Alternative In Vitro Approach for Assessing AHR-Mediated CYP1A

May 15, 2013 - Induction by Dioxins in Wild Cormorant (Phalacrocorax carbo) ... DRC concentrations in the Lake Biwa cormorant population, total TCDD ...
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Alternative In Vitro Approach for Assessing AHR-Mediated CYP1A Induction by Dioxins in Wild Cormorant (Phalacrocorax carbo) Population Leena Mol Thuruthippallil,† Akira Kubota,‡ Eun-Young Kim,*,§ and Hisato Iwata*,† †

Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, United States § Department of Life and Nanopharmaceutical Science and Department of Biology, Kyung Hee University, Hoegi-Dong, Dongdaemun-Gu, Seoul 130-701, Korea ‡

S Supporting Information *

ABSTRACT: Our line of papers revealed that the common (great) cormorant (Phalacrocorax carbo) possesses two isoforms of the aryl hydrocarbon receptor (ccAHR1 and ccAHR2). This paper addresses in vitro tests of the ccAHR signaling pathways to solve two questions: (1) whether there are functional differences in the two ccAHR isoforms, and (2) whether a molecular perturbation, cytochrome P450 1A (ccCYP1A) induction, in the population-level can be predicted from the in vitro tests. The transactivation potencies mediated by ccAHR1 and ccAHR2 were measured in COS-7 cells treated with 15 selected dioxins and related compounds (DRCs), where ccAHR1 or ccAHR2 expression plasmid and ccCYP1A5 promoter/ enhancer-linked luciferase reporter plasmid were transfected. For congeners that exhibited dose-dependent luciferase activities, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) relative potencies (REPs) and induction equivalency factors (IEFs) were estimated. ccAHR1-IEF profile was similar to WHO avian TCDD toxic equivalency factor (TEF) profile except for dioxin-like polychlorinated biphenyls that showed lower IEFs in ccAHR1-driven reporter assay. ccAHR2-IEF profile was different from WHO TEFs and ccAHR1-IEFs. Notably, 2,3,4,7,8-PeCDF was more potent than TCDD for ccAHR2-mediated response. Using ccAHR1- and ccAHR2-IEFs and hepatic DRC concentrations in the Lake Biwa cormorant population, total TCDD induction equivalents (IEQs) were calculated for each ccAHR-mediated response. Nonlinear regression analyses provided significant sigmoidal relationships of ccAHR1- and ccAHR2derived IEQs with hepatic ccCYP1A5 mRNA levels, supporting the results of in vitro ccAHR-mediated TCDD dose−response curves. Collectively, our in vitro AHR reporter assay potentially could be an alternative to molecular epidemiology of the species of concern regarding CYP1A induction by AHR ligands.

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INTRODUCTION Chlorinated dioxins and related compounds (DRCs), including polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (DLPCBs) are environmental contaminants that have been of great concern for the last four decades due to their high biomagnification and toxic potencies to human and wildlife. There is considerable evidence that most, if not all, toxic effects of DRCs in vertebrates are initiated by the activation of aryl hydrocarbon receptor (AHR), a basic helix−loop−helix/PAS ligand-dependent transcription factor.1−3 Despite intensive efforts and attempts to link the exposure to DRCs with impaired wildlife health,4−7 assessing the risk of these contaminants in the wild population, including birds, is still difficult. This is because of the difficulties in evaluating interspecies differences in sensitivity to these chemicals and also because of their existence as complex mixtures in biota, as well as the legal and ethical concerns of wildlife testing. Large interspecies differences in sensitivity of birds to cytochrome © XXXX American Chemical Society

P4501A (CYP1A) induction by 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) and related chemicals have been found, with the domestic chicken (Gallus gallus domesticus) being ranked as the most sensitive species.8,9 The molecular basis for such differential sensitivity to DRCs in birds is not fully understood, but in studies on a variety of avian species, AHR has emerged as the key regulatory protein.10,11 Positive correlations found between the relative potencies of DRCs upon avian AHRmediated transactivation (or CYP1A induction) and embryonic mortalities8,12 imply that CYP1A induction is a key event that precedes the onset of toxic effects by DRCs in birds. To address this issue, TCDD toxic equivalency factor (TEF) concept has been introduced for individual DRCs by the World Health Organization (WHO).13,14 Using the assigned TEFs, total Received: March 20, 2013 Revised: May 13, 2013 Accepted: May 15, 2013

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Figure 1. Dose−responses of ccAHR1-mediated transactivation by (A) PCDDs, (B) PCDFs, and (C) DL-PCBs. Dose−responses of ccAHR2mediated transactivation by (D) PCDDs, (E) PCDFs, and (F) DL-PCBs.

