In Vitro Transactivation Potencies of Black-Footed Albatross

ARTICLE pubs.acs.org/est

In Vitro Transactivation Potencies of Black-Footed Albatross (Phoebastria nigripes) AHR1 and AHR2 by Dioxins To Predict CYP1A Expression in the Wild Population Thuruthippallil Leena Mol,† Eun-Young Kim,‡,§ Hiroshi Ishibashi,† 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: Our previous studies have detected high levels of dioxins and related compounds (DRCs) including polychlorinated dibenzop-dioxins (PCDDs), furans (PCDFs), and coplanar PCBs (Co-PCBs) in the black-footed albatross (BFA), Phoebastria nigripes, from the North Pacific region. We have also cloned two aryl hydrocarbon receptors, AHR1 and AHR2, of the BFA. To evaluate the sensitivity to DRCs in the BFA and to assess the status of cytochrome P450 1A (CYP1A) induction in the wild population, this study investigated the mRNA expression levels of BFA AHR1 and AHR2 and also the transactivation potencies of each AHR by 15 selected DRC congeners. Quantitative real-time PCR of BFA AHR mRNAs showed that hepatic AHR1 is more highly expressed than AHR2. Transactivation by graded concentrations of individual DRCs was measured in COS-7 cells, where BFA AHR1 or AHR2 was transiently transfected. For congeners that exhibited AHR-mediated dose-dependent activities, 50% effective concentration (EC50) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) relative potencies (REPs) were estimated. Based on the estimates of the REPs, TCDD induction equivalency factors (IEFs) were determined. For BFA AHR1, PeCDF was equipotent to TCDD, but other congeners exhibited lower IEFs. For BFA AHR2, PCDD/ F congeners except OCDD/F showed IEFs g 1.0. Using BFA AHR1- or AHR2-IEFs and hepatic concentrations of DRCs in North Pacific BFAs, TCDD induction equivalents (IEQs) were calculated. We further constructed nonlinear regression models on the relationships between BFA AHR1- or AHR2-IEF derived total IEQ or WHO-TEF derived total TEQ and ethoxyresorufinO-deethylase activity (EROD) in the liver of wild BFAs. The results indicated that the relationships of BFA AHR1- and AHR2-based IEQs and EROD were predictable from BFA AHR1- and AHR2-mediated transactivation by TCDD, respectively. Collectively, these results suggest that the in vitro assay incorporating the AHR of species of concern would be a useful tool to predict the sensitivity to DRCs in the species and CYP1A induction in the wild population.

’ INTRODUCTION Dioxins and related compounds (DRCs), including polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (Co-PCBs), induce various toxic and biological effects.1 One such effect is cytochrome P4501A (CYP1A) induction.2,3 Toxic effects of DRCs are triggered by aryl hydrocarbon receptor (AHR), a ligand-dependent transcription factor.4
6 Several in vivo studies have demonstrated that CYP1A induction is a sensitive biomarker for 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) toxicities in avian species.7 Van den Berg et al.8 and Head and Kennedy9 reviewed relationships of relative potencies of DRCs for CYP1A (or its enzymatic activity, ethoxyresorufinO-deethylase [EROD]) induction with avian embryonic mortalities and concluded that there is a positive correlation between them. Thus it is rational to think that CYP1A induction is a key event that can evaluate the potential effects of DRCs. AHR and its homologues are known to be present in a variety of animal species. Fish and birds possess at least two types of r 2011 American Chemical Society

AHRs, designated AHR1 and AHR2, while mammals have only one AHR.10
13 Several studies have indicated species differences in AHR-CYP1A pathways in birds, fish, and mammals from quantitative (as the number of AHR/ARNT/CYP1A isoforms) and qualitative (as the sensitivity and efficacy for transactivations) viewpoints.14
17 Hence, the molecular characterization of avian AHR-CYP1A signaling is of interest, as this would help to unveil the evolutionary history of AHR from fish to mammals. The complex mixture of DRCs complicates the risk and hazard assessments. To address this issue, a concept of TCDD toxic equivalency factors (TEFs) has been introduced by the World Health Organization (WHO). Using the TEFs proposed for individual DRCs, the overall TCDD toxic equivalent (TEQ) is Received: August 13, 2011 Accepted: November 10, 2011 Revised: November 5, 2011 Published: November 10, 2011 525

