Inhibition, Deiodinase 1

Disruption of the thyroid hormone system is increasingly being recognized as an important toxicity pathway, as it can cause many adverse outcomes. Thyroid hormones do not only play an important role in the adult individual, but they are also critical during embryonic development. Thyroid hormones (THs) play an important role in a wide range of biological processes in vertebrates including growth, development, reproduction, cardiac function, thermoregulation, response to injury, tissue repair and homeostasis. Numerous chemicals are known to disturb thyroid function, for example by inhibiting thyroperoxidase (TPO) or deiodinase (DIO), upregulating excretion pathways or modifying gene expression. The two major thyroid hormones are triiodothyronine (T3) and thyroxine (T4), both iodinated derivatives of tyrosine. Most TH actions depend on the binding of T3 to its nuclear receptors. Active and inactive THs are tightly regulated by enzymes called iodothyronine deiodinases (DIO). The activation occurs via outer ring deiodination (ORD), i.e. removing iodine from the outer, phenolic ring of T4 to form T3, while inactivation occurs via inner ring deiodination (IRD), i.e. removing iodine from the inner tyrosol ring of T4 or T3.

Three types of iodothyronine deiodinases (DIO1-3) have been described in vertebrates that activate or inactivate THs and are therefore important mediators of TH action. All deiodinases are integral membrane proteins of the thioredoxin superfamily that contain selenocysteine in their catalytic centre. Type I deiodinase is capable of both ORD and IRD, including the conversion of T4 into T3, as well as the conversion of reverse T3 (rT3) to 3,3'-Diiodothyronine (3,3’ T2). rT3, rather than T4, is the preferred substrate for DIO1. furthermore, DIO1 has a very high Km (µM range, compared to nM range for DIO2) (Darras and Van Herck, 2012). Type II deiodinase (DIO2) is only capable of ORD activity with T4 as a preferred substrate (i.e., activation of T4 to T3). DIO3 can inner ring deiodinate T4 and T3 to the inactive forms of THs, rT3 and 3,3’-T2 respectively. DIO1 is a plasma membrane protein with its catalytic domain facing the cytosol. The relative contribution of the DIOs to thyroid hormone levels varies amongst species, developmental stages and tissues.

At this time, there are no approved OECD or EPA guideline protocols for measurement of DIO inhibition. Deiodination is the major pathway regulating T3 bioavailability in mammalian tissues. In vitro assays can be used to examine inhibition of deiodinase 1 (DIO1) activity upon exposure to thyroid disrupting compounds.

Several methods for deiodinase activity measurements are available. A first in vitro assay measures deiodinase activities by quantifying the radioactive iodine release from iodine-labelled substrates, depending on the preferred substrates of the isoforms of deiodinases (Ferreira et al., 2002; Forhead et al., 2006; Freyberger and Ahr, 2006; Pavelka, 2010; Stinckens et al., 2018). Another assay uses a chromatography-based method coupled to mass spectroscopy to measure products of thyroxin resulting from deiodinase type-1 activity (Butt et al., 2011). A colorimetric method (Renko et al., 2012), the Sandell-Kolthoff method, that measures the release of iodine from T4 is also available. Each of these assays requires a source of deiodinase which can be obtained for example using unexposed pig liver tissue (available from slaughterhouses) or rat liver tissue. Renko et al. (2015), Hornung et al. (2018) and Olker et al. (2019) on the other hand used an adenovirus expression system to produce the DIO1 enzyme and developed an assay for nonradioactive measurement of iodide released using the Sandell-Kolthoff method, a photometric method based on Ce4+ reduction (Renko et al., 2012), in a 96-well plate format. This assay was then used to screen the ToxCast Phase 1 chemical library. The specific synthesis of DIO1 through the adenovirus expression system provides an important advantage over other methods where activity of the different deiodinase isoforms needs to be distinguished in other ways, such as based on differences in enzyme kinetics.

