Evaluation of Relative Potencies for in Vitro Transactivation of the

Jan 4, 2011 - As the response did not reach the maximal plateau, EC50 value for ... as Baikal seal specific TCDD induction equivalency factor (BS IEF)...
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Evaluation of Relative Potencies for in Vitro Transactivation of the Baikal Seal Aryl Hydrocarbon Receptor by Dioxin-Like Compounds Eun-Young Kim,† Tomoko Suda,‡ Shinsuke Tanabe,‡ Valeriy B. Batoev,§ Evgeny A. Petrov,^ and Hisato Iwata*,‡ †

Department of Life and Nanopharmaceutical Science and Department of Biology, Kyung Hee University, Hoegi-Dong, Dongdaemun-Gu, Seoul 130-701, Korea ‡ Center for Marine Environmental Studies, Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan § Baikal Institute of Nature Management, Siberian Branch of Russian Academy of Sciences, Sakhyanova St. 6, Ulan-Ude, Buryatia 670047, Russia ^ The Eastern-Siberian Scientific and Production Fisheries Center “VOSTSIBRYBCENTR”, Hakhalov st. 4, Ulan-Ude, Buryatia, 670034, Russia

bS Supporting Information ABSTRACT: To evaluate the sensitivity and responses to dioxins and related compounds (DRCs) via aryl hydrocarbon receptor (AHR) in Baikal seals (Pusa sibirica), we constructed an in vitro reporter gene assay system. Baikal seal AHR (BS AHR) expression plasmid and a reporter plasmid containing CYP1A1 promoter were transfected in COS-7 cells. The cells were treated with six representative congeners, and dose-dependent responses were obtained for all the congeners. EC50 values of 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 2,3,7,8-TCDF, 2,3,4,7,8-PeCDF, and PCB126 were found to be 0.021, 1.8, 0.16, 2.4, and 2.5 nM, respectively. As the response did not reach the maximal plateau, EC50 value for PCB118 could not be obtained. The TCDD-EC50 for BS AHR was as high as that for dioxin sensitive C57BL/6 mouse AHR. The in vitro dose responses were further analyzed following an established systematic framework and multiple (20, 50, and 80%) relative potencies (REPs) to the maximum TCDD response. The estimates revealed lower REP ranges (20-80%) of PeCDD and PeCDF for BS AHR than for mouse AHR. Average of the 20, 50, and 80% REPs was designated as Baikal seal specific TCDD induction equivalency factor (BS IEF). The BS IEFs of PeCDD, TCDF, PeCDF, PCB126, and PCB118 were estimated as 0.010, 0.018, 0.0078, 0.0059, and 0.00010, respectively. Total TCDD induction equivalents (IEQs) that were calculated using BS IEFs and hepatic concentrations in wild Baikal seals corresponded to only 12-31% of 2005 WHO TEF-derived TEQs. Nevertheless, about 50% of Baikal seals accumulated IEQs over the TCDD-EC50 obtained in this study. This assessment was supported by the enhanced CYP1A1 mRNA expression found in 50% of the specimens contaminated over the TCDD-EC50. These findings suggest that the IEFs proposed from this in vitro assay could be used to predict AHR-mediated responses in wild seals.

’ INTRODUCTION Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related congeners induces a broad spectrum of toxic and biological effects in vertebrates.1,2 Most of these effects are mediated by the aryl hydrocarbon receptor (AHR).3,4 The AHR bound with ligands such as TCDD leads to its translocation into the nucleus, where it forms a heterodimer with AHR nuclear translocator (ARNT). The heterodimer complex activates the transcription of multiple target genes through interaction with xenobiotic response element (XRE) located in the promoter region of these genes.5 Cytochrome P450 (CYP) 1A1 is a member of the most sensitive genes that are regulated by dioxinactivated AHR. Dioxins and related compounds (DRCs) including polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (Co-PCBs), exist in environmental and biological samples as complex mixtures of various congeners. For evaluating the potential risk associated with exposure to mixtures of DRCs, the approach using CYP1A induction equivalency factor (IEF) or toxic equivalency factor r 2011 American Chemical Society

