Inhibition Of Thyroid Peroxidase Research Articles

In vitro assessment of thyroid peroxidase inhibition by chemical exposure: comparison of cell models and detection methods.

Disruption of the thyroid hormone (TH) system is connected with diverse adverse health outcomes in wildlife and humans. It is crucial to develop and validate suitable in vitro assays capable of measuring the disruption of the thyroid hormone (TH) system. These assays are also essential to comply with the 3R principles, aiming to replace the ex vivo tests often utilised in the chemical assessment. We compared the two commonly used assays applicable for high throughput screening [Luminol and Amplex UltraRed (AUR)] for the assessment of inhibition of thyroid peroxidase (TPO, a crucial enzyme in TH synthesis) using several cell lines and 21 compounds from different use categories. As the investigated cell lines derived from human and rat thyroid showed low or undetectable TPO expression, we developed a series of novel cell lines overexpressing human TPO protein. The HEK-TPOA7 model was prioritised for further research based on the high and stable TPO gene and protein expression. Notably, the Luminol assay detected significant peroxidase activity and signal inhibition even in Nthy-ori 3-1 and HEK293T cell lines without TPO expression, revealing its lack of specificity. Conversely, the AUR assay was specific to TPO activity. Nevertheless, despite the different specificity, both assays identified similar peroxidation inhibitors. Over half of the tested chemicals with diverse structures and from different use groups caused TPO inhibition, including some widespread environmental contaminants suggesting a potential impact of environmental chemicals on TH synthesis. Furthermore, in silico SeqAPASS analysis confirmed the high similarity of human TPO across mammals and other vertebrate classes, suggesting the applicability of HEK-TPOA7 model findings to other vertebrates.

A ubiquitous tire rubber additive induced serious eye injury in zebrafish (Danio rerio)

Previous studies have indicated that tire wear particles (TWPs) leachate exposure induced serious eye injury in fish through inhibiting the thyroid peroxidase (TPO) enzyme activity. However, the main TPO inhibitors in the leachate were still unknown. In this study, we identified 2-Mercaptobenzothiazole (MBT) as the potential TPO inhibitor in the TWPs leachate through references search, model prediction based on Danish QSAR and ToxCast database, molecular docking, and in vivo assay. We further explored the toxic mechanism of MBT under environmentally relevant concentrations. The decreased eye size of zebrafish larvae was mainly caused by the decreased lens diameter and cell density in the inner nuclear layer (INL) and outer nuclear layer (ONL) of the retina. Transcriptomics analysis demonstrated that the eye phototransduction function was significantly suppressed by inhibiting the photoreceptor cell proliferation process after MBT exposure. The altered opsin gene expression and decreased opsin protein levels were induced by weakening thyroid hormone signaling after MBT treatment. These results were comparable to those obtained from a known TPO inhibitor, methimazole. This study has identified MBT as the primary TPO inhibitor responsible for inducing eye impairment in zebrafish larvae exposed to TWPs leachate. It is crucial for reducing the toxicity of TWPs leachate in fish.

Development and experimental validation of 3D QSAR models for the screening of thyroid peroxidase inhibitors using integrated methods of computational chemistry

