Influence of Methylene Blue or Dimethyl Sulfoxide on Larval Zebrafish Development and Behavior.

The use of larval zebrafish developmental testing and assessment, specifically larval zebrafish locomotor activity, has been recognized as a higher throughput testing strategy to identify developmentally toxic and neurotoxic chemicals. There are, however, no standardized protocols for this type of assay, which could result in confounding variables being overlooked. Two chemicals commonly employed during early-life stage zebrafish assays, methylene blue (antifungal agent) and dimethyl sulfoxide (DMSO, a commonly used vehicle) have been reported to affect the morphology and behavior of freshwater fish. In this study, we conducted developmental toxicity (morphology) and neurotoxicity (behavior) assessments of commonly employed concentrations for both chemicals (0.6-10.0 µM methylene blue; 0.3%-1.0% v/v DMSO). A light-dark transition behavioral testing paradigm was applied to morphologically normal, 6 days postfertilization (dpf) zebrafish larvae kept at 26°C. Additionally, an acute DMSO challenge was administered based on early-life stage zebrafish assays typically used in this research area. Results from developmental toxicity screens were similar between both chemicals with no morphological abnormalities detected at any of the concentrations tested. However, neurodevelopmental results were mixed between the two chemicals of interest. Methylene blue resulted in no behavioral changes up to the highest concentration tested, 10.0 µM. By contrast, DMSO altered larval behavior following developmental exposure at concentrations as low as 0.5% (v/v) and exhibited differential concentration-response patterns in the light and dark photoperiods. These results indicate that developmental DMSO exposure can affect larval zebrafish locomotor activity at routinely used concentrations in developmental neurotoxicity assessments, whereas methylene blue does not appear to be developmentally or neurodevelopmentally toxic to larval zebrafish at routinely used concentrations. These results also highlight the importance of understanding the influence of experimental conditions on larval zebrafish locomotor activity that may ultimately confound the interpretation of results.

Inconsistencies in variable reporting and methods in larval zebrafish behavioral assays.

New approaches in developmental neurotoxicity (DNT) screening are needed due to the tens of thousands of chemicals requiring hazard assessments. Zebrafish (Danio rerio) are an alternative vertebrate model for DNT testing, but without a standardized protocol for larval behavioral assays, comparison of results among laboratories is challenging. To evaluate the congruence of protocols across laboratories, we conducted a literature review of DNT studies focusing on larval zebrafish behavior assays and cataloged experimental design consistencies. Our review focused on 51 unique method variables in publications where chemical exposure occurred in early development and subsequent larval locomotor evaluation focused on assays that included a light/dark photoperiod transition. We initially identified 94 publications, but only 31 exclusively met our inclusion criteria which focused on parameters that are important to an assay employed by our laboratory. No publication reported 100% of the targeted variables; only 51 to 86% of those variables were reported in the reviewed publications, with some aspects of the experimental design consistent among laboratories. However, no protocol was exactly the same for any two publications. Many of these variables had more than one parameter/design reported, highlighting the inconsistencies among methods. Overall, there is not only a strong need for the development of a standardized testing protocol for larval zebrafish locomotor assays, but there is also a need for a standardized protocol for reporting experimental variables in the literature. Here we include an extensive guideline checklist for conducting larval zebrafish developmental behavior assays.

Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors Within the ToxCast Phase I and II Chemical Libraries.

High-throughput screening for potential thyroid-disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the U.S. Environmental Protection Agency ToxCast screening assay portfolio. To fill 1 critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast phase I and II chemical libraries, comprised of 1074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single-concentration screen were retested in concentration-response. Due to high false-positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed 2 additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using guaiacol as a substrate to confirm the activity profiles of putative TPO inhibitors. This effort represents the most extensive TPO inhibition screening campaign to date and illustrates a tiered screening approach that focuses resources, maximizes assay throughput, and reduces animal use.

Neurotoxicological and thyroid evaluations of rats developmentally exposed to tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) and tris(2-chloro-2-ethyl)phosphate (TCEP).

