Thyroid Follicular Cell Hypertrophy Research Articles

Comparing the effects and potencies of perchlorate and nitrate on amphibian metamorphosis using a modified amphibian metamorphosis assay (AMA).

A modified amphibian metamorphosis assay was performed in which Nieuwkoop and Faber (NF) stage 47 Xenopus laevis larvae were exposed to different concentrations of either perchlorate (ClO4 -) or nitrate (NO3 -) for 32 days. Larvae were exposed to 0.0 (control), 5, 25, 125, 625, and 3125 μg/L ClO4 -, or 0 (control), 23, 71, 217, 660, and 2000 mg/L NO3 -. The primary endpoints were survival, hind limb length (HLL), forelimb emergence and development, developmental stage (including time to NF stage 62 [MT62]), thyroid histopathology, wet weight, and snout-vent length (SVL). Developmental delay as evidenced by altered stage distribution and increased MT62, a higher degree of thyroid follicular cell hypertrophy, and an increase in the prevalence of follicular cell hyperplasia was observed at concentrations ≥125 μg/L ClO4 -. The no observed effect concentration (NOEC) for developmental endpoints was 25.0μg/L ClO4 - and the NOEC for growth endpoints was 3125 μg/L ClO4 -. Exposure to nitrate did not adversely affect MT62, but a decreasing trend in stage distribution and median developmental stage at ≥217 mg/L NO3 - was observed. No histopathologic effects associated with nitrate exposure were observed. An increasing trend in SVL-normalized HLL was observed at 2000 mg/L NO3 -. Nitrate did not alter larval growth. The NOEC for developmental endpoints was 71 mg/L NO3 -, and 2000 mg/L NO3 - for growth endpoints. The present study provided additional evidence that the effects and potency of nitrate and perchlorate on metamorphosis and growth in X.laevis are considerably different.

Inefficacy of dietary test substance administration in Amphibian Metamorphosis Assay (AMA) studies.

Traditionally, the Amphibian Metamorphosis Assay (AMA; OECD TG 231) is performed by exposing Xenopus laevis tadpoles to test substances dissolved in laboratory water. Recently, the use of dietary administration has been proposed to combat poorly soluble test substances in ecotoxicologically-based regulatory endocrine disruption (ED) studies, specifically the AMA warranting an investigation into the efficacy of dietary administration. An efficacy study comprised of two phases: 1) evaluation of the physical influence of the loading process via solvent and 10, 1, and 0.1 mg/l test substance or surrogate (sunflower oil, SFO) on the Sera® Micron Nature (SMN) diet, and 2) performance of a modified AMA in which Nieuwkoop and Faber (NF) stage 51 X. laevis larvae were exposed to dechlorinated tap water using one concentration of the SFO in the diet for 21 days, was performed. In phase 1, the addition of acetone or acetone with bis(2-propylheptyl) phthalate (DPHP) or SFO to SMN with subsequent solvent purge altered the diet reducing the density of the liquified diet and dietary pellet size following centrifugation indicative of alteration of the physical properties of the diet. Treatments used in the modified AMA were acetone alone and 0.1 mg/l SFO dissolved in acetone. These treatments were evaluated against an SMN benchmark using standard AMA endpoints. Both the acetone-treated SMN and 0.1 mg/l SFO-treated diets significantly reduced survival rates, 67 and 70% relative to the SMN benchmark (100%), decreased developmental stage distribution and snout-vent length-normalized hind limb length relative to the SMN benchmark, and slightly increased the prevalence and severity of thyroid follicular cell hypertrophy. Although the acetone-treated diets may have impacted the hypothalamo-pituitary-thyroid axis, clinical signs of gastrointestinal impaction and tail flexure were also observed in the acetone-treated diets, but not the SMN diet alone. Ultimately, test substance exposure via the diet in an AMA study can produce results that may confound data interpretation, which suggests that the traditional aqueous exposure route is generally more appropriate.

An Amphibian Metamorphosis Assay Dietary Restriction Study: Lessons for Data Interpretation.

