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

Abstract

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.

Keywords: Adverse outcome; Endocrine disruption; Pesticides; Thyroid hormone pathway; Thyroid network.

Figure 1

Human health adverse outcome pathway example in the thyroid.

Figure 2

Thyroid hormones and TSH levels in serum of rats treated with selected triazoles and collected after 28 days of treatment (Control: non-treated animals, 6 animals in duplicates measurements; PB: Phenobarbital 500 ppm, 5 animals in duplicates measurements; C: Cyproconazole 1000 ppm, 5 animals in duplicates measurements; Epoxiconazole 900 ppm, 5 animals in duplicates measurements; P: Prochloraz 1000 ppm, 5 animals in duplicates measurements). Values are given as mean ± standard error of mean (SEM). One-Way-ANOVA post hoc (Dunnett’s test) with *(p ≤ 0.05) and ** (p ≤ 0.01).

Figure 3

Specific UGT activity across treatment groups (Control: non-treated animals, 6 animals in duplicates measurements; PB: Phenobarbital, 5 animals in duplicates measurements; C: Cyproconazole 1000 ppm, 5 animals in duplicates measurements; E: Epoxiconazole 900 ppm, 5 animals in duplicates measurements; P: Prochloraz 1000 ppm, 5 animals in duplicates measurements) after 28-days of treatment. Data are expressed as mean ± standard error of mean (SEM). One-Way-ANOVA post hoc (Dunnett’s test) with *(p ≤ 0.05).

Figure 4

Thyroid follicles at different treatment groups. Optical microscopy (50 × , 100 × and 200 × magnifications), H&E staining. (A) Control negative (5×); (B) Cyproconazole 1000 ppm (10×); (C) Epoxiconazole 1000 ppm (10×); (D) Phenobarbital positive control (5×); (E) Prochloraz 900 ppm (10×); (F) Epoxiconazole 1000 ppm (20×).

Figure 5

Thyroid morphometric parameters investigated at different treatment groups after 28-days treatment. Control: non-treated animals, 6 animals in duplicates measurements; PB: Phenobarbital, 4 animals in duplicates measurements; C: Cyproconazole 1000 ppm, 5 animals in duplicates measurements; E: Epoxiconazole 900 ppm, 4 animals in duplicates measurements; P: Prochloraz 1000 ppm, 5 animals in duplicates measurements. Values are given as mean ± standard error of mean (SEM). One-Way-ANOVA post hoc (Dunnett’s test) with *(p ≤ 0.05).

Figure 6

Dio1 and Dehal1 activities in liver and kidney of rats treated with cyproconazole at different doses or PB (Phenobarbital 500 ppm) (mean ± SD, n = 5/group/duplicates); (A) hepatic Dio1 activity; (B) hepatic Dehal1 activity; (C) renal Dio1 activity; (D) renal Dehal1 activity. All measurements used the Sandell-Kolthoff-reaction as readout. One-Way-ANOVA post hoc (Dunnett’s test) with *(p ≤ 0.05).

Figure 7

Testing of cyproconazole in selected in vitro assays reflecting potential MIEs. No obvious effects were seen for DIO1-3, DEHAL1, NIS, TPO and MCT8 function up to the maximal tested concentration of 50 µM (mean + /-SD, N = 2, n = 2). Data on epoxiconazole and prochloraz, including data on positive controls, are found in the supplement (SFig. 1).