Thyroid Axis Disruption in Juvenile Brown Trout (Salmo trutta

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Thyroid Axis Disruption in Juvenile Brown Trout (Salmo trutta) Exposed to the Flame Retardant β-Tetrabromoethylcyclohexane (β-TBECH) via the Diet Bradley J. Park,*,† Vince Palace,†,‡ Kerry Wautier,† Bonnie Gemmill,‡ and Gregg Tomy†,‡ † ‡

Fisheries and Oceans Canada, Freshwater Institute, 501 University Crescent, Winnipeg, Manitoba R3T 2N6 Canada Department of Environment and Geography, University of Manitoba, Winnipeg, Manitoba R3T 2N2 Canada ABSTRACT: Tetrabromoethylcyclohexane (TBECH) is an additive brominated flame retardant used in domestic and industrial applications. It has been detected in wildlife, and there is early evidence that it is an endocrine disruptor. Whereas other brominated flame retardants with similar physicochemical properties have been shown to disrupt the thyroid axis, no such evaluation has been conducted for TBECH. To elucidate this, juvenile brown trout (Salmo trutta) were fed either a control diet or diets containing low, medium, or high doses of βTBECH, the isomer most frequently detected in wildlife, for 56 days (uptake phase) followed by a control diet for an additional 77 days (depuration phase). Eight fish per treatment were lethally sampled on uptake days 7, 14, 21, 35, 49, and 56 and on depuration days 7, 21, 35, 49, and 77 to assess fish condition, circulating free and total triiodothyronine and thyroxine, and thyroid epithelial cell height. Although there was no effect on condition factor, there was a significant reduction in total plasma thyroxine in the high dose group and a significant increase in mean thyroid epithelial cell height in the low, medium, and high dose groups during the uptake phase, whereas there were no differences in the depuration phase. These results indicate that β-TBECH may modulate the thyroid axis in fish at environmentally relevant concentrations.

’ INTRODUCTION Brominated flame retardant chemicals (BFRs) are considered a critical component in the manufacture of a wide variety of industrial and domestic goods because they reduce ignition and combustion in accidental fires, thereby saving lives and reducing economic loss.1 This structurally diverse class of chemicals is commonly incorporated into textiles, electronics, plastics, foams, and construction materials, and is therefore prevalent in the human environment. While global demand for BFRs is increasing, there is concern regarding their environmental fate and behavior, and their potential toxicity to humans and wildlife. Specifically, many BFRs are persistent and bioaccumulative due to their labile and lipophilic nature.2 Biomonitoring efforts are increasingly detecting BFRs in wildlife and humans at elevated levels, and it has been demonstrated that some forms interfere with the vertebrate endocrine system via alterations at various levels in the thyroid and reproductive axes.3,4 While there is also evidence to suggest that some BFRs are immunotoxic and neurotoxic, modulation of the thyroid axis appears to be the most prevalent toxic effect. Previous studies indicate that polybrominated biphenyl ethers (PBDEs),2 hexabromocyclododecane (HBCD),5,6 and tetrabromobisphenol A (TBBPA)7,8 can impact the vertebrate thyroid axis. Concerns over environmental persistence, bioaccumulation, and toxic effects of BFRs have led to voluntary and legislated removal of known deleterious substances (e.g., polybrominated r 2011 American Chemical Society

diphenyl ethers (PBDEs), hexabromocyclododecane (HBCD)), and an expanded search for alternatives. One potential replacement product is tetrabromoethylcyclohexane (TBECH— trade name Saytex BCL-462); an additive flame retardant used in polystyrene foams, adhesives, and electrical cable coatings. Annual production volume estimates in the U.S. range from 4.5 to 226 t/yr for 1986, 1990, 1994, 1998, and 2002.9 There are four possible isomers of TBECH; R- and β-TBECH are present in the technical mixture at equimolar amounts, whereas γ- and δ-TBECH are produced at temperatures >125 °C and constitute very small fractions of the technical product.10 TBECH has recently been detected in biota from the Canadian arctic and the Laurentian great lakes.11,12 Relatively little is known about the potential toxicity of TBECH. Larsson et al. used a combination of modeling and in vitro studies to demonstrate that TBECH is an androgen agonist.13 In a follow up study by the same group, androgen receptor activation was confirmed in a mammalian hepatocellular carcinoma cell line and in a prostate cancer cell line.14 Additional studies have shown that TBECH can bioaccumulate in fish