(TEF) of each congener has been developed during the past several decades.6-8 The IEF and TEF are derived from the potency (REP) of individual DRC congeners for producing biological and/or toxic effects relative to a reference congener, TCDD.8,9 The comprehensive TEFs were initially proposed by the WHO working group in 1994.7 The TEFs were separately assigned for mammals, birds and fish in 1998.8 The WHO mammalian TEF values were further refined in 2005.10 The IEFs and TEFs are applied for estimating the total TCDD CYP1Ainduction equivalents (IEQs) and toxic equivalents (TEQs), respectively, which are defined as the sum of the concentration of each congener multiplied by its corresponding IEF/TEF. The known mammalian TEFs are considered to be applicable for risk assessment on humans, since the TEF values are mainly based on rodent studies.8 However, there are orders of magnitude Received: September 1, 2010 Accepted: December 14, 2010 Revised: December 9, 2010 Published: January 4, 2011 1652

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Environmental Science & Technology variations of IEFs/TEFs, even among closely related mammalian species (e.g., rats and mice). The WHO working group has attempted to harmonize TEFs across different species to the extent possible. Nevertheless, total synchronization of TEFs was not feasible and the need for separate sets of TEFs for wildlife was manifested.8 To apply the TEF concept, knowledge on the mechanisms of action among DRC congeners is a prerequisite. To date, there is sufficient evidence indicating that AHRmediated response is a common mechanism for DRC toxicities in various species.8 Meanwhile, the species differences in IEFs/ TEFs/REPs may be mostly accounted for by the structural and functional difference in AHR.5 However, the sensitivity to DRCs in most of the wild species is not well-known, because legal and/ or ethical concerns preclude the direct testing of toxic chemicals in these animals. Hence, alternative approaches focusing on AHR are necessary for assessing the sensitivity to DRCs and their risk in such animals. Baikal seals (Pusa sibirica) inhabiting the Lake Baikal, Russia, accumulate high levels of DRCs in their tissues.11 Baikal seals may be at high risk by DRCs, as indicated by the mass mortality event in 1987.12 It has been suggested that the viral epizootic affecting seals was triggered by impairment of the seals’ immune system by environmental pollutants like DRCs.13 These reports have led to a great concern over contamination with DRCs and the health of seals. However, the effect of DRCs on Baikal seals has not fully been assessed, because of the lack of investigations on the hazardous potency of DRCs on this species. Our previous study has isolated and sequenced AHR and ARNT cDNAs from the Baikal seal.14,15 Moreover, another study has also indicated that hepatic CYP1A1 is induced by the accumulation of DRCs in this species.16 Following these past investigations, the objective of this study is to provide a means to evaluate the sensitivity and AHRmediated responses to exposure of DRC congeners to Baikal seals. Initially, mRNA expressions of Baikal seal AHR (BS AHR) were certified in various tissues. The binding affinity of TCDD to in vitro synthesized BS AHR was demonstrated. We then estimated 50% effective concentrations (EC50) and REPs of major DRC congeners for BS AHR-mediated responses using an in vitro reporter gene assay. Baikal seal induction equivalency factor (BS IEF) for each DRC congener was determined from the respective REP, and hepatic total IEQs were calculated using the BS IEFs. Based on these results, we discussed the sensitivity to exposure of DRC congeners in this species, and validated the utility of the BS REPs/IEFs.

’ EXPERIMENTAL SECTION DRC Congeners. Standard solutions including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-pentachlorodibenzop-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), 3,30 ,4,40 ,5-pentachlorobiphenyl (PCB126), and 2,30 ,4,40 ,5-pentachlorobiphenyl (PCB118) were purchased from AccuStandard Inc. (New Haven, CT, USA). The congeners examined were selected because they have greater potentials to elicit AHR-mediated toxic responses8,10 and were found to accumulate in wild Baikal seals through the food web.11 Animals. Baikal seals were collected from the Lake Baikal in cooperation with the Limnological Institute of the Russian Academy of Sciences in 1992 and 2005. Biometric and other details of these seals have been reported elsewhere11,16,17 and are also given in the Supporting Information.

