Natural Autoimmunity to Selenoprotein P Impairs Selenium Transport in Hashimoto's Thyroiditis.

The essential trace element selenium (Se) is needed for the biosynthesis of selenocysteine-containing selenoproteins, including the secreted enzyme glutathione peroxidase 3 (GPX3) and the Se-transporter selenoprotein P (SELENOP). Both are found in blood and thyroid colloid, where they serve protective functions. Serum SELENOP derives mainly from hepatocytes, whereas the kidney contributes most serum GPX3. Studies using transgenic mice indicated that renal GPX3 biosynthesis depends on Se supply by hepatic SELENOP, which is produced in protein variants with varying Se contents. Low Se status is an established risk factor for autoimmune thyroid disease, and thyroid autoimmunity generates novel autoantigens. We hypothesized that natural autoantibodies to SELENOP are prevalent in thyroid patients, impair Se transport, and negatively affect GPX3 biosynthesis. Using a newly established quantitative immunoassay, SELENOP autoantibodies were particularly prevalent in Hashimoto's thyroiditis as compared with healthy control subjects (6.6% versus 0.3%). Serum samples rich in SELENOP autoantibodies displayed relatively high total Se and SELENOP concentrations in comparison with autoantibody-negative samples ([Se]; 85.3 vs. 77.1 µg/L, p = 0.0178, and [SELENOP]; 5.1 vs. 3.5 mg/L, p = 0.001), while GPX3 activity was low and correlated inversely to SELENOP autoantibody concentrations. In renal cells in culture, antibodies to SELENOP inhibited Se uptake. Our results indicate an impairment of SELENOP-dependent Se transport by natural SELENOP autoantibodies, suggesting that the characterization of health risk from Se deficiency may need to include autoimmunity to SELENOP as additional biomarker of Se status.

Removing Critical Gaps in Chemical Test Methods by Developing New Assays for the Identification of Thyroid Hormone System-Disrupting Chemicals-The ATHENA Project.

The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood-brain and blood-placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation.

A combined LC-MS/MS and LC-MS3 multi-method for the quantification of iodothyronines in human blood serum.

We report here a novel approach for the extraction and analysis of thyroid hormones (TH) and their metabolites (THM) from human serum samples. Our method features a compact, 96-well micro-titre plate-based pre-analytic extraction/clean-up workflow combined with an isotope dilution LC-MS/MS-MS3 analytical method. In particular, these features make possible the detection of iodothyronines at their endogenous concentrations in serum differing by a factor of ca. 104, with potential to semi-automate the pre-analytics. The method was validated by the assessment of linearity, lower limits of quantification and detection (LLOQ and LLOD respectively), intra- and inter-day accuracy, precision, process efficiency (PE), matrix effect (ME) and relative recovery (RE). Calibration curves were linear in the concentration range in sample matrix from 0.1-250 nM for T3, rT3, T4 and 3-T1AM and from 0.005-1 nM for 3,5-T2 and 3,3'-T2. Using a 200-µL sample volume, the analyte dependant LLOQ were in the range 0.005 (3,5-T2) to 0.25 (T4) nM and LLOD were between 0.002 (3,5-T2) and 0.052 nM (T4). We applied the LC-MS/MS-MS3 method to the analysis of a cross section of patients with disorders of the thyroid hormone axis. T4, T3 and rT3 concentrations (± standard deviation) were 120 ± 18, 1.9 ± 0.4 and 0.45 ± 0.09 nM respectively. 3,3'-T2 concentrations (± standard deviation) were 0.079 ± 0.022 nM; 3,5-T2 concentrations were below the LLOQ and/or LLOD in all but a single sample (0.013 nM). This method expands the analytical spectrum to endogenous thyroid hormone metabolites such as 3,5-T2 which exert biological actions and rT3 which may act as surrogate markers for disturbed thyroid hormone metabolism. Graphical abstract.