The amphibian metamorphosis assay (AMA) is a key in vivo endocrine screen to investigate chemicals with potential thyroid activity. The test guidelines and associated guidance consider that treatment-related effects on thyroid gland histomorphology automatically result in the assay being considered positive for thyroid activity, independent of the direction of change or conflicting results in the other biological endpoints. An AMA study was conducted with five different feeding rations equivalent to 50%, 30%, 20%, 10%, and 5% of the recommended feeding rate. Biological endpoints relating to growth and development, including thyroid gland histopathology, were evaluated, and the specificity of these endpoints for the determination of thyroid activity was assessed. There was no effect on survival or clinical signs of toxicity. Effects related to feed reduction generally occurred in a feeding ration-response manner and included reduced development stage; reduced body weight and body length metrics; decreased prevalence of thyroid follicular cell hyperplasia and hypertrophy, and the occurrence of thyroid atrophy; reduced liver vacuolation; and the occurrence of liver atrophy. The results indicate that treatment-related histopathological changes in the AMA can be induced by Non-chemical factors; therefore histopathological results are not necessarily diagnostically specific for chemically induced thyroid endocrine activity. Consequently, the interpretation of data from AMA studies should be adjusted accordingly. We recommend that the decision logic presented in the test guidelines and associated guidance be changed to reflect a requirement for directional agreement between the thyroid histopathology findings and the growth and developmental endpoints before it is concluded that a test substance has thyroid endocrine activity. Environ Toxicol Chem 2023;42:1061-1074. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Amphibian Metamorphosis Assay: Investigation of the potential effects of five chemicals on the hypothalamic-pituitary thyroid axis of Xenopus laevis

2-Ethylhexyl 4-hydroxybenzoate (2-EHHB), 4-tert-octylphenol (4-OP), 4-nonylphenol-branched (4-NP), benzyl butyl phthalate (BBP) and dibutyl phthalate (DBP) were evaluated using a 21-day Amphibian Metamorphosis Assay (AMA). Xenopus laevis larvae were exposed nominally to each chemical at 3.6, 10.9, 33.0, and 100 μg/L, except 4-NP concentrations were 1.8, 5.5, 16.5 and 50 μg/L. Endpoints included mortality, developmental stage, hind limb length (HLL), snout-vent length (SVL), body weight (BW), and thyroid histopathology. BBP and 4-OP accelerated development compared to controls at the mean measured concentration of 3.5 and 39.8 μg/L, respectively. An increase in developmental stage frequency distribution was observed for 4-OP at 39.8 and 103 µg/L, BBP at all concentrations and DBP at 143 µg/L. Normalized HLL was increased on study day (SD) 21 for all tested substances except 4-NP. Histopathology revealed accelerated development and mild thyroid follicular cell hypertrophy at all BBP concentrations, but moderate severity at 105 μg/L. Increased BW occurred for all chemicals except 4-OP. Increased SVL was observed for 4-NP, BBP and DBP on SD 21. There was insufficient evidence that 4-NP and 2-EHHB affected the hypothalamic-pituitary thyroid axis, however, BBP, DBP and 4-OP showed potential effects on amphibian metamorphosis and thyroid activity, albeit through different lines of evidence.

Toxicological evaluation of brominated vegetable oil in Sprague Dawley rats

Brominated vegetable oil (BVO) has been approved by the US Food and Drug Administration on an interim basis as a food additive. Past studies have raised concerns about potential toxicities from consuming BVO. To investigate further these toxicities, we conducted a 90-day dietary exposure study in Sprague Dawley rats and analyzed tissue distribution of the main metabolites. Six-week-old male and female rats were fed diets containing 0 (control), 0.002%, 0.02%, 0.1%, or 0.5% BVO by weight. Statistically significant increases were observed in the serum bromide in the high-dose group of both sexes and in the incidence of thyroid follicular cell hypertrophy in the two highest dose groups of males and the high-dose group of females. An increase in serum TSH was observed in the high-dose group for both sexes, as well as a decrease in serum T4 in the high-dose males. A clear dose-response was observed in di- and tetra-bromostearic acid levels in the heart, liver, and inguinal fat. These data expand upon previous observations in rats and pigs that oral exposure to BVO is associated with increased tissue levels of inorganic and organic bromine, and that the thyroid is a potential target organ of toxicity.