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Tissue Expression Profiles of AHR and ARNT mRNAs. Detailed procedures are given in the Supporting Information. Plasmid Constructs. Detailed procedures are given in the Supporting Information. In vitro AHR Protein Synthesis and TCDD Binding Assay. Details of procedures have been reported elsewhere18,19 and are also given in the Supporting Information. Luciferase Reporter Gene Assay. All luciferase assays were performed using the Dual-Luciferase reporter gene assay system according to the manufacturer’s protocol (Promega) and also as explained by Lee et al.20 Detailed procedures are given in the Supporting Information. To fulfill the criteria of inclusion in a TEF/IEF scheme proposed by Van den Berg et al.,8 TCDD was included as a reference compound in the same batch of assays, when we measured the transactivation of reporter gene by congeners other than TCDD. The BS AHR reporter assays were carried out 7-8 times for graded concentrations of each congener, and the mouse AHR assays were repeated 4 times. Estimation of EC50, REP, and IFF. The responsiveness of BS AHR and mouse AHR was assessed by luciferase reporter gene assays with graded concentrations of TCDD and other congeners. The transcriptional activation potentials of both AHRs by each congener were evaluated from the dose-response curves. Prior to the analysis of dose-responses, the mean vehicle control response was subtracted from both TCDD and other congener responses, and responses expressed in relative luminescence values were converted to the percentage of the mean maximum response observed for the TCDD standard (% TCDD max) in order to scale the values from 0 to 100% std. max. Responses expressed as % TCDD max were plotted as a function of logarithmically transformed concentrations of the congeners. EC50 of each DRC congener was calculated from the doseresponse curve using Prism 4 (GraphPad, San Diego, CA, USA). Using the systematic framework and REP estimation methods proposed by Villeneuve et al.,21 REPs of individual congeners to TCDD were estimated. REPx was defined as the ratio of concentration of TCDD that induces x% TCDD max (TCDDECx) relative to the concentration of a given congener that induces the response corresponding to x% TCDD max (see Figure S1, Supporting Information). Because the dose-response curves may not be parallel between the examined congeners and TCDD, only the ranges of concentrations at which linearity of responses based on log-doses was observed both for TCDD and a given congener were 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 > 0.90 was obtained by least-squares regression. The linear regression model for each congener was composed of at least three points. Following the recommendation by Villeneuve et al.,21 the range of response over which REPs are calculated was defined as 20-80% (REP20-80 range) of the maximum response achieved for the standard compound, TCDD (20-80% TCDD max). For congeners whose observed maximum response were less than 80% TCDD max, an additional REP estimate was calculated at the observed maximum response. The IEFs of congeners examined were determined as the average values of REP20, REP50, and REP80, since use of a single REP estimate may result in misleading and/or inaccurate interpretations. Hepatic Concentrations of DRCs. Hepatic concentrations of individual congeners of PCDDs/DFs and Co-PCBs in wild Baikal seals have already been reported elsewhere.11 TEQs and 1653

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IEQs were calculated by using 2005 WHO mammalian TEFs10 and BS IEFs of individual congeners proposed in this study, respectively. Hepatic mRNA Levels of CYP1A1. CYP1A1 mRNA expression levels in the liver of Baikal seals have already been given in our previous study.16 Statistical Analyses. Spearman’s rank correlation coefficient was applied to evaluate the strength of the association between BS IEF-based IEQ levels and CYP1A1 mRNA expression levels in the liver of Baikal seals. A p-value of less than 0.05 was regarded to be statistically significant. The StatView ver. 5.0 (SAS Institute, Cary, NC, USA) was used for these statistical analyses.

’ RESULTS AND DISCUSSION Tissue Expression Profiles of AHR and ARNT. The expression profiling certified that BS AHR and ARNT genes were expressed in all tissues examined in Baikal seals (see Figure S2, Supporting Information). The variability of the expression level of each gene between the two animals was small. Relatively high levels of AHR and ARNT mRNAs were observed in the lung. Higher ARNT expression was also detected in the cerebellum. These expression profiles of BS AHR and ARNT were similar to those of rats and humans.22,23 Thus, it can be assumed that, in the tissues where AHR and ARNT are highly expressed, the AHR and ARNT signaling pathway is susceptibly modulated by the exposure to DRCs. TCDD Binding to AHR. A strict criterion for application of the IEF/TEF concept is that the candidate compound must be shown to bind to the AHR.8 Hence, BS AHR protein was synthesized by an in vitro transcription/translation system. A similar expression experiment was also performed for BS ARNT. Gel electrophoresis of the [35S]methionine-labeled translation products represented a single band with the predicted molecular weight for each protein (BS AHR 94.6 kDa and BS ARNT 86.3 kDa) (see Figure S3, Supporting Information). The in vitro translated AHR was then incubated with [3H]TCDD and separated by velocity sedimentation on sucrose density gradients. By comparing the [3H]TCDD-derived signal with nonspecific binding-derived signal detected using an empty vector (UPL), a signal from [3H]TCDD-specific binding to the BS AHR was obtained (see Figure S3, Supporting Information). Construction of Reporter Gene Assay. To confirm whether the transcription of reporter gene can be enhanced in an AHRdependent manner, the transactivation potency of AHR was compared with that of control (empty vector) with “no AHR” (see Figure S4, Supporting Information). The luciferase activities (as relative luciferase unit) were found to become higher in the DMSO- and 14 nM TCDD-treated cells, when each AHR plasmid was transfected. This demonstrates that the elevated transcription of reporter gene is dependent on the transfected AHR. The TCDD-induced luciferase activities in cells transfected with BS or mouse AHR were also greater than those of the respective DMSO controls. On the other hand, BS AHR had about 50% transactivation even in the absence of TCDD, when compared with 14 nM TCDD-induced activity. In this regard, C57BL/6 mouse AHR showed only less than 20% transactivation potency in DMSO-treated cells relative to TCDD-induced activity. This means that the BS AHR assay displayed almost 2-fold maximum induction and resulted in relatively high variability of the transactivation, although the reason that BS AHR had such a high basal transactivation as compared to mouse AHR remains