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estimated as the sum of TCDD equivalent of each congener.8,18 The WHO avian-specific TEFs were assigned based on various in vivo and in vitro studies mainly on the chicken and have been applied to calculate the TEQs in wild birds exposed to a complex mixture of DRCs.8 Some avian populations are exposed to high levels of DRCs, which provides a rationale for investigating whether accumulated DRCs activate AHR-CYP1A signaling pathway in these populations.19,20 On the other hand, it is known that there are dramatic differences among avian species in their sensitivity to DRCs.15,21
23 Hence, investigations are indispensable to evaluate the effects of DRCs via AHR in each species. However, for rare/endangered species, in vivo toxicity experiments are ethically and technically difficult due to the requirement of a large number of birds/eggs. To overcome this, the application of expression plasmids containing AHR cDNA of a given target species to in vitro reporter gene assay has proven to be an alternative approach.13,15,24 Twenty two species of albatrosses are currently in the IUCN red list of endangered species including the black-footed albatross (BFA), Phoebastria nigripes from the North Pacific.25 Contamination by hazardous chemicals is likely to be one of the potential threats to BFA. We have detected high levels of DRCs (120
570 pg/g wet wt.) in the liver of this species. A significant positive correlation was also found between concentrations of some DRC congeners and EROD activities in the North Pacific BFA population, indicating CYP1A induction by DRCs.20 This suggests that individuals with higher TEQs in this population might have experienced a greater threat from DRCs over several decades. Our previous study also demonstrated the presence of AHR1 and AHR2 isoforms in the BFA, their specific binding abilities to [3H] labeled TCDD, and CYP1A5 transactivation potencies by TCDD.13 Together with the vulnerability of albatrosses, these results prompt us to evaluate the sensitivity to DRCs and their risk in wild BFAs. This study investigated the CYP1A transactivation potencies by individual PCDD/Fs and Co-PCBs via each BFA AHR using the in vitro reporter gene system we have constructed.13 We estimated the lowest observable effect concentration (LOEC), 50% effective concentration (EC50), and relative potencies to TCDD (REP) of each DRC congener for BFA AHR1- and AHR2mediated responses. The BFA TCDD induction equivalency factors (IEFs) for individual congeners were determined from the REPs for BFA AHR1 and AHR2, and hepatic total TCDD induction equivalents (IEQs) were calculated using the BFA IEFs. Based on these results, we discussed the sensitivity to DRCs in the BFA and validated the utility of the BFA REPs/IEFs for predicting the status of CYP1A induction by DRCs in the wild population. We also compared the BFA AHR1- and AHR2-IEFs with WHO bird TEFs. Relative abundance of AHR1 and AHR2 mRNA expressions were also measured in the liver tissues of wild BFAs, and the contribution of each AHR to CYP1A induction by DRCs was discussed based on the abundance.

Table S1. It is likely that some impurities in OCDD (1.8%) and OCDF (2.0%) have no or negligible potencies, since no or very low responses have been observed in the cells treated with these standard solutions. The result also showed that all PCBs except PCB126 (only 0.3%) have no impurities. This suggests that the effects of impurities on the transactivation of CYP1A5 promoter sequences are minimal. The congeners examined were selected, as they have greater potentials to elicit AHR-mediated toxic responses, and were found to be accumulated in wild BFA population from the North Pacific.20,26 For all the test compounds, stock solutions were prepared by dissolving each chemical with dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA). For in vitro reporter gene assays, test solutions of individual congeners were prepared by serially diluting the stock solutions with the medium for cell culture. The final DMSO concentration in the test solutions was adjusted to 0.1%. Samples. BFAs caught from the North Pacific Ocean in 1995 as previously described12,13,20 were used in our experiments. After dissection on board the research vessel, subsamples of liver were immediately frozen in liquid nitrogen and stored at
80 °C until total RNA isolation. Liver subsamples from 11 different BFA specimens were used for the measurement of EROD activity,20 and those from selected 6 specimens were for the quantification of AHR mRNA expression level. Plasmid Construction and Luciferase Reporter Gene Assay. The BFA AHR1 or AHR2 and common (great) cormorant (Phalacrocorax carbo) AHR nuclear translocator 1 (ARNT1) expression plasmids were previously constructed by inserting the respective full length cDNAs into pcDNA3.1/Zero(+) vector (Invitrogen).12,15,27 Procedures of the luciferase reporter gene assay for the AHRmediated transactivation have already been reported elsewhere13,15 and are briefly described in the SI. Quantitative RT-PCR. In order to compare the relative mRNA expression levels of AHR1 and AHR2 in BFA livers, a two-step real time RT-PCR was performed with known concentrations of a standard plasmid DNA in which each AHR cDNA fragment was inserted into pGEM-T easy vector. Each plasmid solution was quantified by spectrophotometry. Total RNA extracted from BFA livers was used for determining the absolute levels of mRNA expression of each AHR isoform. Aliquots of the reverse transcription reactions containing cDNA from 200 ng of total RNA were used as templates for the amplification by PCR. The method of quantitative RT-PCR is described in the SI. Estimation of REP and IEF. The responsiveness of BFA AHR1 and AHR2 was assessed by luciferase reporter gene assays with graded concentrations of TCDD and other congeners. The transcriptional activation potentials of each congener for both BFA AHRs were evaluated from the dose
response curves. Prior to the evaluation of dose
responses, the mean vehicle control response (RLU value) from at least three independent experiments was subtracted from both TCDD and other congener responses, and the RLU 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% TCDD max. Responses expressed as % TCDD max were plotted as a function of logarithmically transformed concentrations of the congeners. Using the systematic framework and REP estimation methods proposed by Villeneuve et al. 28 REPs of individual congeners to TCDD were calculated. REPx was defined as the ratio of concentration of TCDD that induces X% TCDD max relative to the concentration of a given congener that induces the

