Relative Potencies of Aroclor Mixtures Derived from Avian in Vitro

Article pubs.acs.org/est

Relative Potencies of Aroclor Mixtures Derived from Avian in Vitro Bioassays: Comparisons with Calculated Toxic Equivalents Rui Zhang,† Gillian E. Manning,‡,§ Reza Farmahin,‡,§ Doug Crump,§ Xiaowei Zhang,*,† and Sean W. Kennedy*,‡,§ †

State Key Laboratory of Pollution Control & Resource Reuse, School of the Environment, Nanjing University, Nanjing, P. R. China, 210023 ‡ Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 § Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 S Supporting Information *

ABSTRACT: The World Health Organization toxic equivalency factors (WHO-TEFs) for birds were developed to simplify risk assessments of environmental mixtures of dioxin-like compounds (DLCs). Under this framework, toxic equivalents (TEQs) are used to represent the toxic potency of DLC mixtures as an equivalent concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Recently, a luciferase reporter gene (LRG) assay, measuring aryl hydrocarbon receptor 1 (AHR1)-mediated gene expression, accurately predicted the relative potency of individual polychlorinated biphenyl (PCB) congeners in different avian species. The study presented here used the LRG assay to predict the relative potency of Aroclors 1016, 1221, 1242, 1248, 1254, and 1260 on induction of LRG activity in cells transfected with chicken, ring-necked pheasant, or Japanese quail AHR1 constructs. LRG assay results were compared to (1) results of ethoxyresorufin-O-deethylase (EROD) assays conducted in chicken hepatocytes and (2) calculated TEQs from the literature. The relative potencies of Aroclors were similar between the LRG and EROD assays, and bioassay-derived TEQs for the chicken closely resembled calculated TEQs. However, LRG assay-derived TEQs for the Japanese quail construct were 1−2 orders of magnitude higher than calculated TEQs for Aroclors 1254 and 1016. These results suggest that the WHO-TEFs are not representative of relative PCB potency for all avian species.

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INTRODUCTION

phenyl [PCB126]), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and some other dioxin-like compounds (DLCs).26−34 Although there is considerable evidence that several of the adverse effects of PCBs are mediated by the aryl hydrocarbon receptor (AHR),35−37 a complete understanding of the mechanism(s) underlying differences in sensitivity is not yet available. However, recent reports provide important information on the role of the avian AHR1 ligand-binding domain (LBD) in interspecies differences to the effects of PCBs.32,38−40 According to these sources, birds can be classified into three main groups of sensitivity to DLCs: chicken-like (type 1), ring-necked pheasant-like (type 2), or Japanese quail-like (type 3). Species sensitivity can be predicted from (1) the identity of the amino acids at two key sites within the AHR1 LBD or (2) by use of a luciferase reporter gene (LRG) assay.31,32,38,39

Aroclors are polychlorinated biphenyl (PCB) mixtures containing many (60−100) PCB congeners.1−3 While the commercial production of Aroclors and other PCB formulations was banned under the Stockholm Convention in 2004,4 PCBs continue to be widespread environmental contaminants. Aroclors were produced by the catalytic chlorination of biphenyl and they contain low concentrations of byproducts including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated naphthalenes (PCNs).5−8 Laboratory exposure of birds to individual PCB congeners and Aroclor mixtures causes various adverse effects, including hepatotoxicity,9−11 lethality,10 embryotoxicity,12,13 reduction in growth,14 immunotoxicity,15 endocrine-disruptive effects,9,16,17 and changes in adult breeding behavior.18−20 Environmental PCB exposure has also been correlated with adverse effects in wild birds.21−25 It is well-established that avian species differ in sensitivity to the toxic effects of two non-ortho-substituted PCBs (3,3′,4,4′tetrachlorobiphenyl [PCB 77] and 3,3′,4,4′,5-pentachlorobi© 2013 American Chemical Society

