(Submitted 9 March 2012; Returned for Revision 29 April 2012; Accepted 2 July 2012)

Transcription

1 Environmental Toxicology and Chemistry, Vol. 31, No. 11, pp , 2012 # 2012 TAC Printed in the USA DOI: /etc.1975 INCIDENCE OF JAW LESIONS AND ACTIVITY AND GENE EXPRESSION OF HEPATIC P4501A ENZYMES IN MINK (MUSTELA VISON) EXPOD TO DIETARY 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN, 2,3,7,8-TETRACHLORODIBENZOFURAN, AND 2,3,4,7,8-PENTACHLORODIBENZOFURAN STEVEN J. BURSIAN,*yz JEREMY MOORE,y JOHN L. NEWSTED, JANE E. LINK,y SCOTT D. FITZGERALD,k# NORA BELLO,yy VIRUNYA S. BHAT,zz DENI KAY, XIAOWEI ZHANG, STEVE WIMAN,kk ROBERT A. BUDINSKY,## JOHN P. GIESY,kk and MATTHEW J. ZWIERNIKy ydepartment of Animal Science, Michigan State University, East Lansing, Michigan, USA zcenter for Integrative Toxicology, Michigan State University, East Lansing Michigan, USA Cardno Entrix, Okemos, Michigan, USA kdepartment of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA #Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, Michigan, USA yydepartment of Statistics, Kansas State University, Manhattan, Kansas, USA zztoxicology Services Department, NSF International, Ann Arbor, Michigan, USA State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, China kkdepartment of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Canada ##Dow Chemical Company, Midland, Michigan, USA (Submitted 9 March 2012; Returned for Revision 29 April 2012; Accepted 2 July 2012) Abstract This study assessed the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8 tetrachlorodibenzofuran (TCDF) on the incidence of jaw lesions and on hepatic cytochrome P4501A (CYP1A) endpoints in mink (Mustela vison). Adult female mink were assigned randomly to one of 13 dietary treatments (control and four increasing doses of TCDD, PeCDF, or TCDF) and provided spiked feed for approximately 150 d (60 d prior to breeding through weaning of offspring at 42 d post-parturition). Offspring were maintained on their respective diets for an additional 150 d. Activity of hepatic CYP1A enzymes in adult and juvenile mink exposed to TCDD, PeCDF, or TCDD was generally greater compared with controls, but changes in other CYP1A endpoints were less consistent. Histopathology of the mandible and maxilla of juvenile mink suggested a doserelated increase in the incidence of jaw lesions. The dietary effective doses (ED) for jaw lesions in 50% of the population (ED50) were estimated to be 6.6, 14, and 149 ng/kg body weight (bw)/d for TCDD, PeCDF, and TCDF, respectively. The relative potencies of PeCDF and TCDF compared with TCDD based on ED10, ED20, and ED50 values ranged from 0.5 to 1.9 and 0.04 to 0.09, respectively. These values are within an order of magnitude of the World Health Organization toxic equivalency factor (TEF WHO ) values of 0.3 and 0.1 for PeCDF and TCDF, respectively. Environ. Toxicol. Chem. 2012;31: # 2012 TAC Keywords Mink Polychlorinated dibenzofuran Biomarker CYP1A Jaw lesion INTRODUCTION Studies indicating the presence of polychlorinated dibenzop-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in the vicinity of the Tittabawassee River, Michigan, USA [1] have led to concerns regarding the potential health risks they may pose to terrestrial and aquatic organisms. The mink (Mustela vison) is a sentinel species of special interest because it inhabits and consumes prey in both terrestrial and aquatic habitats and thus has a great potential for exposure [2]. In addition, laboratory studies have shown that mink are among the most sensitive species to the effects of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) and TCDD-like compounds, not only in terms of impaired reproduction and reduced survival of offspring [3 6], but also in the development of a unique jaw lesion characterized as mandibular and maxillary squamous epithelial proliferation. This jaw lesion has been reported in both laboratory [7 11] and field studies [12,13]. * To whom correspondence may be addressed (bursian@msu.edu). Published online 3 August 2012 in Wiley Online Library (wileyonlinelibrary.com). Several structurally analogous TCDD-like congeners exist that can bind to the aryl hydrocarbon receptor (AhR) with different affinities. The relative proportions of these congeners change as a function of time due to weathering in the environment and selective accumulation and metabolism. Therefore, an equivalency system has been developed to simplify the complexities of exposure to these compounds. In this equivalency approach developed by the World Health Organization (WHO) [14,15], the potency of a mixture of AhR-active congeners can be expressed as an equivalent amount of TCDD, which was considered at the time to be the most potent congener. 2,3,7,8- Tetrachlorodibenzo-p-dioxin equivalents (TEQ WHO ) are calculated as the sum of the product of the concentration of each congener multiplied by its respective consensus TCDD equivalency factor (TEF WHO ), which is based on multiple endpoints. Sampling of wild mink residing in the Tittabawassee River basin demonstrated the presence of AhR-active PCDD and PCDF congeners in their tissues. Using the TEF WHO approach, the mean concentration of TEQ WHO in livers of mink harvested downstream of Midland Michigan, USA, which is the location of the source of PCDDs and PCDFs entering the Tittabawassee River, was 400 ng TEQ WHO /kg, wet weight. In the upstream 2545