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) and tris(2-chloro-2-ethyl)phosphate (TCEP) are organophosphorous flame retardants with widespread usage and human exposures through food, inhalation, and dust ingestion. They have been detected in human tissues including urine and breast milk. Reports of disrupted neural growth in vitro, abnormal development in larval zebrafish, and altered thyroid hormones in several species have raised concern for neurodevelopmental toxicity. This is especially the case for TDCIPP, which is more potent and has more activity in those assays than does TCEP. We evaluated the potential for developmental neurotoxicity of TDCIPP and TCEP in a mammalian model. Pregnant Long-Evans rats were administered TDCIPP (15, 50, or 150 mg/kg/day) or TCEP (12, 40, 90 mg/kg/day) via oral gavage from gestational day 10 to weaning. Corn oil was the vehicle control in both studies. Body weight and righting reflex development were monitored in all pups. A subset of offspring at culling and weaning, and dams at weaning, were sacrificed for serum and organ collection for measurement of brain, liver, and thyroid weights, serum thyroid levels, and serum and brain acetylcholinesterase activities. Brain weights were also measured in a group of adult TDCIPP-treated offspring. One male and one female from each litter were allocated for behavioral testing at several ages: standard locomotor activity (preweaning, postweaning, adults), locomotor activity including a lighting change mid-way (postweaning, adults), elevated zero maze (postweaning, adults), functional observational battery (FOB; postweaning, adults), and Morris water maze (place learning, reference and working memory; adults). Neither chemical produced changes in maternal body weight or serum thyroid hormones, but relative liver weight was increased at the high doses of both TDCIPP and TCEP. In offspring, there were no effects on viability, litter size, or birth weight. With TDCIPP, absolute liver weights were lower at weaning and weight gain was lower in the high-dose offspring until about two months of age. Thyroid hormones and brain weights were not altered and acetylcholinesterase (both brain and serum) was not inhibited by either chemical. TDCIPP-treated offspring showed slight differences in floating in the water maze, hindlimb grip strength, and altered activity habituation, whereas TCEP-treated rats showed differences in quadrant time (probe) and middle-zone preference in the water maze. Regarding these few changes, the effects were minimal, mostly not related to dose, and did not appear treatment-related or biologically significant. Overall, these data do not support the potential for thyrotoxicity or developmental neurotoxicity produced by TDCIPP or TCEP.

Assessment of serum biomarkers in rats after exposure to pesticides of different chemical classes.

There is increasing emphasis on the use of biomarkers of adverse outcomes in safety assessment and translational research. We evaluated serum biomarkers and targeted metabolite profiles after exposure to pesticides (permethrin, deltamethrin, imidacloprid, carbaryl, triadimefon, fipronil) with different neurotoxic actions. Adult male Long-Evans rats were evaluated after single exposure to vehicle or one of two doses of each pesticide at the time of peak effect. The doses were selected to produce similar magnitude of behavioral effects across chemicals. Serum or plasma was analyzed using commercial cytokine/protein panels and targeted metabolomics. Additional studies of fipronil used lower doses (lacking behavioral effects), singly or for 14 days, and included additional markers of exposure and biological activity. Biomarker profiles varied in the number of altered analytes and patterns of change across pesticide classes, and discriminant analysis could separate treatment groups from control. Low doses of fipronil produced greater effects when given for 14 days compared to a single dose. Changes in thyroid hormones and relative amounts of fipronil and its sulfone metabolite also differed between the dosing regimens. Most cytokine changes reflected alterations in inflammatory responses, hormone levels, and products of phospholipid, fatty acid, and amino acid metabolism. These findings demonstrate distinct blood-based analyte profiles across pesticide classes, dose levels, and exposure duration. These results show promise for detailed analyses of these biomarkers and their linkages to biological pathways.

Development of a thyroperoxidase inhibition assay for high-throughput screening.