The intricate network of glands and organs that makes up the endocrine system. Hormones are used to regulate and synchronize the nervous and physiological systems. The agents which perturbate an endocrine system are called endocrine disruptors and they can eventually affect cellular proliferation and differentiation in target tissues. A subclass of endocrine disruptors known as thyroid disruptors (TDs) or thyroid disrupting chemicals (TDCs) influence the hypothalamo-pituitary-thyroid axis or directly interfere with thyroid function by binding to thyroid hormone receptors. Thyroid hormone levels in circulation are now included in more test guidelines (OECD TG 441, 407, 408, 414, 421/422, 443/416). Although these might be adequate to recognize thyroid adversity, they are unable to explain the underlying mechanism of action. Thyroid peroxidase (TPO) and sodium iodide symporter (NIS), two proteins essential in the biosynthesis of thyroid hormones, are well-accepted molecular targets for inhibition. The screening of a large number of molecules using high throughput screening (HTS) requires a minimum quantity of sample, cost, and time consuming. Whereas 3-dimensional quantitative structure-activity relationship (3D-QSAR) analysis can screen the TDCs before synthesizing a compound. In the present study, the human TPO (hTPO) and NIS (hNIS) structures were modelled using homology modeling and the quality of the structures was validated satisfactorily using MD simulation for 100ns. Further, 190 human TPO inhibitors with IC50 were curated from Comptox and docked with the modelled structure of TPO using D238, H239 and D240 centric grid. The binding conformation of a molecule with low binding energy was used as a reference and the rest other molecules were aligned after generating the possible conformers. The activity-stratified partition was performed for aligned molecules and training set (139), test set (51) were defined. The machine learning models such as k Nearest Neighbor (kNN) and Random Forest (RF) models were built and validated using external experimental dataset containing 10 molecules. Among the 10 molecules, all 10 molecules were identified as TPO inhibitors and demonstrated 100 % accuracy qualitatively. To confirm the selective TPO inhibition all 10 molecules were docked with the modelled structure of hNIS and the results have demonstrated the selective TPO inhibition.

A metabolomics approach to reveal the mechanism of developmental toxicity in zebrafish embryos exposed to 6-propyl-2-thiouracil

A crucial component of a substance registration and regulation is the evaluation of human prenatal developmental toxicity. Current toxicological tests are based on mammalian models, but these are costly, time consuming and may pose ethical concerns. The zebrafish embryo has evolved as a promising alternative model to study developmental toxicity. However, the implementation of the zebrafish embryotoxicity test is challenged by lacking information on the relevance of observed morphological alterations in fish for human developmental toxicity. Elucidating the mechanism of toxicity could help to overcome this limitation. Through LC-MS/MS and GC-MS metabolomics, we investigated whether changes to the endogenous metabolites can indicate pathways associated with developmental toxicity. To this aim, zebrafish embryos were exposed to different concentrations of 6-propyl-2-thiouracil (PTU), a compound known to induce developmental toxicity. The reproducibility and the concentration-dependence of the metabolome response and its association with morphological alterations were studied. Major morphological findings were reduced eye size, and other craniofacial anomalies; major metabolic changes included increased tyrosine, pipecolic acid and lysophosphatidylcholine levels, decreased methionine levels, and disturbance of the ‘Phenylalanine, tyrosine and tryptophan biosynthesis’ pathway. This pathway, and the changes in tyrosine and pipecolic acid levels could be linked to the mode of action of PTU, i.e., inhibition of thyroid peroxidase (TPO). The other findings suggested neurodevelopmental impairments. This proof-of-concept study demonstrated that metabolite changes in zebrafish embryos are robust and provide mechanistic information associated with the mode of action of PTU.

In silico prediction models for thyroid peroxidase inhibitors and their application to synthetic flavors

Systematic toxicity tests are often waived for the synthetic flavors as they are added in a very small amount in foods. However, their safety for some endpoints such as endocrine disruption should be concerned as they are likely to be active in low levels. In this case, structure–activity-relationship (SAR) models are good alternatives. In this study, therefore, binary, ternary, and quaternary prediction models were designed using simple or complex machine-learning methods. Overall, hard-voting classifiers outperformed other methods. The test scores for the best binary, ternary, and quaternary models were 0.6635, 0.5083, and 0.5217, respectively. Along with model development, some substructures including primary aromatic amine, (enol)ether, phenol, heterocyclic sulfur, and heterocyclic nitrogen, dominantly occurred in the most highly active compounds. The best predicting models were applied to synthetic flavors, and 22 agents appeared to have a strong inhibitory potential towards TPO activities.