Figure 1. Transcriptional activities of Baikal seal and mouse AHRs in cells treated with individual DRC congeners. COS-7 cells were cotransfected with expression plasmids for BS AHR (A) or mouse AHR (B), and for BS ARNT and a reporter plasmid containing four XREs of mouse Cyp1a1 gene, pGudLuc6.1. After the transfections, cells were treated with DMSO or graded concentrations of each DRC congener. Y-axis represents relative luminescence values of tested congeners that were converted to a percentage of the mean maximum response observed for TCDD standard (% TCDD max). The responses are plotted as a function of logarithmically transformed congener concentrations (nM). Each plot represents means ( SD of responses of each congener from 7 or 8 experiments for BS AHR, and 4 experiments for mouse AHR.

unresolved. To reduce the uncertainties in the estimates of EC50 values and REPs that may arise from high variability in the BS AHR-mediated responses, these estimates for individual congeners were drawn from 7 or 8 repeated BS AHR reporter assays, while the mouse AHR assays were repeated 4 times. EC50 for AHR Transactivation by DRC Congeners. For all congeners examined in this study, BS and mouse AHRs were activated in a dose-dependent manner. Differences in the dose responses among congeners were apparent. To visualize the relative potency of each congener, the activation potential of each AHR was evaluated by a dose-response curve that was plotted as a relative unit to the maximum response of TCDD (% TCDD max) (Figure 1). TCDD-EC50 for BS AHR was estimated to be 0.021 nM (6.8 pg/g wet wt) which was lower than that (0.057 nM, 18 pg/g wet wt) of C57BL/6 mouse AHR (Table 1). This indicates that the Baikal seal may be at least as sensitive to TCDD as the mouse. This result agrees well with some previous reports showing that TCDD-binding affinity of harbor seal AHR, which has 98% amino acid identity with BS AHR, was as high as that of C57BL/6 mouse AHR.14,18 Similarly, the luciferase activities of other dioxin-like congeners were measured (Figure 1, Table 1). PeCDD increased XREdriven reporter gene via both AHRs. PeCDD-EC50 values of Baikal seal and mouse AHRs were 1.8 nM (640 pg/g wet wt) and 0.14 nM (50 pg/g wet wt), respectively, showing 13-fold higher 1654

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values for Baikal seal AHR than mouse AHR. TCDF-EC50 values for Baikal seal and mouse AHRs were estimated to be 0.16 nM (49 pg/g wet wt) and 0.22 nM (67 pg/g wet wt), respectively. PeCDF also transactivated Baikal seal and mouse AHRs in a dose-dependent manner, but the EC50 values were 11-fold higher for BS AHR (2.4 nM, 820 pg/g wet wt) than for mouse AHR (0.21 nM, 71 pg/g wet wt). PCB126-EC50 values of Baikal seal and mouse AHRs were almost comparable, representing 2.5 nM (820 pg/g wet wt) and 4.1 nM (1300 pg/g wet wt), respectively. As for PCB118, the EC50 values for both AHRs were not obtained because the responses did not reach the maximal plateau even at the highest dose tested. Overall, EC50 values of TCDD, TCDF, and PCB126 for BS AHR were as low as those of the dioxin-sensitive C57BL/6 mouse AHR, but PeCDD- and PeCDF-EC50 for BS AHR were apparently higher than those of the mouse AHR. REPs and IEFs. For estimating REPs of the Baikal seal and mouse, the dose-response data were further analyzed using the systematic framework and multiple-point REP estimation methods proposed by Villeneuve et al.21 We initially made each dose-response curve fit for a linear regression model. Results showed that responses of all congeners were greater than 20% TCDD max for AHRs of both species (Figure 1). For BS AHR, maximal plateau responses were achieved for TCDD, PeCDD, TCDF, PeCDF, and PCB126, but not for PCB118, one of the mono-ortho Co-PCB congeners. The mouse AHR assay also