High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein, we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluorescent peroxidase substrate, Amplex UltraRed (AUR), were employed in an end-point assay for comparison to the existing kinetic guaiacol (GUA) oxidation assay. Following optimization of assay metrics, including Z', dynamic range, and activity, using methimazole (MMI), the assay was tested with a 21-chemical training set. The potency of MMI-induced TPO inhibition was greater with AUR compared to GUA. The dynamic range and Z' score with MMI were as follows: 127-fold and 0.62 for the GUA assay, 18-fold and 0.86 for the 96-well AUR assay, and 11.5-fold and 0.93 for the 384-well AUR assay. The 384-well AUR assay drastically reduced animal use, requiring one-tenth of the rat thyroid microsomal protein needed for the GUA 96-well format assay. Fourteen chemicals inhibited TPO, with a relative potency ranking of MMI > ethylene thiourea > 6-propylthiouracil > 2,2',4,4'-tetrahydroxy-benzophenone > 2-mercaptobenzothiazole > 3-amino-1,2,4-triazole > genistein > 4-propoxyphenol > sulfamethazine > daidzein > 4-nonylphenol > triclosan > iopanoic acid > resorcinol. These data demonstrate the capacity of this assay to detect diverse TPO inhibitors. Seven chemicals acted as negatives: 2-hydroxy-4-methoxybenzophenone, dibutylphthalate, diethylhexylphthalate, diethylphthalate, 3,5-dimethylpyrazole-1-methanol, methyl 2-methyl-benzoate, and sodium perchlorate. This assay could be used to screen large numbers of chemicals as an integral component of a tiered TH-disruptor screening approach.

Cross-species analysis of thyroperoxidase inhibition by xenobiotics demonstrates conservation of response between pig and rat.

Thyroperoxidase (TPO), the enzyme that catalyzes the synthesis of thyroid hormone, is a known target for thyroid-disrupting chemicals. In vivo toxicological evidence supporting TPO-inhibition as one molecular-initiating event that leads to thyroid disruption is derived largely from rat models; however, a significant fraction of research on the inhibition of TPO by xenobiotics has been conducted using porcine TPO. The current work tested the hypothesis that porcine and rat thyroid microsomes exposed to TPO-inhibiting chemicals would demonstrate different responses in a guaiacol oxidation assay. A primary objective of this work is to establish the degree of concordance between rat and porcine TPO inhibition data. Microsomes were isolated from both rat and pig thyroid glands, and the guaiacol oxidation assay was performed for a training set of 12 chemicals, including previously reported TPO inhibitors, thyroid-disrupting chemicals thought to perturb other targets, and several previously untested chemicals, to determine the relative TPO inhibition responses across species. Concentration-response curves were derived for methimazole (MMI), dibutylphthalate (DBP), diethylhexylphthalate (DEHP), diethylphthalate (DEP), 3,5-dimethylpyrazole-1-methanol (DPM), iopanoic acid (IOA), 2-mercaptobenzothiazole (MBT), sodium perchlorate (PERC), p-nonylphenol (PNP), 4-propoxyphenol (4POP), 6-propylthiouracil (PTU), and triclosan (TCS). MMI, PTU, MBT, DPM, 4POP, and at extremely high concentrations, PERC, inhibited TPO activity. Results demonstrated a strong qualitative concordance of response between the two species. All chemicals that inhibited TPO in porcine microsomes also inhibited TPO in rat microsomes. Hill model-derived IC50 values revealed approximate 1.5- to 50-fold differences in relative potency to MMI between species for positive chemicals. DPM, MBT, 4POP, and PTU exhibited greater relative potency to MMI using rat TPO versus porcine TPO, but rank order potency for inhibition was similar for the other test chemicals, with: PTU>MBT>DPM>4POP>PERC for rat TPO and MBT>PTU>DPM>4POP>PERC for porcine TPO. These data support the extrapolation of porcine TPO data to potential thyroid-disrupting activity in rodent models to evaluate TPO-inhibiting chemicals.

An animal model of marginal iodine deficiency during development: the thyroid axis and neurodevelopmental outcome.