Report from the BfR expert hearing on practicability of hormonal measurements: recommendations for experimental design of toxicological studies with integrated hormonal end points.

This publication summarizes discussions that were held during an international expert hearing organized by the German Federal Institute for Risk Assessment (BfR) in Berlin, Germany, in October 2017. The expert hearing was dedicated to providing practical guidance for the measurement of circulating hormones in regulatory toxicology studies. Adequate measurements of circulating hormones have become more important given the regulatory requirement to assess the potential for endocrine disrupting properties for all substances covered by the plant protection products and biocidal products regulations in the European Union (EU). The main focus was the hypothalamus-pituitary-thyroid axis (HPT) and the hypothalamus-pituitary-gonadal axis (HPG). Insulin, insulin-like growth factor 1 (IGF-1), parathyroid hormone (PTH) and vitamins A and D were also discussed. During the hearing, the experts agreed on specific recommendations for design, conduct and evaluation of acceptability of studies measuring thyroid hormones, thyroid stimulating hormone and reproductive hormones as well as provided some recommendations for insulin and IGF-1. Experts concluded that hormonal measurements as part of the test guidelines (TGs) of the Organisation for Economic Co-operation and Development (OECD) were necessary on the condition that quality criteria to guarantee reliability and reproducibility of measurements are adhered to. Inclusion of the female reproductive hormones in OECD TGs was not recommended unless the design of the study was modified to appropriately measure hormone concentrations. The current report aims at promoting standardization of the experimental designs of hormonal assays to allow their integration in OECD TGs and highlights research needs for better identification of endocrine disruptors using hormone measurements.

Selenium status in neonates with connatal infection.

Infectious diseases impair Se metabolism, and low Se status is associated with mortality risk in adults with critical disease. The Se status of neonates is poorly characterised, and a potential impact of connatal infection is unknown. We hypothesised that an infection negatively affects the Se status of neonates. We conducted an observational case-control study at three intensive care units at the Charité-Universitätsmedizin Berlin, Germany. Plasma samples were collected from forty-four neonates. On the basis of clinical signs for bacterial infection and concentrations of IL-6 or C-reactive protein, neonates were classified into control (n 23) and infected (n 21) groups. Plasma Se and selenoprotein P (SePP) concentrations were determined by X-ray fluorescence and ELISA, respectively, at day of birth (day 1) and 48 h later (day 3). Se and SePP showed a positive correlation in both groups of neonates. Se concentrations indicative of Se deficit in adults (500 ng/l). During antibiotic therapy, SePP increased significantly from day 1 (1·03 (sd 0·10) mg/l) to day 3 (1·34 (sd 0·10) mg/l), indicative of improved hepatic Se metabolism. We conclude that both Se and SePP are suitable biomarkers for assessing Se status in neonates and for identifying subjects at risk of deficiency.

Lipopolysaccharide responsiveness is an independent predictor of death in patients with chronic heart failure.

BACKGROUND: The origin of pro-inflammatory activation in chronic heart failure (HF) remains a matter of debate. Lipopolysaccharide (LPS) may enter the blood stream through the morphologically altered and leaky gut barrier. We hypothesized that lower LPS reactivity would be associated with worse survival as compared to normal or higher LPS reactivity. METHODS: LPS responsiveness was studied in 122 patients with chronic HF (mean±SD: age 67.3±10.3 years, 24 female, New York Heart Association class [NYHA] class: 2.5±0.8, left ventricular ejection fraction [LVEF]: 33.5±12.5%) and 27 control subjects of similar age (63.7±7.7 years, p>0.05). Reference LPS was added at increasing doses to ex vivo whole blood samples and necrosis factor-α (TNFα) was measured. Patients were subgrouped into good- and poor-responder status according to their potential to react to increasing doses of LPS (delta TNFα secretion). The optimal cut-off value was calculated by receiver-operator characteristic curve (ROC) analysis. RESULTS: A total of 56 patients with chronic HF died from any cause during follow-up. At 24 months, cumulative mortality was 16.4% (95% confidence interval 16.0-16.7%). The delta TNFα value representing the optimal cut-off for the prediction of mortality was 1522 pg/mL (24 months) with a sensitivity of 49.3% (95% confidence interval 37.2-61.4%) and specificity of 81.5% (95% confidence interval 61.9-93.6%). LPS responder status remained an independent predictor of death after multivariable adjustment (hazard ratio 0.09 for good- vs. poor-responders, 95% confidence interval 0.01-0.67, p<0.05). CONCLUSIONS: LPS responsiveness in patients with chronic HF is an independent predictor of death.