’ MATERIALS AND METHODS Standard Solutions of DRC Congeners. Standard solutions of a total of 15 DRC congeners including 5 PCDDs, 5 PCDFs, and 5 Co-PCBs, purchased from AccuStandard Inc. (Connecticut, USA), were used as test compounds in this study. The purity of each tested compound that has been verified with a gas chromatograph
mass spectrometry by the provider is given in 526

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response corresponding to X% TCDD max.24 Since 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 used. 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. The linear regression model for each congener was composed of at least three points. Following the recommendation by Villeneuve et al.,28 the range of response over which REPs are calculated was defined as 20 to 80% (REP20
80 range) of the maximum response achieved for the standard compound, TCDD (20
80% TCDD max). Only in the case that the efficacy of dosedependent response of a given congener was more than 20% TCDD max, REPs were calculated. Congeners that had less than 20% TCDD max were regarded to be inactive for the BFA AHR transactivation, and those REPs were not given. In the case that the observed maximum response for a given congener was less than 80% of TCDD max, but more than 20%, extrapolation beyond the range of the empirical linearity was made to estimate REP80. For parallel dose responses, REP estimates are anticipated to be ideally independent of the response level selected. Hence, the REP20/REP80 ratio of each congener was determined to validate the parallelism of the linear dose
response between the congener and TCDD. A limited range of REP20/REP80 ratio (0.1
10) indicates that the slope of the regression line of a given congener is nearly parallel to that of TCDD standard, suggesting a less uncertainty for the REP. In contrast, a diverse range of REP20/REP80 ratio (10) reflects a large uncertainty for the REP due to the deviation from parallelism to the slope of the regression line for TCDD. When the parallelism of the dose response for the examined congener was confirmed (REP20/ REP80 ratio = 0.1
10), REP50 was regarded as TCDD IEF. For congeners whose parallelism was not confirmed (REP20/REP80 ratio 10), REP50 was given as a predicted IEF with large uncertainties, since the use of a single REP estimate may result in misleading and/or inaccurate interpretations. The IEFs obtained for AHR1 and AHR2 of the BFA were compared with WHO avian TEFs. Estimation of LOEC and EC50. To estimate LOEC of each congener, RLU values were compared between solvent (DMSO) control and individual dosage groups by Posthoc Dunnett’s test. EC50 of each DRC congener for AHR1 or AHR2 transactivation (as RLU value converted to % TCDD max) was calculated from a nonlinear regression analysis (log-transformed dose vs response, variable slope mode) using Prism 5.0 (GraphPad, San Diego, CA, USA). Estimation of IEQs. Total BFA AHR1- and AHR2-IEQs were calculated by summing up the concentrations of individual congeners in the BFA livers20 multiplied by the respective AHR1and AHR2-IEFs. Similarly, total TEQ was obtained using the congener’s concentrations and WHO avian TEFs. Statistical Analyses. Details of statistical analyses are described in the SI.

Figure 1. Hepatic mRNA expression levels of BFA AHR 1 and AHR2. Messenger RNA expression level of each AHR in the liver of BFAs was quantified by two-step real-time RT-PCR. A significant difference in expression levels between AHR1 and AHR2 was detected by Student’s t test.

that AHR1 mRNA level was about 20-fold higher than the AHR2 mRNA, indicating that AHR1 is the dominant AHR isoform in the liver of BFAs (Figure 1). The relative prevalence of AHR1 expression in BFA livers implies that the differences in sensitivity to TCDD may depend primarily on the function of AHR1 in this species. However, we do not rule out that BFA AHR2 showed clear dose-dependent responses to most of the congeners, as noted in detail below. Although AHR2 mRNA expression levels in BFA livers were about 20-fold lower than AHR1, such a quantitative relation may be tissue- and/or cell-specific; if some tissues possess AHR2 as a dominant isoform, it is likely that AHR2 would participate in the induction of DRC toxicities. There is no information yet on the tissue expression profile for each AHR mRNA in the BFA. The tissue expression profiles of cormorant AHR1 and AHR2 have been investigated in our previous study. Higher and similar AHR1 mRNA expression levels were detected in the cormorant tissues other than the muscle and pancreas.13 On the other hand, AHR2 mRNA was mainly expressed in the liver and was detectable in the gonad, brain, and intestine. Knowledge on the expression of both BFA AHRs will lead to a better understanding of possible distinct roles in dioxin sensitivity and toxicity. To date, it is obvious that the expression level of AHR has not been incorporated as a factor when IEQ/TEQ is calculated.8,18 However, the presence of multiple AHRs with different expression levels in birds and fish may need a further refinement of IEQ/TEQ concepts. AHR1- and AHR2-Mediated Transactivation Potencies. BFA AHR1-mediated transactivation potency was measured in COS-7 cells treated with individual PCDD/F and Co-PCB congeners. The EC50 values for these congeners were determined from the dose-dependent response curves (Figure 2A-C). TCDD and PeCDD generated clear dose-dependent responses via AHR1, and the EC50 values were estimated as 0.077 nM (25 pg/g wet wt) and 1.8 nM (640 pg/g wet wt), respectively. Although TCDF and PeCDF showed higher efficacies than TCDD and induced responses in a dose-dependent manner, complete sigmoidal curves were not attained even at the highest doses. Hence the EC50 values were not estimated for these congeners. HxCDD, HxCDF, and HpCDD showed responses only at the respective highest doses. HpCDF dose-dependently enhanced the luciferase activity with 0.25 nM (100 pg/g wet wt) of EC50, but the efficacy was lower than that of TCDD. OCDD and OCDF exhibited no responses. Non-ortho coplanar PCB126