exhibited that only PCB118 did not attain the maximal plateau. Because of the limited solubility of the congeners in tested solution, assays were not performed at higher concentrations. Using the linear regression models of TCDD and other congeners, multiple point estimates (REP20, REP50, and REP80) were then performed to generate REP20-80 ranges for each congener (Table 2). The REP20-80 ranges were valid for PeCDD, PCB126, and PCB118 in BS AHR-mediated responses. TCDF and PeCDF congeners did not reach 80% TCDD max. Alternatively, a slightly lower value of 72% TCDD max was attained for both congeners. For mouse AHR, REP20-80 ranges were available for congeners other than PCB118, and instead 62% TCDD max was given for PCB118. As a result, for BS AHR, the REP20-80 ranges of PeCDD, PCB126, and PCB118 were very small (ratios of REP20/REP80 = 1.1-2.3), suggesting parallel slopes of regression lines for the dose responses between these congeners and TCDD. In contrast, the REP20-80 ranges of TCDF and PeCDF were relatively large (REP20/REP80 22 for TCDF and 66 for PeCDF), reflecting the fact that there are uncertainties up to 1 order of magnitude for these REPs due to deviations from parallelism to the slope of the regression line for TCDD standard. For mouse REPs, the REP20-80 ranges of tested congeners were fairly small (REP20/REP80 = 0.2-12), indicating only limited uncertainties for the REPs. For mouse AHR in the present study, REP values of PeCDD, TCDF, PeCDF, PCB126, and PCB118 were similar to those in the 2004 database for REPs estimated by in vitro CYP1A induction bioassay9 (Table 2). Average REPs of congeners excluding PeCDF were within the ranges of REP values summarized in the 2004 database, and average REP value (0.14) and REP20 of PeCDF were also very close to the lower range of REP distribution (0.28). The REP values of mono-ortho PCBs have shown 4-5 orders of magnitude of variation in the literature.9 Nevertheless, the REP for PCB118 obtained in this study is comparable to the 50th percentile of REP distribution of this congener in the 2004 database.9 These comparisons thus likely ensure the quality of our present methodology and data. REPs of tested congeners for BS AHR were as follows: TCDD > PeCDD, TCDF, PeCDF, PCB126 > PCB118, whereas REPs for mouse AHR were in the order TCDD, PeCDD > TCDF, PeCDF > PCB126 > PCB118 (Table 2). REP20-80 of PCB118 for BS AHR were higher than those for mouse AHR. REP20-80 of TCDF and PCB126 for BS AHR were comparable to those for

Table 1. EC50 Values of Six Selected Congeners Based on Baikal Seal and Mouse AHR-Mediated Transcriptional Activation in in vitro Reporter Gene Assays EC50 (nM) congener

Baikal seal

mouse

2,3,7,8-TCDD

0.021

0.057

1,2,3,7,8-PeCDD

1.8

0.14

2,3,7,8-TCDF

0.16

0.22

2,3,4,7,8-PeCDF PCB126

2.4 2.5

0.21 4.1

NEa

PCB118

NE

a

NE: EC50 was not estimated because the response did not reach the maximum plateau even at the highest dose tested.

Table 2. Comparison of in vitro REPs of Six Selected Congeners Obtained in This and Other Studiesa congener