A geospatial modeling approach to quantifying risk of exposure to environmental chemical mixtures via a common molecular initiating event

BACKGROUND AND AIM: The exposome, a measure of all chemical exposures experienced through a lifetime, plays an important role in human health outcomes. Thus, identifying and quantifying lifetime exposure to chemicals present in the environment is important for risk assessment. Linking health outcomes to environmental chemicals exposures is challenging, as real-world exposures involve multiple chemicals and unresolved spatial and temporal distributions. To address these challenges, we propose a geospatially resolved biological target-based risk assessment for exposure to chemical mixtures. This approach models the geospatial distribution of environmental chemicals based on common activation of toxicological adverse outcome pathways (AOPs) as indicated by Tox21 high throughput assays. METHODS: We exemplify this with a USA-wide proof-of-concept that quantifies the predicted geospatial likelihood of thyroid peroxidase (TPO) inhibition. This enzyme is necessary for producing the thyroid hormone T3 and T4, and inhibition has been associated with decreased cognitive function. First, environmental chemicals were identified that both 1) inhibit TPO based on Tox21 data, and 2) were measured by the USGS and EPA in environmental samples (surface water). Next, the geospatial chemical distributions were modeled with combined land-use regression and Gaussian process models. The individual chemical exposure surfaces can be combined into an aggregate risk surface using an unweighted approach or weighted by chemical potency from the toxicological assay concentration-response data. RESULTS:Maps of the aggregate risk surface will identify regions where thyroid peroxidase inhibition is more likely based on combined environmental chemical exposures and chemical potency. CONCLUSIONS:This work will help advance risk assessment methods by providing a method for assessing the geospatial risk of exposure to environmental chemical mixtures and identifying regions with a higher cumulative risk of activating the selected molecular pathways, which may lead to adverse health outcomes. KEYWORDS: Mixtures, External exposome, Exposure assessment-general, risk assessment, Spatial statistics, big data

Mechanistic Computational Model for Extrapolating InVitro Thyroid Peroxidase (TPO) Inhibition Data to Predict Serum Thyroid Hormone Levels in Rats.

High-throughput in vitro assays are developed to screen chemicals for their potential to inhibit thyroid hormones (THs) synthesis. Some of these experiments, such as the thyroid peroxidase (TPO) inhibition assay, are based on thyroid microsomal extracts. However, the regulation of thyroid disruption chemicals is based on THs in vivo serum levels. This necessitates the estimation of thyroid disruption chemicals in vivo tissue levels in the thyroid where THs synthesis inhibition by TPO takes place. The in vivo tissue levels of chemicals are controlled by pharmacokinetic determinants such as absorption, distribution, metabolism, and excretion, and can be described quantitatively in physiologically based pharmacokinetic (PBPK) models. An integrative computational model including chemical-specific PBPK and TH kinetics models provides a mechanistic quantitative approach to translate thyroidal high-throughput in vitro assays to in vivo measures of circulating THs serum levels. This computational framework is developed to quantitatively establish the linkage between applied dose, chemical thyroid tissue levels, thyroid TPO inhibition potential, and in vivo TH serum levels. Once this link is established quantitatively, the overall model is used to calibrate the TH kinetics parameters using experimental data for THs levels in thyroid tissue and serum for the 2 drugs, propylthiouracil and methimazole. The calibrated quantitative framework is then evaluated against literature data for the environmental chemical ethylenethiourea. The linkage of PBPK and TH kinetics models illustrates a computational framework that can be extrapolated to humans to screen chemicals based on their exposure levels and potential to disrupt serum THs levels in vivo.

Metribuzin-induced non-adverse liver changes result in rodent-specific non-adverse thyroid effects via uridine 5′-diphospho-glucuronosyltransferase (UDPGT, UGT) modulation