Low thyroxine serves as an upstream regulator of ecophysiological adaptations in Ansell's mole-rats.

Introduction: About 10% of all rodent species have evolved a subterranean way of life, although life in subterranean burrows is associated with harsh environmental conditions that would be lethal to most animals living above ground. Two key adaptations for survival in subterranean habitats are low resting metabolic rate (RMR) and core body temperature (Tb). However, the upstream regulation of these traits was unknown thus far. Previously, we have reported exceptionally low concentrations of the thyroid hormone (TH) thyroxine (T4), and peculiarities in TH regulating mechanisms in two African mole-rat species, the naked mole-rat and the Ansell's mole-rat. Methods: In the present study, we treated Ansell's mole-rats with T4 for four weeks and analyzed treatment effects on the tissue and whole organism level with focus on metabolism and thermoregulation. Results: We found RMR to be upregulated by T4 treatment but not to the extent that was expected based on serum T4 concentrations. Our data point towards an extraordinary capability of Ansell's mole-rats to effectively downregulate TH signaling at tissue level despite very high serum TH concentrations, which most likely explains the observed effects on RMR. On the other hand, body weight was decreased in T4-treated animals and Tb was upregulated by T4 treatment. Moreover, we found indications of the hypothalamus-pituitary-adrenal axis potentially influencing the treatment effects. Conclusion: Taken together, we provide the first experimental evidence that the low serum T4 concentrations of Ansell's mole-rats serve as an upstream regulator of low RMR and Tb. Thus, our study contributes to a better understanding of the ecophysiological evolution of the subterranean lifestyle in African mole-rats.

Screening for endocrine disrupting chemicals inhibiting monocarboxylate 8 (MCT8) transporter facilitated thyroid hormone transport using a modified nonradioactive assay.

Early neurodevelopmental processes are strictly dependent on spatial and temporally modulated of thyroid hormone (TH) availability and action. Thyroid hormone transmembrane transporters (THTMT) are critical for regulating the local concentrations of TH, namely thyroxine (T4) and 3,5,3'-tri-iodothyronine (T3), in the brain. Monocarboxylate transporter 8 (MCT8) is one of the most prominent THTMT. Genetically induced deficiencies in expression, function or localization of MCT8 are associated with irreversible and severe neurodevelopmental adversities. Due to the importance of MCT8 in brain development, studies addressing chemical interferences of MCT8 facilitated T3 uptake are a crucial step to identify TH system disrupting chemicals with this specific mode of action. Recently a non-radioactive in vitro assay has been developed to rapidly screen for endocrine disrupting chemicals (EDCs) acting upon MCT8 mediated transport. This study explored the use of an UV-light digestion step as an alternative for the original ammonium persulfate (APS) digestion step. The non-radioactive TH uptake assay, with the incorporated UV-light digestion step of TH, was then used to screen a set of 31 reference chemicals and environmentally relevant substances to detect inhibition of MCT8-depending T3 uptake. This alternative assay identified three novel MCT8 inhibitors: methylmercury, bisphenol-AF and bisphenol-Z and confirmed previously known MCT8 inhibitors.

Identification of Human TRIAC Transmembrane Transporters.