Received: Revised: Accepted: Published: 8852

March July 1, July 1, July 1,

26, 2013 2013 2013 2013

dx.doi.org/10.1021/es401340t | Environ. Sci. Technol. 2013, 47, 8852−8861

Environmental Science & Technology

Article

CYP1A5 reporter construct, 0.75 ng of Renilla luciferase vector (Promega), and 32.2 ng of salmon sperm DNA (Invitrogen). Cells were dosed 5 h after transfection with DMSO (solvent control) or DMSO solutions of TCDD or Aroclors. A positive control (0.1 μg/mL TCDD) was also included when cells were dosed with Aroclors. LRG activity was measured 20 h after dosing using Dual-Glo luciferase assay kits (Promega) in a LuminoSkan Ascent luminometer (Thermo Fisher Scientific Inc., Waltham, MA). Additional details are provided elsewhere.38 LRG Data Analysis. For the LRG assay, triplicate concentration−response curves were obtained from three independent experiments for each AHR1 construct and Aroclor treatment. Four technical replicates per dilution of Aroclor, TCDD, or DMSO were included with each experiment. LRG activity was converted to a ratio of firefly luciferase units to Renilla luciferase units (luciferase ratio). This normalization removes variability resulting from differences in cell plating, transfection efficiency, pipetting inconsistencies, and toxicity.45 The luciferase ratio was then normalized to a percent response value expressed relative to the response elicited by the 0.1 μg/mL TCDD positive control, according to the protocol described in OECD guideline 455.46 The data were fitted to a four-parameter logistic model using GraphPad (GraphPad Prism 5.0 software, San Diego, CA).47 The model integrates the EC50, slope, baseline response, and maximal response as parameters. EC50 values could not be calculated in cases where the concentration−response curves did not reach a plateau or where a plateau could not be estimated accurately by curve fitting and were therefore not presented in these instances. The concentrations of Aroclors that elicited a response equal to 10%, 20%, 50%, and 80% of the positive control response, referred to as positive control (PC)10, PC20, PC50, and PC80 values, were determined for each replicate concentration− response curve. EC50, PC10, PC20, PC50, PC80, and maximal response values were determined for each replicate concentration−response curve using logistic curve fitting and are presented as the mean ± standard error (SE). When concentration−response curves did not reach a plateau, the highest observed response was reported as the maximal response. Chicken Embryo Hepatocyte (CEH) Cultures and EROD Assays. CEH cultures were prepared in 48-well plates from 19day old White Leghorn chicken embryos as described elsewhere.48 Fertile, unincubated chicken eggs were obtained from the Canadian Food Inspection Agency (Ottawa, Ontario, Canada), and all procedures were conducted according to protocols approved by the Animal Care Committee at the National Wildlife Research Centre. In brief, eggs were incubated at 37.5 °C and 60% relative humidity until 1 to 3 days prehatch. Eggs were candled periodically and infertile eggs or eggs containing dead embryos were discarded. Chicken embryos were euthanized by decapitation, and livers were removed, pooled, and digested with collagenase. Cells were plated at concentrations that resulted in total protein concentrations of 60 μg/well in 48-well culture plates. Each well contained 500 μL of Waymouth’s medium supplemented with insulin (1 μg/mL) and thyroxine (1 μg/mL), and incubated for 24 h at 37 °C in a humidified incubator with 5% CO2. After 24 h, cells were treated in triplicate with DMSO (solvent control) or DMSO solutions of TCDD or Aroclors. Inwell TCDD concentrations ranged from 10−7 to 3 μg/mL, and concentrations of Aroclors 1016, 1221, 1242, 1248, 1254, and 1260 ranged from 10−4 to 5 μg/mL. After another 24 h incubation period, the medium was removed, and plates were

The LRG assay is useful for predicting the relative potency (ReP) values of PCB congeners in different avian species and the relative sensitivity (ReS) values of avian species to the effects of individual PCBs,32,38,39 but the assay had not been used to study the effects of PCB mixtures. The objectives of the present study were the following: (1) use the LRG assay to characterize the concentration-dependent effects of Aroclors 1016, 1221, 1242, 1248, 1254, and 1260 on induction of LRG activity in COS-7 cells transfected with chicken, ring-necked pheasant, or Japanese quail AHR1 expression constructs; (2) determine the ReS values of the different avian AHR1 constructs for each Aroclor and the ReP values of Aroclors for each AHR1 construct using the data derived from LRG assays; (3) compare LRG-derived RePs for chicken AHR1 with ethoxyresorufin O-deethylase (EROD)derived RePs in chicken embryo hepatocyte (CEH) cultures; (4) compare calculated toxic equivalents (TEQs) for the Aroclors (derived using the World Health Organization toxic equivalency factors (TEFs) for birds) with bioassay-derived TEQs from this study.

