Short-term Exposure to Triclosan Decreases Thyroxine In Vivo via Upregulation of Hepatic Catabolism in Young Long-Evans Rats

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)-phenol) is a chlorinated phenolic antibacterial compound found in consumer products. In vitro human pregnane X receptor activation, hepatic phase I enzyme induction, and decreased in vivo total thyroxine (T4) suggest adverse effects on thyroid hormone homeostasis. Current research tested the hypothesis that triclosan decreases circulating T4 via upregulation of hepatic catabolism and transport. Weanling female Long-Evans rats received triclosan (0–1000 mg/kg/day) by gavage for 4 days. Whole blood and liver were collected 24 h later. Total serum T4, triiodothyronine (T3), and thyroid-stimulating hormone (TSH) were measured by radioimmunoassay. Hepatic microsomal assays measured ethoxyresorufin-O-deethylase, pentoxyresorufin-O-deethylase (PROD), and uridine diphosphate glucuronyltransferase enzyme activities. The messenger RNA (mRNA) expression of cytochrome P450s 1a1, 2b1/2, and 3a1/23; UGTs 1a1, 1a6, and 2b5; sulfotransferases 1c1 and 1b1; and hepatic transporters Oatp1a1, Oatp1a4, Mrp2, and Mdr1b was measured by quantitative reverse transcriptase PCR. Total T4 decreased dose responsively, down to 43% of control at 1000 mg/kg/day. Total T3 was decreased to 89 and 75% of control at 300 and 1000 mg/kg/day. TSH did not change. Triclosan dose dependently increased PROD activity up to 900% of control at 1000 mg/kg/day. T4 glucuronidation increased nearly twofold at 1000 mg/kg/day. Cyp2b1/2 and Cyp3a1/23 mRNA expression levels were induced twofold and fourfold at 300 mg/kg/day. Ugt1a1 and Sult1c1 mRNA expression levels increased 2.2-fold and 2.6-fold at 300 mg/kg/day. Transporter mRNA expression levels were unchanged. These data denote important key events in the mode of action for triclosan-induced hypothyroxinemia in rats and suggest that this effect may be partially due to upregulation of hepatic catabolism but not due to mRNA expression changes in the tested hepatic transporters

Developmental triclosan exposure decreases maternal, fetal, and early neonatal thyroxine: A dynamic and kinetic evaluation of a putative mode-of-action

This work tests the mode-of-action (MOA) hypothesis that maternal and developmental triclosan (TCS) exposure decreases circulating thyroxine (T4) concentrations via up-regulation of hepatic catabolism and elimination of T4. Time-pregnant Long-Evans rats received TCS po (0–300 mg/kg/day) from gestational day (GD) 6 through postnatal day (PND) 21. Serum and liver were collected from dams (GD20, PND22) and offspring (GD20, PND4, PND14, PND21). Serum T4, triiodothyronine (T3), and thyroid stimulating hormone (TSH) concentrations were measured by radioimmunoassay. Ethoxy-O-deethylase (EROD), pentoxyresorufin-O-depentylase (PROD) and uridine diphosphate glucuronyltransferase (UGT) enzyme activities were measured in liver microsomes. Custom Taqman® qPCR arrays were employed to measure hepatic mRNA expression of select cytochrome P450s, UGTs, sulfotransferases, transporters, and thyroid-hormone responsive genes. TCS was quantified by LC/MS/MS in serum and liver. Serum T4 decreased approximately 30% in GD20 dams and fetuses, PND4 pups and PND22 dams (300 mg/kg/day). Hepatic PROD activity increased 2- to 3-fold in PND4 pups and PND22 dams, and UGT activity was 1.5-fold higher in PND22 dams only (300 mg/kg/day). Minor up-regulation of Cyp2b and Cyp3a expression in dams was consistent with hypothesized activation of the constitutive androstane and/or pregnane X receptor. T4 reductions of 30% for dams and GD20 and PND4 offspring with concomitant increases in PROD (PND4 neonates and PND22 dams) and UGT activity (PND22 dams) suggest that up-regulated hepatic catabolism may contribute to TCS–induced hypothyroxinemia during development. Serum and liver TCS concentrations demonstrated greater fetal than postnatal internal exposure, consistent with the lack of T4 changes in PND14 and PND21 offspring. These data support the MOA hypothesis that TCS exposure leads to hypothyroxinemia via increased hepatic catabolism; however, the minor effects on thyroid hormone metabolism may reflect the low efficacy of TCS as thyroid hormone disruptor or highlight the possibility that other MOAs may also contribute to the observed maternal and early neonatal hypothyroxinemia