Received: May 4, 2011 Accepted: August 8, 2011 Revised: August 4, 2011 Published: August 18, 2011 7923

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Environmental Science & Technology exposed via the diet and that the assimilated doses can be maternally transferred to young via the eggs.15,16 To our knowledge, there have been no evaluations of the potential impacts of TBECH on the fish thyroid axis. To address this, we exposed juvenile brown trout (Salmo trutta) via the diet to environmentally relevant concentrations of β-TBECH for 56 days followed by a 77-day depuration phase. The juvenile life stage was targeted because it is a potentially sensitive period of TH-dependent growth and development. This experimental approach has been employed previously in other studies of thyroid-active flame retardants.5,6 The β-isomer was selected because it is the form most frequently detected in previous wildlife monitoring studies.11,12 We have reported results on toxicokinetics from this exposure elsewhere;17 here, we report effects on the thyroid axis.

’ MATERIALS AND METHODS Details on the chemicals, food preparation, fish exposure, and extraction methods can be found in Gemmill et al.17 Briefly, four experimental diets were prepared: one control diet and three with different concentrations of the β-TBECH isomer (Rac(1R,2R)-1,2-dibromo-(4S)-4-((1S)-1,2-dibromoethyl)cyclohexane, C8H12Br4, MW = 427.8 g/mol). Unpublished data from our group demonstrates that the β-isomer was detected in fish from Lake Winnipeg, Canada, at 6 pmol/g lw. The low and medium doses in the experimental diet were chosen to bracket this concentration. It was not possible to incorporate a positive control in the current study, because we had no previous knowledge of a potential thyroid response from TBECH exposure. In lieu of a positive control, a high dose treatment was incorporated; this dose is roughly an order of magnitude higher than previously published environmental levels. Mean β-TBECH concentrations (+SE) in the diets were as follows (pmol/g, lipid basis): control = not detected, low = 2.02 ( 0.4, medium = 14.7 ( 0.9, high = 118.4 ( 3.1. Four hundred juvenile brown trout (Salmo trutta, approximately 60 g) were randomized into four 800-L flow-through tanks containing dechlorinated water held at 12
15 °C (flow rate = 1.5 L/min). All experimental procedures were in accordance with Guidelines of the Canadian Council on Animal Care (Freshwater Institute Animal Use Protocol FWI-ACC-2009-2010-006). Fish were fed the control diet and acclimated for 1 week before initiating the exposure. Each tank was then given a different experimental diet for the 56-day uptake phase, followed by control diet for the 77-day depuration phase. Feeding rate was 1% of the total fish weight per tank each day. Eight fish per treatment were euthanized on uptake days 7, 14, 21, 35, 49, and 56 and on depuration days 7, 21, 35, 49, and 77. Fresh weight and length were recorded, and condition factor was calculated as follows: weight (g)/length3 (cm)  100. Blood was collected from the caudal vein by heparin-rinsed syringe, centrifuged at 6000g for 10 min, and plasma was collected and frozen at
85 °C until analysis. The lower jaw (thyroid region) was removed for histological processing as described below. The remaining carcass was homogenized, and subsamples of homogenate were analyzed for TBECH isomers and metabolites (data not shown). Plasma samples were analyzed for free and total T3 (3,5,30 triiodo-L-thyronine) and T4 (L-thyroxine) by coated tube radioimmunoassay (MP Biomedical, Santa Ana, CA, USA). Due to limited plasma volumes, not all parameters could be measured on all dates. Priority was given to total T4 (assessed on uptake days

