010-0012150). ’ REFERENCES (1) Giesy, J. P.; Kannan, K. Global distribution of perfluorooctane sulfonate in wildlife. Environ. Sci. Technol. 2001, 35, 1339–1342. (2) Kannan, K.; Koistinen, J.; Beckmen, K.; Evans, T.; Gorzelany, J. F.; Hansen, K. J.; Jones, P. D.; Helle, E.; Nyman, M.; Giesy, J. P. Accumulation of perfluorooctane sulfonate in marine mammals. Environ. Sci. Technol. 2001, 35, 1593–1598. (3) Kannan, K.; Corsolini, S.; Falandysz, J.; Fillmann, G.; Kumar, K. S.; Loganathan, B. G.; Mohd, M. A.; Olivero, J.; Van Wouwe, N.; Yang, J. H.; Aldous, K. M. Perfluorooctanesulfonate and related fluorochemicals in human blood from several countries. Environ. Sci. Technol. 2004, 38, 4489–4495. (4) Van de Vijver, K. I.; Hoff, P. T.; Das, K.; Van Dongen, W.; Esmans, E. L.; Jauniaux, T.; Bouquegneau, J. M.; Blust, R.; de Coen, W. Perfluorinated chemicals infiltrate ocean waters: link between exposure levels and stable isotope ratios in marine mammals. Environ. Sci. Technol. 2003, 37, 5545–5550. (5) Martin, J. W.; Smithwick, M. M.; Braune, B. M.; Hoekstra, P. F.; Muir, D. C.; Mabury, S. A. Identification of long-chain perfluorinated acids in biota from the Canadian Arctic. Environ. Sci. Technol. 2004, 38, 373–380. (6) Butt, C. M.; Mabury, S. A.; Muir, D. C.; Braune, B. M. Prevalence of long-chained perfluorinated carboxylates in seabirds from the Canadian Arctic between 1975 and 2004. Environ. Sci. Technol. 2007, 41, 3521–3528. (7) Houde, M.; Martin, J. W.; Letcher, R. J.; Solomon, K. R.; Muir, D. C. Biological monitoring of polyfluoroalkyl substances: A review. Environ. Sci. Technol. 2006, 40, 3463–3673.

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