’ RESULTS AND DISCUSSION Hepatic AHR mRNA Levels. To understand the quantitative relationship of AHR1 and AHR2 mRNA expression levels, both AHR transcript levels in 6 liver samples from the North Pacific population were determined. The quantitative assessment revealed 527

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Figure 2. Dose
responses of DRCs for BFA AHR1- and AHR2-mediated transactivation. (A-C) BFA AHR1-mediated transactivation, (D-F) BFA AHR2-mediated transactivation. (A) PCDDs, (B) PCDFs, (C) Co-PCBs, (D) PCDDs, (E) PCDFs, (F) Co-PCBs.

In the present study, the in vitro reporter gene assay is comprised of constructs from 3 bird species; albatross AHR, cormorant ARNT, and chicken CYP1A5 promoter. Since the study emphasizes species-specific responses, this experimental design warrants attention. Our previous study clearly demonstrated that the structural differences in CYP1A5 promoters between the chicken and the cormorant less affected the response to TCDD.15 The study also showed that the TCDD-EC50 is dependent on transfected AHR isoforms regardless of the CYP1A5 promoter construct used. Moreover, the comparison of TCDDEC 50 in chicken AHR1-, cormorant ARNT1-, and chicken CYP1A5 promoter-transfected COS-7 cells with that from chicken primary hepatocytes which express chicken ARNT indicated a similar TCDD-EC50 value in both assays.15 This suggests that the transactivation of CYP1A5 promoter sequences is determined irrespective of the species from which the ARNT gene originates. Thus, the transactivation potencies may be mostly governed by the transfected AHR, not by the ARNT. On the other hand, there are other variables that are not being taken into account to conclude that transactivation potencies depend mostly on AHR. Further experiments are needed to back this up. REPs and IEFs. To estimate the relative potencies of BFA AHR1- and AHR2-dependent transactivation, REP values were calculated. The estimates showed that no REP values could be given to OCDD, OCDF, PCB118, and PCB 156 for AHR1 and OCDD, PCB118, and PCB 156 for AHR2, because responses with more than 20% TCDD max (REP20) were not obtained for these congeners. For congeners of which REPs were given, we calculated REP20/REP80 ratios to verify the parallelism of the respective dose
response curves. For AHR1 mediated responses, a limited range of REP20/ REP80 ratios (0.1
10) was obtained for all PCDD/F and Co-PCB congeners except HpCDF which showed a high REP20/REP80 ratio (Table 1). The evaluation of the parallelism indicates that

and PCB169 showed clear responses, whereas PCB77 had a weaker response. Mono-ortho coplanar PCB118 and PCB156 failed to produce any responses (data not shown). The LOEC of TCDD that could trigger the minimum transactivation via BFA AHR1 was estimated to be 0.014 nM (4.5 pg/g wet wt). For BFA AHR2 (Figure 2D-F), treatment with TCDD showed a dose dependent increase in luciferase activities, but the plateau of the response was not attained even at the highest dose examined. Likewise, the plateau in the dose
response curves of HxCDD, HpCDD, TCDF, and HxCDF was not evident, although these congeners showed potencies to induce luciferase activities. For PeCDD, PeCDF, and HpCDF, the luciferase activities were dose-dependently induced and reached a plateau, and the EC50 values were estimated to be 1.5 nM (540 pg/g wet wt), 4.2 nM (1400 pg/g wet wt), and 4.0 nM (1600 pg/g wet wt), respectively. OCDD had little response, and OCDF exhibited a weak response. Among the Co-PCB congeners examined, only PCB126 showed a clear dose dependent response, while PCB77 and PCB169 showed weaker responses. PCB118 and PCB156 showed no response at all. The TCDD-LOEC for BFA AHR2mediated transactivation was 1.4 nM (450 pg/g wet wt). From a viewpoint of ecotoxicology, there has been a growing interest in the activation of AHR by DRCs in birds. It was recently reported that there is a positive correlation between EC50 values for induction of EROD activity and embryo mortalities in avian species.9 Hence, the integrated knowledge indicates that accurate measurements of AHR-mediated responses including CYP1A may help in assessing the risk of DRCs in wild birds. The present study investigated the transactivation potencies of BFA AHR1 and AHR2 isoforms for DRCs. Our in vitro reporter gene assay revealed that transient transfection of BFA AHR1 and AHR2 isoforms enhanced the transcriptional activities of luciferase gene under the control of CYP1A5 promoter sequences by most of the DRCs in a dose-dependent manner, suggesting that both AHR isoforms play functional roles in CYP1A induction. 528

