Elsevier

Toxicology in Vitro

Volume 27, Issue 4, June 2013, Pages 1320-1346
Toxicology in Vitro

Review
Mechanism-based testing strategy using in vitro approaches for identification of thyroid hormone disrupting chemicals

https://doi.org/10.1016/j.tiv.2013.02.012Get rights and content

Abstract

The thyroid hormone (TH) system is involved in several important physiological processes, including regulation of energy metabolism, growth and differentiation, development and maintenance of brain function, thermo-regulation, osmo-regulation, and axis of regulation of other endocrine systems, sexual behaviour and fertility and cardiovascular function. Therefore, concern about TH disruption (THD) has resulted in strategies being developed to identify THD chemicals (THDCs). Information on potential of chemicals causing THD is typically derived from animal studies. For the majority of chemicals, however, this information is either limited or unavailable. It is also unlikely that animal experiments will be performed for all THD relevant chemicals in the near future for ethical, financial and practical reasons. In addition, typical animal experiments often do not provide information on the mechanism of action of THDC, making it harder to extrapolate results across species. Relevant effects may not be identified in animal studies when the effects are delayed, life stage specific, not assessed by the experimental paradigm (e.g., behaviour) or only occur when an organism has to adapt to environmental factors by modulating TH levels. Therefore, in vitro and in silico alternatives to identify THDC and quantify their potency are needed. THDC have many potential mechanisms of action, including altered hormone production, transport, metabolism, receptor activation and disruption of several feed-back mechanisms. In vitro assays are available for many of these endpoints, and the application of modern ‘-omics’ technologies, applicable for in vivo studies can help to reveal relevant and possibly new endpoints for inclusion in a targeted THDC in vitro test battery. Within the framework of the ASAT initiative (Assuring Safety without Animal Testing), an international group consisting of experts in the areas of thyroid endocrinology, toxicology of endocrine disruption, neurotoxicology, high-throughput screening, computational biology, and regulatory affairs has reviewed the state of science for (1) known mechanisms for THD plus examples of THDC; (2) in vitro THD tests currently available or under development related to these mechanisms; and (3) in silico methods for estimating the blood levels of THDC. Based on this scientific review, the panel has recommended a battery of test methods to be able to classify chemicals as of less or high concern for further hazard and risk assessment for THD. In addition, research gaps and needs are identified to be able to optimize and validate the targeted THD in vitro test battery for a mechanism-based strategy for a decision to opt out or to proceed with further testing for THD.

Introduction

Endocrine disruption (ED) by chemicals is not restricted to the sex hormone system, but also includes thyroid hormone (TH) disruption (THD). THD is defined herein as a change in hormone production, transport, function or metabolism resulting in impaired homeostasis. When the homeostasis is not impaired it is called a hormone modulator. THD can be induced by a variety of causes including diet, disease, and exposure to environmental chemicals.

TH are involved in several important physiological processes such as regulation of energy metabolism (Cheng et al., 2010), growth and differentiation, development and maintenance of brain function and the sympathetic nervous system (Bernal, 2007, Horn and Heuer, 2010, Reinehr, 2010, Warner and Mittag, 2012), thermo-regulation (Ribeiro, 2008), osmo-regulation and renal function (Vargas et al., 2006), regulation of onset and proper function of other endocrine systems including the estrogen system, sexual behaviour and fertility, and cardiovascular functioning (Danzi and Klein, 2012, Krassas et al., 2010, Wagner et al., 2008). Whether during development of the organism, differentiation of cells and tissues, maintenance or alteration of physiological functions of adult individuals, in many cases TH effects can best be characterized as ‘permissive hormone action’. This indicates that the TH status of cells, tissues, and organisms provides the background and platform for other biological signals – hormonal, neural, immunological, nutritive and environmental – that are critical for maintenance of both development and homeostasis of the organism as a whole (Lopez-Juarez et al., 2012, Pascual and Aranda, 2012, Sirakov et al., 2012).

The predominant TH in the circulation in the euthyroid situation is 3,3′,5,5′-tetraiodothyronine (thyroxine, T4), which is the precursor for the most active form of TH (3,3′,5-triiodothyronine; T3). Most of the known functions of TH are mediated by the interaction of T3 with the nuclear T3-receptors (TRs), which act as ligand-modulated transcription factors. While almost none of the genes regulated by T3 are exclusively responsive to T3, virtually all molecular, cellular and metabolic events are more or less sensitive to TH (Grimaldi et al., 2012, König and Moura Neto, 2002, Oetting and Yen, 2007).