Adversity Considerations for Thyroid Follicular Cell Hypertrophy and Hyperplasia in Nonclinical Toxicity Studies: Results From the 6th ESTP International Expert Workshop.

The European Society of Toxicologic Pathology organized an expert workshop in May 2018 to address adversity considerations related to thyroid follicular cell hypertrophy and/or hyperplasia (FCHH), which is a common finding in nonclinical toxicity studies that can have important implications for risk assessment of pharmaceuticals, food additives, and environmental chemicals. The broad goal of the workshop was to facilitate better alignment in toxicologic pathology and regulatory sciences on how to determine adversity of FCHH. Key objectives were to describe common mechanisms leading to thyroid FCHH and potential functional consequences; provide working criteria to assess adversity of FCHH in context of associated findings; and describe additional methods and experimental data that may influence adversity determinations. The workshop panel was comprised of representatives from the European Union, Japan, and the United States. Participants shared case examples illustrating issues related to adversity assessments of thyroid changes. Provided here are summary discussions, key case presentations, and panel recommendations. This information should increase consistency in the interpretation of adverse changes in the thyroid based on pathology findings in nonclinical toxicity studies, help integrate new types of biomarker data into the review process, and facilitate a more systematic approach to communicating adversity determinations in toxicology reports.

Evaluation of chronic toxicity of cyclocreatine, a creatine analog, in Sprague Dawley rat after oral gavage administration for up to 26 weeks

Cyclocreatine (LUM-001), a creatine analog, was evaluated for its nonclinical toxicity in Sprague Dawley (SD) rats. Deionized water as a vehicle control article or cyclocreatine was administered by oral gavage twice daily (approximately 12 ± 1 h apart) at 30, 100 and 300 mg/kg/dose levels in rats up to 26 weeks followed by a 28-day recovery period. Due to an increased incidence of seizures, the 600 mg/kg/day dose group males were dosed only for 16-weeks followed by a 14-week recovery period. Thirteen males and four females from 600 mg/kg/day dose group were sacrificed at interim on Day 113 to study plausible brain lesions and not due to moribundity. There was a dose dependent increase in the number of seizure incidences in ≥60 mg/kg/day males and 600 mg/kg/day females. Microscopically, higher incidences of vacuoles in the brain at 600 mg/kg/day in both sexes, thyroid follicular atrophy and follicular cell hypertrophy at ≥200 mg/kg/day in males and 600 mg/kg/day in females, and seminiferous tubular degeneration and/or interstitial edema in testes at ≥200 mg/kg/day were observed. Mean plasma half-life of cyclocreatine was between 3.5 and 6.5 h. In conclusion, chronic administration of cyclocreatine by oral gavage in Sprague Dawley rats induced the seizures and microscopic lesions in the brain, testes and thyroid. Based on the results of this study the highest tested dose of 600 mg/kg/day (mean Cmax of 151.5 μg/mL; AUC0-24 of 1970 h*μg/mL) was considered the maximum tolerated dose (MTD) in SD rats.

Thyroid-disrupting effects and mechanism of thiazole-Zn-induced thyroid cell hypertrophy and hyperplasia in male Sprague-Dawley rats.

Thiazole-Zn is a systemic fungicide synthesized and developed in China that has been used for the prevention and treatment of bacterial and fungal diseases on fruits and vegetables. Thiazole-Zn is a new thyroid disruptor chemical. The purpose of this study was to clarify the thyroid-disrupting property of thiazole-Zn and the mechanism responsible for thyroid hormone (TH) biosynthesis inhibition in male rats induced by thiazole-Zn. First, the effects of different thiazole-Zn doses and exposure times on the thyroid weights, thyroid morphology and serum hormone levels of rats were investigated. The results showed that thiazole-Zn increased thyroid weights and serum thyroid-stimulating hormone (TSH) levels and induced thyroid cell hypertrophy and hyperplasia in a dose-related and time-related manner. Furthermore, measurement of thyroid radioiodine uptake in vivo in rats confirmed that thiazole-Zn inhibited active iodide uptake into the thyroid, which reduced circulating levels of serum T3 and T4. Decreases in circulating THs resulted in a compensatory increase in serum TSH levels through a negative feedback system. Subsequently, sustained excessive stimulation of the thyroid gland by TSH led to thyroid follicular cell hypertrophy and hyperplasia. In addition, thiazole-Zn increased sodium/iodide symporter (NIS) expression in the rat thyroid, and the increased NIS expression promoted and restored iodide uptake into the thyroids of rats. The risk of iodine intake inhibition by thiazole-Zn to humans, especially susceptible individuals, such as children and pregnant women, warrants additional attention.