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EXPERIMENTAL SECTION Preparation of TCDD and Aroclor Solutions. A detailed description of the preparation of TCDD solutions is provided elsewhere.41 In brief, serial dilutions of TCDD were prepared from stock solutions in dimethyl sulfoxide (DMSO; SigmaAldrich, St. Louis, MO), and concentrations were determined by high-resolution gas chromatography high-resolution mass spectrometry using isotope dilution following EPA method 1613.42 Stock solutions of each Aroclor were prepared by dissolving a weighed amount (9−10 mg; weighed on a Sartorius analytical balance, Model ME215P, 0.01 mg accuracy) of each Aroclor in DMSO to obtain a concentration of 20 000 μg/mL. Stock solutions were diluted to nominal concentrations ranging from 0.6 to 2000 μg/mL for the LRG assay and from 0.0001 to 5 μg/mL for the EROD assay. Aroclor 1242 was obtained from Supelco (lot no. LB86404 V, Bellefonte, PA), and the other Aroclors (1016 - lot no. NT01016; 1221 - lot no. NT01017; 1248 - lot no. not available; 1254 - lot no. not available; 1260 - lot no. not available) were purchased from Ultra Scientific, (North Kingstown, RI). For the LRG assays, Aroclor or TCDD test solutions were prepared by dissolving the serially diluted solutions with the cell culture medium. The final in-well concentration of DMSO in the LRG assay was 0.5%. For the CEH cultures, DMSO solutions (2.5 μL) of each Aroclor or TCDD were added to 48-well plates to yield a final DMSO concentration of 0.5% in each well. COS-7 Cell Culture, Transfection, and the LRG Assay. COS-7 cells, provided by Dr. R. Haché (University of Ottawa, Ottawa, ON, Canada), were plated at a concentration of 10 000 cells per well in 96-well plates. Details for the preparation of fulllength chicken, ring-necked pheasant, and Japanese quail AHR1 constructs are provided elsewhere.38 A firefly luciferase reporter vector containing the common cormorant (Phalacrocorax carbo) CYP1A5 promoter region and a common cormorant ARNT1 vector were generously provided by Dr. Hisato Iwata (Ehime University, Japan).43,44 AHR1 constructs were transfected 18 h after the plating of COS-7 cells. DNA and Fugene 6 transfection reagent (Promega, Madison, WI) were diluted in Opti-MEM (Invitrogen, Burlington, ON, Canada), and 6 μL of this transfection mixture was added to each well. The amounts of transfected DNA per 6 μL of transfection mixture were 8 ng of chicken, ring-necked pheasant, or Japanese quail AHR1 expression construct, 1.55 ng of cormorant ARNT, 7.5 ng of 8853



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Figure 1. (A) Concentration-dependent effects of TCDD and Aroclors 1248, 1242, 1254, 1260, 1016, and 1221 on AHR1-mediated LRG activity in COS-7 cells transfected with chicken, ring-necked pheasant, or Japanese quail AHR1 constructs. Data are presented as percent response values relative to that of a 0.1 μg/mL TCDD positive control for each of the avian constructs. Concentration−response curves for the Aroclors are only presented for constructs that showed a significant (p < 0.05), concentration-dependent increase in LRG activity relative to the DMSO response. Points represent mean, positive control-normalized luciferase ratios obtained from three independent experiments, each with four technical replicates per concentration of Aroclor or TCDD. Bars represent standard error. (B) Concentration-dependent effects of TCDD and Aroclors 1248, 1242, 1254, 1260, 1016, and 1221 on EROD activity in chicken embryo hepatocyte cultures. Points represent mean EROD activity obtained from three replicates on different cell culture plates ± standard error, each with three technical replicates. (C) Linear regression analysis comparing log-transformed chicken EROD RePavg values with log-transformed LRG assay RePavg values for the chicken AHR1 construct.

flash-frozen in powdered dry ice and stored at −80 °C until the time of analysis. The Calcein-AM assay, described elsewhere,41 was used to measure cell viability. No significant differences between the viability of Aroclor-treated, DMSO-treated, and untreated hepatocytes were observed.

A detailed description of the EROD assay is provided elsewhere.48 In brief, three replicate plates of hepatocytes per chemical were incubated at 37.5 °C in the presence of nicotinamide adenine dinucleotide phosphate (NADPH, reduced) and 7-ethoxyresorufin for 7 min. During assay 8854



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Table 1. Endpoints Determined for AHR1-Mediated LRG Activity in COS-7 Cells Transfected with Chicken, Ring-Necked Pheasant, or Japanese Quail AHR1 Constructs and Exposed to TCDD, or Aroclors 1248, 1242, 1254, 1260, 1016, and 1221 for 20 h*† AHR1 chicken

compound TCDD

pheasant

Aroclor 1248 Aroclor 1242 Aroclor 1254 Aroclor 1260 Aroclor 1016 Aroclor 1221 TCDD

quail

Aroclor 1248 Aroclor 1242 Aroclor 1254 Aroclor 1260 Aroclor 1016 Aroclor 1221 TCDD Aroclor 1248 Aroclor 1242 Aroclor 1254 Aroclor 1260 Aroclor 1016 Aroclor 1221

EC50 ± SE (μg/mL) 10−5 ± −6a

PC10 ± SE (μg/mL) 10−5 ± −6a

PC20 ± SE (μg/mL) 10−5 ± −6a

PC50 ± SE (μg/mL) 10−5 ± −6a

PC80 ± SE (μg/mL) 10−5 ± −6

max. response ± SE (% PC)