Background: 3,5,3'-Triiodothyroacetic acid (TRIAC) is a T3-receptor agonist pharmacologically used in patients to mitigate T3 resistance. It is additionally explored to treat some symptoms of patients with inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8, SLC16A2). MCT8 is expressed along the blood-brain barrier, on neurons, astrocytes, and oligodendrocytes. Hence, pathogenic variants in MCT8 limit the access of TH into and their functions within the brain. TRIAC was shown to enter the brain independently of MCT8 and to modulate expression of TH-dependent genes. The aim of the study was to identify transporters that facilitate TRIAC uptake into cells. Methods: We performed a whole-genome RNAi screen in HepG2 cells stably expressing a T3-receptor-dependent luciferase reporter gene. Validation of hits from the primary and confirmatory secondary screen involved a counter screen with siRNAs and compared the cellular response to TRIAC to the effect of T3, in order to exclude siRNAs targeting the gene expression machinery. MDCK1 cells were stably transfected with cDNA encoding C-terminally myc-tagged versions of the identified TRIAC-preferring transporters. Several individual clones were selected after immunocytochemical characterization for biochemical characterization of their 125I-TRIAC transport activities. Results: We identified SLC22A9 and SLC29A2 as transporters mediating cellular uptake of TRIAC. SLC22A9 encodes the organic anion transporter 7 (OAT7), a sodium-independent organic anion transporter expressed in the plasma membrane in brain, pituitary, liver, and other organs. Competition with the SLC22A9/OAT7 substrate estrone-3-sulfate reduced 125I-TRIAC uptake. SLC29A2 encodes the equilibrative nucleoside transporter 2 (ENT2), which is ubiquitously expressed, including pituitary and brain. Coincubation with the SLC29A2/ENT2 inhibitor nitrobenzyl-6-thioinosine reduced 125I-TRIAC uptake. Moreover, ABCD1, an ATP-dependent peroxisomal pump, was identified as a 125I-TRIAC exporter in transfected MDCK1 cells. Conclusions: Knowledge of TRIAC transporter expression patterns, also during brain development, may thus in the future help to interpret observations on TRIAC effects, as well as understand why TRIAC may not show a desirable effect on cells or organs not expressing appropriate transporters. The identification of ABCD1 highlights the sensitivity of our established screening assay, but it may not hold significant relevance for patients undergoing TRIAC treatment.

In vitro and in vivo investigation of a thyroid hormone system-specific interaction with triazoles.

Alterations in thyroid hormones (TH) and thyroid-stimulating hormone levels are frequently found following exposure to chemicals of concern. Dysregulation of TH levels can severely perturb physiological growth, metabolism, differentiation, homeostasis in the adult and developmental processes in utero. A frequently identified mode of action for this interaction is the induction of hepatic detoxification mechanisms (e.g. SULTs and UGTs), which lead to TH conjugation and elimination and therefore interfere with hormonal homeostasis, fulfilling the endocrine disruptors (EDs) definition. A short-term study in rats with dietary exposure to cyproconazole, epoxiconazole and prochloraz was conducted and hepatocyte hypertrophy, hepatic UGT activity and Phase 1/2 gene expression inductions were observed together with changes in TH levels and thyroid follicular hypertrophy and hyperplasia. To test for specific interaction with the thyroid hormone system, in vitro assays were conducted covering thyroidal I-uptake (NIS), TH transmembranal transport via MCT8 and thyroid peroxidase (TPO) function. Assays for iodothyronine deiodinases (DIO1-DIO3) and iodotyrosine deiodinase (DEHAL1) were included, and from the animal experiment, Dio1 and Dehal1 activities were measured in kidney and liver as relevant local indicators and endpoints. The fungicides did not affect any TH-specific KEs, in vitro and in vivo, thereby suggesting hepatic conjugation as the dominant MoA.

ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection.

AIMS: Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15 kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown. METHODS AND RESULTS: Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modelling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced up-regulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modelling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system. CONCLUSION: Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction.

High throughput drug screening identifies resveratrol as suppressor of hepatic SELENOP expression.