Metribuzin is a herbicide that inhibits photosynthesis and has been used for over 40 years. Its main target organ is the liver and to some extent the kidney in rats, dogs, and rabbits.Metribuzin shows a specific thyroxine (T4) profile in rat studies with T4 increases at low doses and T4 decreases at higher doses. Only the T4 decreases occur together with histopathological changes in the thyroid and weight changes of liver and thyroid.A set of experiments was conducted to investigate metribuzin's endocrine disruptor potential according to European guidance and regulations.The results indicate that a liver enzyme modulation, i.e. of the uridine 5′-diphospho-glucuronosyltransferase (UDPGT, UGT), is most likely responsible for both increased and decreased plasma thyroxine level and for thyroid histopathological observations. Animals with high T4 levels show low UGT activity, while animals with low T4 levels show high UGT activity. A causal relationship was inferred, since other potentially human-relevant mode of action (MOA) pathways were excluded in dedicated studies, i.e. inhibition of deiodinases (DIO), inhibition of thyroid peroxidase (TPO) or of the sodium importer system (NIS). This liver metabolism-associated MOA is considered not relevant for human hazard assessment, due to species differences in thyroid homeostasis between humans and rats and, more importantly, based on experimental data showing that metribuzin affects UGT activity in rat but not in human hepatocytes. Further, we discuss whether or not increased T4 levels in the rat, in the absence of histopathological changes, should be considered as adverse and therefore used as an appropriate hazard model for humans.Based on a weight of evidence approach, metribuzin should not be classified as an endocrine disruptor with regard to the thyroid modality.

In vitro assays for characterization of distinct multiple catalytic activities of thyroid peroxidase using LC-MS/MS

A diverse set of environmental contaminants have raised a concern about their potential adverse effects on endocrine signaling. Robust and widely accepted battery of in vitro assays is available to assess the disruption of androgenic and estrogenic pathways. However, such definitive systems to investigate effects on the disruption of thyroid pathways by the xenobiotics are not yet well established. One of the major “Molecular Initiating Events” (MIEs) in thyroid disruption involves targeting of thyroid peroxidase (TPO), a key enzyme involved in thyroid hormone synthesis. TPO catalyzes mono- and diiodination of L-Tyrosine (L-Tyr) to generate 3-Iodo-l-tyrosine (MIT) and 3,5-Diiodo-l-tyrosine (DIT), respectively, followed by the coupling of iodinated tyrosine rings to generate thyroid hormones, 3,3’5-Triiodo-l-thyronine (T3) and Levothyroxine (T4).We sought to develop a robust, sensitive, and rapid in vitro assay systems to evaluate the effects of test chemicals on the multiple catalytic activities of thyroid peroxidase. Simple in vitro assays were designed to study TPO mediated distinct reactions using a single LC-MS/MS method.Herein, we describe a battery of assays to investigate the iodination of L-Tyr to MIT and DIT, MIT to DIT as well as, T3 to T4 catalyzed by rat thyroid TPO. Importantly, two sequential reactions involving mono- and diiodination of L-Tyr could be analyzed in a single assay. The assay that monitors in vitro conversion of DIT to T4 was developed to study the coupling of tyrosine rings. Enzyme kinetics studies revealed distinct characteristics of multiple reactions catalyzed by TPO. Further, the known TPO inhibitors were used to assess their potency towards individual TPO substrates and reactions. The resultant half maximum inhibitory concentration (IC50) values highlighted differential targeting of TPO catalyzed reactions by the same inhibitor. Overall results underscore the need to develop more nuanced approaches that account for distinct multiple catalytic activities of TPO.

Toxicogenomic fin(ger)prints for thyroid disruption AOP refinement and biomarker identification in zebrafish embryos

Endocrine disruption (ED) can trigger far-reaching effects on environmental populations, justifying a refusal of market approval for chemicals with ED properties. For the hazard assessment of ED effects on the thyroid system, regulatory decisions mostly rely on amphibian studies. Here, we used transcriptomics and proteomics for identifying molecular signatures of interference with thyroid hormone signaling preceding physiological effects in zebrafish embryos. For this, we analyzed the thyroid hormone 3,3′,5-triiodothyronine (T3) and the thyroid peroxidase inhibitor 6-propyl-2-thiouracil (6-PTU) as model substances for increased and repressed thyroid hormone signaling in a modified zebrafish embryo toxicity test. We identified consistent gene expression fingerprints for both modes-of-action (MoA) at sublethal test concentrations. T3 and 6-PTU both significantly target the expression of genes involved in muscle contraction and functioning in an opposing fashion, allowing for a mechanistic refinement of key event relationships in thyroid-related adverse outcome pathways in fish. Furthermore, our fingerprints identify biomarker candidates for thyroid disruption hazard screening approaches. Perspectively, our findings will promote the AOP-based development of in vitro assays for thyroidal ED assessment, which in the long term will contribute to a reduction of regulatory animal tests.