2 2546 Environ. Toxicol. Chem. 31, 2012 S.J. Bursian et al. locations, the mean TEQ WHO concentration in liver was 20 ng TEQ WHO /kg, wet weight [16]. Of the total TEQ WHO measured in downstream mink, approximately 73% was contributed by PCDFs and 5% was contributed by PCDDs. The individual congeners contributing the greatest proportion of TEQ WHO were 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8- tetrachlorodibenzofuran (TCDF). Estimates of exposure based on measurements of PCDDs/ PCDFs in both the diet and tissues of mink were such that, based on the current understanding of the toxicological potency of these mixtures derived from laboratory studies, mink should be experiencing adverse effects with hazard quotients (ratio of estimated chemical intake [dose] and a reference dose below which adverse health effects are unlikely) ranging from less than 1.0 to 10 [10,11,17,18]. No pathology was reported, however, in any of the 48 wild mink collected from within the basin, and measures of the population, including abundance and demographics, indicated that mink populations are stable and at or close to carrying capacity for the Tittabawassee River [19]. Reasons for the observed inconsistency between the apparently healthy population and hazard quotients greater than 1.0 are unclear. Possible explanations include (1) TEF WHO that are too conservative, resulting in an overestimation of risk; (2) the toxicity reference values used to estimate hazard quotients for mink are inaccurate due to a lack of toxicological information for the two dominant PCDF congeners, necessitating use of toxicity reference values derived for other classes of chemicals such as TCDD-like PCBs; and (3) pharmacokinetics and rates of metabolism of TCDF and PeCDF were different compared with those of TCDD or TCDD-like PCBs that have been studied in mink [3,4,6,20]. Through a series of studies, several of the uncertainties outlined above were addressed. Specifically, the pharmacokinetics of TCDF and PeCDF in mink were examined [20] and the toxicological potency of PCDD and PCDF at the molecular and organismal level in mink was evaluated [21,22]. Uncertainties regarding the potencies of TCDF and PeCDF were also addressed by conducting a dietary reproduction study, the overall objective of which was to assess the effects of TCDD, PeCDF, or TCDF on reproductive performance of adult female mink and the survival and growth of their offspring. The effects of TCDD, PeCDF, and TCDF on reproduction, survival, and growth are reported in Moore et al. [23]. Additional objectives of the reproduction study, which are the subject of the present study, were to (1) determine the relationships between dietary exposure to TCDD, PeCDF, or TCDF on mrna transcription and protein synthesis of CYP1A1 and CYP1A2, as well as ethoxyresorufin-o-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities in the liver, which are sensitive, functional biomarkers of exposure, in adult mink and their 27-week-old offspring; (2) determine the incidence and severity of the jaw lesion in 27-week-old juvenile mink; and (3) determine the relative potencies of PeCDF and TCDF to TCDD based on CYP1A endpoints and jaw lesion responses. MATERIALS AND METHODS Chemicals and reagents 2,3,7,8-Tetrachlorodibenzo-p-dioxin, TCDF, and PeCDF were obtained from AccuStandard and dissolved in hexane (OmniSolv, EMD Chemicals) to produce a stock solution for each congener. For enzyme analyses, 7 ethoxyresorufin was obtained from Molecular Probes, and 7-methoxyresorufin and resorufin were obtained from Sigma-Aldrich. All other biochemical reagents, including reduced nicotinamide adenine dinucleotide phosphate, were obtained from Sigma-Aldrich and were reagent grade or better, unless stated otherwise. For the molecular analyses, the key reagents used included the Agilent Total RNA Isolation Mini Kit (Agilent Technologies), Superscript III first-strand synthesis SuperMix (Invitrogen), and SYBR Green master mix (Applied Biosystems). For immunoblots of the CYP1A1 and CYP1A2 proteins, protease inhibitor cocktail and bicinchoninic acid reagent were purchased from Sigma-Aldrich. All electrophoresis reagents and molecular weight markers were from Bio-Rad. b-actin antibody (mouse anti b-actin monoclonal antibody) and CYP1A1 þ 1A2 antibody (goat-anti CYP1A1 þ 1A2 polyclonal antibody) were from Abcam. The secondary antibodies to b-actin and CYP1A1 þ 1A2 were alkaline phosphatase conjugated to either goat anti-mouse IgG for b-actin (Abcam) or rabbit anti-goat for CYP1A1 þ 1A2 (Abcam). Nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate salt were obtained from Fisher Scientific. Dietary treatments The study, which was approved by the Michigan State University (MSU) Institutional Animal Care and Use Committee (Animal Use Form 12/ ), was conducted at the MSU Experimental Fur Farm. Housing of mink complied with guidelines specified in the Standard Guidelines for the Operation of Mink Farms in the United States [24]. A standard dietary mix was used throughout the study with the specific ingredients, mix ratios, and nutritional data presented in Moore et al. [23]. The base diet was used as the control diet, with treatment diets differing only in the supplemental TCDD, TCDF, or PeCDF added. The dosing regime was set to bracket a wide range of concentrations designed to elicit responses in one or more measurement endpoints. Lower doses bracketed environmentally relevant concentrations and were expected to result in no observable effect levels for all but the most sensitive biological responses. The highest dose for each congener was greater than the median predicted environmental exposure for the Tittabawassee River of 3.85 ng TEQ WHO /kg body weight (bw)/d. Based on TEF WHO -normalized PCB and PCB-mixture feeding studies [6,10,11,17,25 27], the highest dose of each compound was expected to result in complete reproductive failure. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, TCDF, or PeCDF were dissolved in hexane to produce stock solutions, and aliquots of the stock were then diluted with approximately 100 ml corn oil. The corn oil solutions were then added to the mink diet and mixed well prior to adding other feed ingredients. The feed was mixed for approximately 30 min and then stored in 3.8-L aluminum containers. Grab samples were taken from each batch of feed and stored in I-Chem jars for congener concentration determination. All samples were stored in a freezer ( 208C) until analysis. Animal husbandry Adult female mink were housed individually in wire breeder cages (76 cm L 46 cm W 38 cm H) suspended above the ground in an open-sided mink shed. A wooden nest box (38 cm L 25 cm W 29 cm H) bedded with aspen shavings and excelsior (wood wool) was attached to the outside of each cage. When the resulting offspring were 18 weeks of age, they were moved to wire grower cages (61 cm L 25 cm W 38 cm H) suspended above the ground in an open-sided mink shed until the end of the study. A wooden nest box