INTRODUCTION: Selenium (Se) is an essential trace element that exerts its effects mainly as the proteinogenic amino acid selenocysteine within a small set of selenoproteins. Among all family members, selenoprotein P (SELENOP) constitutes a particularly interesting protein as it serves as a biomarker and serum Se transporter from liver to privileged tissues. SELENOP expression is tightly regulated by dietary Se intake, inflammation, hypoxia and certain substances, but a systematic drug screening has hitherto not been performed. METHODS: A compound library of 1861 FDA approved clinically relevant drugs was systematically screened for interfering effects on SELENOP expression in HepG2 cells using a validated ELISA method. Dilution experiments were conducted to characterize dose-responses. A most potent SELENOP inhibitor was further characterized by RNA-seq analysis to assess effect-associated biochemical pathways. RESULTS: Applying a 2-fold change threshold, 236 modulators of SELENOP expression were identified. All initial hits were replicated as biological triplicates and analyzed for effects on cell viability. A set of 38 drugs suppressed SELENOP expression more than three-fold, among which were cancer drugs, immunosuppressants, anti-infectious drugs, nutritional supplements and others. Considering a 90% cell viability threshold, resveratrol, vidofludimus, and antimony potassium-tartrate were the most potent substances with suppressive effects on extracellular SELENOP concentrations. Resveratrol suppressed SELENOP levels dose-dependently in a concentration range from 0.8 µM to 50.0 µM, without affecting cell viability, along with strong effects on key genes controlling metabolic pathways and vesicle trafficking. CONCLUSION: The results highlight an unexpected direct effect of the plant stilbenoid resveratrol, known for its antioxidative and health-promoting effects, on the central Se transport protein. The suppressive effects on SELENOP may increase liver Se levels and intracellular selenoprotein expression, thereby conferring additional protection to hepatocytes at the expense of systemic Se transport. Further physiological effects from this interaction require analyses in vivo and by clinical studies.

Comparative analysis of thyroid hormone systems in rodents with subterranean lifestyle.

African mole-rats are subterranean rodents inhabiting underground burrows. This habitat entails risks of overheating, hypoxia, and scarce food availability. Consequently, many subterranean species have evolved low basal metabolism and low body temperature, but the regulation of these traits at the molecular level were unknown. Measurements of serum thyroid hormone (TH) concentrations in African mole-rats have revealed a unique TH phenotype, which deviates from the typical mammalian pattern. Since THs are major regulators of metabolic rate and body temperature, we further characterised the TH system of two African mole-rat species, the naked mole-rat (Heterocephalus glaber) and the Ansell's mole-rat (Fukomys anselli) at the molecular level in a comparative approach involving the house mouse (Mus musculus) as a well-studied laboratory model in TH research. Most intriguingly, both mole-rat species had low iodide levels in the thyroid and naked mole-rats showed signs of thyroid gland hyperplasia. However, contrary to expectations, we found several species-specific differences in the TH systems of both mole-rat species, although ultimately resulting in similar serum TH concentrations. These findings indicate a possible convergent adaptation. Thus, our study adds to our knowledge for understanding adaptations to the subterranean habitat.

A microphysiological system for studying human bone biology under simultaneous control of oxygen tension and mechanical loading.

Throughout life, continuous remodelling is part of human bone biology and depends on the simultaneous action of physicochemical parameters such as oxygen tension and varying mechanical load. Thus, suitable model systems are needed, which allow concomitant modulation of these factors to recapitulate in vivo bone formation. Here, we report on the development of a first microphysiological system (MPS) that enables perfusion, environment-independent regulation of the oxygen tension as well as precise quantification and control of mechanical load. To demonstrate the use of the MPS for future studies on the (patho-)biology of bone, we built a simplified 3D model for early de novo bone formation. Primary human osteoblasts (OBs), which are the key players during this process, were seeded onto type I collagen scaffolds and cultured in the MPS. We could not only monitor cell viability and metabolism of OBs under varied physicochemical conditions, but also visualise the mineralisation of the extracellular matrix. In summary, we present a MPS that uniquely combines the independent control of physicochemical parameters and allows investigation of their influence on bone biology. We consider our MPS highly valuable to gain deeper insights into (patho-)physiological processes of bone formation in the future.

