Table 2.

Identification of known and putative molecular targets [i.e., molecular initiating events (MIEs) and key events (KEs) being treated as MIEs] of chemical-induced thyroid disruption.

Molecular initiating event	Toxicological mechanisma	In vitro HTS assay readinessb	Potential adverse outcomesc	References
TH synthesis (thyroid gland)
Sodium–iodide symporter (NIS)	Regulates serum iodide uptake into thyroid follicular cells and other tissues. Inhibition of NIS-iodide transport disrupts T4 and T3 synthesis. Well-characterized chemical target in thyroid pathway.	Existing: Hallinger et al. 2017; Wang et al. 2018	Mammals: Developmental neurotoxicity, cognitive defects	In vivo studies: Gilbert and Sui 2008; Goleman et al. 2002; McNabb et al. 2004; Tietge et al. 2005, 2010; York et al. 2004
			Amphibians: Impaired metamorphosis
			Birds: Delayed hatching, increased mortality, decreased growth
				In vitro studies: Chen et al. 2008 Reviews: NRC 2005
	Chemicals: perchlorate, chlorate, nitrate, thiocyanate, small ion drugs		AOPs 54, 110, 134, 176
Thyroperoxidase (TPO)	Catalyzes oxidation of iodide, nonspecific iodination of tyrosyl residues of thyroglobulin (Tg), and coupling of iodotyrosyls to form Tg-bound T3 and T4. Inhibition of TPO activity disrupts TH synthesis. Well-characterized chemical target in thyroid pathway. Chemicals: methimazole, PTU, pronamide, soy isoflavones, mancozeb, resorcinol, triclosan	Existing: Paul Friedman et al. 2016	Mammals: Visual deficits, developmental neurotoxicity, cognitive defects; MOA/AOP developmental neurotoxicity in rat	In vivo studies: Ausó et al. 2004; Boyes et al. 2018; Degitz et al. 2005; Fort et al. 2000; Gilbert 2011; Gilbert et al. 2013, 2016; Goodman and Gilbert 2007; Lasley and Gilbert 2011; Nelson et al. 2016; O’Shaughnessy et al. 2018a, 2018b; Stinckens et al. 2016; Zoeller and Crofton 2005
			Rat: Thyroid cancer
			Amphibians: Impaired metamorphosis
			Fish: Reduced swim bladder inflation AOPs 42, 119, 159, 175, 271
				In vitro studies: Davidson et al. 1978
				Reviews: Dellarco et al. 2006; Hurley 1998
Iodotyrosine deiodinase (IYD)	Scavenges/recycles iodide in the thyroid by catalyzing deiodination to T1 and T2. Limited evidence of chemical inhibition.	Promising	Amphibians: Impaired metamorphosis AOP 188	In vivo studies: Olker et al. 2018b
				In vitro studies: Shimizu et al. 2013
	Chemicals: 3-nitro-L-tyrosine, OH-PCBs, OH-PBDEs, rose bengal
Pendrin	Transports iodide from cytosol of thyroid follicular cell into lumen for organification. No reports of chemical interactions; research limited.	Early	Not yet characterized AOP 192	—
	Chemicals: Unknown
Dual oxidase (DUOX)	Generates peroxide necessary for TH synthesis. No reports of chemical interactions. Research limited.	Early	Not yet characterized AOP 193	—
	Chemicals: Unknown
TH transport (serum)
Transthyretin (TTR); Thyroid binding globulin (TBG); Albumin	Bind and distribute TH in circulation. TTR and TBG are known chemical targets. Albumin is the most abundant, but TH binding is nonspecific with low affinity.	Existing: Marchesini et al. 2006; Montaño et al. 2012	Not yet characterized AOP 152	In vivo studies: Hallgren and Darnerud 2002; Hedge et al. 2009
	Chemicals: OH-PCBs, OH-BDEs, PFAS, TBBPA, TCBPA, genistein, dioxins			In vitro studies: Cheek et al. 1999; Hamers et al. 2006; Lans et al. 1994
				Reviews: Brouwer et al. 1998
TH metabolism and excretion (liver and other target tissues)
Iodothyronine deiodinase(DIO) Type 1 (DIO1); DIO Type 2 (DIO2); DIO Type 3 (DIO3)	Control the activation and inactivation of T4 in a tissue-specific and temporal manner. With the exception of FD&C red dye no. 3 that has been shown to induce thyroid tumors in rats, no studies to date have shown chemicals that exert effects on DIO expression and/or activity to directly manifest in adverse outcomes.	Existing: DIO1: Hornung et al. 2018 DIO2, DIO3: Olker et al. 2019	Not yet characterized AOPs 156-158, 189-191	In vivo studies: Borzelleca et al. 1987; Hood and Klaassen 2000; Mol et al. 1999; Morse et al. 1993; Noyes et al. 2011, 2013; Szabo et al. 2009
				In vitro studies: Butt et al. 2011; Capen and Martin 1989; Ferreira et al. 2002; Olker et al. 2019; Renko et al. 2015
	Chemicals: FD&C red dye no. 3, phenobarbital, PCN PTU, PCBs, PBDEs
Constitutive androstane receptor (CAR); Pregnane X receptor (PXR); Aryl hydrocarbon receptor (AhR)	Xenobiotic nuclear receptors that up-regulate expression of phase I and II metabolic enzymes and phase III uptake and efflux transporters, some of which may accelerate TH catabolism and clearance.	Existing: ToxCast/Tox21 He et al. 2011; Maglich et al. 2003; Romanov et al. 2008; Rosenfeld et al. 2003	See UDPGTs and SULTs	See UDPGTs and SULTs
	Chemicals: See UDPGTs and SULTs