RePavg

ReP range

4.59 × 4.9 × 10 0.56 ± 0.05b

1.82 × 6.8 × 10 0.23 ± 0.04b

2.45 × 7.2 × 10 0.38 ± 0.04b

4.29 × 7.6 × 10 1.92 ± 0.56b

7.41 × 7.4 × 10 NE

60.4 ± 5.7

1.12 ± 0.06c

0.30 ± 0.07b

0.61 ± 0.07b

3.73 ± 1.4b

NE

65.0 ± 4.8b

3.7 × 10−5

1.37 ± 0.15cd

0.41 ± 0.19b,c

0.63 ± 0.20b

3.40 ± 1.1b

NE

70.4 ± 6.7b

3.2 × 10−5

2.43 ± 0.30d

1.69 ± 0.13c,d

2.94 ± 0.25c

NE

NE

33.7 ± 6.6c

9.6 × 10−6

NC

2.36 ± 0.19d

9.79 ± 0.73d

NE

NE

19.03 ± 0.57‡ c

5.1 × 10−6

1.27 ± 0.33cd

NE

NE

NE

NE

9.94 ± 2.6c

NA

2.2 × 10−5 ∼7.9 × 10−5 1.2 × 10−5 ∼6.1 × 10−5 1.3 × 10−5 ∼4.4 × 10−5 8.3 × 10−6 ∼1.1 × 10−5 2.5 × 10−6 ∼7.7 × 10−5 NA

7.32 × 10−4 ± 1.9 × 10−4a 2.22 ± 0.40b

1.06 × 10−04 ± 3.4 × 10−4 NE

2.04 × 10−4 ± 5.8 × 10−5 NE

6.47 × 10−4 ± 1.7 × 10−4 NE

1.96 × 10−3 ± 5.4 × 10−4 NE

107 ± 1.6a

1.0

1.0−1.0

7.22 ± 0.20b

NA

NA

2.28 ± 0.50§b

NE

NE

NE

NE

8.88 ± 1.0b

NA

NA

2.45 ± 0.27b

NE

NE

NE

NE

6.14 ± 2.3b

NA

NA

>10

NE

NE

NE

NE

no induction

NA

NA

>10

NE

NE

NE

NE

no induction

NA

NA

>10

NE

NE

NE

NE

no induction

NA

NA

6.49 × 10−3 ± 1.2 × 10−3a NC

1.42 x10−3 ± 4.2 × 10−4a 1.72 ± 0.27b

2.47 × 10−3 ± 6.4 × 10−4a 4.74 ± 1.7b

6.28 × 10−3 ± 1.3 × 10−3 NE

1.56 × 10−2 ± 2.5 × 10−3 NE

103 ± 2a

1.0

1.0−1.0

32.3 ± 5.9‡ b

6.7 × 10−4

NC

2.56 ± 0.88b,c

4.56 ± 1.5b

NE

NE

33.3 ± 5.7‡ b

5.5 × 10−4

NC

3.43 ± 0.70b,c

6.05 ± 1.4b

NE

NE

29.75 ± 8.8‡ bd

4.1 × 10−4

3.05 ± 0.23b

NE

NE

NE

NE

4.21 ± 0.72cd

NA

5.2 × 10−4 ∼8.3 × 10−4 5.4 × 10−4 ∼5.6 × 10−4 4.1 × 10−4 ∼4.1 × 10−4 NA

NC

4.92 ± 0.54c

NE

NE

NE

19.3 ± 2.7‡ bcd

2.9 × 10−4

2.78 ± 0.59b

NE

NE

NE

NE

5.29 ± 0.18d

NA

107 ± 8

a

1.0 b

1.0−1.0 −5

5.5 × 10

NA

*

EC50, PC10, PC20, PC50, PC80, and maximal response values represent the average of three replicates ± standard error (SE) obtained from three 96well plates for each compound. PC10, PC20, PC50, and PC80 values were not calculated if the maximum observed response was below 10% of the positive control. The average relative potency (RePavg) values and ReP ranges were calculated from PC10-, PC20-, PC50- and PC80-based ReP values (Table S1, Supporting Information). No range of ReP values was presented when only the RePPC10 could be calculated. †LRG activity values were normalized to responses of 0.1 μg/mL TCDD positive control (PC). Maximal response values were obtained from the curve fit, unless otherwise indicated. Superscript roman letters indicate significant differences among treatments (p < 0.05) within each AHR1 construct. NA: ReP estimates not available to calculate the value. NC: Not calculated because the maximal response was not reached. NE: Not estimated because the maximum observed response was below 10%, 20%, 50%, or 80% of positive control response. ‡A plateau was not reached. Values represent the highest observed response.