Lessons learned from decades of biomedical studies of iodide deficiency, congenital hypothyroidism, genetic diseases related to defective TH function as well as data from various animal models corroborate evidence for the hypothesis that transient or persistent THD could alter maintenance of homeostasis within the hypothalamus–pituitary–thyroid-periphery (HPTP) axis and modulate the peripheral thyroid hormone dependent functions. This might lead to temporary loss of homeostasis or even alter set points leading to long-term TH dysregulation and physiological consequences, including thyroid pathology and altered metabolism and perinatal development. Alterations of the TH system are associated with several serious human diseases (Table 1).

TH are particularly important in perinatal development. They are involved in several critical processes for neurodevelopment: neuronal proliferation, migration, synaptogenesis, synaptic plasticity and myelination processes (Horn and Heuer, 2010, Howdeshell, 2002). In humans, TH production starts at approximately 11 gestational weeks and increases with the development of the fetal HPTP axis (Howdeshell, 2002). Trans-placental transfer of maternal TH to the fetus is critical for neurodevelopment (Bernal, 2007, Zoeller and Rovet, 2004), as impaired psychomotor development, behavioural changes and effects on visuo-spatial processing have been observed in children born to mothers with (subclinical) hypothyroidism (de Cock et al., 2012, Haddow et al., 1999, Pop et al., 1999). Because alterations in TH balance can lead to altered development, even temporary THD during the perinatal period can have long-term consequences on human health (Zoeller and Rovet, 2004). But also later in life alterations in the TH system have been shown to play a role in several mental illnesses such as Alzheimer’s disease, bipolar disorder and major depressive disorder (Bauer et al., 2008, Carta et al., 2002, Cooper-Kazaz and Lerer, 2008, de Jong et al., 2009, Hogervorst et al., 2008, Lovell et al., 2008, Tan and Vasan, 2009).

THDCs may interact with a number of molecular components of the HPTP axis and the functioning of the peripheral tissues: TH synthesis, TH storage and release by the thyroid gland, feedback mechanisms within the HPT, protein-binding and TH distribution, cellular TH uptake, intracellular TH metabolism, catabolism of TH, classical ‘nuclear’ T3 receptor binding, as well as other target proteins (‘receptors’) in the cell membrane, mitochondria and other subcellular structures (Brix et al., 2011, Brucker-Davis, 1998, Capen, 1994, Cheng et al., 2010, DeVito et al., 1998, Köhrle, 2008, Miller et al., 2009). THDCs may also react with more than one component of the TH system, possibly at different internal concentrations, which may give an overall in vivo effect at blood concentrations that are lower than in vitro studies might suggest. This is expected to be the case with polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) in the blood of mothers and infants that have been associated with changes in thyroid hormone status and developmental endpoints and fertility (Chevrier et al., 2010, Harley et al., 2010, Koopman-Esseboom et al., 1994, Langer, 1998, Langer et al., 1998, Morse et al., 1993).

In addition to the direct impact of chemicals on the TH system, biological and environmental stressors may also increase the sensitivity of a number of physiological processes to THDCs. Iodine deficiency, which is commonly observed worldwide (Andersson et al., 2012, de Benoist et al., 2008, Walker et al., 2007, Zimmermann and Andersson, 2012), is a well-known risk factor for increased sensitivity to adverse effects from thyroid-disrupting chemicals (Blount et al., 2006, Pearce and Braverman, 2009). Such effects are known as well from co-exposure to goitrogens originating from the diet (e.g., (iso-)flavones like genistein in soy, goitrin contained in millet and cassava) in areas of iodine deficiency (Delange, 1994, Doerge and Sheehan, 2002, Köhrle, 2008).