Thyroid hormones (THs) are essential for brain development, and iodine is required for TH synthesis. Environmental chemicals that perturb the thyroid axis result in modest reductions in TH, yet there is a paucity of data on the extent of neurological impairments associated with low-level TH disruption. This study examined the dose-response characteristics of marginal iodine deficiency (ID) on parameters of thyroid function and neurodevelopment. Diets deficient in iodine were prepared by adding 975, 200, 125, 25, or 0 µg/kg potassium iodate to the base casein diet to produce five nominal iodine levels ranging from ample (Diet 1: 1000 µg iodine/kg chow, D1) to deficient (Diet 5: 25 µg iodine/kg chow, D5). Female Long Evans rats were maintained on these diets beginning 7 weeks prior to breeding until the end of lactation. Dams were sacrificed on gestational days 16 and 20, or when pups were weaned on postnatal day (PN) 21. Fetal tissue was harvested from the dams, and pups were sacrificed on PN14 and PN21. Blood, thyroid gland, and brain were collected for analysis of iodine, TH, and TH precursors and metabolites. Serum and thyroid gland iodine and TH were reduced in animals receiving two diets that were most deficient in iodine. T4 was reduced in the fetal brain but was not altered in the neonatal brain. Neurobehavior, assessed by acoustic startle, water maze learning, and fear conditioning, was unchanged in adult offspring, but excitatory synaptic transmission was impaired in the dentate gyrus in animals receiving two diets that were most deficient in iodine. A 15% reduction in cortical T4 in the fetal brain was sufficient to induce permanent reductions in synaptic function in adults. These findings have implications for regulation of TH-disrupting chemicals and suggest that standard behavioral assays do not readily detect neurotoxicity induced by modest developmental TH disruption.

Developmental triclosan exposure decreases maternal, fetal, and early neonatal thyroxine: a dynamic and kinetic evaluation of a putative mode-of-action.

This work tests the mode-of-action (MOA) hypothesis that maternal and developmental triclosan (TCS) exposure decreases circulating thyroxine (T4) concentrations via up-regulation of hepatic catabolism and elimination of T4. Time-pregnant Long-Evans rats received TCS po (0-300mg/kg/day) from gestational day (GD) 6 through postnatal day (PND) 21. Serum and liver were collected from dams (GD20, PND22) and offspring (GD20, PND4, PND14, PND21). Serum T4, triiodothyronine (T3), and thyroid-stimulating hormone (TSH) concentrations were measured by radioimmunoassay. Ethoxy-O-deethylase (EROD), pentoxyresorufin-O-depentylase (PROD) and uridine diphosphate glucuronyltransferase (UGT) enzyme activities were measured in liver microsomes. Custom Taqman(®) qPCR arrays were employed to measure hepatic mRNA expression of select cytochrome P450s, UGTs, sulfotransferases, transporters, and thyroid hormone-responsive genes. TCS was quantified by LC/MS/MS in serum and liver. Serum T4 decreased approximately 30% in GD20 dams and fetuses, PND4 pups and PND22 dams (300mg/kg/day). Hepatic PROD activity increased 2-3 fold in PND4 pups and PND22 dams, and UGT activity was 1.5 fold higher in PND22 dams only (300mg/kg/day). Minor up-regulation of Cyp2b and Cyp3a expression in dams was consistent with hypothesized activation of the constitutive androstane and/or pregnane X receptor. T4 reductions of 30% for dams and GD20 and PND4 offspring with concomitant increases in PROD (PND4 neonates and PND22 dams) and UGT activity (PND22 dams) suggest that up-regulated hepatic catabolism may contribute to TCS-induced hypothyroxinemia during development. Serum and liver TCS concentrations demonstrated greater fetal than postnatal internal exposure, consistent with the lack of T4 changes in PND14 and PND21 offspring. These data support the MOA hypothesis that TCS exposure leads to hypothyroxinemia via increased hepatic catabolism; however, the minor effects on thyroid hormone metabolism may reflect the low efficacy of TCS as thyroid hormone disruptor or highlight the possibility that other MOAs may also contribute to the observed maternal and early neonatal hypothyroxinemia.

Developmental triclosan exposure decreases maternal and neonatal thyroxine in rats.

Disruption of maternal thyroid hormones during fetal developmental may result in irreversible neurological consequences in offspring. The present study tested the hypothesis that perinatal triclosan exposure of dams decreases thyroxine in dams and offspring prior to weaning. Pregnant Long-Evans rats received triclosan by oral gavage (0-300 mg/kg/d) in corn oil from gestational day (GD)6 through postnatal day (PND)21. Serum was obtained from pups on PND4, 14, and 21, and from dams on PND22. Serum thyroxine (T4) was reduced 31% in dams on PND22. In pups, a unique pattern of hypothyroxinemia was observed; serum T4 decreased 27% in PND4 pups with no significant reduction observed on PND14 or PND21. Comparable reductions of approximately 30% in serum T4 at 300 mg/kg/d for dams and PND4 neonates and a lack of effect at PND14 and PND21 suggest that toxicokinetic or toxicodynamic factors may have contributed to a reduced exposure or a reduced toxicological response during the lactation period.