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Table 1. Transactivation Potencies of BFA AHR1 and AHR2 by DRC Congeners AHR1 congener

% TCDD maxa

AHR2

REP20-REP80 rangeb (REP20/REP80 ratio)

% TCDD maxa

REP20-REP80 rangeb (REP20/REP80 ratio)

PCDDs 2,3,7,8-TCDD

100

1

100

1,2,3,7,8-PeCDD

173 ( 11

0.33
0.63 (0.52)

60 ( 16

1 4.3
1.0 (4.3)

1,2,3,4,7,8-HxCDD

55 ( 28

0.0028
0.0012 (2.3)

218 ( 37

0.37
4.4 (0.084)

1,2,3,4,6,7,8-HpCDD

95 ( 18

0.070
0.027 (2.6)

125 ( 24

0.50
2.6 (0.19)

1,2,3,4,6,7,8,9-OCDD

NAc

NAc

NAc

NAc

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

229 ( 10 143 ( 8.7

0.020
0.18 (0.11) 0.30
0.99 (0.30)

311 ( 94 72 ( 18

0.28
3.3 (0.085) 1.1
1.3 (0.85)

PCDFs

1,2,3,4,7,8-HxCDF

74 ( 18

0.0015
0.011 (0.14)

510 ( 44

0.38
6.9 (0.055)

1,2,3,4,6,7,8-HpCDF

67 ( 14

0.12
0.0030 (40)

135 ( 9.0

7.1
10 (0.70)

1,2,3,4,6,7,8,9-OCDF

NAc

NAc

60 ( 26

0.26
0.27 (0.96)

3,30 ,4,40 -TeCB (PCB77)

98 ( 16

0.000040
0.00024 (0.17)

87 ( 53

0.016
0.014 (1.1)

3,30 ,4,40 ,5-PeCB (PCB126)

172 ( 4.4

0.0023
0.0079 (0.29)

330 ( 46

0.085
0.76 (0.11)

3,30 ,4,40 ,5,50 -HxCB (PCB169) 2,30 ,4,40 ,5-PeCB (PCB118)

51 ( 6.1 NAc

0.00081
0.00011 (7.4) NAc

80 ( 24 NAc

0.021
0.0060 (3.5) NAc

2,3,30 ,4,40 ,5-HxCB (PCB156)

NAc

NAc

NAc

Coplanar PCBs

NAc

a

b

Response of a given congener expressed as a percentage of the maximum response observed for TCDD standard. Range of REP estimates generated over responses from 20% to 80% TCDD max. c NA: not available because of incomplete or no dose-dependent responses.

the uncertainties of IEFs for PeCDD, HxCDD, HpCDD, TCDF, PeCDF, HxCDF, PCB77, PCB126, and PCB169 were substantially within the range of 1 order of magnitude. As WHO TEFs incorporate 1 order of magnitude uncertainty,18 dose
responses of these congeners can be regarded as being mostly parallel to that of TCDD. The result of HpCDF indicated a greater uncertainty of the IEF (40 as REP20/REP80 ratio). IEF obtained for PeCDF (0.54) was similar with TCDD-IEF, indicating that this congener is equipotent to TCDD. IEFs of other congeners were lower than that of TCDD by 1
4 orders of magnitude (Table 2). In the case of BFA AHR2-mediated responses, REP20/REP80 ratios for most of PCDD/F and Co-PCB congeners were within the range of 0.1
10 (Table 1). Only HxCDD, TCDF, and HxCDF showed REP20/REP80 ratios that were slightly lower than 0.1. Efficacies of DRC congeners other than OCDD, PCB118, and PCB156, which exhibited no dose-dependent responses, were more than 20% TCDD max. Evaluations on the REP20/REP80 ratio and efficacy indicate that the uncertainties of IEFs from the single point estimate (REP50) were substantially limited for most of the congeners that exhibited dose-dependent responses. IEFs of PeCDD, HxCDD, HpCDD, TCDF, PeCDF, HxCDF, and HpCDF for BFA AHR2 were similar or higher than TCDD-IEF, whereas IEFs of OCDF, PCB77, PCB126, and PCB169 were lower than TCDD-IEF. IEFs could not be estimated for OCDD, PCB118, and PCB156 for BFA AHR2, because no response was generated by the treatment with these congeners. BFA AHR1-derived IEFs for most of the PCDD and PCDF congeners were within 1 order of magnitude of the WHO-TEFs proposed for avian species, whereas IEFs for Co-PCBs were more than 1 order of magnitude lower. Albatross AHR2-derived IEFs of HxCDD, HpCDD, HxCDF, HpCDF, and OCDF were apparently higher than the WHO-TEFs, but the trend of IEFs among