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35, 49, and 56 and depuration days 7 and 21) followed by free T4 (uptake days 35 and 49; depuration day 21), total T3 (uptake days 35 and 49; depuration day 7 and 21) and Free T3 (21, 35 and 49; depuration day 35). Histological samples were fixed in Bouin solution for 48 h, dehydrated in graded alcohols, cleared in toluene, and embedded in paraffin for routine sectioning. Sagittal sections (5 μm) were stained with hematoxylin and eosin. Histological analyses were conducted on samples from uptake days 35, 49 and 56, and depuration day 21. To quantify epithelial cell activity, cell heights were measured on 15 follicles per fish, at four cardinal points per follicle, and the mean of these values was taken as the epithelial cell height for a given individual. For plasma hormones (free and total T3 and T4) and epithelial cell heights, ANOVA was used to test for differences among treatment groups, followed by Dunnett’s multiple comparison procedure (R = 0.05, SAS version 8.02). Due to limited sample sizes for plasma hormones, multiple uptake sampling dates were combined, and 2-way ANOVA was used to test for treatment and time effects for free and total T3 and T4.

’ RESULTS AND DISCUSSION Toxicokinetics of β-TBECH from the current study have been described previously.17 Briefly, the β-isomer was analyzed in whole-body homogenates, and was not detected in control fish, but was detected in the low dose treatment at 14 days, and in the medium and high dose treatments at 7 days. Steady state conditions were achieved in all TBECH-exposed groups by day 21. There was no evidence of biotransformation of the β-isomer to the other isomers (R-, γ-, or δ-TBECH), and no debrominated or dehydrobrominated metabolites were detected. The highest mean tissue concentrations of β-TBECH were as follows (pmol/g lw, ( standard error): low 3.8 ( 0.4; medium 222.1 ( 58.2; high 1965 ( 196. In previous studies of biota from environmental exposures, the β-isomer was detected in arctic beluga whale tissues at 2.57
21.74 pmol/g lw,11 and ΣTBECH (predominantly β-isomer) in herring gull eggs from the North American Great Lakes was detected at up to 8.04 pmol/g wet weight.12 Thus, it appears that tissue concentrations of TBECH from the low and medium dose groups in the current study bracket those previously detected in other aquatic biota. However, given that these compounds biomagnify, it is impossible to make direct comparisons between tissue concentrations of TBECH in fish from our study with those of wildlife in other trophic levels. To our knowledge there are no published studies indicating tissue concentrations of this novel contaminant in wild fish. Endogenous thyroid hormones regulate development, growth, and aspects of reproduction in fish via widespread and diverse permissive actions in target tissues. In teleost fish, the thyroid typically comprises scattered follicles in the basibranchial region. Follicular epithelial cells synthesize and secrete T4 into the bloodstream under the influence of the hypothalamus and pituitary. Thyroxine is a prohormone, and is converted to the physiologically active T3 in various peripheral tissues such as the liver via enzymatic deiodination. Both T3 and T4 can circulate in the plasma either free (unbound) or bound to transport proteins.18 Thyroid axis disruption has been previously documented in fish, birds, and mammals exposed to structurally diverse BFRs, with the majority of reported effects being reductions in circulating TH.3 The relative sensitivity of various vertebrate taxa has not 7924

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Figure 1. Plasma concentrations of free and total triiodothyronine (T3) and thyroxine (T4) of fish fed control diets or diets enriched with three concentrations of β-TBECH for 56 days (uptake), followed by control diet for 77 days (depuration). Data are presented as mean + standard error (n = 3 to 8).