Short-term exposure to triclosan decreases thyroxine in vivo via upregulation of hepatic catabolism in Young Long-Evans rats.

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)-phenol) is a chlorinated phenolic antibacterial compound found in consumer products. In vitro human pregnane X receptor activation, hepatic phase I enzyme induction, and decreased in vivo total thyroxine (T4) suggest adverse effects on thyroid hormone homeostasis. Current research tested the hypothesis that triclosan decreases circulating T4 via upregulation of hepatic catabolism and transport. Weanling female Long-Evans rats received triclosan (0-1000 mg/kg/day) by gavage for 4 days. Whole blood and liver were collected 24 h later. Total serum T4, triiodothyronine (T3), and thyroid-stimulating hormone (TSH) were measured by radioimmunoassay. Hepatic microsomal assays measured ethoxyresorufin-O-deethylase, pentoxyresorufin-O-deethylase (PROD), and uridine diphosphate glucuronyltransferase enzyme activities. The messenger RNA (mRNA) expression of cytochrome P450s 1a1, 2b1/2, and 3a1/23; UGTs 1a1, 1a6, and 2b5; sulfotransferases 1c1 and 1b1; and hepatic transporters Oatp1a1, Oatp1a4, Mrp2, and Mdr1b was measured by quantitative reverse transcriptase PCR. Total T4 decreased dose responsively, down to 43% of control at 1000 mg/kg/day. Total T3 was decreased to 89 and 75% of control at 300 and 1000 mg/kg/day. TSH did not change. Triclosan dose dependently increased PROD activity up to 900% of control at 1000 mg/kg/day. T4 glucuronidation increased nearly twofold at 1000 mg/kg/day. Cyp2b1/2 and Cyp3a1/23 mRNA expression levels were induced twofold and fourfold at 300 mg/kg/day. Ugt1a1 and Sult1c1 mRNA expression levels increased 2.2-fold and 2.6-fold at 300 mg/kg/day. Transporter mRNA expression levels were unchanged. These data denote important key events in the mode of action for triclosan-induced hypothyroxinemia in rats and suggest that this effect may be partially due to upregulation of hepatic catabolism but not due to mRNA expression changes in the tested hepatic transporters.

Lack of alterations in thyroid hormones following exposure to polybrominated diphenyl ether 47 during a period of rapid brain development in mice.

Thyroid alterations have been shown to occur following exposure to polybrominated diphenyl ether (PBDE) mixtures, possibly indicating that disruptions in thyroid hormone levels may underlie behavior deficits observed in animals following postnatal PBDE exposure. This study determined whether acute postnatal exposure to PBDE-47 would alter thyroid hormones. Mice were dosed with PBDE-47 on postnatal day 10, and serum collected either 1, 5, or 10 days after the dose. No effect was observed on thyroxine and triiodothyronine levels at any age examined. This suggests that the neurological abnormalities reported in mice exposed to PBDE-47 are not due to acute changes in circulating thyroid hormones at these observed periods.

Low-dose effects of ammonium perchlorate on the hypothalamic-pituitary-thyroid axis of adult male rats pretreated with PCB126.

The objective of this research was to characterize the disturbances in the hypothalamic-pituitary-thyroid (HPT) axis resulting from exposure to a binary mixture, 3,3',4,4',5-pentachlorobiphenyl (PCB126) and perchlorate (ClO(4)(-)), known to cause hypothyroidism by different modes of action. Two studies were conducted to determine the HPT axis effects of ClO(4)(-) on adult male Sprague-Dawley rats pretreated with PCB126. In dosing study I, rats were administered a single oral dose of PCB126 (0, 7.5, or 75 microg/kg) on day 0 and 9 days later ClO(4)(-) (0, 0.01, 0.1, or 1 mg/kg day) was added to the drinking water until euthanasia on day 22. Significant dose-dependent trends were found for all thyroid function indices measured following ClO(4)(-) in drinking water for 14 days. Seventy-five micrograms PCB126/kg resulted in a significant increase in hepatic T(4)-glucuronide formation, causing a decline in serum thyroxine and fT(4), and resulting in increased serum thyroid-stimulating hormone (TSH). Serum TSH was also increased in animals that received 7.5 microg PCB126/kg; no other HPT axis alterations were found in these animals. When pretreated with PCB126, the ClO(4)(-) dose trends disappeared, suggesting a less than additive effect on the HPT axis. In dosing study II, animals were given lower doses of PCB126 (0, 0.075, 0.75, or 7.5 microg/kg) on day 0, and followed with ClO(4)(-) (0 or 0.01 mg/kg day) in drinking water beginning on day 1 and continuing for several days to explore transient HPT axis effects. No statistical effects were seen for PCB126 or ClO(4)(-) alone, and no perturbations were found when administered sequentially in dosing study II. In conclusion, these studies demonstrate that HPT axis disturbances following exposure to ClO(4)(-) are less than additive when pretreated with relatively high doses of PCB126. At relatively low doses, at or near the no-observed-effect-level for PCB126 and ClO(4)(-), no interactions between the chemicals occur.