to protein content and expressed as the rate of resorufin production (pmol·min−1·mg protein−1). EROD activity data were imported into GraphPad and fitted to a modified Gaussian curve as described elsewhere.48 Triplicate concentration− response curves were obtained from three independent experiments conducted on three separate 48-well cell culture plates for each compound. Three technical replicates were included in each experiment. The concentrations of TCDD and Aroclors that elicited a response equal to 10%, 20%, 50%, and 80% of the TCDD maximal response (TCDD10, TCDD20, TCDD50, and TCDD80), EC50, and maximal response values were estimated

development, time-course reactions determined that the timedependent increase in resorufin was linear for 10 min.48 Reactions were stopped by the addition of ice-cold methanol. Standard curves of resorufin and protein were prepared on each 48-well plate for each run. Plates were analyzed for both EROD activity (excitation wavelength: 530 nm, emission wavelength: 590 nm) and total protein concentration (excitation wavelength: 400 nm, emission wavelength: 460 nm) using a Cytofluor 2300 fluorescence plate reader (Millipore, Bedford, MA). EROD Data Analysis. For the EROD assays, concentrations of resorufin were calculated from linear regression of calibration standards after blank correction. EROD activity was normalized 8855



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from the curve fit and are presented as the mean ± SE obtained from the triplicate curves. Calculation of Relative Sensitivity (ReS) and Relative Potency (ReP) Values. Because the chicken is considered to be the most sensitive avian species to the toxic and biochemical effects of most DLCs,31,41 the sensitivity of different AHR1 constructs is expressed relative to data obtained from cells transfected with the chicken AHR1 construct in the LRG assay. PC10 and EC50 values were used to calculate ReS values. However, in cases where a plateau was not reached, only PC10based ReS values (ReSPC10) were calculated. If no induction of LRG activity was observed, ReSEC50 values were estimated by dividing the chicken values by the maximum concentration tested (10 μg/mL for all Aroclors), and the ReS was expressed as being less than the calculated value. The ReS values were calculated as follows: PC10 or EC50 of compound X in chicken AHR1 ÷ PC10 or EC50 of compound X in the AHR1 construct of interest. For the LRG and EROD assays, ReP values were calculated using the systematic framework proposed by Villeneuve et al. 49 with some modifications. ReP values based on EC50, PC10/ TCDD10, PC20/TCDD20, PC50/TCDD50, and PC80/TCDD80 values (RePEC50, RePPC10/RePTCDD10, RePPC20/RePTCDD20, RePPC50/RePTCDD50, and RePPC80/RePTCDD80) obtained from the regression models were calculated. The average (RePavg) and range of ReP values were calculated from RePPC and RePTCDD values. RePEC50 values were excluded because these values can overestimate potency, especially when significant differences in efficacies exist among the compounds being compared,50 as was observed in the present study. ReP values were calculated as follows: EC50, PC10/TCDD10, PC20/TCDD20, PC50/TCDD50, or PC80/TCDD80 of TCDD in species X ÷ EC50, PC10/TCDD10, PC20/TCDD20, PC50/TCDD50, or PC80/TCDD80 of the Aroclor of interest in species X. Statistical Analysis. Significant differences between maximal response, log EC50, log PC10/log TCDD10, log PC20/log TCDD20, log PC50/log TCDD50 and log PC80/log TCDD80 values for different Aroclors or different AHR1 constructs/ species were determined using a t test (p ≤ 0.05) or a one-way ANOVA (p ≤ 0.05) followed by Tukey’s Multiple Comparison Test (p ≤ 0.05). Linear regression analyses were conducted to compare logtransformed TCDD10 and EC50 values from chicken EROD assays with log-transformed PC10 and EC50 values from chicken LRG assays. Linear regression analyses were also performed to compare bioassay-derived TEQ values with calculated TEQs for the different Aroclors obtained from Burkhard and Lukasewycz.37 ReP values (in (μg/mL TCDD)/(μg/mL Aroclor)) were multiplied by a factor of 109 to convert the values to bioassay-derived TEQ values, measured in (ng TCDD equivalents)/(g Aroclor).51