Manipulating plasma thyroid hormone levels at hatching alters development of endothermy and ventilation in Pekin duck (Anas platyrhynchos domestica).

At hatching in precocial birds, there are rapid physiological and metabolic phenotypic changes associated with attaining endothermy. During the transition to ex ovo life, thyroid hormone levels naturally increase, peaking at hatching, and then decline. To better understand the role of the natural increase in thyroid hormone at hatching in regulating the developmental trajectory of the Pekin duck's endothermic phenotype, we examined development of O2 consumption (V̇O2 ) and ventilation (frequency, tidal volume and minute ventilation) while inhibiting the developmental increase in thyroid hormones that occurs at hatching via administration of the thyroid-peroxidase inhibitor methimazole (MMI) or accelerating the developmental increase via triiodothyronine (T3) supplementation. Animals were dosed only on day24 of a 28-day incubation period and studied on incubation day25, during external pipping (EP) and 1 day post-hatching (dph). On day25, there was an increase in V̇O2 in the hyperthyroid treatment compared with the other two treatments. During the EP stage, there was a significant effect of thyroid status on V̇O2 ,with hyperthyroid V̇O2 being highest and hypothyroid V̇O2 the lowest. By 1 dph, the supplemented T3 and control animals had similar V̇O2 responses to cooling with comparable thermal neutral zones followed by increased V̇O2 Hypothyroid 1 dph hatchlings had a lower resting V̇O2 that did not increase to the same extent as the supplemented T3 and control animals during cooling. During EP, inhibiting the rise in T3 resulted in embryos with lower ventilation frequency and tidal volume than control and supplemented T3 embryos. At 1 dph, ventilation frequency of all animals increased during cooling, but tidal volume only increased in supplemented T3 and control hatchlings. Our data support the role of the late incubation increase in T3 in regulating the systemic development of endothermic metabolic capacity and associated control of ventilation occurring at hatching of the Pekin duck.

Mechanism of Action and Interactions between Thyroid Peroxidase and Lipoxygenase Inhibitors Derived from Plant Sources.

This study focused on the effect of kaempferol, catechin, apigenin, sinapinic acid, and extracts from plants (i.e., parsley, cumin, mustard, green tea, and green coffee) on thyroid peroxidase (TPO) and lipoxygenase (LOX) activity, antiradical potential, as well as the result of interactions among them. Catechin, sinapinic acid, and kaempferol acted as a competitive TPO inhibitors, while apigenin demonstrated an uncompetitive mode of inhibitory action. Ethanol extracts from all plants acted as competitive TPO inhibitors, while, after in vitro digestion, TPO activation was found especially in the case of mustard (24%) and cumin (19.85%). Most importantly, TPO activators acted synergistically. The TPO effectors acted as LOX inhibitors. The most effective were potentially bioaccessible compounds from green tea and green coffee (IC50 = 29.73 mg DW/mL and 30.43 mg DW/mL, respectively). The highest free radical scavenging ability was determined for catechin and sinapinic acid (IC50 = 78.37 µg/mL and 84.33 µg/mL, respectively) and potentially bioaccessible compounds from mustard (0.42 mg DW/mL) and green coffee (0.87 mg DW/mL). Green coffee, green tea, cumin, and mustard contain potentially bioaccessible TPO activators that also act as effective LOX inhibitors, which indicate their potentially health-promoting effects for people suffering from Hashimoto’s disease.