Baikal seal REP20

REP50

REP databaseb

mouse REP80

REP20

REP50

REP80 1 1.3

min 1 0.095

max

2,3,7,8-TCDD 1,2,3,7,8-PeCDD

1 0.015

1 0.0095

1 0.0063

1 0.30

1 0.64

1 1.1

2,3,7,8-TCDF

0.045

0.0082

0.0020c

0.34

0.098

0.029

0.0070

0.63

2,3,4,7,8-PeCDF

0.021

0.0025

0.00031c

0.28

0.10

0.038

0.28

2.0

PCB 126

0.0063

0.0059

0.0056

0.0050

0.0056

0.0062

0.0036

0.64

PCB 118

0.00013

0.00010

0.000081

0.000049

0.000026

0.000014d

0.0000020

0.00080

a Following the REP estimation methods proposed by Villeneuve et al.,21 REPx was calculated as the ratio of concentration of TCDD that induces x% TCDD max (TCDD-ECx) relative to concentration of a given congener that induces the response corresponding to x% TCDD max. For congeners whose observed maximum response were less than 80% TCDD max, an additional REP estimate was calculated at the observed maximum response. b Data summarized by Haws et al. 9 In vitro REPs for CYP1A induction including AHH, EROD, and CALUX, etc. are extracted. c REP80 was replaced by the REP72 values because the maximum respose did not reach 80% TCDD max. d REP80 was replaced by the REP62 value because the maximum respose did not reach 80% TCDD max.

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Table 3. Comparison of Baikal Seal IEFs, Mouse IEFs, and 2005-WHO TEFs of Six Selected Congeners congener 2,3,7,8-TCDD 1,2,3,7,8-PeCDD 2,3,7,8-TCDF 2,3,4,7,8-PeCDF

Baikal seal IEF

mouse IEF

1

1

0.010 ( 0.0042

0.76 ( 0.52

2005-WHO TEF 1 1

0.018 ( 0.023

0.16 ( 0.16

0.1

0.0078 ( 0.011

0.14 ( 0.13

0.3

PCB126

0.0059 ( 0.00036

0.0056 ( 0.00062

PCB118

0.00010 ( 0.000025

0.000029 ( 0.000018

mouse AHR. On the other hand, REP20-80 of PeCDD and PeCDF for BS AHR were remarkably lower than those for mouse AHR. Average value of 20, 50, and 80% REPs for each congener was defined as IEF (Table 3). BS IEFs (mean ( standard deviation) of PeCDD, TCDF, PeCDF, PCB126, and PCB118 were estimated to be 0.010 ( 0.0042, 0.018 ( 0.023, 0.0078 ( 0.011, 0.0059 ( 0.00036, and 0.00010 ( 0.000025, respectively. For the mouse, the estimated IEFs were 0.76 ( 0.52 for PeCDD, 0.16 ( 0.16 for TCDF, 0.14 ( 0.13 for PeCDF, 0.0056 ( 0.00062 for PCB126, and 0.000029 ( 0.000018 for PCB118. Compared to the mouse IEFs, the BS IEFs for PeCDD, TCDF, and PeCDF were 1-2 orders of magnitude lower, while the BS IEFs for PCB126 and PCB118 were similar to and higher than the corresponding mouse IEFs, respectively. These results imply that the relative contribution of effects by PeCDD, TCDF, and PeCDF in Baikal seals would be lesser than those in mouse AHRtype mammals. We then compared BS IEFs with mammalian 2005-WHO TEFs (Table 3). BS IEFs for PeCDD, TCDF, PeCDF, and PCB126 were 1-2 orders of magnitude lower than the WHO TEFs, while PCB118 had an IEF similar to the WHO TEF. The differences between these two sets of IEFs/TEFs may be possibly accounted for by the following reasons: (1) the mammalian WHO TEF values are mainly based on rodent and human studies, (2) uncertainty of REPs derived from deviations from parallelism to the regression line of TCDD dose response, (3) in vivo toxicity data were given more weight than in vitro data, and toxic effects were more prioritized than biochemical effects, (4) pharmacokinetic properties are ruled out in in vitro assay, although WHO TEFs are assigned on the basis of intake level (administered dose) rather than residue level (tissue concentration). For TCDF and PeCDF, the difference may be partly accounted for by the second reason, deviations from the parallelism. We discussed earlier that BS REPs of TCDF and PeCDF involve orders of magnitude of uncertainties. For PeCDD and PCB126, the difference could not be explained by deviations from the parallelism, because the dose-responses of these congeners were almost parallel to that of TCDD (REP20/ REP80 ratios were 2.3 for PeCDD and 1.1 for PCB126). Besides, there is a difference in similar (1-2 orders of) magnitude even between BS REPs and in vitro REPs that are summarized by Haws et al.9 In addition, there is only a small difference between in vitro and in vivo REP values of these congeners.9 Given these observations, it can be presumed that the differences in IEFs/ TEFs of PeCDD/PCB126 are arising from the species difference in responses. Risk Assessment in Wild Baikal Seal Population. Studies addressing mixtures of DRCs have indicated that their toxicity can be predicted fairly well and also support the use of the TEF methodology.24,25 These previous findings led us to apply the