been conclusively demonstrated. However, amphibians and some fish species undergo TH-dependent metamorphosis; the transition from larva to adult is typically considered sensitive due to the massive restructuring of organ systems, and can be inhibited by environmental chemicals. In anadromous fish, the physiological adaptations required prior to migration from freshwater to saltwater are mediated by the thyroid axis. Disruption of this process has been documented in fish exposed to BFRs.19 There was no significant difference among treatments in free or total T3 or free T4, but total T4 was significantly reduced in the high dose group relative to controls during the uptake phase (p = 0.045, uptake days 35, 49, and 56 combined, Figure 1). On day 56, total T4 concentrations were 4.91 ( 0.62 and 3.80 ( 0.61 ng/mL ( SE in control and high dose fish, respectively. This reduction was transient, as there was no apparent treatment effect in the early depuration phase. Reduced circulating T4 may be the result of chemical interference with iodide uptake, peroxidase activity, secretion from the thyroid gland, plasma binding proteins, or peripheral metabolism. A full suite of targeted assays would be required to elucidate which of these mechanisms may play a role in TBECH-related thyroid axis disruption. Thyroid epithelial cell hypertrophy is a sensitive indicator of thyroid axis disruption.20 Epithelial cells are responsible for all aspects of TH synthesis and secretion, thus changes in cell size are indicative of overall gland activity.18 Histological appearance of the thyroid tissue in fish from the current study was typical, consisting of colloid-containing ovoid follicles with cuboidal epithelium. There was no evidence of proliferative thyroid lesions or other overt pathologies. However, quantitative analysis revealed evidence of thyroid epithelial cell hypertrophy in treated

Figure 2. Thyroid epithelial cell height of fish fed control diets or diets enriched with three concentrations of β-TBECH for 56 days (uptake), followed by control diet for 77 days (depuration). Data are presented as mean + standard error (n = 8).

groups during the late uptake phase (Figure 2). Cell heights were significantly elevated in all treated groups relative to controls from uptake day 35 to 56. On day 56, mean epithelial cell height ((SE) was 5.35 ((0.11) μm for controls, and 6.18 ((0.15), 5.97 ((0.20), and 6.33 ((0.13) μm for low, medium, and high dose fish, respectively. This effect appeared to be transient, as there was no difference in cell heights in the depuration phase. Similar transient alterations in circulating thyroid hormone concentrations and ECH have been demonstrated in fish exposed to other BFRs, with limited epithelial cell height changes identified in periods when tissue concentrations are at their highest.6 7925

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’ ACKNOWLEDGMENT We thank Fisheries and Oceans Canada and the Research Affiliate Program for funding to B.G. ’ REFERENCES

Figure 3. Condition factor of fish fed control diets or diets enriched with three concentrations of β-TBECH for 56 days (uptake), followed by control diet for 77 days (depuration). Data are presented as mean + standard error (n = 8).

The reductions in circulating total T4 and concomitant epithelial cell hypertrophy during exposure in the high dose group indicate that TBECH altered TH synthesis or metabolism at some level, and that epithelial cell activity was likely up-regulated as a compensatory mechanism in the maintenance of thyroid homeostasis. Similar results have been reported in HBCD-exposed fish.6 In the medium and low dose groups, epithelial cell heights showed a similar increase, whereas there was no significant effect on plasma thyroid hormones. Thus, biochemical responses in the epithelial cells may have been sufficient to maintain TH homeostasis at this lower level of exposure. Further studies would be required to determine whether the endocrinedisrupting effects seen in the current study would be detrimental to wild salmonids or other free-ranging taxa. Thyroid axis disruption may ultimately impair growth and development in fish from contaminated sites, however, a clear cause-and-effect relationship remains to be demonstrated.21,22 In the current study there was no significant difference among treatments in condition factor at the end of the exposure or the depuration phase (Figure 3); therefore effects on circulating T4 and thyroid histology did not impact growth in the fish. Although the β-TBECH concentrations in fish from the high dose group were greater than previously reported environmental concentrations,11,12 it should be noted that the fate and behavior of TBECH in the environment remains to be elucidated. In the current study fish were exposed to a single isomer, whereas in the wild they may be exposed to multiple isomers. Finally, cellularlevel changes were detected in the more ecologically relevant low and medium dose groups. To our knowledge, this is the first study to demonstrate that TBECH can modulate the thyroid axis in fish. Total exposure time was 56 days in the current study, whereas wild fish in contaminated environments may be exposed for longer periods. Additional work to determine if exposure over a full life cycle could result in more pronounced impacts is warranted. This is especially true given that TBECH can bioaccumulate and has been shown to be maternally transferred; fish in contaminated environments may be exposed to significant doses during potentially sensitive thyroid and sex steroiddependent physiological processes such as embryogenesis, organogenesis/neurological development, and gonad differentiation.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]; phone: 204-983-5006; fax: 204-984-6587.

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