Effects of multiple life stage exposure to the fungicide prochloraz in Xenopus laevis: Manifestations of antiandrogenic and other modes of toxicity

The Larval Amphibian Growth and Development Assay (LAGDA) is an internationally harmonized testing guideline for evaluating effects of chronic chemical exposure in amphibians. In order to evaluate the effects of chronic exposure to an antiandrogenic chemical in an amphibian model, prochloraz was tested using a variation of the LAGDA design. Exposure was initiated with <1d post-fertilization embryos at nominal concentrations of 0, 6.7, 20, 60 and 180 μg/L (0, 18, 53, 159, 478 nM) and continued in flow-through conditions until two months following the median time that controls completed metamorphosis. Growth, developmental rate, circulating thyroid hormone and thyroid gland histopathology were evaluated in a subsample at completion of metamorphosis. There were no effects on growth or development at this stage, but circulating thyroid hormone was elevated in the 20, 60 and 180 μg/L treatments and minimal to mild thyroid follicular cell hypertrophy was observed histologically in the 180 μg/L treatment. Growth, overt toxicity, and reproductive development were evaluated at test termination. There were no effects on growth in either gender, but livers and kidneys exhibited treatment-related pathologies consistent with organ toxicity related to metabolism and presumably impaired excretion of prochloraz metabolites. Histological assessments of female ovaries resulted in minimal pathologies only in the 180 μg/L treatment while male testes exhibited numerous treatment-related pathologies that are consistent with previously reported antiandrogenic effects of prochloraz in other species. The most severe testis pathologies occurred in the 180 μg/L treatment; however, incidences of treatment-related pathologies occurred in all prochloraz treatments. Müllerian duct regression in males was inhibited by prochloraz exposure while Müllerian duct maturation in females was accelerated, characteristic of a feminizing effect. Gene expression levels of potential biomarkers of testis function were also measured. Relative abundance of cyp17a1 transcripts was generally unaffected by prochloraz exposure whereas the Insl3 orthologue, rflcii, was elevated by 3 and >5-fold in the 60 and 180 μg/L treatments, respectively, indicating impaired Leydig cell maturation and testosterone signaling. Overall, prochloraz exposure caused effects characteristic of an antiandrogenic mode of action, which is consistent with previously reported results in other species and supports the utility of the LAGDA design for chemical testing.

Image analysis for TSH mRNA in situ hybridization in pituitary glands from rats with thyroid follicular cell hypertrophy after treatment with three different test compounds

The goal of this in situ hybridization and image analysis technique is to study the effects of new pharmacological/chemical entities on the thyroid and pituitary gland in rats, reveal the pathogenesis of thyroid follicular cell hypertrophy and to retrospectively exclude the risk of thyroid tumor development in humans.In the present study, we describe the increase of thyroid-stimulating hormone- (TSH-) beta subunit mRNA in the pars distalis of the pituitary gland and the quantitative measurement of TSH mRNA positive cells from rats of three 4-week toxicity studies treated with three different test compounds inducing thyroid follicular cell and hepatocellular hypertrophy in rats. Compared to immunohistochemistry (IHC), in situ hybridization (ISH) for TSH was found to be more sensitive.With this technique we are able to exclude a direct effect of the test compound on the thyroid gland by showing the activation of thyrotrope cells from the pituitary gland and therefore this technique retrospectively enables us to exclude a possible risk for humans at an early stage of drug development. Also in case blood serum samples for evaluation of TSH are not available anymore or hepatocellular hypertrophy is not present (close metabolic relationship between thyroid gland and liver in rodents), the described method allows retrospective investigations on thyroid follicular cell hypertrophy or hyperplasia. This can be of high relevance in human safety assessment for certain drugs in order to exclude a primary effect on the thyroid gland especially when it comes to thyroid neoplasia in rodents as previously described.