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All six Aroclor mixtures induced LRG activity in chicken and Japanese quail AHR1-transfected cells, but only Aroclors 1248, 1242, and 1254 significantly induced LRG activity in pheasant AHR1-transfected cells. The EC50, PCx values, maximal responses, and ReP values for each Aroclor and AHR1 construct are provided in Table 1. Aroclors 1248, 1242, and 1254 induced the highest responses in all three AHR1 constructs, while Aroclors 1260, 1016, and 1221 generally induced lower responses. With the exception of Aroclor 1016, concentration−response curves achieved a plateau for chicken AHR1transfected cells dosed with all other Aroclors. Concentration− response curves also reached plateaus in ring-necked pheasant AHR1-transfected cells exposed to Aroclors 1248, 1242, and 1254, and in Japanese quail AHR1-transfected cells exposed to Aroclors 1260 and 1221, but all responses were below 10% of the positive control. Induction of EROD Activity in CEH Cultures. TCDD and all Aroclors significantly induced EROD activity in a concentration-dependent manner in CEH cultures (Figure 1B). EC50, TCDDx values, and maximal responses for each DLC treatment are presented in Table 2. Aroclors 1248, 1242, and 1254 induced the highest EROD responses, and no significant differences were observed among their maximal responses. No significant differences were observed between the maximal responses for Aroclors 1260, 1016, and 1221, but their responses were significantly lower than those induced by Aroclors 1248, 1242, and 1254. Comparisons between Chicken AHR1-Mediated LRG Activity and EROD Activity in CEH Cultures. Linear regression analyses comparing log-transformed EC50 values (Figure S1A, Supporting Information) or log-transformed TCDD10 and PC10 values (Figure S1B) from the chicken EROD and LRG assays, respectively, were performed. There was a significant or near-significant correlation between each pair of log-transformed endpoints, except when the EC50 for Aroclor 1221 was included (R2 = 0.31, P = 0.61). The LRG assay-based EC50 for Aroclor 1221 likely affected the linear regression, as it was similar to the EC50 values for Aroclors 1242 and 1254 even though Aroclor 1221 induced significantly lower responses in the chicken-AHR1 construct compared to the other Aroclors (Table 1). A much better relationship was observed between EROD and LRG assay EC50 values when the data point for Aroclor 1221 was excluded (Figure S1A; R2 = 0.89; P = 0.054). Log-transformed TCDD10 and PC10 values from the EROD and LRG assays (Figure S1B; R2 = 0.94, P = 0.0071) had a much better correlation than EROD and LRG EC50 values. TCDD10 and PC10 values are therefore a more useful endpoint for measuring Aroclor-mediated induction of EROD activity and LRG activity, respectively, because these endpoints are not affected by differences in the maximal responses achieved by different Aroclors and can also be calculated using partial concentration− response curves. No previously published study has systematically compared the toxic potencies of Aroclors in birds. However, induction of EROD activity and LRG activity by individual PCB congeners and other DLCs was shown to be significantly correlated with in ovo toxicity in several avian species.39,52 This, combined with the significant correlation observed between EROD and LRG activity in the present study, highlights the potential to use the EROD and LRG assays to predict the embryolethal effects of Aroclor mixtures in avian species, as has been previously demonstrated with other PCB congeners.39,52

RESULTS AND DISCUSSION

Induction of LRG Activity in COS-7 Cells. The concentration-dependent effects of TCDD and Aroclor mixtures on LRG activity in COS-7 cells transfected with chicken, ringnecked pheasant, or Japanese quail AHR1 constructs are shown in Figure 1A. LRG activity induced by TCDD reached a plateau in the chicken, ring-necked pheasant, and Japanese quail AHR1 constructs. Overall, the results indicated that the 0.1 μg/mL concentration of TCDD was an appropriate positive control for the normalization of LRG activity data. 8856