Short-term in vivo exposure to the water contaminant triclosan: Evidence for disruption of thyroxine.

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) is a chlorinated phenolic antibacterial compound found as an active ingredient in many personal care and household products. The structural similarity of triclosan to thyroid hormones and recent studies demonstrating activation of the human pregnane X receptor (PXR) and inhibition of diiodothyronine (T(2)) sulfotransferases, have raised concerns about adverse effects on thyroid homeostasis. The current research tested the hypothesis that triclosan alters circulating concentrations of thyroxine. The hypothesis was tested using a 4-day oral triclosan exposure (0-1000mg/kg/day) in weanling female Long-Evans rats, followed by measurement of circulating levels of serum total thyroxine (T(4)). Dose-dependent decreases in total T(4) were observed. The benchmark dose (BMD) and lower bound on the BMD (BMDL) for the effects on T(4) were 69.7 and 35.6mg/kg/day, respectively. These data demonstrate that triclosan disrupts thyroid hormone homeostasis in rats.

Effect of PCB 126 on hepatic metabolism of thyroxine and perturbations in the hypothalamic-pituitary-thyroid axis in the rat.

The objective of this research was to examine the time- and dose- dependent disturbances in the hypothalamic-pituitary-thyroid (HPT) axis of adult male rats administered a potent coplanar (non-ortho) PCB, 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Adult male Sprague-Dawley rats were administered a single oral bolus dose of 0, 7.5, 75, or 275 microg PCB 126/kg bw dissolved in corn oil. The rats were sacrificed periodically over 22 days. The 7.5-microg/kg dose induced hepatic ethoxyresorufin-O-deethylation EROD activity, but no changes were observed in hepatic uridine diphosphate glucuronyl transferases (UDPGTs) activity or serum TSH, T4, or fT4 concentrations. The two highest doses caused a modest decline in weight gain, induced hepatic EROD and UDPGT activities, increased serum TSH concentrations, and decreased serum T4 and fT4 concentrations. The amount of thyroxine glucuronide formed daily (pM/mg protein) increased linearly with the area-under-the-concentration-curve (AUCC) for PCB 126 in liver (microg/kg/day) and then slowed at the 275-microg/kg PCB 126 dose. Perturbations in the HPT axis were nonlinear with respect to PCB 126 dosing. As expected, an inverse relationship between the AUCC for serum T4 (microg/dl/day) and the AUCC for serum TSH (ng/dl/day) was observed; however, the relationship was highly nonlinear. These data support a mode of action for PCB 126 involving induction of hepatic UDPGTs by the aryl hydrocarbon receptor AhR. However, the dose-response characteristics of the HPT axis are nonlinear and complex, requiring sophisticated tools, such as PBPK models, to characterize dose response.

Thyroid-hormone-disrupting chemicals: evidence for dose-dependent additivity or synergism.