Measurements of in vivo deiodinase activity in tissues collected from animal experiments are scarce. Noyes et al. (2011) showed decreased rate of outer ring deiodination (mediated by DIO1 and DIO2) in whole fish microsomes after exposure to BDE-209. After incubation with the substrate, thyroid hormone levels were measured using LC-MS/MS. Houbrechts et al. (2016) confirmed decreased DIO1 activity in a DIO1-DIO2 knockdown zebrafish at the ages of 3 and 7 days post fertilization. Decreased T3 levels are often used as evidence of DIO inhibition, for example after exposure to iopanoic acid, in fish species such as zebrafish (Stinckens et al., 2020) and fathead minnow (Cavallin et al., 2017). It should be noted that it is difficult to make the distinction between decreased T3 levels caused by outer ring deiodination mediated by DIO2 inhibition or DIO1 inhibition. Renko et al. (2022) showed tissue-specific changes in DIO1 activity in hyper- and hypothyroid mice.

Taxonomic: Deiodination by DIO enzymes is known to exist in a wide range of vertebrates and invertebrates. Therefore, this KE is plausibly applicable across vertebrates. Studies reporting DIO1 inhibition have used human liver (Kuiper et al., 2006), human recombinant DIO1 enzyme (Olker et al., 2019), rat (Rattus norvegicus) liver (Klaren et al., 2005; Freyberger and Ahr, 2006; Kuiper et al., 2006; Pavelka, 2010) and thyroid gland (Ferreira et al., 2002), mouse (Mus musculus) brain (hernandez et al., 2006), hog (Sus scrofa domesticus) liver (Stinckens et al., 2018), sheep (Ovis orientalis aries) fetal hepatic, renal and perirenal adipose tissue (Forhead et al., 2006), tadpole (Xenopus laevis) liver (Kuiper et al., 2006), fathead minnow (Pimephales promelas) whole fish (Noyes et al., 2011), Nile tilapia (Oreochromis niloticus) liver (Walpita et al., 2007), Gilthead Seabream (Sparus aurata) kidney (Klaren et al., 2005), and killifish (Fundulus heteroclitus) liver (Orozco et al., 2003) among others. The latter teleostean DIO1 enzymes as well as amphibian enzymes differ from other vertebrate DIO1 enzymes in their lower sensitivity to propylthiouracil (PTU), a typical DIO1 inhibitor in mammals.

In mammals, DIO2 is thought to control the intracellular concentration of T3, while DIO1 is thought to be more important in determining systemic T3 levels. (Marsili et al., 2011), and the same appears to be true for  birds. However, this hypothesis has been challenged. For example, Maia et al. (2005) determined that in a normal physiological situation in humans the contribution of DIO2 to plasma T3 levels is twice that of DIO1. Only in a hyperthyroid state was the contribution of DIO1 higher than that of DIO2. A DIO1 knockout mouse showed normal T3 levels and a normal general phenotype and DIO1 was rather found to play a role in limiting the detrimental effects of conditions that alter normal thyroid function, including hyperthyroidism and iodine deficiency (Schneider et al., 2006). van der Spek et al. concluded that the primary role of DIO1 in vivo is to degrade inactivated TH (van der Spek et al., 2017).

By contrast, DIO1 function in teleostean and amphibian T3 plasma regulation is less clear (Finnson et al. 1999, Kuiper et al. 2006). The presence of DIO1 in the liver of teleosts has been a controversial issue, and both the high level of DIO2 activity and its expression in the liver of teleosts are unique among vertebrates (Orozco and Valverde, 2005). This could explain why DIO2 inhibition seems to be more important than DIO1 inhibition in determining the adverse outcome in zebrafish (Stinckens et al., 2018).

Life stage: Deiodinase activity is important for all vertebrate life stages. Already during early embryonic development, deiodinase activity is needed to regulate thyroid hormone concentrations and coordinate developmental processes. However, the role of DIO1 and DIO2 seems to be distinct. The fact that DIO1 knockdown during zebrafish development only causes developmental defects when combined with DIO2 knockdown (Walpita et al., 2010), suggests that DIO1 is only important in cases of increased TH need during specific stages of development, as supported by increased expression during such stages (Vergauwen et al., 2018), and in cases of thyroid hormone depletion in fish. There can also be differences in sensitivity between sexes. There is evidence for sex- and age-differences of Dio1 expression in mice (Schomburg et al., 2007).

Sex: This KE is plausibly applicable to both sexes. Deiodinases are important for TH homeostasis and identical in both sexes. There can however be differences in sensitivity between sexes. There is evidence for sex- and age-differences of Dio1 expression in mice (Schomburg et al., 2007).

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