Uridine diphosphate glucuronosyltransferase (UDPGTs; e.g., UGT1A1, UGT1A6); sulfotransferases (SULTs; e.g., SULT2A1)	Major phase II chemical conjugation pathways that also regulate TH catabolism. Chemical up-regulation in the expression and activity of UDPGTs and SULTs increase T4 glucuronidation and sulfation, respectively. There are numerous isoforms of UDPGTs and SULTs, with UGT1A1, UGT1A6, and SULT2A1 having been shown to metabolize T4.	Promising	UDPGTs: Mammalian cochlear damage and hearing loss; MOA/AOP hearing deficits via up-regulated TH catabolism. AOPs 8, 194	In vivo studies: Barter and Klaassen 1992, 1994; Haines et al. 2018; Klaassen and Hood 2001; Szabo et al. 2009; Vansell and Klaassen 2002; Visser et al. 1993; Wong et al. 2005; Yu et al. 2009
	Chemicals: OH-BDEs, OH-PCBs, PAHs, PFAS, BPA, triclosan, dioxins, propiconazole, phthalates, phenobarbital, rifampicin, PCN
			SULTs: Not yet characterized
				In vitro studies: Butt and Stapleton 2013; Larson et al. 2011; Rotroff et al. 2010; Schuur et al. 1998
				Reviews: Crofton and Zoeller 2005; Konno et al. 2008; Wang and James 2006
Alanine side-chain reactions	T4 and T3 alanine side-chains can be metabolized by oxidative decarboxylation or deamination, producing thyroanimines and thyroacetic acids, respectively.	Early	Not yet characterized	Reviews: Scanlan 2009; Wu et al. 2005
	Chemicals: Unknown
Peroxisome proliferator-activated receptor (PPARα, PPARβ/δ, PPARγ)	Key regulators controlling lipid and carbohydrate metabolism, as well as in mediating cellular differentiation and proliferation, and reproductive development. PPARs and TRs bind to DNA response elements as heterodimers with the RXR and other NRs and have been shown to compete for binding with RXR as well as for TR-transcriptional coactivators and corepressors.	Existing: ToxCast/Tox21: R Huang et al. 2016; Martin et al. 2010; Romanov et al. 2008; van Raalte et al. 2004	Not yet characterized	In vivo studies: Lake et al. 2016; Springer et al. 2012
				In vitro studies: Huang et al. 2011; Juge-Aubry et al. 1995
				In silico studies: Nolte et al. 1998 Reviews: Hyyti and Portman 2006; Lu and Cheng 2010; Miller et al. 2009; White et al. 2011
	Chemicals: PFAS, PBDEs, phthalates, BPA, TBBPA, TCBPA, organochlorine pesticides, EE2, fibrate drugs, Wy14,643, rosiglitazone, thiazolidinediones
TH transport (cellular)
Monocarboxylate transporter (MCT8, MCT10); organic anion transporter polypeptide (e.g., OATP1C1; OATP1A4);	MCT8 is a specific cellular transporter of TH, and MCT8 mutations produce hypothyroidism and severe neurological impairments. MCT10, OATP1C1, and OATP1A4 mediate transport TH and other ligands. There are numerous other transporters that have been shown to transport TH, including several other subtypes of OATPs and L-type amino acid transporter (LAT1, LAT2). Limited evidence that some chemicals may alter expression. Chemicals: tyrosine kinase inhibitors, fenamate drugs, TRIAC, PBDEs	Early	Not yet characterized	In vivo studies: Braun et al. 2012; Heuer et al. 2005; Noyes et al. 2013; Richardson et al. 2008; Roberts et al. 2008; Sharlin et al. 2018; Song et al. 2016; Westholm et al. 2009 In vitro studies: Dong and Wade 2017; Friesema et al. 2003 Reviews: Visser et al. 2011
Multidrug resistance protein (MDR1); multidrug resistance associated protein (MRP2)	Phase III hepatic efflux transporters that mediate hepatobiliary efflux of xenobiotics and TH. Importance as a chemical target is unclear.	Early	Not yet characterized	In vivo studies: Richardson et al. 2008; Szabo et al. 2009; Wong et al. 2005
	Chemicals: PBDEs, anxiolytic/antiepileptic drugs
Receptor–ligand binding
TRH receptor	Controls synthesis and release of TSH; TRH mutations lead to hypothyroidism.	Promising: ToxCast/Tox21	Not yet characterized	In silico studies: Engel et al. 2008; Knudsen et al. 2011; Sipes et al. 2013
	Chemicals: Unknown
				Reviews: Beck-Peccoz et al. 2006
TSH receptor	When activated, stimulates adenyl cyclase and formation of cAMP that increases iodide uptake and TH synthesis in thyroid follicular cells.	Promising: ToxCast/Tox21	Not yet characterized	In vitro studies: Jomaa et al. 2013; Neumann et al. 2009; Santini et al. 2003; Titus et al. 2008 In silico studies: Paul Friedman et al. 2017; Shobair et al. 2019 Reviews: Gershengorn and Neumann 2012
	Chemicals: Unknown
TR binding and transactivation (TRα, TRβ)	Transcription factors that have ligand (T3)-dependent and -independent activity. In humans, THRA genes encode TRα1, TRα2, and TRα3 and truncated isoforms. THRB genes encode TRβ1, TRβ2, and TRβ3 (possibly rat only) and truncated isoforms. Only TRα1, TRβ1, TRβ2, and TRβ3 can bind T3 and TREs; TRβ1 and TRβ2 regulate TRH in the hypothalamus. Some chemicals bind TRs as antagonists and/or modify transcription; however, screens of chemical libraries suggest binding is restricted.	Existing: ToxCast/Tox21: Freitas et al. 2011, 2014	Not yet characterized	In vivo studies: Dupré et al. 2004
				In vitro studies: Cheek et al. 1999; Clerget-Froidevaux et al. 2004; Freitas et al. 2011, 2014; Gauger et al. 2007; Hofmann et al. 2009; Huang et al. 2011; Kitamura et al. 2002, 2005; Kojima et al. 2009; Moriyama et al. 2002; Schriks et al. 2006; Sun et al. 2009; You et al. 2006 In silico studies: Knudsen et al. 2011; Politi et al. 2014; Romanov et al. 2008; Sipes et al. 2013 Reviews: Zoeller 2005
	Chemicals: TBBPA, TCBPA, BPA, OH-PCBs, OH-BDEs, triclosan