3 Biomarkers in mink exposed to TCDD-like chemicals Environ. Toxicol. Chem. 31, (25 cm L 20 cm W 29 cm H) bedded with aspen shavings was suspended within each grower cage. Feed (125 g) was placed on a grid on the top of the cage on a daily basis. Water was available ad libitum. Experimental design A total of 117 first-year (virgin) and second-year (proven breeder), natural dark, female mink were assigned randomly to one of 13 treatment groups (Table 1). Nine animals per treatment group were assigned randomly to a bank of nine cages separated from the next bank of nine cages by an empty cage. Assigning treatments to banks of cages was done to prevent cross-contamination between doses and compounds. Untreated, natural dark, male mink were used for breeding purposes only. Mink were fed the control feed for 10 d prior to being fed treated diets. All surviving adults were euthanized (CO 2 ) when kits were 6 weeks old. The adult females were necropsied, and samples of selected tissues were collected for analytical and histological assessment. Nine to 29 kits from three to six litters for each of the 13 treatment groups were maintained on their respective diets until they were 27 weeks old, at which time they were euthanized and processed as above. Necropsy Immediately following euthanasia, body weight and length were recorded. A board-certified veterinary pathologist assessed the overall physical condition of the animal as well as gross appearance of organs. The thyroid gland, thymus, heart, adrenal glands, kidneys, spleen, mesenteric lymph node, reproductive organs (uterus with ovaries/testes), liver, and brain were removed, weighed, and placed in buffered formalin for subsequent histological assessment. A 2-g subsample of liver was distributed between two cryovials (Corning Costar) that were placed in liquid nitrogen for subsequent determination of microsomal EROD and MROD activities and two vials containing RNAlater (Qiagen) for subsequent CYP1A1 and CYP1A2 gene expression analysis. The flesh of each skull was removed, and the skull was placed in formalin for subsequent histological examination of the maxilla and mandible. EROD and MROD quantification Liver microsomes were prepared by homogenizing 0.5 g of liver in Tris buffer (0.05 M Tris and 1.15% KCl, ph 7.5) and centrifuging to obtain the microsomal fraction. The microsomal pellet was resuspended in microsomal stabilization buffer (20% glycerol, 0.1 M KH 2 PO 4, 1 mm EDTA, and 1 mm dithiothreitol, ph 6.25), and aliquots were stored at 808C. Ethoxyresorufin- O-deethylase and MROD activities were measured using a modification of methods described by Kennedy and Jones [28] and as detailed in Moore et al. [22]. The assays were optimized and conducted in 96-well plates (Corning Costar), where both microsomal cytochrome P450 activity and protein concentration were measured simultaneously using a Fluoroscan Ascent microplate fluorometer (Thermo Fisher Scientific). Ethoxyresorufin O-deethylase and MROD activities were determined kinetically from the linear range of the time-curves for each well, and the results were expressed as pmol substrate converted per minute, per millegram protein (pmol/min/mg). Total RNA isolation and reverse transcription polymerase chain reaction Total RNA was extracted from the livers of individual mink using the Agilent Total RNA Isolation Mini Kit according to the manufacturer s protocol. Purified RNA was stored at 808C until analysis. First-strand cdna synthesis and reverse transcription was performed as described in Zhang et al. [21]. The cdna synthesis reactions were stored at 208C until further analysis. Real-time polymerase chain reaction Quantitative real-time polymerase chain reactions (Q-RT- PCRs) were performed using an ABI 7900 high-throughput real-time PCR System in 384-well PCR plates (Applied Biosystems). Polymerase chain reaction primers used in these reactions were: CYP1A1 sense CCT AAG CAC CTG GAA AGC; CYP1A1 anti-sense CTA AGT GTC AGA GGG ATT GG; CYP1A2 sense ACA GCA GRG AGC ACA GAT GG; CYP1A2 anti-sense CCA GAG TAC CAG GCA GAA GAG; b- actin sense GAT GTG GAT CAG CAA GCA GGA G; b-actin anti-sense GCC AGC AGT CCG TTT AGA AGC [21]. Poly- Table 1. Concentrations of TCDD, PeCDF, and TCDF in the diet and corresponding doses Dietary concentration Consumed dose a n b ng/kg feed ng TEQ WHO /kg feed c ng/kg bw/d ng TEQ WHO /kg bw/d TCDD d PeCDF TCDF a Consumed dose based on estimated feed consumed from a daily allotment of 125 g feed and mean body weights (bw) of adult female mink through week 15 of the study. b n refers to the number of dietary samples analyzed. c TEQ WHO were determined using mammalian toxic equivalency factors of 1.0, 0.3, and 0.1 for TCDD, PeCDF, and TCDF, respectively [17]. d Data are presented as mean standard deviation. TCDD ¼ 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF ¼ 2,3,4,7,8-pentachlorodibenzofuran; TCDF ¼ 2,3,7,8-tetrachlorodibenzofuran; TEQ WHO ¼ TCDDtoxic equivalents based on World Health Organization (WHO) toxic equivalency factors.

4 2548 Environ. Toxicol. Chem. 31, 2012 S.J. Bursian et al. merase chain reaction mixtures for 100 reactions contained 500 ml of SYBR Green master mix (Applied Biosystems), 2 ml of 10 mm sense/anti-sense gene-specific primers, and 380 ml of nuclease-free distilled water. A final reaction volume of 10 ml was made up with 2 ml of diluted cdna and 8 ml of PCR reaction mixtures using a Biomek automation system (Beckman Coulter). The PCR reaction mix was denatured at 958C for 10 min before the first PCR cycle. The thermal cycle profile was as follows: denaturation for 15 s at 958C; annealing for 30 s at 608C; and extension for 30 s at 728C. A total of 45 PCR cycles was used. Polymerase chain reaction efficiency, uniformity, and linear dynamic range of each Q-RT-PCR assay were assessed by the construction of standard curves using DNA standards. The CYP1A1 and CYP1A2 mrna expression levels in the liver of mink were measured by semiquantitative RT-PCR using b-actin as the reference gene. Polymerase chain reaction efficiency and linear dynamic range of each Q-RT-PCR assay was assessed by construction curves using DNA standard [21]. Western blot analysis Samples of microsomes isolated from mink livers were prepared in the following manner for use in Western analyses of CYP1A proteins: The concentrations of protein in the microsomes were determined by the bicinchoninic