NA

Relative Sensitivity (ReS) of AHR1 Constructs to Aroclor Exposure. PC10- and EC50-based ReS values are presented in Table 3. ReS values indicated that the chicken AHR1 was the most sensitive, the ring-necked pheasant was slightly less (2 to 4 times) sensitive than chicken, and the Japanese quail AHR1 was significantly less sensitive than the chicken (8 times) and ring-necked pheasant AHR1 constructs to induction of LRG activity by Aroclors 1248, 1242, and 1254. The chicken and Japanese quail AHR1 constructs had similar sensitivity to induction by Aroclor 1260, but the chicken and Japanese quail ReS values differed by approximately 2-fold for Aroclors 1016 and 1221. Ring-necked pheasant ReS values for Aroclors 1260, 1016, and 1221 could not be calculated because no significant induction in LRG activity was observed. On the basis of the highest concentration tested (10 μg/mL), the ringnecked pheasant was estimated to be at least 4 and 8 times less sensitive than the chicken AHR1 construct to induction of LRG activity by Aroclors 1260 and 1221, respectively. The sensitivity of avian species to DLCs is associated with the identity of amino acids at positions 324 and 380 within the AHR1 LBD,29,31−33,39,41 and species can be classified into three general groups according to these amino acids. The rank order of sensitivity for AHR1 constructs exposed to TCDD, 2,3,7,8tetrachlorodibenzofuran, PCB 126, and PCB 77 was chicken-like constructs (Ile324_Ser380) > ring-necked pheasant-like constructs (Ile324_Ala380) > Japanese quail-like constructs (Val324_Ala380).31,32,38,39 Similar trends were also observed for Aroclors 1248, 1242, and 1254. However, the ring-necked pheasant AHR1 appeared to be less sensitive than the Japanese quail AHR1 to induction of LRG activity by Aroclors 1260, 1016, and 1221. In addition, no significant differences were observed between the EC50 values for the Japanese quail and chicken AHR1 constructs exposed to Aroclor 1260. When exposed to certain mono-ortho PCB congeners, namely PCBs 105 and 118, Japanese quail embryo hepatocyte cultures (JEH) were also found to be equally sensitive or slightly more sensitive to induction of EROD activity than CEH and ring-necked pheasant embryo hepatocytes.53 As was likely the case with PCBs 105 and 118 in avian hepatocyte cultures, the Japanese quail AHR1 may interact as strongly or more strongly with the components of Aroclor 1260 than the chicken and ring-necked pheasant constructs, respectively. The Japanese quail AHR1 also appears to interact more strongly with the components of Aroclors 1016 and 1221 than the ring-necked pheasant AHR1 construct. Relative Potency (ReP) of Aroclors in Different AHR1 Constructs and in CEH Cultures. Based on ReP values (Table 1, Table S1), visual inspection of the concentration−response curves (Figure 1A), and the results of statistical analyses between LRG assay endpoints (Table 1), the rank order of Aroclor potency was Aroclor 1248 ≥ 1242 = 1254 >1260 ≥ 1016> 1221 in the chicken AHR1 construct, Aroclor 1248 = 1242 = 1254 > 1016, 1221, and 1260 in the ring-necked pheasant construct, and Aroclor 1248 = 1242 = 1254 ≥ 1016 > 1260 = 1221 in the Japanese quail construct. Similar trends in the rank order of potency were observed for the chicken, ring-necked pheasant, and Japanese quail AHR1 constructs: Aroclors 1248, 1242, and 1254 had approximately the same potency and were more potent than Aroclors 1016, 1221, and 1260. There were no significant differences among PC10, PC20, or PC50 values for Aroclors 1248, 1242, and 1254 in cells transfected with the chicken or Japanese quail AHR1 constructs. PC10 and PC20 values for Aroclors 1248, 1242, and 1254 were often significantly smaller than those of 1260, 1016, and 1221 derived from the chicken or Japanese quail

EC50, TCDD10, TCDD20, TCDD50, TCDD80, and maximal responses represent the average of three replicates ± standard error (SE) from three independent experiments conducted on three separate 48well cell culture plates for each compound. The average relative potency (RePavg) values and ReP ranges were calculated from TCDD10-, TCDD20-, TCDD50-, and TCDD80-based ReP values (Table S2, Supporting Information). No range of ReP values was presented when only the RePTCDD10 could be calculated. †Superscript roman letters indicate significant differences among treatments (p < 0.05). NA: ReP estimates not available to calculate the value. NE: Not estimated because the maximum observed response was below 10%, 20%, 50%, or 80% of the TCDD maximal response.

2.1 × 10−5∼3.9 × 10−5 1.3 × 10−05−2.5 × 10−5 7.3 × 10−6∼1.3 × 10−5

3.0 × 10−5 1.9 × 10−5 1.0 × 10−5 9.2 × 10−7 1.5 × 10−6 NA 227 ± 23b 225 ± 14b 189 ± 17b 109 ± 3.0c 101 ± 4.0c 63.0 ± 5.9c Aroclor 1248 Aroclor 1242 Aroclor 1254 Aroclor 1260 Aroclor 1016 Aroclor 1221

TCDD chicken

Article

*

1.00−1.00

4.01 × 7.5 × 10 NE NE NE NE NE NE ± 5.51 × 5.9 × 10 NE NE NE NE NE NE 5.05 × ± 2.2 × 10 0.0130 ± 0.0030b 0.0202 ± 0.0008b,c 0.0383 ± 0.0047c NE NE NE 1.29 × ± 1.9 × 10 0.00602 ± 0.0012b 0.00968 ± 0.0001c 0.0177 ± 0.0018d 0.140 ± 0.0090e 0.0842 ± 0.0085f NE

5.47 × ± 2.3 × 10 0.0157 ± 0.0011b 0.0251 ± 0.0012c 0.0355 ± 0.0026c 0.135 ± 0.013d 0.0756 ± 0.0009e 0.574 ± 0.076f