Various environmental contaminants have been shown to disrupt thyroid homeostasis via several mechanisms (Brucker-Davis, 1998, Capen, 1997, Cavalieri and Pitt-Rivers, 1981, Crofton, 2008, Hurley, 1998, Jugan et al., 2010, Köhrle, 2008, Surks and Sievert, 1995, Zoeller, 2007). Disruption of the TH system has been shown as one of the major toxic effects for chemicals ranging from halogenated aromatic chemicals such as PCBs to inorganic anions such as perchlorate and nitrate (Brouwer et al., 1998, Brucker-Davis, 1998, Hallgren and Darnerud, 2002, Wolff, 1998), arsenic (Ciarrocca et al., 2012) or perfluorinated chemicals (Lopez-Espinosa et al., 2012). This results in alteration of important biological processes under control of THs (Lopez-Juarez et al., 2012, Pascual and Aranda, 2012, Sirakov et al., 2012).

Historically, information on the potency of THDCs has been derived from animal studies, mostly using rodents or amphibians (Biegel et al., 1995, Christenson et al., 1996, Hallgren and Darnerud, 2002, Davey et al., 2008, Grimaldi et al., 2012). However, this information is limited to a small number of the 10,000+ chemicals that need assessments of potential risk (EPA, 2012, Judson et al., 2009, NRC, 1984, NRC, 2007, Wagner, 2000). Performing in vivo experiments to assess possible THD for such a large number of chemicals is unlikely to happen in the near future for practical, as well as ethical and financial reasons. In addition, a scientific argument to replace animal experiments is that apical endpoints provide little insight in underlying mechanisms (NIEHS, 2007), may not be predictive for the human situation or may not identify relevant life-stage specific effects, especially since unexpected species differences may exist, and current rodent experiments are not optimized for the identification of THD. In addition, the fact that THDC could react with more than one component of the TH system, possibly at different internal concentrations, could result in confusing non-monotonic in vivo dose–response curves. A more mechanistically driven in vitro approach can help to discriminate between chemicals without indication for THD, with a strong indication for THD and with weak or not conclusive indication for THD.

The relevance of such an approach is illustrated by the fact that the U.S. EPA is considering screening a universe of approximately 10,000 chemicals for endocrine disrupting properties as part of the endocrine disruptor screening program. To facilitate rapid screening of endocrine-related endpoints, further priority setting will be done using high throughput in vitro assays and in silico models (EPA, 2012). In Europe, different regulations contain specific provisions regarding endocrine disruptors: REACH (EU, 2007), plant protection products (EU, 2009a), cosmetics (EU, 2009b) and biocides (EU, 2012). The OECD has developed several test methodologies and testing strategies relating to EDCs but not all of these methods are currently part of the standard information requirements of the above mentioned regulations. Considering the large number of substances that might need to be prioritized for endocrine-related endpoints, both in the US and in the EU, there is a need for an efficient, less-animal intensive screening method to identify potential THDCs.

Over the last two decades there has been a growing interest in defining the mechanisms of toxicity in in vitro systems (Eisenbrand et al., 2002). Furthermore, there has been an explosion of technology that has greatly increased not only the breadth of information that can be extracted from biological samples, but also the throughput by which samples can be analysed and interpreted. Thus, genomics, proteomics and metabolomics are becoming commonplace tools in biological research, as are genetically modified cells, transgenic animal models, model organisms such as zebra fish, and stem cell models. Many of these approaches have been adapted to the process of drug discovery (Houck and Kavlock, 2008), which has facilitated their adoption in toxicological studies including endocrine disruption, based on knowledge of the interaction of chemicals with biological pathways. While there has been much attention to develop pathway-based assays for estrogen and androgen function, approaches to the assessment of thyroid function have lagged behind because of the multiplicity of ways in which TH function can (and has) been altered by environmental chemicals (OECD, 2006). This paper focuses on the implementation of these approaches in testing strategies to the assessment of the effect of chemicals on the thyroid system.

The current review is based on the outcome of a workshop organized within the framework of the ASAT initiative (Assuring Safety without Animal Testing) (Fentem et al., 2004). The general aim of the ASAT initiative is to develop “a radical new approach to assessing the risk posed by exposure to chemicals that would not involve testing of animals, taking advantage of the rapid advances in science and technology.” Experts in thyroid endocrinology, toxicologists with experience with endocrine disruption and neurotoxicology, computational experts, high-throughput screening (HTS) and regulatory experts reviewed the state of science for (1) known mechanisms for THD plus examples of THDC; (2) in vitro THD tests currently available or under development related to these mechanisms; and (3) in silico methods for estimating the blood levels of THDC.