acid protein assay method using bovine serum albumin as the standard according to the manufacturer s instructions (Sigma-Aldrich). Samples were adjusted to a final concentration of 2 mg/ml, and 40 mg of protein was separated on 8% polyacrylamide gels using the discontinuous buffer system of Laemmli [21]. Proteins were transferred (20 V for 20 min) onto a 0.45-mM nitrocellulose membrane (Bio-Rad) using Trans-blot SD semidry electrophoretic transfer cell (Bio-Rad). A 5% solution of nonfat dry milk in 1 TTBS (2 mm Tris, 30 mm NaCl, 0.01% Tween 20, ph 7.5) was used as a blocking agent (1 h at room temperature) and for diluting antibodies. The blots were incubated with primary antibodies for 1 h at room temperature, followed by 1-h incubation with an alkaline phosphatase conjugated secondary antibody. The membranes were washed after incubation in either primary (2 15 min washes in TTBS [2 mm Tris, 30 mm NaCl, ph 7.5, 0.1% Tween-50]) or secondary antibodies (2 15 min in TTBS followed by 1 10 min in TBS). The antibody used in the present study was raised against CYP1A1 and CYP1A2 in rats and was not raised specifically against CYP1A1 and CYP1A2 in mink. A preliminary analysis using microsomes isolated from livers of rats and mink showed that the banding patterns in the two samples were identical. In the western blots, there were two clearly distinguishable bands representing CYP1A1 and CYP1A2. Proteins of interest were detected using 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium color substrate (Bio-Rad). Images were captured on a VersaDoc Imaging system (Bio-Rad). The protein bands were quantified using Quantity One Software (Bio-Rad). Quantification of chemicals To ensure that co-contaminants were not a factor in the study, the concentrations of 17 2,3,7,8-substituted PCDF and PCDD congeners and 12 PCB congeners were measured in the dietary items, feed samples, and liver tissue as described in Zwiernik et al. [20]. Concentrations of TEQs were calculated as the sum of the products of the concentrations of congeners multiplied by their respective TEF WHO [17]. A surrogate value of one-half the method detection limit (MDL) was used for concentrations less than the MDL. Liver tissues were extracted following a modification of U.S. Environmental Protection Agency (U.S. EPA) Method 1613B. Histological analysis Histological examination of tissues was performed at MSU s Diagnostic Center for Population and Animal Health. A boardcertified veterinary pathologist examined slides of the maxilla and mandible of each mink sampled at necropsy for squamous epithelial proliferation, which was scored using a modification of the scoring system described in [12] where 0 ¼ normal morphology, no squamous epithelial cell proliferation; 1 ¼ mild lesion, focal (one or two) squamous epithelial cysts invading jaw bone, restricted to a single region of the dental arcade (molar, premolar, incisor); 2 ¼ moderate lesion, multiple squamous epithelial cysts (three or more), adjacent to several teeth or in two or more regions of the dental arcade; and 3 ¼ severe lesion, multiple sites of squamous epithelial proliferation that are larger in size or coalescing (Fig. 1). Histological results for other tissues sampled in the study are presented in Moore et al. [23]. Statistical analysis Dietary concentrations of compounds are presented in Table 1 as means standard deviations for the number of times the particular diet was mixed and analyzed. Due to the unbalanced experimental design (unequal sample sizes), least square mean estimates and their corresponding estimated standard errors are reported for the various endpoint analyses. Statistical models for adult female liver and adipose TEQ WHO concentrations, and for hepatic cytochrome P4501A endpoints, included the fixed effects of treatment and dose within treatment; for juveniles, the statistical models included the fixed effects of treatment, dose within treatment, and sex, as well as the random effect of litter. When treatment effects were statistically significant, differences among treatment groups were tested with the Tukey Kramer test to prevent inflation of type I error rate while accounting for differences in sample size among the treatments. Differences among treatments were considered significant at p < The MIXED procedure of SAS (ver 9.2, SAS Institute) was used for these analyses. The presence or absence of jaw lesions in the 27-week-old kits was analyzed using a generalized linear mixed model fitted to the binary outcome using a logit link function. The model included the fixed effects of chemical, dose (in TEQ WHO ), and their two-way interaction in order to evaluate chemical-specific susceptibility in mink. World Health Organization TEFs of 1.0, 0.3, and 0.1 were used to scale the doses of TCDD, PeCDF, and TCDF, respectively, to TEQ WHO. Also included in the statistical models was the random effect of litter nested within treatment to account for technical replication in the design and to recognize litter as the experimental unit for treatment. Differences between treatment groups were considered significant at p < The GLIMMIX procedure of SAS was used for this analysis. United States Environmental Protection Agency benchmark dose (BMD) software (ver 2.2; index.html) was used to determine effect doses or concentrations (as different percentages of the population; ED10/EC10, ED20/EC20, and ED50/EC50) for jaw lesion incidence in 27-week-old juvenile mink, with exposure metrics being average daily dose or liver concentrations (when appropriate) that were expressed as analytical concentration or TEQ WHO. The best-fit model for each compound was chosen collectively considering the following criteria: the highest p value, chi-squared