ReP range RePavg

1.0 ±

428 ± 10

a

10−5 −6 10−6 −7 10−7 −8a 10−7 −9a 10−6 −7

maximal response ± SE (pmol/min/mg protein) TCDD80 ± SE (μg/mL) TCDD50 ± SE (μg/mL) TCDD20 ± SE (μg/mL) TCDD10 ± SE (μg/mL) EC50 ± SE (μg/mL) compound species

Table 2. Endpoints Determined for EROD Activity in Chicken Embryo Hepatocyte Cultures Exposed to TCDD or Aroclors 1248, 1242, 1254, 1260, 1016, and 1221*,†


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Table 3. Relative Sensitivity (ReS) Values for the Chicken, Ring-Necked Pheasant, and Japanese Quail AHR1 Constructs Exposed to TCDD or Aroclors 1248, 1242, 1254, 1260, 1016, and 1221*,† TCDD

Aroclor 1248

Aroclor 1242

Aroclor 1254

Aroclor 1260

Aroclor 1016

Aroclor 1221

AHR1 construct

ReSEC50

ReSPC10

ReSEC50

ReSPC10

ReSEC50

ReSPC10

ReSEC50

ReSPC10

ReSEC50

ReSPC10

ReSEC50

ReSPC10

ReSEC50

ReSPC10

chicken pheasant quail

1.0a 0.063b 0.0071c

1.0a 0.17a 0.013b

1.0a 0.25b NC

1.0a NC 0.13b

1.0a 0.49b NC

1.0a NC 0.12b

1.0a 0.56a NC

1.0a NC 0.12b

1.0a 1016 > 1221. As shown in Table 2, EROD assay-derived TCDD10 values for Aroclors 1248, 1242, and 1254 were approximately 1 order of magnitude lower than those of 1260 and 1016. A TCDD10 value could not be calculated for Aroclor 1221, because induction of EROD activity was below 10% of the TCDD positive control. LRG assay-derived RePavg values for the chicken AHR1 were similar to those obtained from EROD assays in CEH. Although the RePavg value for Aroclor 1260 in the LRG assay was approximately 10 times higher than the EROD-derived value, a significant correlation was observed between log-transformed chicken RePavg values from both assays (Figure 1C; R2 = 0.88, P = 0.018). Comparisons between Calculated Toxic Equivalents (TEQs) and Bioassay-Derived TEQs for Aroclors. Although the congener composition of Aroclor mixtures vary from lot to lot even in products from the same manufacturer,54 Burkhard and Lukasewycz 37 found that the TEQs were similar across different PCB product lines for mixtures of similar chlorine content. Calculated avian TEQs from Burkhard and Lukasewycz 37 and bioassay-derived TEQs for the Aroclor mixtures investigated in this study are presented in Table S3, Supporting Information. TEQs for the LRG and EROD assays were

Figure 2. Linear regression analyses comparing calculated TEQ values for birds from Burkhard and Lukasewycz37 with (A) chicken ERODTEQs, (B) chicken LRG-TEQs, and (C) Japanese quail LRG-TEQs. Data points for Aroclors 1221 and 1260 are excluded in Figure 2A−C and 2C, respectively, because RePavg values were not available to calculate their TEQ values. To be consistent with the units (ng/g) of the calculated TEQs from Burkhard and Lukasewycz,37 RePavg values derived from the LRG and EROD assays were multiplied by a factor of 109, as described in Statistical Analysis. All the TEQs are logtransformed values. 8858



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(Figure S2, Supporting Information; R2 = 0.00001−0.04). Differences between the calculated and chicken bioassay-derived TEQs for each Aroclor were within 1 order of magnitude. The chicken LRG assay, EROD assay, and calculated TEQs had approximately the same span in relative potency (Table S3). These results demonstrate that the WHO-TEFs for birds55 used to calculate the avian TEQs found in Burkhard and Lukasewycz37 gave reasonable ReP estimates for Aroclor mixtures that were in accordance with ReP values derived from EROD and LRG assays in the chicken. A linear regression analysis also revealed a significant relationship between calculated avian TEQs and Japanese quail LRG-TEQs (Figure 2C) (R2 = 0.98, P = 0.025). However, the LRG assay-derived TEQs for the Japanese quail were 1 to 2 orders of magnitude higher than the calculated TEQs for Aroclors 1254 and 1016, respectively. Furthermore, LRG assayderived TEQs for the Japanese quail were at least 1 order of magnitude higher than those for the chicken AHR1 for all Aroclors. This occurred because, while the chicken AHR1 was 100 times more sensitive to TCDD than the Japanese quail AHR1 in the LRG assay, smaller differences in sensitivity (

Relative Potencies of Aroclor Mixtures Derived from Avian in Vitro

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