Based on this scientific review, the panel recommends a battery of test methods to be able to classify chemicals as of less or high concern for further hazard and risk assessment for THD. In addition, research gaps and needs are identified in order to optimize and validate the targeted THD in vitro test battery. This validation can lead to a mechanism-based strategy used for deciding on whether to opt out or to proceed with further THD testing.

To this aim, an inventory was taken of (1) the most relevant molecular targets and pathways that underlie thyroid functional disturbances, which should be included in THD testing, (2) presently available or preliminary in vitro and in silico approaches that cover the most relevant mechanisms, and (3) areas in which assay development is needed to fill data gaps in a comprehensive in vitro THD testing strategy.

Section snippets

Thyroid hormone system and endpoints for THD

Current knowledge indicates that the majority of THD effects are mediated via influences on the HPTP axis rather on direct interference with nuclear receptor function in the target tissues. For a conceptual framework, the workshop expanded earlier systems biology models of thyroid disruption (Capen, 1997) using an approach recently developed (Keune et al., 2012, Ravnum et al., 2012, Smita et al., 2012, Zimmer et al., 2012) (Boxes 1–6; Fig. 1). THDC can interfere with the central regulation and

Chemical bio-activation and availability in vitro

In addition to chemicals directly interfering with endpoints of the TH system, other potential THDCs such as PCBs and PBDEs have to undergo metabolic activation to become a THD hazard. This bio-activation into hydroxylated chemicals occurs in vivo by Phase I metabolic enzymes (Marsh et al., 2006, Morse and Brouwer, 1995). These OH-metabolites can show remarkable structural similarities with TH thereby causing direct effects on the TH homeostasis by mimicking T4 and to a lesser extent T3 (

Suggested testing strategy

The goal of this review was to identify a mechanism-based battery of non-animal tests that can be applied for THD hazard assessment. To minimize the risk of false negatives as well as of false positives, the in vitro assays should cover all relevant endpoints of THD to distinguish between chemicals of less concern and chemicals that need further study. Prioritization of chemicals based on a well-chosen in vitro assay battery, possibly extended with in silico approaches, will be much more

Advantages of in vitro testing

Although in vivo bioassays are generally perceived as being the most reliable tests for assessing the hazard of chemicals, including THDC, there also are several limitations for this approach. The functioning of the TH system depends on life-stage, species and health condition. Healthy animals may cope with certain exposures by compensation via HPT feedback and effects may be unnoticed, while people with specific health conditions, such as hypo- or hyperthyroidism or malnutrition, may be more

Further outlook: using computational modelling to interpret in vitro assays for in vivo effects

Although it was not the ambition of this review to present an in vitro approach to be able to predict in vivo effects, this could at a later stage be further elaborated. Despite the many advantages of an in vitro testing battery, estimating in vivo toxicity from in vitro results can be a daunting task, as it is not possible to fully reproduce the in vivo situation in vitro. There are two major uncertainties in extrapolation from in vitro to in vivo. One is the need to determine the ability of

Recommendations for assay use and development

The overall goal of this paper is to summarize the potential thyroid targets of environmental chemicals and in vitro assays that cover the important mechanisms for TH disruption by environmental chemicals. From the above review it is clear that the state of the science for these assays ranges from those already being used in medium-throughput screening (MTS) or HTS, to potential targets that lack cost efficient testing methods. To help focus research and development efforts we provide a list of

Summarizing conclusions

This review presents a ‘best expert judgement’ for a set of in vitro testing assays that together will cover the most important mechanisms for TH disruption by chemicals. For these endpoints, currently available or desired in vitro assays are discussed that are or could be suitable for medium to high throughput testing. Bio-activation of the tested chemicals is a critical consideration in the in vitro testing, as several cases are known when testing of the parent chemical alone results in false

Conflict of interest statement

There are no conflicts of interest issues.

Disclaimer

The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Acknowledgements

This paper is based on the results of a workshop organized by the Institute for Risk Assessment Sciences, Utrecht University, for which financial support was kindly provided by “Proefdiervrij” (the Dutch Society for the Replacement of Animal Testing (dsRAT) www.proefdiervrij.nl/english) and the ASAT Foundation (www.asat-initiative.eu/). We want to thank Maurico Montaño for the nicer version he made of Fig. 1.

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