Endocrine disruption from environmental contaminants has been linked to a broad spectrum of adverse outcomes. One concern about endocrine-disrupting xenobiotics is the potential for additive or synergistic (i.e., greater-than-additive) effects of mixtures. A short-term dosing model to examine the effects of environmental mixtures on thyroid homeostasis has been developed. Prototypic thyroid-disrupting chemicals (TDCs) such as dioxins, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers have been shown to alter thyroid hormone homeostasis in this model primarily by up-regulating hepatic catabolism of thyroid hormones via at least two mechanisms. Our present effort tested the hypothesis that a mixture of TDCs will affect serum total thyroxine (T4) concentrations in a dose-additive manner. Young female Long-Evans rats were dosed via gavage with 18 different polyhalogenated aromatic hydrocarbons [2 dioxins, 4 dibenzofurans, and 12 PCBs, including dioxin-like and non-dioxin-like PCBs] for 4 consecutive days. Serum total T4 was measured via radioimmunoassay in samples collected 24 hr after the last dose. Extensive dose-response functions (based on seven to nine doses per chemical) were determined for individual chemicals. A mixture was custom synthesized with the ratio of chemicals based on environmental concentrations. Serial dilutions of this mixture ranged from approximately background levels to 100-fold greater than background human daily intakes. Six serial dilutions of the mixture were tested in the same 4-day assay. Doses of individual chemicals that were associated with a 30% TH decrease from control (ED30), as well as predicted mixture outcomes were calculated using a flexible single-chemical-required method applicable to chemicals with differing dose thresholds and maximum-effect asymptotes. The single-chemical data were modeled without and with the mixture data to determine, respectively, the expected mixture response (the additivity model) and the experimentally observed mixture response (the empirical model). A likelihood-ratio test revealed statistically significant departure from dose additivity. There was no deviation from additivity at the lowest doses of the mixture, but there was a greater-than-additive effect at the three highest mixtures doses. At high doses the additivity model underpredicted the empirical effects by 2- to 3-fold. These are the first results to suggest dose-dependent additivity and synergism in TDCs that may act via different mechanisms in a complex mixture. The results imply that cumulative risk approaches be considered when assessing the risk of exposure to chemical mixtures that contain TDCs.

Assessment of DE-71, a commercial polybrominated diphenyl ether (PBDE) mixture, in the EDSP male and female pubertal protocols.

DE-71, a commercial mixture, was used to test the sensitivity of the female and male pubertal protocol to detect thyroid active chemicals. These protocols are being evaluated for the U.S. EPA's Endocrine Disruptor Screening Program as part of a Tier I Screening Battery. To examine the ability of these protocols to screen for chemicals that induce the clearance of thyroid hormone, we examined male and female Wistar rats following DE-71 exposure. Rats were gavaged daily with 0, 3, 30, or 60 mg/kg DE in corn oil from postnatal day (PND) 23-53 in the male or PND 22-41 in the female. The temporal effects of DE-71 on liver enzymes and thyroid hormones were measured in another group of males and females following only 5 days of dosing (PND 21 to 26 in females and PND 23 to 28 in males). Serum T4 was significantly decreased at 30 and 60 mg/kg following the 5-day exposures and in the 21-day exposed females. Doses of 3, 30, and 60 mg/kg decreased T4 in 31-day exposed males. Serum T3 was decreased and TSH elevated by 30 and 60 mg/kg in the 31-day exposed males only. Decreased colloid area and increased follicular cell heights (indicative of the hypothyroid state) were observed in thyroids of the 60 mg/kg groups of 20- and 31-day exposed female and males. Increased liver-to-body weight ratios coincided with a significant induction of uridinediphosphate-glucuronosyltransferase (UDGPT; two to four-fold), and ethoxy- and pentoxy-resorufin-O-deethylase (EROD and PROD) at the two highest doses in all exposures. Of the androgen dependent tissues in the 31-day exposed males, seminal vesicle (SV) and ventral prostate (VP) weights were reduced at 60 mg/kg, while testes and epididymal weights were not affected. Preputial separation (PPS) was also significantly delayed by doses of 30 and 60 mg/kg. In the female, the 60 mg/kg dose also caused a significant delay in the age of vaginal opening. Based upon the thyroid hormone response data, this study provides evidence that the 31-day alternative Tier 1 male protocol is a more sensitive test protocol than the 5-day or female pubertal protocol for thyrotoxic agents that act via up-regulation of hepatic metabolism. This apparent greater sensitivity may be due a greater body burden attained following the longer dosing regimen as compared with that of the female protocol, or to gender specific differences in thyroid hormone metabolism. Also, the delay in PPS and reduction in SV and VP weights may indicate a modification or inhibition of endogenous androgenic stimulation directly by DE-71 or a secondary effect that occurs in response to a DE-induced change in thyroid hormones.