0.1 0.00003

TEF/IEF methodology for assessing the risk of DRCs in wild Baikal seal population. The present TEF scheme and TEQ methodology are primarily meant for estimating exposure via dietary intake because TEFs are largely based on oral uptake studies through diet.10 Hence, application of these “intake or ingestion” TEFs for calculating the TEQ in animal tissues has limited use for risk assessment, unless pharmacokinetic aspects of DRC congeners is taken into account. To overcome the limited application and to enable TEQ estimates on the basis of tissue concentrations, in vitro-derived TEFs can potentially be useful rather than in vivo-derived TEFs. For most toxicological studies on wild species other than the human, it would be more realistic to measure the tissue concentrations than the ingestion amounts. Since we have measured hepatic concentrations of DRCs in wild Baikal seals,11 we attempted to assess the risk of DRCs by using the in vitroderived IEFs obtained in this study. In the present study, we focused on the risk of DRCs in the liver of Baikal seals because we have already clarified that some DRC congeners are sequestered in the liver and CYP1A is induced in the microsome fractions.11,16 Hepatic total IEQ levels in Baikal seals were calculated by summing up the concentration of each congener (TCDD, TCDF, PeCDD, PeCDF, PCB126, and PCB118) multiplied by the respective BS IEF. We compared the IEQs with the TEQs derived from mammalian 2005-WHO TEFs (see Figure S5, Supporting Information). The IEQs corresponded to only 12-31% of the WHO TEFs-derived TEQs. The estimates of IEQs clearly indicate that total TEQ levels in this species are overestimated when the classic WHO TEFs are used in combination with tissue concentrations of DRCs. To evaluate the risk of DRCs, the frequency distribution of the hepatic total IEQs (pg/g on wet weight basis) in the Baikal seal population were compared with the TCDD-EC50 (6.8 pg/g on wet weight basis) proposed in the present study (see Figure S6, Supporting Information). Although the BS IEF-based IEQs were much lower than the WHO-based TEQs, the IEQs in about 50% of the wild seals used in the present study exceeded the TCDDEC50 for BS AHR. These results indicate that half of Baikal seal population might have been exposed to the DRC levels that are sufficient for AHR activation. This in vitro approach is further supported by data viewed from another angle: the BS IEF-based IEQs had a significant positive correlation (p < 0.01) with CYP1A1 mRNA expression levels in the wild Baikal seal population (Figure 2). This figure also indicates that ideally CYP1A1 mRNA expression is higher in 50% of specimens contaminated over the TCDD-EC50. These findings suggest that the IEFs generated from the established in vitro assay could be used as an appropriate predictor of AHR-mediated responses in wild seals. There are no data on pathologies that are likely caused by DRCs in this seal species. However, it is accepted that AHR mediates a 1656

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’ ASSOCIATED CONTENT

bS

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

’ AUTHOR INFORMATION Corresponding Author

*Mail: Hisato Iwata, Center for Marine Environmental Studies, Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan; e-mail: [email protected]. Figure 2. Relationship between BS IEF-derived total IEQ levels and CYP1A1 mRNA expression levels in the liver of wild Baikal seals. The total IEQs (on wet weight basis) were obtained by summing up the hepatic concentration of each of six congeners (TCDD, TCDF, PeCDD, PeCDF, PCB126, and PCB118) multiplied by the respective BS IEF. Data on CYP1A1 mRNA expression levels are cited from Hirakawa et al.16 The correlation analysis was performed by Spearman’s rank correlation test. TCDD-EC50 (6.8 pg/g on wet weight basis) estimated for BS AHR in this study is indicated by an arrow.