Note: Table adapted from Murk et al. (2013) and OECD (2014). —, Not applicable; BPA, bisphenol A; EE2, 17α-ethinylestradiol (synthetic estrogen); MOA, mode of action; NR, nuclear receptor; OH-BDE, hydroxylated bromodiphenyl ether (PBDE metabolites); OH-PCB, hydroxylated polychlorinated biphenyl ether (PCB metabolites); PAH, polycyclic aromatic hydrocarbon; PBDE, polybrominated diphenyl ether; PCB, polychlorinated biphenyl ether; PCN, pregnenolone-16α-carbonitrile; PFAS, per- and polyfluoroalkyl substances; PTU, propylthiouracil; RXR, retinoid X receptor; T3, triiodothyronine; T4, thyroxine; TBBPA, tetrabromobisphenol A; TCBPA, tetrachlorobisphenol A; TRIAC, triiodothyroacetic acid; TR, thyroid hormone receptor; TRE, thyroid hormone response element; TRH, thyrotropin releasing hormone; TSH, thyroid stimulating hormone; UDPGT, uridine diphosphate glucuronosyltransferase.

^aThe toxicological mechanism column highlights the role of MIEs in thyroid hormone (TH) signaling along with chemicals shown to interact with them.

^bIn vitro HTS assay development denoted as “promising” indicates MIEs in the thyroid axis for which there is interest and/or activity in developing in vitro HTS approaches, typically with supportive in vivo and slow- or medium-throughput in vitro toxicity studies indicating chemical interactions. In vitro HTS readiness denoted as “early” indicates putative MIEs but with limited toxicity evidence and with little current activity to develop high-throughput alternatives.

^cMIEs in the thyroid axis with evidence of linkages to adverse outcomes. Additional information on individual AOPs in development and completed can be found at https://aopwiki.org/.