A rapid assay of human thyroid peroxidase activity

Impaired synthesis or action of thyroid hormones (THs) during critically sensitive periods of development can have long term adverse effects on health. Development of rapid assays to identify chemicals that impair THs physiology is an important goal for reducing risks from chemical use. Thyroid peroxidase (TPO) is a key enzyme regulating THs synthesis in thyroid gland and a vulnerable target for chemicals that disrupt THs synthesis. To develop a human-relevant, rapid assay for TPO inhibition, we have engineered two cell lines (CHO and LentiX- 293) to express active human TPO (hTPO) enzyme and applied them in a recently-described assay using a stable fluorescent product (Amplex UltraRed). Assay performance was assessed by comparing activity of 19 reference chemicals with known strong, weak or no TPO inhibitory activity. The assay using hTPO from either cell line consistently identified the relative potency of strong to moderate inhibitors and chemicals known to be inactive. Results were less consistent for chemicals reported to be weak inhibitors of rodent TPO, possibly suggesting some species specificity. Our studies support the use of hTPO from stably transfected cell lines to substitute for animal-derived thyroid microsomes for rapid high throughput screening assays to identify and characterize TPO inhibitors.

Thyroid Peroxidase Activity is Inhibited by Phenolic Compounds-Impact of Interaction.

The aim of this study was to estimate the mode of thyroid peroxidase (TPO) inhibition by polyphenols: Chlorogenic acid, rosmarinic acid, quercetin, and rutin. All the tested polyphenols inhibited TPO; the IC50 values ranged from 0.004 mM to 1.44 mM (for rosmarinic acid and rutin, respectively). All these pure phytochemical substances exhibited different modes of TPO inhibition. Rutin and rosmarinic acid showed competitive, quercetin—uncompetitive and chlorogenic acid—noncompetitive inhibition effect on TPO. Homology modeling was used to gain insight into the 3D structure of TPO and molecular docking was applied to study the interactions of the inhibitors with their target at the molecular level. Moreover, the type and strength of mutual interactions between the inhibitors (expressed as the combination index, CI) were analyzed. Slight synergism, antagonism, and moderate antagonism were found in the case of the combined addition of the pure polyphenols. Rutin and quercetin as well as rutin and rosmarinic acid acted additively (CI = 0.096 and 1.06, respectively), while rutin and chlorogenic acid demonstrated slight synergism (CI = 0.88) and rosmarinic acid with quercetin and rosmarinic acid with chlorogenic acid showed moderate antagonism (CI = 1.45 and 1.25, respectively). The mixture of chlorogenic acid and quercetin demonstrated antagonism (CI = 1.79). All the polyphenols showed in vitro antiradical ability against 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), ABTS. The highest ability (expressed as IC50) was exhibited by rosmarinic acid (0.12 mM) and the lowest value was ascribed to quercetin (0.45 mM).

An assay for screening xenobiotics for inhibition of rat thyroid gland peroxidase activity

1. A number of chemicals have been shown to produce disruption of the thyroid gland, resulting in reduced thyroid hormone synthesis, by a mechanism involving inhibition of thyroid peroxidase (TPO) activity (EC 1.11.1.8).2. An assay was developed for rat thyroid gland microsomal TPO activity, employing L-tyrosine as the physiological substrate, with analysis of the formation of the 3-iodo-L-tyrosine (3MIT) metabolite by ultra-performance liquid chromatography-mass spectrometry-mass spectrometry.3. Formation of 3MIT was linear with respect to both rat thyroid gland microsomal protein concentration and incubation time, whereas only small quantities of 3,5-diodo-L-tyrosine were formed.4. Studies were performed with nine known TPO inhibitors. The most potent inhibitors were 3-amino-1,2,4-triazole, ethylene thiourea, methimazole and 6-propyl-2-thiouracil which had IC50 values (i.e. concentration to produce a 50% inhibition of enzyme activity) of 0.059, 0.791, 1.07 and 1.96 μM, respectively, whereas the least potent inhibitor was sodium perchlorate which had an IC50 value of 13,800 µM.5. For five inhibitors, where literature data were available, the observed IC50 values obtained in this study employing rat thyroid gland microsomes and L-tyrosine as substrate were similar to those previously reported using the spectrophotometric guaiacol oxidation assay.

Specific alteration of gene expression profile in rats by treatment with thyroid toxicants that inhibit thyroid hormone synthesis.