Toxicologic effects of 28-day dietary exposure to the flame retardant 1,2-dibromo-4-(1,2-dibromoethyl)-cyclohexane (TBECH) in F344 rats.

The brominated flame retardant TBECH is used as an additive to delay ignition and inhibit fires in construction materials and consumer goods. Trends in human exposure are not clear, although humans may be exposed to TBECH via indoor dust and air. In birds and fish there is some evidence of disruption in endocrine and reproductive parameters due to TBECH. In vitro studies indicate that TBECH is an androgen receptor agonist. In this study rats were exposed to 0, 10, 50, 250, 1250 or 5000mg/kg technical TBECH for 28days in diet, corresponding to 0, 0.9, 4.2, 21.3, 98.0 or 328.9mg TBECH/kg bw/d in males and 0, 0.8, 3.9, 19.4, 91.7 or 321.4mg TBECH/kg bw/d in females. Dose-dependent increases in α- and β- TBECH were detected in serum, liver and adipose. Rats in the 5000mg/kg group lost weight rapidly and were euthanized after 15-18days. At study termination rats displayed dose-dependent clinical and histopathological changes consistent with mild hepatic and renal inflammation. In male rats, evidence of gender-specific alpha2u-globulin nephropathy was not considered predictive of renal toxicity in humans. Frank immunosuppression or inappropriate immunostimulation were not apparent, nor was there a primary effect of TBECH on adaptive immunity. Some evidence of hormone disruption was observed, including changes in serum testosterone levels in males and changes in serum T3 and T4 levels in females. Apparent increases in thyroid follicular cell hypertrophy and hyperplasia in male and female rats were not statistically significant. Benchmark dose (BMD) modelling indicated that clinical changes indicative of mild nephrotoxicity and increased blood monocyte numbers indicative of inflammation and tissue damage were the most sensitive outcomes of TBECH exposure that could be modelled. Preliminary evidence of hormone disruption supports the need for rodent studies using more sensitive models of growth, development and reproduction.

Development of the Larval Amphibian Growth and Development Assay: Effects of benzophenone-2 exposure in Xenopus laevis from embryo to juvenile.

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized chemical testing guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of Environment to support risk assessment. The assay is employed as a higher tiered approach to evaluate effects of chronic chemical exposure throughout multiple life stages in a model amphibian species, Xenopus laevis. To evaluate the utility of the initial LAGDA design, the assay was performed using a mixed mode of action endocrine disrupting chemical, benzophenone-2 (BP-2). X. laevis embryos were exposed in flow-through conditions to 0, 1.5, 3.0 or 6.0 mg l-1 BP-2 until 2 months post-metamorphosis. Overt toxicity was evident throughout the exposure period in the 6.0 mg l-1 treatment due to elevated mortality rates and observed liver and kidney pathologies. Concentration-dependent increases in severity of thyroid follicular cell hypertrophy and hyperplasia occurred in larval tadpoles indicating BP-2-induced impacts on the thyroid axis. Additionally, gonads were impacted in all treatments with some genetic males showing both testis and ovary tissues (1.5 mg l-1 ) and 100% of the genetic males in the 3.0 and 6.0 mg l-1 treatments experiencing complete male-to-female sex reversal. Concentration-dependent vitellogenin induction occurred in both genders with associated accumulations of protein in the livers, kidneys and gonads, which was likely vitellogenin and other estrogen-responsive yolk proteins. This is the first study that demonstrates the endocrine effects of this mixed mode of action chemical in an amphibian species and demonstrates the utility of the LAGDA design for supporting chemical risk assessment. Copyright © 2016 John Wiley & Sons, Ltd.

Development of the Larval Amphibian Growth and Development Assay: effects of chronic 4-tert-octylphenol or 17β-trenbolone exposure in Xenopus laevis from embryo to juvenile.