5 Biomarkers in mink exposed to TCDD-like chemicals Environ. Toxicol. Chem. 31, Fig. 1. View of a normal mink jaw (A). Cysts (cy) of squamous epithelial proliferation within the jaw adjacent to teeth (T), scored as mild (B); moderate (C); or severe (D). (50 magnification). residual values less than 2, the lowest Akaike s Information Criterion value, the smallest margin between the benchmark dose (BMD) and benchmark dose lower 95% confidence limit (BMDL) curves, and a good visual fit of the BMD and BMDL curves. Prior to calculation of EC values, the hepatic concentrations of TCDD, PeCDF, or TCDF as a function of dietary dose were modeled using the U.S. EPA BMD software. For congeners having an adequate dose response relationship based on measures of statistical tests of fit for continuous models (chisquared parameters and likelihood ratios), EC values were then determined. RESULTS Exposure and tissue concentrations Dietary concentrations and consumed doses of TCDD, TCDF, or PeCDF expressed as ng chemical and ng TEQ WHO are presented in Table 1. Calculations of dose were based on the average estimated feed consumption and body mass of adult females in each treatment group through the first 15 weeks of the trial [23]. Concentrations of TEQ WHO contributed by TCDD, PeCDF, and TCDF in livers and adipose tissue of adult and juvenile mink are presented in Tables 2 and 3, respectively. In general, TEQ concentrations increased in a dose-related manner in liver and adipose tissue of adult and juvenile mink exposed to TCDD or PeCDF. Hepatic TEQ concentrations in adult females exposed to TCDF were significantly greater compared with controls, but there was no evidence that concentrations increased with increasing dose. In contrast, hepatic TEQ concentrations of juveniles exposed to TCDF increased in a dose-related manner. Adipose TEQ concentrations in adults and juveniles exposed to TCDF were significantly greater compared with controls only at the greatest dose (246 ng TCDF/kg bw/d or 25 TEQ WHO-TCDF /kg bw/d. EROD and MROD activity Hepatic EROD and MROD activities in adult mink (Table 4) exposed to dietary TCDD or PeCDF via the diet were greater than activities in nonexposed controls, but, in general, there was no evidence for a dose-dependent increase in enzyme activities in any of the treatment groups with the exception of MROD activity in females exposed to TCDD. Activity of MROD was significantly greater in animals exposed to 6.0 and 8.4 ng TCDD/kg bw/d (6.0 and 8.4 ng TEQ WHO-TCDD /kg bw/d) compared with those exposed to 2.1 and 4.6 ng TCDD/kg bw/d (2.1 and 4.6 ng TEQ WHO-TCDD /kg bw/d). In adult females exposed to TCDF, EROD activity was significantly greater compared with control activity only at 214 ng TCDF/kg bw/d (21 ng TEQ WHO-TCDF /kg bw/d), and MROD activity was significantly greater compared with control activity at 52 and 214 ng TCDF/ kg bw/d (5.2 and 21 ng TEQ WHO-TCDF /kg bw/d) but not at 116 and 246 ng TCDF/kg bw/d (12 and 25 ng TEQ WHO-TCDF /kg bw/d). In 27-week-old juvenile mink (Table 5), EROD activity was significantly greater compared with control activity at all doses of TCDD, PeCDF, and TCDF, but there was no evidence of a dose response relationship. Activity of MROD was significantly greater compared with control activity at all doses of TCDD (no evidence of a dose response relationship) and at all doses of PeCDF except 30 ng PeCDF/kg bw/d (9.0 ng TEQ WHO-PeCDF )/kg bw/d. The activity of MROD was also significantly greater compared with control activity for all doses of TCDF, with activity at 214 ng TCDF/kg bw/d (21 ng TEQ WHO-TCDF /kg bw/d) being greater than activity at 52 ng TCDF/kg bw/d (5.2 ng TEQ WHO-TCDF /kg bw/d). Hepatic CYP1A mrna expression Exposure of adult female mink to PeCDF resulted in a significant increase in hepatic CYP1A1 and CYP1A2 mrna

6 2550 Environ. Toxicol. Chem. 31, 2012 S.J. Bursian et al. Table 2. Concentrations of TEQ WHO in liver of adult and 27-week-old juvenile mink exposed to TCDD, PeCDF, or TCDF via the diet a Consumed dose (ng TEQ WHO /kg bw/d) Liver (ng TEQ WHO /kg, wet wt) b n Adults n Juveniles Control A A 79.5 TCDD B B BC C BC D C E 14.0 PeCDF B B BC BC C C D D 113 TCDF B B B C B D B D 1.18 a TEQ WHO determined using mammalian toxic equivalency factors of 1.0, 0.3, and 0.1 for TCDD, PeCDF, and TCDF, respectively [17]. b Data are presented as least squares mean and standard error (). Adult female mink and juvenile mink were sampled approximately 9 and 27 weeks postpartum, respectively. Means within the same column with different uppercase letters are significantly different at p < TEQ WHO ¼ TCDD-toxic equivalents based on World Health Organization (WHO) toxic equivalency factors; TCDD ¼ 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF ¼ 2,3,4,7,8-pentachlorodibenzofuran; TCDF ¼ 2,3,7,8-tetrachlorodibenzofuran. Table 3. Concentrations of TEQ WHO in adipose tissue of adult and 27-week-old juvenile mink exposed to TCDD, PeCDF, or TCDF via the diet a Consumed dose (ng TEQ WHO /kg bw/d) Adipose (ng TEQ WHO /kg, wet wt) b n Adults n Juveniles Control A A 57.8 TCDD B B B C C ,105 CD ,418 D ,560 D 113 PeCDF B B BC C CD C D D 64.1 TCDF A A A A AB AB B B 2.99 a TEQ WHO were determined using mammalian toxic equivalency factors of 1.0, 0.3, and 0.1 for TCDD, PeCDF, and TCDF, respectively [17]. b Data are presented as least squares mean and standard error (). Adult female mink and juvenile mink were sampled approximately 9 and 27 weeks postpartum, respectively. Means within the same column with different uppercase letters are significantly different at p < TEQ WHO ¼ TCDD-toxic equivalents based on World Health Organization (WHO) toxic equivalency factors; TCDD ¼ 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF ¼ 2,3,4,7,8-pentachlorodibenzofuran; TCDF ¼ 2,3,7,8-tetrachlorodibenzofuran. Table 4. Hepatic EROD and MROD activities in adult female mink exposed to TCDD, PeCDF, or TCDF via the diet Consumed dose a (ng TEQ WHO /kg bw/d) n EROD b (pmol/min/mg) MROD b (pmol/min/mg) Control A A 4.28 TCDD AB B B AB B C B C 4.94 PeCDF B B B B B B B B 6.05 TCDF A B A AB B B A AB 4.28 a TEQ WHO were determined using mammalian toxic equivalency factors of 1.0, 0.3, and 0.1 for TCDD, PeCDF, and TCDF, respectively [17]. b Data are presented as least square mean and standard error (). Means within the same column with different letters are significantly different from one another ( p < 0.05). Adult female mink were sampled at approximately 9 weeks postpartum. EROD ¼ ethoxyresorufin-o-deethylase; MROD ¼ methoxyresorufin-o-demethylase; TCDD ¼ 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF ¼ 2,3,4,7,8- pentachlorodibenzofuran; TCDF ¼ 2,3,7,8-tetrachlorodibenzofuran; TEQ WHO ¼ TCDD-toxic equivalents based on World Health Organization (WHO) toxic equivalency factors.