Transcriptomics technologies have been used for risk assessment of chemicals, mainly to predict the modes of action (MOAs) of chemicals or identify biomarkers. Transcriptomics data may also be helpful to understand MOAs of chemicals at the molecular level in more detail. As an example of the known MOAs, there are two MOAs of thyroid toxicity: inhibition of thyroid hormone synthesis ("direct" effect) and hypermetabolism of thyroid hormone by enzyme induction in liver ("indirect" effect). In the present study, global profiles of gene expression were analyzed in rats treated with chemicals acting directly on the thyroid (thyroid peroxidase inhibitors such as propylthiouracil and methimazole) and chemicals acting indirectly on the thyroid (hepatic enzyme inducers such as phenobarbital and pregnenolone-16α-carbonitrile) using microarrays. Using a subtraction method between these two types of chemicals, we identified characteristic gene expression changes on the thyroid hormone synthesis pathway by direct-acting chemicals. Based on the functions of these genes, alterations of their expression seem to indicate the results of thyroid peroxidase inhibition, and might be helpful in more accurate evaluation of MOAs for thyroid toxicity.

Interaction between iodine and glucosinolates in rutabaga sprouts and selected biomarkers of thyroid function in male rats

Rutabaga sprouts belong to the Brassicaceae family and may exert a negative influence on thyroid function, because they are a rich in glucosinolates. These sprouts are also valuable source of iodine (6.5 ± 0.6 μg/100 g of fresh weight). Sprouts were tested in a long-term experiment with young male rats as an element of their diet, combined with two models of hypothyroidism, the first – deficit of iodine and the second – sulfadimethoxine ingestion as a pharmacological agent caused inhibition of thyroid peroxidase. Evaluations were performed for the serum TSH and thyroid hormones together with analyzes of thyroid histopathology, cytosolic glutathione peroxidase (GPX1), thioredoxin reductase in the thyroid, plasma GPX3 and CAT, erythrocyte GPX1. Rutabaga sprouts’ intake by healthy rats did not cause any harmful effect on their health, including thyroid function. For animals with hypothyroidism, rutabaga sprouts enhanced the adverse effect of iodine deficiency or ingestion of sulfadimethoxine on the organism. According to the results obtained for young male rats thyroid function, the interpretation of data for human exposure to rutabaga sprouts has to be avoided. Furthermore, unless new scientific data confirms a lack of the negative effect of brassica sprouts on thyroid function in human, they should not be excluded from the group of goitrogenic products.

Testing the correlation between experimentally-induced hypothyroidism during pregnancy and autistic-like symptoms in the rat offspring

Thyroid hormones are important for the development of the central nervous system. Since the fetal thyroid gland is not functioning until mid-gestation, transport of maternal thyroid hormones across the placenta is essential during the early phases of gestation. Maternal thyroid deficiency has been associated with a higher incidence of neurodevelopmental disorders in the newborns. The relationship between maternal hypothyroidism and the onset of autism spectrum disorders (ASD) in the offspring, however, is still debated. To address this issue, we used a validated animal model of prenatal hypothyroidism based on the administration of the thyroid peroxidase inhibitor methimazole (MMI, 0.02g/100ml in tap water) to rat dams from gestational day 9 up to delivery. The offspring was tested in behavioral tasks during infancy (PNDs 5, 9, 13) and adolescence (PND 35-40) to capture some of the core and associated symptoms of ASD. MMI-exposed pups were able to vocalize as controls when separated from the nest, and showed intact social discrimination abilities in the homing behavior test. At adolescence, the offspring from both sexes did not show an anxious-phenotype in the elevated plus maze and showed intact object recognition. However, MMI-exposed male rats showed increased novelty-directed exploratory behaviors: they solicited their partner to play more and showed more interest for novel rather than familiar objects compared to control rats. Our results show that prenatal MMI-induced hypothyroidism does not cause in the rat offspring behaviors that resemble core and associated ASD symptoms, like deficits in communication and social interaction and anxiety.

Assessment of petroleum streams for thyroid toxicity.