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized test guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of the Environment. The LAGDA was designed to evaluate apical effects of chronic chemical exposure on growth, thyroid-mediated amphibian metamorphosis and reproductive development. During the validation phase, two well-characterized endocrine-disrupting chemicals were tested to evaluate the performance of the initial assay design: xenoestrogen 4-tert-octylphenol (tOP) and xenoandrogen 17β-trenbolone (TB). Xenopus laevis embryos were exposed, in flow-through conditions, to tOP (nominal concentrations: 0.0, 6.25, 12.5, 25 and 50 µg l-1 ) or TB (nominal concentrations: 0.0, 12.5, 25, 50 and 100 ng l-1 ) until 8 weeks post-metamorphosis, at which time growth measurements were taken, and histopathology assessments were made of the gonads, reproductive ducts, liver and kidneys. There were no effects on growth in either study and no signs of overt toxicity, sex reversal or gonad dysgenesis. Exposure to tOP caused a treatment-related decrease in circulating thyroxine and an increase in thyroid follicular cell hypertrophy and hyperplasia (25 and 50 µg l-1 ) during metamorphosis. Müllerian duct development was affected after exposure to both chemicals; tOP exposure caused dose-dependent maturation of oviducts in both male and female frogs, whereas TB exposure caused accelerated Müllerian duct regression in males and complete regression in >50% of the females in the 100 ng l-1 treatment. Based on these results, the LAGDA performed adequately to evaluate apical effects of chronic exposure to two endocrine-active compounds and is the first standardized amphibian multiple life stage toxicity test to date. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Comprehensive Assessment of a Chlorinated Drinking Water Concentrate in a Rat Multigenerational Reproductive Toxicity Study

Some epidemiological studies report associations between drinking water disinfection byproducts (DBPs) and adverse reproductive/developmental effects, e.g., low birth weight, spontaneous abortion, stillbirth, and birth defects. Using a multigenerational rat bioassay, we evaluated an environmentally relevant "whole" mixture of DBPs representative of chlorinated drinking water, including unidentified DBPs as well as realistic proportions of known DBPs at low-toxicity concentrations. Source water from a water utility was concentrated 136-fold, chlorinated, and provided as drinking water to Sprague-Dawley rats. Timed-pregnant females (P0 generation) were exposed during gestation and lactation. Weanlings (F1 generation) continued exposures and were bred to produce an F2 generation. Large sample sizes enhanced statistical power, particularly for pup weight and prenatal loss. No adverse effects were observed for pup weight, prenatal loss, pregnancy rate, gestation length, puberty onset in males, growth, estrous cycles, hormone levels, immunological end points, and most neurobehavioral end points. Significant, albeit slight, effects included delayed puberty for F1 females, reduced caput epidydimal sperm counts in F1 adult males, and increased incidences of thyroid follicular cell hypertrophy in adult females. These results highlight areas for future research, while the largely negative findings, particularly for pup weight and prenatal loss, are notable.

The effects of clobazam treatment in rats on the expression of genes and proteins encoding glucronosyltransferase 1A/2B (UGT1A/2B) and multidrug resistance‐associated protein-2 (MRP2), and development of thyroid follicular cell hypertrophy

Clobazam (CLB) is known to increase hepatobiliary thyroxine (T4) clearance in Sprague–Dawley (SD) rats, which results in hypothyroidism followed by thyroid follicular cell hypertrophy. However, the mechanism of the acceleration of T4-clearance has not been fully investigated.In the present study, we tried to clarify the roles of hepatic UDP-glucronosyltransferase (UGT) isoenzymes (UGT1A and UGT2B) and efflux transporter (multidrug resistance–associated protein-2; MRP2) in the CLB-induced acceleration of T4-clearance using two mutant rat strains, UGT1A-deficient mutant (Gunn) and MRP2-deficient mutant (EHBR) rats, especially focusing on thyroid morphology, levels of circulating hormones (T4 and triiodothyronine (T3)) and thyroid-stimulating hormone (TSH), and mRNA or protein expressions of UGTs (Ugt1a1, Ugt1a6, and Ugt2b1/2) and MRP2 (Mrp).CLB induced thyroid morphological changes with increases in TSH in SD and Gunn rats, but not in EHBR rats. T4 was slightly decreased in SD and Gunn rats, and T3 was decreased in Gunn rats, whereas these hormones were maintained in EHBR rats. Hepatic Ugt1a1, Ugt1a6, Ugt2b1/2, and Mrp2 mRNAs were upregulated in SD rats. In Gunn rats, UGT1A mRNAs (Ugt1a1/6) and protein levels were quite low, but UGT2B mRNAs (Ugt2b1/2) and protein were prominently upregulated. In SD and Gunn rats, MRP2 mRNA and protein were upregulated to the same degree. These results suggest that MRP2 is an important contributor in development of the thyroid cellular hypertrophy in CLB-treated rats, and that UGT1A and UGT2B work in concert with MRP2 in the presence of MRP2 function to enable the effective elimination of thyroid hormones.