7 Biomarkers in mink exposed to TCDD-like chemicals Environ. Toxicol. Chem. 31, Table 5. Hepatic EROD and MROD activities in 27-week-old juvenile mink exposed to TCDD, PeCDF, or TCDF via the diet Consumed dose a (ng TEQ WHO /kg bw/d) n EROD b (pmol/min/mg) MROD b (pmol/min/mg) Control A A 4.76 TCDD B B B B B B B B 4.76 PeCDF B B B B B AB B B 5.82 TCDF B B B BC B C B BC 5.21 a TEQ WHO were determined using mammalian toxic equivalency factors of 1.0, 0.3, and 0.1 for TCDD, PeCDF, and TCDF, respectively [17]. b Data presented as least squares mean and standard error (). Means within the same column with different letters are significantly different from one another ( p < 0.05). Juvenile mink sampled at approximately 27 weeks post partum. EROD ¼ ethoxyresorufin-o-deethylase; MROD ¼ methoxyresorufin-o-demethylase; TCDD ¼ 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF ¼ 2,3,4,7,8-pentachlorodibenzofuran; TCDF ¼ 2,3,7,8-tetrachlorodibenzofuran; TEQ WHO ¼ TCDD-toxic equivalents based on World Health Organization (WHO) toxic equivalency factors. expression compared with controls at 13 and 30 ng PeCDF/kg bw/d (4.0 and 9.0 ng TEQ WHO-PeCDF /kg bw/d), but not at 25 and 49 ng PeCDF/kg bw/d (7.6 and 15 ng TEQ WHO-PeCDF /kg bw/d). CYP1A1 and CYP1A2 mrna levels in TCDD- and TCDFtreated mink were not significantly different from those in control mink (Table 6). For juvenile mink, expression of CYP1A1 and CYP1A2 mrna was significantly greater compared with controls in animals exposed to TCDD or PeCDF, but not TCDF (Table 7). In animals exposed to TCDD, expression of both CYP1A1 and CYP1A2 was greater compared with controls at doses greater than 2.1 ng TCDD/kg bw/d (2.1 ng TEQ WHO-TCDD /kg bw/d). In animals exposed to PeCDF, expression of CYP1A1 and CYP1A2 mrna was significantly greater compared with controls at doses of 13 and 30 ng PeCDF/ kg bw/d (4.0 and 9.0 ng TEQ WHO-PeCDF /kg bw/d), but not at 25 and 49 ng PeCDF/kg bw/d (7.6 and 15 ng TEQ WHO-PeCDF /kg bw/d). CYP1A protein in liver No evidence was found for any consistent effect of exposure to TCDD, PeCDF, or TCDF on concentrations of CYP1A protein (Tables 6 and 7). In adult female mink treated with TCDD, only the 4.6- and 8.4-ng TCDD/kg bw/d (4.6 and 8.4 ng TEQ WHO-TCDD /kg bw/d) groups had CYP1A protein concentrations that were greater than controls. For PeCDF, mink dosed with the greatest concentration (49 ng PeCDF/kg bw/d or 15 ng TEQ WHO-PeCDF /kg bw/d) had significantly greater protein concentrations compared with controls and with animals in the 13-ng PeCDF/kg bw/d (4.0 ng TEQ WHO-PeCDF /kg bw/d) group. Exposure to TCDF had no significant effect on CYP1A1 protein concentrations in the liver. In juvenile mink, there was no evidence that exposure to TCDD or TCDF had an effect on CYP1A protein concentrations. Exposure to the greatest dose of PeCDF (49 ng PeCDF/kg bw/d or 15 ng TEQ WHO-PeCDF /kg Table 6. Hepatic CYP1A endpoints in adult female mink exposed to TCDD, PeCDF, or TCDF via the diet Consumed dose a (ng TEQ WHO /kg bw/d) n CYP1A1 mrna expression b (fold-change) CYP1A2 mrna expression b (fold-change) CYP1A protein b (fold-change) Control A A A 0.66 TCDD A A A A A B A A A A A B 0.76 PeCDF B B A AB AB AB B B AB AB AB B 0.93 TCDF A A A A A A A A A A A A 0.66 a TEQ WHO were determined using mammalian toxic equivalency factors of 1.0, 0.3, and 0.1 for TCDD, PeCDF, and TCDF, respectively [17]. b Fold change in gene expression was calculated by the delta cycle threshold (Ct) method where CYP1A1 and CYP1A2 levels were first normalized to the b-actin Ct values and then all samples were normalized to the mean of the biological control samples. Data are presented as least square mean and standard error (). Means within the same column with different letters are significantly different from one another ( p < 0.05). Adult female mink were sampled at approximately 9 weeks postpartum. CYP1A ¼ cytochrome P4501A; TCDD ¼ 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF ¼ 2,3,4,7,8-pentachlorodibenzofuran; TCDF ¼ 2,3,7,8-tetrachlorodibenzofuran; TEQ WHO ¼ TCDD-toxic equivalents based on World Health Organization (WHO) toxic equivalency factors.

8 2552 Environ. Toxicol. Chem. 31, 2012 S.J. Bursian et al. Table 7. Hepatic CYP1A endpoints in 27-week-old juvenile mink exposed to TCDD, PeCDF, or TCDF via the diet Consumed dose a (ng TEQ WHO /kg bw/d) n CYP1A1 mrna expression b (fold-change) CYP1A2 mrna expression b (fold-change) CYP1A protein b (fold-change) Control A A A 0.58 TCDD A A A B B A B B A B B A 0.47 PeCDF B AB AB BC BC AB BC B AB C C B 0.41 TCDF A A A A A A A A A A A A 0.41 a TEQ WHO were determined using mammalian toxic equivalency factors of 1.0, 0.3, and 0.1 for TCDD, PeCDF, and TCDF, respectively [17]. b Fold change in gene expression was calculated by the delta cycle threshold (Ct) method where CYP1A1 and CYP1A2 levels were first normalized to the b-actin Ct values and then all samples were normalized to the mean of the biological control samples. Data presented as least squares mean and standard error (). Means within the same column with different letters are significantly different from one another ( p < 0.05). Juvenile mink sampled at approximately 27 weeks post partum. CYP1A ¼ cytochrome P4501A; TCDD ¼ 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF ¼ 2,3,4,7,8-pentachlorodibenzofuran; TCDF ¼ 2,3,7,8-tetrachlorodibenzofuran; TEQ WHO ¼ TCDD-toxic equivalents based on World Health Organization (WHO) toxic equivalency factors. bw/d) resulted in a significant increase in hepatic CYP1A protein concentrations in juvenile mink compared with controls. Jaw lesions Histopathological assessment of the mandible and maxilla of 27-week-old juvenile mink indicated the presence of jaw lesions in mink exposed to TCDD, TCDF, or PeCDF. Lesion severity was predominantly mild to moderate (lesion scores of 1 and 2, respectively). One animal in the 49-ng PeCDF/kg bw/d (15 ng TEQ WHO-PeCDF /kg bw/d) group had a lesion score of 3 (severe) (Fig. 2). A significant p value ( p ¼ ) for the interaction between chemical treatments and dose on the probability of jaw lesion indicates that the toxicity response in terms of TEQ doses is specific to each chemical (Fig. 3). The effective consumed doses resulting in 10, 20, and 50% of the animals having an increased incidence of the lesion over background expressed as estimated ED10, ED20, and ED50 values are presented in Table 8. Assessment of dose response relationships between consumed dose and hepatic concentrations of TCDD, PeCDF, or TCDF indicated that concentrations of TCDD and PeCDF in the liver could be used as a reliable internal dose metric for jaw lesions (as a surrogate for external consumed dose), but hepatic TCDF concentration could not because there was no evidence of a dose response relationship. Thus, EC10, EC20, and EC50 values for TCDD and PeCDF only are presented in Table 8. Fig. 2. Observed incidence and severity of maxillary and mandibular squamous epithelial proliferation in 27-week-old juvenile mink treated with TCDD, PeCDF, or TCDF (ng TEQ WHO /kg bw/d). Total number of animals per treatment: n ¼ 16 for control; n ¼ 10 for PeCDF 4.0, TCDF 12 and 25; n ¼ 9 for TCDD 2.1, 4.6, 6.0, 8.4, PeCDF 9.0, 15, TCDF 5.2 and 21; and n ¼ 7 for PeCDF 12 ng TEQ WHO /kg bw/d. TCDD ¼ 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF ¼ 2,3,4,7,8-pentachlorodibenzofuran; TCDF ¼ 2,3,7,8-tetrachlorodibenzofuran; TEQ WHO ¼ TCDD-toxic equivalents based on World Health Organization (WHO) toxic equivalency factors.