may contribute to establishing an in vitro approach for risk assessment of DRCs in the wild population of this species. This paper addresses in vitro tests of the ccAHR signaling pathways to solve two questions: (1) whether there are functional differences in the two ccAHR isoforms, and (2) whether a molecular perturbation, ccCYP1A induction, in the population-level can be predicted from in vitro tests. To address these questions, we initially investigated the CYP1A transactivation potencies by individual PCDD/Fs and DLPCBs via each ccAHR using the in vitro reporter gene assay system that was previously constructed.15,16 We then estimated the relative potencies to TCDD (REPs) of each DRC congener from ccAHR1- and AHR2-mediated dose−response curves (ccAHR1- and ccAHR2-REPs). The TCDD induction equivalency factors (IEFs) for individual congeners were determined from the ccAHR1- and ccAHR2-REPs (ccAHR1- and ccAHR2IEFs), and hepatic total TCDD induction equivalents (IEQs) were calculated using the IEFs and residue levels of the respective congeners in the population from Lake Biwa (ccAHR1- and ccAHR2-IEQs). Based on these results, we discussed the sensitivity to ccAHR-mediated transactivation by DRCs in the cormorant and validated the utility of the in vitro approach for predicting the status of CYP1A induction by DRCs in the wild population.

TCDD toxic equivalence (TEQ) is estimated as the sum of TCDD equivalence of each congener. However, the WHO bird TEFs of individual DRC congeners is mostly assigned on the basis of results obtained from chicken studies, while less effort has been devoted to validate the WHO TEFs by extrapolating to other species. Since wild birds are exposed to a complex mixture of DRCs, it is critical to understand which DRC congener and to what extent can trigger AHR-mediated signaling in the species of concern. To overcome ethical and experimental difficulties due to the requirement of large number of birds/eggs in toxicological studies, development of an in vitro assay system to evaluate the AHR-mediated signaling for individual DRC congeners is indispensable. We have established an in vitro assay system where the AHR expression plasmid of species of interest was introduced to a reporter gene transactivation assay that has proven to be an alternative approach to wildlife testing for toxicological studies.15−18 Other groups have also demonstrated the alternative of the in vitro AHR assay.19−21 The common (great) cormorant (Phalacrocorax carbo), a fish-eating bird, is a top predator of Lake Biwa, the largest freshwater lake in Japan. High and steady levels of DRCs (12− 1900 pg TEQ/g wet wt) have been detected in the liver of cormorants collected in 2001−2008.22,23 The induction of common cormorant CYP1A (ccCYP1A) expression and ccCYP1A-dependent O-dealkylation activities of methoxy-, ethoxy-, pentoxy-, and benzyloxy-resorufin (MROD, EROD, PROD, and BROD, respectively) by accumulated DRCs were also suggested.4,23,24 The ccCYP1A4 and 1A5 mRNA induction was also confirmed by in ovo TCDD injection test. 25 From the liver of common cormorants, we previously isolated two AHR isoforms, ccAHR1 and ccAHR2.15 Both ccAHRs were found to have TCDD binding capacities and to mediate CYP1A5 transactivation induced by TCDD.15,16 These earlier achievements thus evoke that determining the relative potencies of individual DRC congeners for ccAHR-mediated transactivation

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MATERIALS AND METHODS Standard Solutions of DRC Congeners. Details are given in the Supporting Information (SI). Sample Collection and Hepatic CYP1A Expression Levels. Details are given in the SI. Plasmid Construction and ccAHR-Driven Luciferase Reporter Gene Assay. The ccAHR1, ccAHR2, and ccAHR nuclear translocator 1 (ccARNT1) expression plasmids were previously constructed by inserting the respective full-length cDNAs into pcDNA3.1/Zero(+) vector (Invitrogen).15,16

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Table 1. Potencies of DRC Congeners for ccAHR1- and ccAHR2-Mediated Transactivation AHR1 congener

% TCDD maxb

2,3,7,8-TCDD 1,2,3,7,8-PeCDD 1,2,3,4,7,8-HxCDD 1,2,3,4,6,7,8-HpCDD 1,2,3,4,6,7,8,9-OCDD

100 78 ± 39 69 ± 15 41 ± 4.0 NAa

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

88 ± 42 72 ± 41 96 ± 36 NA NA

3,3′,4,4′-TeCB (77) 3,3′,4,4′,5-PeCB (126) 3,3′,4,4′,5,5′-HxCB (169) 2,3′,4,4′,5-PeCB(118) 2,3,3′,4,4′,5-HxCB (156)

NA 136 ± 8.2 NA NA NA

AHR2

REP20−REP80 rangec (REP20/REP80 ratio) PCDDs 1 0.13−0.18 (0.72) 0.013−0.011 (1.1) 0.037−0.0000022 (1.7×104) NA PCDFs 0.41−0.16 (2.5) 1.2−0.28 (4.3) 0.014−0.024 (0.58) NA NA DL-PCBs NA 0.00090−0.00061 (1.2) NA NA NA