variety of toxic responses associated with DRC exposure, and that CYP1A induction in the AHR signaling pathway is a sensitive indicator that can integrate the potency of a mixture of DRCs to elicit toxicities. Taken together, we conclude that Baikal seals should have experienced threats from DRCs through the AHR. To our knowledge, this is the first report on REPs and IEFs based on the transactivation of AHR from aquatic mammalian species. The present study demonstrated that the BS IEF profile is likely to be distinct from that of mammalian WHO TEF, although the mechanism that can account for the difference between these two data sets remains unknown. Comparative studies on the affinity to AHR, binding to XRE, and cofactor recruitment among congeners may provide mechanistic data to understand the different transactivation potencies toward certain DRCs. Given that the TCDD-EC50 in Baikal seals is relatively low among the species tested so far and that the amino acid corresponding to Ala at position 375 in a dioxinsensitive mouse (C57BL/6) AHR is also Ala in BS AHR,14 the BS AHR may be highly sensitive to TCDD. On the other hand, seals may be less sensitive to other congeners that were examined in this study. This consequently led to lower IEQs in this study than TEQs that can be predicted from WHO assigned TEFs. Nevertheless, since TCDD-EC50 for the seal AHR transactivation was low, the IEQs in about 50% of the wild seals used in the present study exceeded the TCDD-EC50. Although the WHO TEF approach has been conventionally useful for hazard and risk assessment of DRCs in humans (e.g., ref 26), we propose here that a more feasible IEF/TEF approach is necessary to weigh the species-specific response and susceptibility to DRCs in various wild animals. As an alternative application in animals in which direct-dosing tests are ethically and economically difficult, the in vitro reportergene assay constructed with AHR cDNA from the species of concern can be a valuable tool for evaluating the responses and susceptibility to DRCs. Moreover, in vitro-derived TEFs/IEFs would be more practical for assessing the risk of DRCs on a tissue concentration basis than on a food intake basis.

’ ACKNOWLEDGMENT We thank Prof. An. Subramanian for critical reading of this manuscript. This study was supported by Grants-in-Aid for Scientific Research (S) (21221004 and 20221003). Financial assistance was also provided by Global COE Program from the Ministry of Education, Culture, Sports, Science and Technology and JSPS, Japan, and by Basic Research in ExTEND2005 from the Ministry of the Environment of 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) Fernandez-Salguero, P.; Pineau, T.; Hilbert, D. M.; McPhail, T.; Lee, S. S. T.; Kimura, S.; Nebert, D. W.; Rudikoff, S.; Ward, J. M.; Gonzalez, F. J. Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 1995, 268 (5211), 722–726. (2) Smith, A. G.; Clothier, B.; Carthew, P.; Childs, N. L.; Sinclair, P. R.; Nebert, D. W.; Dalton, T. P. Protection of the Cyp1a2(-/-) null mouse against uroporphyria and hepatic injury following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 2001, 173 (2), 89–98. (3) Poland, A.; Glover, E.; Kende, A. S. Stereospecific, high-affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. J. Biol. Chem. 1976, 251 (16), 4936–4946. (4) Sogawa, K.; Fujii-Kuriyama, Y. Ah receptor, a novel ligandactivated transcription factor. J. Biochem. 1997, 122 (6), 1075–1079. (5) Hahn, M. E. The aryl hydrocarbon receptor: A comparative perspective. Comp. Biochem. Physiol., Part C: Pharmacol., Toxicol. Endocrinol. 1998, 121 (1-3), 23–53. (6) Safe, S. H. Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans. Annu. Rev. Pharmacol. Toxicol. 1986, 26, 371–399. (7) Ahlborg, U. G.; Becking, G. C.; Birnbaum, L. S.; Brouwer, A.; Derks, H. J. G. M.; Feeley, M.; Golor, G.; Hanberg, A.; Larsen, J. C.; Liem, A. K. D.; et al. Toxic equivalency factors for dioxin-like PCBs: Report on WHO-ECEH and IPCS consultation. Chemosphere 1994, 28 (6), 1049–1067. (8) Van den Berg, M.; Birnbaum, L.; Bosveld, A. T.; Brunstrom, B.; Cook, P.; Feeley, M.; Giesy, J. P.; Hanberg, A.; Hasegawa, R.; Kennedy, S. W.; et al. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ. Health Perspect. 1998, 106 (12), 775–92. (9) Haws, L. C.; Su, S. H.; Harris, M.; Devito, M. J.; Walker, N. J.; Farland, W. H.; Finley, B.; Birnbaum, L. S. Development of a refined database of mammalian relative potency estimates for dioxin-like compounds. Toxicol. Sci. 2006, 89 (1), 4–30. (10) Van den Berg, M.; Birnbaum, L. S.; Denison, M.; De Vito, M.; Farland, W.; Feeley, M.; Fiedler, H.; Hakansson, H.; Hanberg, A.; Haws, L.; et al. The 2005 World Health Organization reevaluation of human 1657

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