9 Biomarkers in mink exposed to TCDD-like chemicals Environ. Toxicol. Chem. 31, Fig. 3. Incidence of jaw lesion by dose. Because the dose is expressed in TEQ WHO, a significant p value for the interaction between dose and chemical treatments ( p ¼ ) indicates evidence for heterogeneous slopes in the toxicity response to dose for each chemical. TCDD ¼ 2,3,7,8- tetrachlorodibenzo-p-dioxin; PeCDF ¼ 2,3,4,7,8-pentachlorodibenzofuran; TCDF ¼ 2,3,7,8-tetrachlorodibenzofuran; TEQ WHO ¼ TCDD-toxic equivalents based on World Health Organization (WHO) toxic equivalency factors. Cytochrome P450 DISCUSSION Given the ability of AhR agonists such as TCDD, TCDF, or PeCDF to upregulate CYP1A genes [29,30], three different measures of CYP1A expression were evaluated in the current study. These included mrna transcription levels, concentrations of catalyst proteins, and enzymatic activities. The purpose of evaluating these endpoints was to further characterize their utility as functional indicators of exposure to TCDD-like compounds in mink [30]. In the current study, only EROD and MROD activities were increased by exposure to each of the three chemicals in both adult and juvenile mink, and only exposure to PeCDF resulted in an increase in all three endpoints in adults and juveniles, although there was no evidence that the increases were related to dose. The absence of a consistent effect of exposure to TCDD, PeCDF, or TCDF on CYP1A metrics may have been due to several factors including metabolism or sequestration, or both, of these compounds in the liver, thus altering their biological activity at the site of action. The experimental design also could have influenced effects on CYP1A metrics. For example, in the present study, the time from the start of exposure until collection of tissues was relatively long in that both adult and juvenile mink were exposed for longer than 150 d. This might have allowed concentrations of the test chemicals in the liver to reach a steady state at which CYP1A expression was near or at maxima. In a dietary pharmacokinetic study [20], a time-dependent accumulation of TCDF and PeCDF in livers of female mink was reported with concentrations approaching an apparent steady state by 90 d for both compounds. Furthermore, although the time to steady state was similar for both TCDF and PeCDF, tissue concentration data and pharmacokinetic model parameters showed that there were significant differences in accumulation of TCDF and PeCDF in the liver of exposed mink that presumably involved induction of CYP1A gene expression. For instance, the rapid clearance of TCDF from the liver (half-life less than 15 h) is related to the induction of CYP1A1 enzymatic activity, whereas for PeCDF, the selective uptake and sequestration into the liver is related to induction and binding to CYP1A2 protein [31,32]. As a result, exposure to these compounds not only results in the induction of CYP1A genes, but also influences the accumulation of these compounds in the liver, which alters the dose response relationships in a concen- Table 8. ED10/EC10, ED20/EC20, and ED50/EC50 values for incidence of jaw lesions in juvenile mink based on consumed dose or liver concentrations (as an internal dose metric) of TCDD, PeCDF, or TCDF and corresponding TEQ WHO a Consumed dose (ng/kg bw/d) Consumed dose expressed as TEQ WHO b (ng TEQ WHO /kg bw/d) ED10 ED20 ED50 ED10 ED20 ED50 Best-fit model p value TCDD Log probit PeCDF 2.1 (1.9) c 4.4 (1.1) 14 (0.5) Gamma TCDF 45 (0.09) 73 (0.06) 149 (0.04) Weibull Liver concentration (ng/kg, wet wt) Liver concentration expressed as TEQ WHO (ng TEQ WHO /kg, wet wt) EC10 EC20 EC50 EC10 EC20 EC50 Best-fit model p value TCDD Log probit PeCDF 299 (0.50) 634 (0.29) 1969 (0.13) Gamma TCDF d a ED10/EC10, ED20/EC20, and ED50/EC50 were calculated using the U.S. Environmental Protection Agency Benchmark Dose (BMD) software ( Values in the table correspond to the BMD. The best-fit model for each compound was determined by collectively considering the following criteria: the highest p value, chi-squared residual values of less than 2, the lowest Akaike s Information Criterion (AIC) value, the smallest margin between the BMD and the BMDL (lower limit of a one-sided 95% confidence limit of the BMD) curves, and a good visual fit of the BMD and BMDL curves. b TEQ WHO were determined using mammalian toxic equivalency factors of 1.0, 0.3, and 0.1 for TCDD, PeCDF, and TCDF, respectively [17]. c Numbers in parentheses refer to the relative potency of PeCDF or TCDF compared with TCDD based on the corresponding ED or EC values. d EC values were not calculated for TCDF because liver concentrations did not increase with consumed dose as indicated by inadequate statistical fit using the EPA BMD software. The lack of a dose response relationship suggests that hepatic TCDF concentration is not a reliable internal dose metric for jaw lesions. EDx ¼ effective dose resulting in x% increase in the incidence of the jaw lesion over background; ECx ¼ effective concentration resulting in x% increase in the incidence of the jaw lesion over background; TCDD ¼ 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF ¼ 2,3,4,7,8-pentachlorodibenzofuran; TCDF ¼ 2,3,7,8- tetrachlorodibenzofuran; TEQ WHO ¼ TCDD-toxic equivalents based on World Health Organization (WHO) toxic equivalency factors.