% TCDD maxb

REP20−REP80 rangec (REP20/REP80 ratio)

100 92 ± 28 26 ± 6.0 47 ± 22 NA

1 0.094−0.079(1.2) 0.00089−0.00013 (6.9) 0.18−0.13 (1.4) NA

79 ± 46 199 ± 34 92 ± 40 58 ± 16 NA

0.023−0.036 (0.63) 0.34−5.4 (0.064) 0.71−0.37(1.9) 0.19−0.078 (2.4) NA

114 ± 32 209 ± 47 108 ± 26 40 ± 21 NA

0.00031−0.0011 (0.28) 0.0049−0.0093 (0.53) 0.00024−0.00095 (0.25) 0.00013−0.00000021 (630) NA

a

NA denotes no data available because of incomplete or no dose-dependent responses. bResponse of a given congener expressed as a percentage of the maximum response observed for TCDD standard. cRange of REP estimates generated over responses from 20 to 80% TCDD max.

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RESULTS AND DISCUSSION ccAHR-Mediated Transactivation Potencies. PCDD, PCDF, and DL-PCB congeners were examined for the dosedependent responses of ccAHR1- or ccAHR2-mediated reporter gene expression in COS-7 cells. Expression vectors of ccAHR1 or ccAHR2 and ccARNT1 were transfected into COS-7 cells together with reporter vectors of pGL4-ccCYP1A5-7XREs and pGL4-Renilla transfection control, following the method established by Lee et al.16 EC50 values for the tested DRCs were determined from the dose−response curves (Figure 1A− C). TCDD, PeCDD, TCDF, and PeCDF generated complete dose-dependent responses via ccAHR1, and the EC50 values were 0.36 nM (110 pg/g wet wt), 2.2 nM (620 pg/g), 2.5 nM (820 pg/g), and 0.24 nM (71 pg/g), respectively. The TCDDEC50 in this study was close to that obtained by the same ccAHR1 reporter assay in our previous study (0.29 nM).16 HxCDD and HxCDF generated responses only at the maximum tested concentration, and hence the EC50 values were not given. HpCDD had a dose-dependent response, but lower efficacy than TCDD, showing an EC50 of 2.5 nM (588 pg/g wet wt). OCDD, HpCDF, and OCDF exhibited no responses. Among the DL-PCBs tested, PCB126 showed responses only at the highest and second-highest concentrations, but failed to reach a maximum plateau. Other DLPCBs (PCB77, 169, 118, and 156) had weaker or no responses. The LOEC of TCDD that could trigger the minimum transactivation via ccAHR1 was estimated to be 0.14 nM (45 pg/g wet wt). For ccAHR2-mediated responses (Figure 1D−F), treatment with TCDD, PeCDD, HpCDD, TCDF, PeCDF, and HpCDF showed dose-dependent increases in XRE-driven luciferase activities and the EC50 values were 0.23 nM (71 pg/g wet wt), 1.6 nM (450 pg/g), 0.20 nM (47 pg/g), 3.5 nM (1100 pg/g), 0.29 nM (85 pg/g), and 0.28 nM (68 pg/g), respectively. As for the TCDD-EC50, a similar value has been reported by the same ccAHR2 reporter assay (0.29 nM).16 PeCDF showed a higher efficacy to induce luciferase activity than TCDD. Since HxCDD

Procedures of the luciferase reporter gene assay for AHRmediated transactivation have already been reported elsewhere,15−18 and are briefly described in the SI. Dose−Responses. For the response of each DRC, the luciferase activities were obtained from four replicates of DMSO- and each congener-treated well. The mean of relative luciferase unit value (RLU) from DMSO-treated wells was subtracted from the RLUs from each congener-treated well. Triplicate dose−response curves of each DRC were obtained for each ccAHR-mediated transactivation. REPs and IEFs. The detailed procedures of REP and IEF estimation have already been reported elsewhere.17,18 Briefly, RLU values obtained from the dose−response curves 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. The normalized RLU values were then plotted against logarithmically transformed DRC concentrations. Prism 5.0 (GraphPad, San Diego, CA, USA) was used for curve fitting, and data were fit to a four parameter logistic model. Three points, corresponding to EC20, EC50, and EC80 values that were obtained for TCDD, were used to estimate REP20, REP50, and REP80 values for individual congeners examined. This is because the single point estimate (REP50) may veil a large uncertainty of IEF for the congener that exhibits a nonparallel dose-dependent response to TCDD. For congeners whose parallelism was confirmed (10 > REP20/ REP80 ratio > 0.1), the REP50 was regarded as the 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. IEQs. Total ccAHR1- and ccAHR2-IEQs in wild cormorants from Lake Biwa, Japan were calculated by summing up hepatic concentrations of individual congeners22,23 multiplied by the respective ccAHR1- and ccAHR2-IEFs. Similarly, total TEQ was obtained using the concentrations of congeners and WHO avian TEFs.13 Statistical Analyses. Details are given in the SI. C