New approach methods to improve human health risk assessment of thyroid hormone system disruption-a PARC project.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available-and are currently in the process of regulatory validation-there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

Tentative Application of a Streamlined Protocol to Determine Organ-Specific Regulations of Deiodinase 1 and Dehalogenase Activities as Readouts of the Hypothalamus-Pituitary-Thyroid-Periphery-Axis.

In animal studies, both in basic science and in toxicological assessment of potential endocrine disruptors, the state of the thyroid hormone (TH) axis is often described and defined exclusively by the concentrations of circulating THs and TSH. Although it is known that the local, organ-specific effects of THs are also substantially regulated by local mechanisms such as TH transmembrane transport and metabolism of TH by deiodinases, such endpoint parameters of the axis are rarely assessed in these experiments. Currently developed in vitro assays utilize the Sandell-Kolthoff reaction, a photometric method of iodide determination, to test the effect of chemicals on iodotyrosine and iodothyronine deiodinases. Furthermore, this technology offers the possibility to determine the iodine content of various sample types (e.g., urine, ex vivo tissue) in a simple way. Here, we measured deiodinase type 1 and iodotyrosine dehalogenase activity by means of the Sandell-Kolthoff reaction in ex vivo samples of hypo- and hyperthyroid mice of two age groups (young; 3 months and old; 20 months). In thyroid, liver and kidney, organ-specific regulation patterns emerged across both age groups, which, based on this pilot study, may serve as a starting point for a deeper characterization of the TH system in relevant studies in the future and support the development of Integrated Approach for Testing and Assessment (IATA).

Design and Characterization of a Fluorescent Reporter Enabling Live-cell Monitoring of MCT8 Expression.

The monocarboxylate transporter 8 (MCT8) is a specific thyroid hormone transporter and plays an essential role in fetal development. Inactivating mutations in the MCT8 encoding gene SLC16A2 (solute carrier family 16, member 2) lead to the Allan-Herndon-Dudley syndrome, a condition presenting with severe endocrinological and neurological phenotypes. However, the cellular distribution pattern and dynamic expression profile are still not well known for early human neural development. OBJECTIVE: Development and characterization of fluorescent MCT8 reporters that would permit live-cell monitoring of MCT8 protein expression in vitro in human induced pluripotent stem cell (hiPSC)-derived cell culture models. METHODS: A tetracysteine (TC) motif was introduced into the human MCT8 sequence at four different positions as binding sites for fluorescent biarsenical dyes. Human Embryonic Kidney 293 cells were transfected and stained with fluorescein-arsenical hairpin-binder (FlAsH). Counterstaining with specific MCT8 antibody was performed. Triiodothyronine (T3) uptake was indirectly measured with a T3 responsive luciferase-based reporter gene assay in Madin-Darby Canine Kidney 1 cells for functional characterization. RESULTS: FlAsH staining and antibody counterstaining of all four constructs showed cell membrane expression of all MCT8 constructs. The construct with the tag after the first start codon demonstrated comparable T3 uptake to the MCT8 wildtype. CONCLUSION: Our data indicate that introduction of a TC-tag directly after the first start codon generates a MCT8 reporter with suitable characteristics for live-cell monitoring of MCT8 expression. One promising future application will be generation of stable hiPSC MCT8 reporter lines to characterize MCT8 expression patterns during in vitro neuronal development.

Impact of metabolic stress induced by diets, aging and fasting on tissue oxygen consumption.