Table 2. Comparisons of BFA AHR1- and AHR2-Derived IEFs with WHO Bird TEFs of DRC Congeners BFA AHR1

BFA AHR2

WHO bird

IEFa

IEFa

TEF

congener PCDDs 2,3,7,8-TCDD

1

1

1

1,2,3,7,8-PeCDD 1,2,3,4,7,8-HxCDD

0.46 0.0018

2.1 1.3b

1 0.05

1,2,3,4,6,7,8-HpCDD

0.044

1.1

PCB77 > PCB169 . PCB118/PCB156) were similar to that of the WHO-TEFs (Table 2). These results revealed that both of BFA AHR1 and AHR2 are transcriptionally 529

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Table 3. BFA AHR1 and AHR2 IEF-Derived IEQs and WHO Bird TEF-Derived TEQs in the Liver of BFAs from the North Pacific average ( SD congener

a

no. of samples

BFA AHR1- IEQ

BFA AHR2- IEQ

WHO bird-TEQ

PCDDs 2,3,7,8-TCDD

11

4.0 ( 1.3

4.0 ( 1.3

4.0 ( 1.3

1,2,3,7,8-PeCDD 1,2,3,4,7,8-HxCDD

11 11

22 ( 9.0 0.028 ( 0.016

100 ( 41 21 ( 11

48 ( 19 0.79 ( 0.44

1,2,3,4,6,7,8-HpCDD

11

0.52 ( 0.35

13 ( 9.0

NAb

1,2,3,4,6,7,8,9-OCDD

b

10

NA

NA

0.00020 ( 0.00012 12 ( 6.4

b

PCDFs 2,3,7,8-TCDF

11

0.73 ( 0.39

12 ( 6.2

2,3,4,7,8-PeCDF

11

45 ( 20

100 ( 44

83 ( 36

1,2,3,4,7,8-HxCDF

11

0.15 ( 0.097

59 ( 37

4.0 ( 2.3

1,2,3,4,6,7,8-HpCDF 1,2,3,4,6,7,8,9-OCDF

11 1

0.11 ( 0.063 NAb

48 ( 28 0.27

0.056 ( 0.033 0.00011

3,30 ,4,40 -TeCB (PCB77)

11

0.061 ( 0.021

9.0 ( 3.1

31 ( 11

3,30 ,4,40 ,5-PeCB (PCB126)

11

5.7 ( 2.3

330 ( 140

130 ( 54

3,30 ,4,40 ,5,50 -HxCB (PCB169)

11

0.26 ( 0.14

9.4 ( 5.1

0.85 ( 0.47

2,30 ,4,40 ,5-PeCB (PCB118)

11

NAb

NAb

2.0 ( 0.55

2,3,30 ,4,40 ,5-HxCB (PCB156)

11

NAb

NAb

3.1 ( 1.4

79 ( 14

700 ( 86

330 ( 40

Coplanar PCBs

Total IEQ/TEQ a

Number of samples that showed detectable concentrations of the respective congener. b NA: not available because no IEF/TEF was obtained. IEQs and TEQs of individual and total congeners were expressed as pg/g liver wet wt.

active, but the transactivation potencies of AHR2 are different from those of AHR1 in terms of REPs and efficacy. Hepatic IEQs/TEQs in the Wild Population. In this study, two types of total IEQ values were obtained using two sets of IEFs that were generated from BFA AHR1- and AHR2-mediated transactivation potencies (Table 3). For IEQ calculation, BFA AHR1- or AHR2-derived IEFs of individual DRC congeners were multiplied by the respective hepatic concentrations in the wild BFA population from the North Pacific.20 The IEQs of individual congeners were summed up for the total IEQs. Hepatic total IEQ levels for BFA AHR1 transactivation ranged from 24 to 140 pg/g wet wt (79 ( 14 pg/g wet wt on average). The highest IEQ-contributed congener was PeCDF (45 ( 20 pg/g wet wt), followed by PeCDD (22 ( 9.0 pg/g wet wt) and PCB126 (5.7 ( 2.3 pg/g wet wt). Total IEQs for BFA AHR2 transactivation were in the range of 220
1300 pg/g wet wt (700 ( 86 pg/g wet wt on average). PCB126 (330 ( 140 pg/g wet wt), PeCDD (100 ( 41 pg/g wet wt), and PeCDF (100 ( 44 pg/g wet wt) greatly contributed to the total IEQ. On the other hand, the WHO-derived TEQs were 120
520 pg/g wet wt (330 ( 40 pg/g wet wt on average). The difference in AHR1- and AHR2-derived IEQs is primarily based on the fact that AHR2 is not fully responsive to TCDD and IEFs of other congeners are hence inflated relative to the TCDD response. However, this result does not violate the assumptions of the toxic equivalency concept. It is meaningless to make a comparison between the AHR1-derived IEQs and AHR2derived IEQs, when the AHR1- and AHR2-mediated responses are two different signaling pathways and responses to a standard congener, TCDD, are different between the paralogs. Since the function (pleiotropy, redundancy, or the combination) of the paralogs is not fully understood, more information is necessary to