The thyroid gland, and its associated endocrine hormones, is a growing area of interest in regulatory toxicology due to its important role in metabolism, growth and development. This report presents a review of the toxicology data on chemically complex petroleum streams for thyroid hormone effects. Toxicological summaries and studies from all available published and un-published sources were considered, drawing upon the European REACH regulatory submissions for 19 petroleum streams, with in depth review of 11 individual study reports and 31 published papers on related products or environmental settings. Findings relevant to thyroid pathology or thyroid hormone homeostasis were specifically sought, summarized, and discussed. A total of 349 studies of 28-days or longer duration were considered in the review, including data on mice, rats, rabbits, dogs, humans, and fish. The thyroid was almost invariably not a target organ in these studies. Three rodent studies did find thyroid effects; one on a jet fuel product (JP-8), and two studies on a heavy fuel oil product (F-179). The JP-8 product differs from other fuels due to the presence of additives, and the finding of reduced T4 levels in mice in the study occurred at a dose that is above that expected to occur in environmental settings (e.g. 2000mg/kg). The finding for F-179 involved thyroid inflammation at 10-55mg/kg that co-occurred with liver pathology in rats, indicating a possible secondary effect with questionable relevance to humans. In the few cases where findings did occur, the polycyclic aromatic hydrocarbon (PAH) content was higher than in related substances, and, in support of one possible adverse outcome pathway, one in-vitro study reported reduced thyroid peroxidase (TPO) activity with exposure to some PAH compounds (pyrene, benzo(k)fluoranthene, and benzo(e)pyrene). However, it could not be determined from the data available for this review, whether these specific PAH compounds were substantially higher in the JP-8 or F-179 products than in studies in which thyroid effects were not observed. Thus, a few products may carry a weak potential to affect the thyroid at high doses in rodents, possibly through secondary effects on the rodent liver or possibly through a pathway involving the inhibition of TPO by specific members of the PAH family. Human epidemiology evidence found weak and inconsistent effects on the thyroid but without identification of specific chemicals involved. Two studies in petroleum workers, which found a lower rate of morbidity and mortality overall, reported a statistically significant increase in thyroid cancer, but the small number of cases could not exclude confounding variables as possible explanations for the statistical findings. Overall, the available data indicates a low potential for thyroid hormone effects from exposure to petroleum streams, especially when the aromatic content is low. Because regulatory studies for most chemicals do not include detailed thyroid function or receptor studies, it remains possible that subclinical effects on this system may exist that were not detectable using conventional pathology or hormone measurements.

Estimating Margin of Exposure to Thyroid Peroxidase Inhibitors Using High-Throughput in vitro Data, High-Throughput Exposure Modeling, and Physiologically Based Pharmacokinetic/Pharmacodynamic Modeling.

Some pharmaceuticals and environmental chemicals bind the thyroid peroxidase (TPO) enzyme and disrupt thyroid hormone production. The potential for TPO inhibition is a function of both the binding affinity and concentration of the chemical within the thyroid gland. The former can be determined through in vitro assays, and the latter is influenced by pharmacokinetic properties, along with environmental exposure levels. In this study, a physiologically based pharmacokinetic (PBPK) model was integrated with a pharmacodynamic (PD) model to establish internal doses capable of inhibiting TPO in relation to external exposure levels predicted through exposure modeling. The PBPK/PD model was evaluated using published serum or thyroid gland chemical concentrations or circulating thyroxine (T4) and triiodothyronine (T3) hormone levels measured in rats and humans. After evaluation, the model was used to estimate human equivalent intake doses resulting in reduction of T4 and T3 levels by 10% (ED10) for 6 chemicals of varying TPO-inhibiting potencies. These chemicals were methimazole, 6-propylthiouracil, resorcinol, benzophenone-2, 2-mercaptobenzothiazole, and triclosan. Margin of exposure values were estimated for these chemicals using the ED10 and predicted population exposure levels for females of child-bearing age. The modeling approach presented here revealed that examining hazard or exposure alone when prioritizing chemicals for risk assessment may be insufficient, and that consideration of pharmacokinetic properties is warranted. This approach also provides a mechanism for integrating in vitro data, pharmacokinetic properties, and exposure levels predicted through high-throughput means when interpreting adverse outcome pathways based on biological responses.