Thyroid-Hormone–Disrupting Chemicals: Evidence for Dose-Dependent Additivity or Synergism

Endocrine disruption from environmental contaminants has been linked to a broad spectrum of adverse outcomes. One concern about endocrine-disrupting xenobiotics is the potential for additive or synergistic (i.e., greater-than-additive) effects of mixtures. A short-term dosing model to examine the effects of environmental mixtures on thyroid homeostasis has been developed. Prototypic thyroid-disrupting chemicals (TDCs) such as dioxins, polychlorinated biphenyls (PCBs), and poly-brominated diphenyl ethers have been shown to alter thyroid hormone homeostasis in this model primarily by up-regulating hepatic catabolism of thyroid hormones via at least two mechanisms. Our present effort tested the hypothesis that a mixture of TDCs will affect serum total thyroxine (T(4)) concentrations in a dose-additive manner. Young female Long-Evans rats were dosed via gavage with 18 different polyyhalogenated aromatic hydrocarbons [2 dioxins, 4 dibenzofurans, and 12 PCBs, including dioxin-like and non-dioxin-like PCBs] for 4 consecutive days. Serum total T(4) was measured via radioimmunoassay in samples collected 24 hr after the last dose. Extensive dose–response functions (based on seven to nine doses per chemical) were determined for individual chemicals. A mixture was custom synthesized with the ratio of chemicals based on environmental concentrations. Serial dilutions of this mixture ranged from approximately background levels to 100-fold greater than background human daily intakes. Six serial dilutions of the mixture were tested in the same 4-day assay. Doses of individual chemicals that were associated with a 30% TH decrease from control (ED(30)), as well as predicted mixture outcomes were calculated using a flexible single-chemical-required method applicable to chemicals with differing dose thresholds and maximum-effect asymptotes. The single-chemical data were modeled without and with the mixture data to determine, respectively, the expected mixture response (the additivity model) and the experimentally observed mixture response (the empirical model). A likelihood-ratio test revealed statistically significant departure from dose additivity. There was no deviation from additivity at the lowest doses of the mixture, but there was a greater-than-additive effect at the three highest mixtures doses. At high doses the additivity model underpredicted the empirical effects by 2- to 3-fold. These are the first results to suggest dose-dependent additivity and synergism in TDCs that may act via different mechanisms in a complex mixture. The results imply that cumulative risk approaches be considered when assessing the risk of exposure to chemical mixtures that contain TDCs

Development of a Thyroperoxidase Inhibition Assay for High-Throughput Screening

High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein, we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluorescent peroxidase substrate, Amplex UltraRed (AUR), were employed in an end-point assay for comparison to the existing kinetic guaiacol (GUA) oxidation assay. Following optimization of assay metrics, including <i>Z</i>′, dynamic range, and activity, using methimazole (MMI), the assay was tested with a 21-chemical training set. The potency of MMI-induced TPO inhibition was greater with AUR compared to GUA. The dynamic range and <i>Z</i>′ score with MMI were as follows: 127-fold and 0.62 for the GUA assay, 18-fold and 0.86 for the 96-well AUR assay, and 11.5-fold and 0.93 for the 384-well AUR assay. The 384-well AUR assay drastically reduced animal use, requiring one-tenth of the rat thyroid microsomal protein needed for the GUA 96-well format assay. Fourteen chemicals inhibited TPO, with a relative potency ranking of MMI > ethylene thiourea > 6-propylthiouracil > 2,2′,4,4′-tetrahydroxy-benzophenone > 2-mercaptobenzothiazole > 3-amino-1,2,4-triazole > genistein > 4-propoxyphenol > sulfamethazine > daidzein > 4-nonylphenol > triclosan > iopanoic acid > resorcinol. These data demonstrate the capacity of this assay to detect diverse TPO inhibitors. Seven chemicals acted as negatives: 2-hydroxy-4-methoxybenzophenone, dibutylphthalate, diethylhexylphthalate, diethylphthalate, 3,5-dimethylpyrazole-1-methanol, methyl 2-methyl-benzoate, and sodium perchlorate. This assay could be used to screen large numbers of chemicals as an integral component of a tiered TH-disruptor screening approach

Development of a tiered screening strategy for a molecular-initiating event: thyroperoxidase inhibition

<table><tbody><tr> <td>Presented at the Annual Society of Toxicology meeting</td></tr></tbody></table