Role of iodine in diiodomethyl-p-tolylsulfone induced reproductive toxicity in rats: Proposed mode of action

The biocide diiodomethyl-p-tolylsulfone (DIMPTS) caused dystocia, decreased neonatal survival and hypothyroidism in rat reproduction studies resembling the effects caused by iodine. One molecule of DIMPTS contains two iodine moieties that are hydrolyzed upon ingestion and systemically absorbed, suggesting iodine toxicity as a probable mode of action for the effects observed in rats. This study compared the effects induced by DIMPTS and an equimolar concentration of its de-iodinated analogue, methyl-p-tolylsulfone (MPTS). Groups of 20 female Sprague Dawley rats were fed diets supplying 80mg DIMPTS/kg/day, 32mg MPTS/kg/day or control feed from prior to breeding through lactation and gonadal function, mating performance, conception, gestation, parturition, lactation, survival, growth and development of pups evaluated through postnatal day 7. Serum thyroid hormones and iodine levels in milk and sera were also determined. Females given DIMPTS had increased incidence of vulvar discharge and dystocia, decreased litter size, decreased body weights and feed consumption, increased thyroid weights, thyroid follicular cell hypertrophy with decreased colloid, decreased triidothyronine, and increased thyroid stimulating hormone levels. DIMPTS pups had decreased neonatal survival and body weights. These effects were associated with elevated levels of iodine in milk and sera. In contrast, MPTS did not produce similar effects in adult females or their offspring. These data support the hypothesis that the dystocia, altered neonatal survival and hypothyroidism following repeated dietary administration of DIMPTS were due to excessive iodine released from DIMPTS during absorption and metabolism.

In vivo effects of a human thyroid-stimulating monoclonal autoantibody (M22) and a human thyroid-blocking autoantibody (K1-70).

PurposeTo study in vivo effects of the human monoclonal TSH receptor (TSHR) autoantibodies M22 (stimulating type) and K1-70 (blocking type) on thyroid hormone levels in rats.MethodsSerum levels of total T4, free T4, M22 and K1-70 were measured following intramuscular injection of M22 IgG (2–4 μg/animal), K1-70 IgG (10–200 μg/animal) or both into rats. Thyroid pathology was assessed in M22-injected rats.ResultsSerum levels of total T4 and free T4 increased in a dose-dependent manner following injection of M22 IgG. Thyroid follicular cell hypertrophy was dependent on the dose of M22 IgG. K1-70 IgG caused a dose dependent decrease of total T4 and free T4 levels in rats receiving K1-70 only. The stimulating effects of M22 IgG on T4 levels in rats were completely inhibited by K1-70 IgG.ConclusionM22 is a potent stimulator of thyroid hormone secretion in vivo. In contrast, K1-70 inhibits thyroid hormone secretion in vivo. Furthermore, K1-70 has the ability to inhibit the stimulating activity of M22 in vivo and as such has potential as a new drug to block TSHR stimulation by autoantibodies in Graves’ disease.