OBJECTIVE: Alterations in mitochondrial function play an important role in the development of various diseases, such as obesity, insulin resistance, steatohepatitis, atherosclerosis and cancer. However, accurate assessment of mitochondrial respiration ex vivo is limited and remains highly challenging. Using our novel method, we measured mitochondrial oxygen consumption (OCR) and extracellular acidification rate (ECAR) of metabolically relevant tissues ex vivo to investigate the impact of different metabolic stressors on mitochondrial function. METHODS: Comparative analyses of OCR and ECAR were performed in tissue biopsies of young mice fed 12 weeks standard-control (STD), high-fat (HFD), high-sucrose (HSD), or western diet (WD), matured mice with HFD, and 2year-old mice aged on STD with and without fasting. RESULTS: While diets had only marginal effects on mitochondrial respiration, respiratory chain complexes II and IV were reduced in adipose tissue (AT). Moreover, matured HFD-fed mice showed a decreased hepatic metabolic flexibility and prolonged aging increased OCR in brown AT. Interestingly, fasting boosted pancreatic and hepatic OCR while decreasing weight of those organs. Furthermore, ECAR measurements in AT could indicate its lipolytic capacity. CONCLUSION: Using ex vivo tissue measurements, we could extensively analyze mitochondrial function of liver, AT, pancreas and heart revealing effects of metabolic stress, especially aging.

Mixture Risk Assessment of Complex Real-Life Mixtures-The PANORAMIX Project.

Humans are involuntarily exposed to hundreds of chemicals that either contaminate our environment and food or are added intentionally to our daily products. These complex mixtures of chemicals may pose a risk to human health. One of the goals of the European Union's Green Deal and zero-pollution ambition for a toxic-free environment is to tackle the existent gaps in chemical mixture risk assessment by providing scientific grounds that support the implementation of adequate regulatory measures within the EU. We suggest dealing with this challenge by: (1) characterising 'real-life' chemical mixtures and determining to what extent they are transferred from the environment to humans via food and water, and from the mother to the foetus; (2) establishing a high-throughput whole-mixture-based in vitro strategy for screening of real-life complex mixtures of organic chemicals extracted from humans using integrated chemical profiling (suspect screening) together with effect-directed analysis; (3) evaluating which human blood levels of chemical mixtures might be of concern for children's development; and (4) developing a web-based, ready-to-use interface that integrates hazard and exposure data to enable component-based mixture risk estimation. These concepts form the basis of the Green Deal project PANORAMIX, whose ultimate goal is to progress mixture risk assessment of chemicals.

Selenium controls the sex-specific immune response and selenoprotein expression during the acute-phase response in mice.

Selenium modifies inflammatory reactions in rodents and humans. The liver controls metabolism and transport of selenium via hepatically-derived SEPP (selenoprotein P). Intracellular SEPS (selenoprotein S) modifies endoplasmic-reticulum function and immune-cell activity. Polymorphisms in SEPS have been associated with cytokine levels and inflammatory diseases in a subset of clinical studies. In the present study, we hypothesized that sex and selenium represent decisive parameters controlling the immune response and regulation of SEPS expression in vivo. Male and female mice fed a selenium-poor diet were supplemented or not with selenite for 3 days and injected with saline or LPS (lipopolysaccharide) 24 h before analysis. Selenium supplementation mitigated the LPS-induced rise in circulating cytokines in male mice. Serum SepP and selenium concentrations decreased in response to LPS, whereas hepatic SepS was specifically up-regulated despite declining selenium concentrations in the liver. Hepatic SepS induction was mainly controlled by post-transcriptional mechanisms and attributed to hepatocytes by analysing transgenic mice. Notably, selenium supplementation was essential for an optimal SepS induction. We conclude that selenoprotein biosynthesis becomes redirected in hepatocytes during the acute-phase response at the expense of dispensable selenoproteins (e.g. SepP) and in favour of SepS expression, thereby causing declining serum selenium and improving liver function. The selenium status and sex control SepS expression and modify cytokine response patterns in serum, which might explain contradictory results on associations of SEPS genotype and inflammatory diseases in clinical studies.