evaluate whether AHR2-mediated effects must be treated with a different paradigm from AHR1-mediated effects. Measuring the total transactivation potency in cells that are transfected with both AHR1 and AHR2 plasmids in a 20 to 1 ratio in concentrations similar to the ones found in BFA livers may help to solve the paradigm issue. Relationships of Total IEQs/TEQs with EROD Activities in the Wild Population. To assess the effects of DRCs on CYP1A induction via AHR isoforms, Spearman’s rank correlation analyses were carried out between hepatic DRC levels and EROD activities in wild BFAs of the North Pacific population. For the correlation analyses, we examined the relationships of EROD activities with three types of total IEQs/TEQs, which were derived from BFA AHR1-, AHR2-IEFs, and WHO bird TEFs. The correlation analysis showed that EROD activities were positively correlated with BFA AHR1-IEF (p = 0.023, r = 0.72) and AHR2-IEF derived IEQs (p = 0.041, r = 0.65) at significant levels. A similar positive correlation with WHO bird TEF derived-TEQs was also found, but it was not statistically significant (p = 0.058, r = 0.60). This indicates that hepatic EROD activity is induced in wild specimens with high accumulation levels of DRCs and also suggest that the BFA specific IEFs obtained from this study may be suitable as well as the WHO bird TEFs for predicting the status of AHR-mediated transactivation in wild BFA populations. Together with the rank correlation analysis, nonlinear regression analyses for the relationships between three types of total IEQs/TEQs and EROD activities in the liver of BFA wild population were performed with an equation of sigmoidal dose
response (Figure 3). Results showed that the relationship between total IEQs/TEQs and EROD activities fits in a sigmoidal curve. R2 values for BFA AHR1 IEF- and AHR2 IEF-derived total IEQs and for WHO TEF-derived TEQs that indicate the 530

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In addition, TCDD-EC50 (9.7 pg/g wet wt as the concentration in cell culture medium) for the in vitro chicken AHR1-mediated transactivation in COS-7 cells and TCDD-EC50 (15 pg/g wet wt as the concentration in cell culture medium) for in vitro chicken endogenous AHR-mediated transactivation in chicken hepatocyte cells15 showed only a 7.2 and a 4.7 fold difference with TCDD-EC50 (70 pg/g wet wt as the concentration in the liver tissue of embryos) for CYP1A5 mRNA induction in TCDDtreated chicken embryos (unpublished data), respectively. On the other hand, WHO bird TEF-derived TEQs-EC50 was estimated to be 260 pg IEQ/g liver wet wt. This EC50 differs considerably from either of the AHR1 or AHR2 derived total IEQ-EC50 values. It is important to note that the available WHO bird TEFs were mostly determined on the basis of the toxicological studies on the chicken. Our findings highlight the importance of assessing the parameters such as REPs and EC50 of DRCs in avian species other than the chicken and also in AHR1 and AHR2. Implications of Critical Amino Acids in AHRs To Determine Sensitivity to DRCs. As birds differ in sensitivity to DRCs among species,21 we compared the results of the present study with the previous findings in an avian model, the chicken. Comparisons of TCDD-EC50 value obtained for the chicken hepatocytes29,30 and ckAHR1 transactivation15 with that for BFA AHR1 transactivation showed that the BFA is less sensitive to TCDD than the chicken. In contrast, the comparison with studies on the pheasant, tern, and herring gull21,22 showed that TCDD is more potent in the BFA than in the other avian species. The differential TCDD sensitivity in avian species has been explained by the differences in two amino acids in the ligand binding domain (LBD) of avian AHRs.23 Whereas AHR1 of the chicken, a dioxin sensitive species has Ile324 and Ser380 in the LBD, BFA AHR1 has Ile325 and Ala381 in the corresponding sites. We have already suggested that BFA AHR1 responds to TCDD in a moderately sensitive manner due to the conservation of one amino acid, Ile325.13 According to the 00 two amino acids00 rule, amino acid residues of Val325 and Ala381 in BFA AHR2 may account for this low sensitivity to TCDD in this isoformmediated response; the combination of amino acids in BFA AHR2 is identical to those in AHR1 in TCDD-resistant avian species. The result of BFA AHR2-mediated response to TCDD seems to follow the 00 two amino acids00 rule. Interestingly, EC50 values of PeCDD were similar for BFA AHR1 (1.8 nM) and BFA AHR2 (1.5 nM), although a large difference in TCDD-EC50 between BFA AHR isoforms was noticed. The present study also showed that REPs of PeCDD, HxCDD, HpCDD, TCDF, PeCDF, HxCDF, and HpCDF for BFA AHR2 were equal to or higher than that of TCDD. Thus, the EC50 and REP of congeners other than TCDD could not be due to the two amino acid residues in the LBD. This suggests that critical amino acids involved in ligand binding specificity can shift depending on the structure of ligands (e.g., TCDD vs PeCDD). In addition, the structural differences in the transactivation domain (TAD) between AHR1 and AHR2 may result in the differences in the ligand profiles. It has been suggested that the glutamine (Q)
rich region in the TAD of AHR may be involved in the transactivation function.17 We have previously shown that avian AHR1s possess a Q-rich region in the TAD, but there is no prominent Q-rich region in avian AHR2s.13 Hence, for evaluating the AHR-mediated transactivation potency by a variety of congeners other than TCDD, sites other than the two amino acid residues may also be critical.