Characterization of Xenobiotic-Induced Hepatocellular Enzyme Induction in Rats

During routine safety evaluation of RO2910, a non-nucleoside reverse transcriptase inhibitor for HIV infection, histopathology findings concurrent with robust hepatocellular induction occurred in multiple organs, including a unique, albeit related, finding in the pituitary gland. For fourteen days, male and female rats were administered, by oral gavage vehicle, 100, 300, or 1000 mg/kg/day of RO2910. Treated groups had elevated serum thyroid-stimulating hormone and decreased total thyroxine, and hypertrophy in the liver, thyroid gland, and pituitary pars distalis. These were considered consequences of hepatocellular induction and often were dose dependent and more pronounced in males than in females. Hepatocellular centrilobular hypertrophy corresponded with increased expression of cytochrome P450s 2B1/2, 3A1, and 3A2 and UGT 2B1. Bilateral thyroid follicular cell hypertrophy occurred concurrent to increased mitotic activity and sometimes colloid depletion, which were attributed to changes in thyroid hormone levels. Males had hypertrophy of thyroid-stimulating hormone-producing cells (thyrotrophs) in the pituitary pars distalis. All findings were consistent with the well-established adaptive physiologic response of rodents to xenobiotic-induced hepatocellular microsomal enzyme induction. Although the effects on the pituitary gland following hepatic enzyme induction-mediated hypothyroidism have not been reported previously, other models of stress and thyroid depletion leading to pituitary stimulation support such a shared pathogenesis.

Thyroid Follicular Lesions Induced by Oral Treatment for 2 Years with 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Dioxin-like Compounds in Female Harlan Sprague-Dawley Rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and structurally-similar dioxin-like compounds affect thyroid function and morphology and thyroid hormone metabolism in animals and humans. The National Toxicology Program conducted eight 2-year gavage studies in female Harlan Sprague-Dawley rats to determine the relative potency of chronic toxicity and carcinogenicity of TCDD, 3,3',4,4',5-pentachlorobiphenyl (PCB126), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), 2,3',4,4',5-pentachlorobiphenyl (PCB118), 2,2',4,4',5,5'-hexachloro-biphenyl (PCB153), a tertiary mixture of TCDD/PCB126/PeCDF, and two binary mixtures (PCB126/PCB153 and PCB126/PCB118). Administration of these compounds was associated with increased incidences of thyroid follicular cell hypertrophy, variably observed in the 14-, 31-, and 53-week interim and 2-year sacrifice groups. In all studies, the incidences of follicular cell adenoma and carcinoma were not increased. Decreased levels of serum thyroxine were primarily noted in the 14-or-later -week interim groups of all chemicals. Serum triiodothyronine (T3) levels were increased in the TCDD, PCB126, PeCDF, TCDD/PCB126/PeCDF, and PCB126/PCB153 studies, while decreased levels were noted in the PCB153 and PCB126/PCB118 studies. TCDD, PCB126, PCB126/PCB153, and PCB126/PCB118 increased levels of serum thyroid-stimulating hormone almost in a dose-dependent manner in the 14-week groups. These data suggest that although dioxin-like compounds alter thyroid hormones and increase follicular cell hyperplasia, there is not an increase in thyroid adenoma or carcinoma in female Sprague-Dawley rats.

Safety assessment of EPA-rich triglyceride oil produced from yeast: Genotoxicity and 28-day oral toxicity in rats

The 28-day repeat-dose oral and genetic toxicity of eicosapentaenoic acid triglyceride oil (EPA oil) produced from genetically modified Yarrowia lipolytica yeast were assessed. Groups of rats received 0 (olive oil), 940, 1880, or 2820mg EPA oil/kg/day, or fish oil (sardine/anchovy source) by oral gavage. Lower total serum cholesterol was seen in all EPA and fish oil groups. Liver weights were increased in the medium and high-dose EPA (male only), and fish oil groups but were considered non-adverse physiologically adaptive responses. Increased thyroid follicular cell hypertrophy was observed in male high-dose EPA and fish oil groups, and was considered to be an adaptive response to high levels of polyunsaturated fatty acids. No adverse test substance-related effects were observed on body weight, nutritional, or other clinical or anatomic pathology parameters. The oil was not mutagenic in the in vitro Ames or mouse lymphoma assay, and was not clastogenic in the in vivo mouse micronucleus test. In conclusion, exposure for 28days to EPA oil derived from yeast did not produce adverse effects at doses up to 2820mg/kg/day and was not genotoxic. The safety profile of the EPA oil in these tests was comparable to a commercial fish oil.