10 2554 Environ. Toxicol. Chem. 31, 2012 S.J. Bursian et al. tration-dependent manner. This was observed in EROD activities that increased up to maximal values until concentrations of PeCDF and TCDF in liver reached concentrations of approximately 350 to 400 ng TEQ WHO-TCDF /kg and 5 to 6 ng TEQ WHO-TCDF /kg wet weight, respectively [20]. In the present study, concentrations of these two congeners in liver either approached or exceeded these values in both adult and juvenile mink. Jaw lesion The most consistent histopathological lesion observed in the present study was the presence of the mandibular and maxillary squamous epithelial proliferation that occurred in all treatment groups except the control and 2.1-ng TCDD/kg bw/d (2.1 ng TEQ WHO-TCDD /kg bw/d) groups. Initial studies from our laboratory have shown that young mink fed diets containing PCB 126 and TCDD at concentrations of 2,400 ng TEQ WHO /kg feed (300 ng TEQ WHO /kg bw/d) beginning at 6 to 12 weeks of age exhibited histological signs of a lesion associated with the jaw [7 9]. Gross lesions consisted of mandibular and maxillary nodular proliferation of the gingiva and loose teeth. The maxilla and mandible were porous because of loss of alveolar bone. Histologically, this osteoporosis was caused by proliferation of squamous cells that formed infiltrating cords. Subsequent studies demonstrated histological evidence of the lesion in mink exposed to PCBs derived from contaminated fish both in utero and during the growth phase [11,17] as well as in mink trapped in the wild [12,13]. Mandibular and maxillary squamous epithelial proliferation was induced in 27-week-old juveniles in the present study at doses as small as 6.0 ng TCDD (6.0 ng TEQ WHO-TCDD )/kg bw/d, 13 ng PeCDF (4.0 ng TEQ WHO-PeCDF )/kg bw/d, or 52 ng TCDF (5.2 ng TEQ WHO-TCDF )/kg bw/d. Juvenile mink that were fed diets containing as little as 6.6 ng TEQ WHO-PCBs/TCDDs/PCDFs /kg feed (0.83 ng TEQ WHO-PCBs/TCDDs/PCDFs /kg bw/d) provided by fish collected from the Housatonic River, Berkshire County, Massachusetts, USA [11] or 36 ng TEQ WHO-PCBs/PCDDs/PCDFs / kg feed (4.5 ng TEQ WHO-PCBs/TCDDs/PCDFs /kg bw/d) provided by fish collected from the Saginaw River, Michigan, USA [17] had histological evidence of mandibular and maxillary squamous epithelial proliferation. Comparison of the various feeding studies indicate that the jaw lesion has typically been observed in juvenile or adult mink at a TEQ WHO dose approximating 3.4 ng TEQ WHO /kg bw/d, which is the geometric mean of the five lowest observed adverse effect levels from the present study, the Housatonic River study [11], and the Saginaw River study [17]. In the present study, the jaw lesion was induced by TCDD, PeCDF, and TCDF at doses for which there was no evidence of significant effects on reproduction and offspring growth and survivability [23]. These results are similar to results from the Housatonic River study [11] and Saginaw River study [17] in that the jaw lesion occurred at doses less than the dose that resulted in reduced kit survivability (Housatonic River) or in the absence of evidence for effects on reproduction and offspring growth and survivability (Saginaw River). These results suggest that proliferation of mandibular and maxillary squamous epithelial cells may be used as a sensitive biomarker of exposure and effect in relation to TCDD-like chemicals. In the present study, the dietary ED50s expressed on a TEQ WHO basis were 6.6 ng TEQ WHO-TCDD /kg bw/d, 4.1 ng TEQ WHO-PeCDF /kg bw/d, and 15 ng TEQ WHO-TCDF /kg bw/d for TCDD, PeCDF, and TCDF, respectively. The ED50s in the present study are 2.6- to 9.4-fold greater than the estimated ED50 (13 ng TEQ WHO-PCBs/TCDDs/PCDFs /kg feed or 1.6 ng TEQ WHO-PCBs/TCDDs/PCDFs /kg bw/d) for the Housatonic River study [11]. In the Housatonic River study, PCB 126 contributed 81% of the dietary TEQ WHO. Relative potency factors The use of TEF WHO to evaluate potential risks of TCDD-like compounds to wildlife such as mink must be understood in terms of the underlying assumptions and uncertainties that are inherent in this approach. Assumptions include (1) that the most dominant effects of TCDD-like chemicals are mediated through the AhR; (2) that all individual congeners are full agonists with parallel dose response curves; (3) that TEFs are assumed to be equivalent for all exposure scenarios and endpoints; and (4) that the relative ranking in potencies remains the same, even though the pharmacokinetics of the different congeners may differ between wildlife species [14,15,33]. However, the toxicity literature related to TCDD-like chemicals in wildlife species, including traditional laboratory mammals, indicates that the uncertainties associated with these assumptions when examined within the context of individual species responses are considerable. These uncertainties are of particular relevance for mink given their importance in ecological risk assessments of TCDDlike compounds and the fact that few studies have been conducted that specifically examine the appropriateness of these assumptions. For instance, the AhR has yet to be characterized in any mustelid, and sequence information for CYP1A1 and CYP1A2 has only recently become available in the literature [21,22]. Although some inferences regarding AhR responsiveness can be ascertained from CYP1A enzyme activities that have been reported in several mink studies, the use of these data in a quantitative manner to better understand the sensitivity of the mink to TCDD-like chemicals is difficult due to differences in experimental design, including exposure scenarios, that can contribute to age- and sex-based variation in chemical accumulation and organismal responsiveness. In the present study, the assumption that there is direct proportional bioequivalence between AhR agonists is brought into question in that the interaction between chemical and consumed dose expressed on a TEQ basis on the probability of jaw lesion was significant ( p ¼ ), indicating that the biological response (presence of the jaw lesion) in terms of TEQ dose is specific to each of the chemicals. The current TEF WHO-TCDF value for TCDF is 0.1 [15], which is slightly greater than the relative potencies (RePs) of 0.09, 0.06, and 0.04 calculated in the present study based on the ED10s, ED20s, and ED50s, respectively, for the incidence of jaw lesions in juvenile mink (Table 8). However, it is important to remember that TEF WHO values are consensus values that are half order of magnitude increments on a logarithmic scale of 0.03, 0.1, 0.3, etc. [15]. Furthermore, TEF WHO are given as point estimates that are presented without any confidence intervals to better understand the variability associated with these values. A review of the in vivo and in vitro data used for establishing TEFs [34] found that the ReP values for TCDF in mammalian species are variable and range from to This span in ReP values encompasses the ReP values determined in the present study. Thus, although the use of the current TEF WHO-TCDF would tend to slightly overestimate the potential risks associated with TCDF to mink, the magnitude of this overestimate would be less than a half-logarithmic unit if one used the ReP values calculated in the present study. Relative potency factors for PeCDF have been proposed based on numerous toxicity endpoints that include acute lethal-