Figure 3. Results of nonlinear regression analyses for the relationships between total IEQs/TEQs and EROD activities in the liver of BFAs from the North Pacific. (A) BFA AHR1 IEF-derived IEQs vs EROD, (B) BFA AHR2 IEF-derived IEQs vs EROD, and (C) WHO bird TEFderived TEQs vs EROD. The relationship fits in a sigmoidal curve in all the cases. R2 values for BFA AHR1 IEF- and AHR2 IEF-derived total IEQs and for WHO TEF-derived TEQs that indicate the “Goodness of Fit” were 0.58, 0.52, and 0.46, respectively. The EC50 values of total IEQs/TEQs from AHR1, AHR2, and WHO IEFs/TEFs were estimated to be 63, 550, and 260 pg IEQ/g liver wet wt, respectively.

“Goodness of Fit” were 0.58, 0.52 and 0.46, respectively. The EC50 values of total IEQs/TEQs from AHR1, AHR2, and WHO IEFs/TEFs were estimated to be 63, 550, and 260 pg/g liver wet wt, respectively (Figure 3). The comparison between the AHR1-derived total IEQ-EC50 (63 pg IEQ/g liver wet wt) and the TCDD-EC50 (25 pg/g wet wt) obtained from in vitro BFA AHR1-transfected reporter gene assay (Figure 2) showed only a 2.6 fold difference. Although the lack of TCDD-EC50 from in vitro BFA AHR2-transfected reporter gene assay (Figure 2) precluded the comparison with the AHR2-derived total IEQ-EC50 (550 pg IEQ/g liver wet wt) in BFA livers, the total IEQ-EC50 was reasonably higher than the TCDD-LOEC (450 pg/g wet wt) for in vitro BFA AHR2mediated transactivation. The consistencies of these effective concentrations derived from our field investigation (total IEQs vs EROD activity) and from the in vitro experiment (TCDD dosage vs CYP1A5 promoter transactivation) suggest that the concentrations of DRCs in cell culture medium can be equated to their tissue concentrations, although further confirmation is necessary. 531

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In the present study, we revealed that AHR2-mediated transactivation profiles by a variety of DRC congeners are different from those of AHR1. Since no TEF/IEF profile of DRC congeners is given for AHR2 transactivation of other avian species and fish, studies on AHR2 ligand profiles in more diverse species may provide clues regarding the evolution of AHR and its signaling pathways. Further studies on the ligand profile of avian AHR2 would aid at unfolding the distinct toxicological and physiological roles of this isoform. Our in vitro reporter gene assay system which has been constructed using the expression vectors of AHRs from the species of interest can potentially be a valuable tool for evaluating the species-specific susceptibility to DRCs and also for assessing the status of CYP1A induction in wild population of various vertebrate species, especially endangered species.

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’ ASSOCIATED CONTENT

bS

Supporting Information. Detailed description of experimental procedures including “plasmid construction and luciferase reporter gene assay”, “quantitative RT-PCR”, and “statistical analysis”, and the information on the purity of each tested compound (Table S1) and sequences of primers and probes used for the quantification of BFA AHR mRNAs (Table S2). This material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*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 the Japan Society for the Promotion of Science and Global COE Program (G-COE) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. 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 the 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. (20100012150). ’ REFERENCES (1) Safe, S. H. Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans. Annu. Rev. Pharmacol. Toxicol. 1986, 26, 371–399. (2) Schmidt, J. V.; Bradfield, C. A. Ah receptor signaling pathways. Annu. Rev. Cell Dev. Biol. 1996, 12, 55–89. (3) Knutson, J. C.; Poland, A. Response of murine epidermis to 2,3,7,8-tetrachlorodibenzo-p-dioxin: interaction of the ah and hr loci. Cell 1982, 30 (1), 225–234. (4) Fernandez-Salguero, P. M.; Hilbert, D. M.; Rudikoff, S.; Ward, J. M.; Gonzalez, F. J. Aryl hydrocarbon receptor-deficient mice are resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin induced toxicity. Toxicol. Appl. Pharmacol. 1996, 140 (1), 173–179. (5) Mimura, J.; Yamashita, K.; Nakamura, K.; Morita, M.; Takagi, T.; Nakao, K.; Ema, M.; Sogawa, K.; Yasuda, M.; Katsuki, M.; Fujii-Kuriyama, 532

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