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Table 2. Comparisons of ccAHR1- and ccAHR2-IEFs with WHO Bird TEFs of DRC Congener

showed a response only at the highest concentration and OCDD/F exhibited only a weak response, no EC50 values were thus obtained. DL-PCBs, PCB77, 126, and 169 showed dosedependent responses, but the plateau of the responses was not attained even at the highest dose examined. PCB118 and 156 showed weak or no responses. The TCDD-LOEC for ccAHR2mediated transactivation was 0.14 nM (45 pg/g wet wt), which was the same as that of the TCDD-LOEC obtained for ccAHR1. REPs and IEFs. REP values were obtained from relative luciferase activities that were normalized to the maximum transcriptional activity elicited by TCDD for each ccAHR isoform. The estimates showed no REP values for OCDD, HpCDF, OCDF, PCB77, PCB169, PCB118, and PCB156 for ccAHR1, and OCDD, OCDF, PCB77, PCB169, PCB118, and PCB156 for ccAHR2, because no responses with more than 20% TCDD max were obtained for these congeners. To evaluate the uncertainty of IEF of each congener, we checked the parallelism of dose−response curves between TCDD and the congener by calculating REP20/REP80 ratios (Table 1). A narrow range of REP20/REP80 ratios (0.1−10) was obtained for PCDD/F or DL-PCB congeners except HpCDD in ccAHR1-mediated responses (Table 1). The evaluation of the parallelism indicates that the uncertainties of IEFs for PeCDD, HxCDD, TCDF, PeCDF, HxCDF, and PCB126 were within the range of 1 order of magnitude. As WHO-TEFs incorporate 1 order of magnitude uncertainty,13 the uncertainties of IEFs of these congeners are small and the IEFs are thus definitive. The result of HpCDD indicated greater uncertainties of the IEF (1.7 × 104 as REP20/REP80 ratio). This is due to the gentle slope of dose−response for this congener, compared to that of TCDD. IEF obtained for PeCDF (0.58) was similar to TCDD-IEF, indicating that this congener is equipotent to TCDD for ccAHR1-mediated transactivation. IEFs of other congeners were lower than that of TCDD by 1−4 orders of magnitude (Table 2). For ccAHR2-mediated responses, REP20/REP80 ratios for most of PCDD/F and DL-PCB congeners were within the range of 0.1−10, except PeCDF and PCB118 which showed a lower (0.064) and a higher (630) ratio, respectively (Table 1). Efficacies of all the tested DRC congeners except OCDD, OCDF, and PCB156, which exhibited no dose-dependent responses, were more than 20% TCDD max. Evaluations of the REP20/REP80 ratios and efficacies indicate that the uncertainties of IEFs were substantially limited for the congeners that exhibited dose-dependent responses. The IEFs of PeCDF and HxCDF were higher than or equivalent to that of TCDD, whereas IEFs of other PCDD/F and DL-PCB congeners used were lower than TCDD-IEF. One of the most interesting results was that PeCDF showed equipotency to TCDD for ccAHR1 and a higher potency than TCDD for ccAHR2. In our previous study on black-footed albatross AHRs (bfaAHRs), we observed similar effects of PeCDF on bfaAHR-mediated transactivation potencies.18 In comparison with WHO TEFs proposed for avian species, ccAHR1-IEFs of PCDD/F congeners were within 1 order of magnitude of the WHO-assigned values, but the IEF of PCB126 was 2 orders of magnitude lower (Table 2). ccAHR2IEF profiles of PCDD/F congeners were different from the WHO TEFs; the IEFs of HxCDD and TCDF were 2 orders of magnitudes lower, while the IEFs of HpCDD and HpCDF were more than an order of magnitude higher. For DL-PCBs,

congener

ccAHRlIEFb

ccAHR2IEFb

WHO bird TEF

PCDDs 2,3,7,8-TCDD 1,2,3,7,8-PeCDD 1,2,3,4,7,8-HxCDD 1,2,3,4,6,7,8-HpCDD 1,2,3,4,6,7,8,9-OCDD 2,3,7,8-TCDF 2,3,4,7,8-PeCDF 1,2,3,4,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF 1,2,3,4,6,7,8,9-OCDF 3,3′,4,4′-TeCB (77) 3,3′,4,4′,5-PeCB (126) 3,3′,4,4′,5,5′-HxCB (169) 2,3′,4,4′,5-PeCB (118) 2,3,3′,4,4′,5-HxCB (156)

1 0.15 0.012 0.00030c NAa PCDFs 0.26 0.58 0.019 NA NA DL-PCBs NA 0.00074 NA NA NA

1 0.087 0.00034 0.15 NA

1 1 0.05