Arsenic as an endocrine disruptor: arsenic disrupts retinoic acid receptor-and thyroid hormone receptor-mediated gene regulation and thyroid hormone-mediated amphibian tail metamorphosis

Abstract

Background: Chronic exposure to excess arsenic in drinking water has been strongly associated with increased risks of multiple cancers, diabetes, heart disease, and reproductive and developmental problems in humans. We previously demonstrated that As, a potent endocrine disruptor at low, environmentally relevant levels, alters steroid signaling at the level of receptor-mediated gene regulation for all five steroid receptors.

Objectives: The goal of this study was to determine whether As can also disrupt gene regulation via the retinoic acid (RA) receptor (RAR) and/or the thyroid hormone (TH) receptor (TR) and whether these effects are similar to previously observed effects on steroid regulation.

Methods and results: Human embryonic NT2 or rat pituitary GH3 cells were treated with 0.01-5 microM sodium arsenite for 24 hr, with or without RA or TH, respectively, to examine effects of As on receptor-mediated gene transcription. At low, noncytotoxic doses, As significantly altered RAR-dependent gene transcription of a transfected RAR response element-luciferase construct and the native RA-inducible cytochrome P450 CYP26A gene in NT2 cells. Likewise, low-dose As significantly altered expression of a transfected TR response element-luciferase construct and the endogenous TR-regulated type I deiodinase (DIO1) gene in a similar manner in GH3 cells. An amphibian ex vivo tail metamorphosis assay was used to examine whether endocrine disruption by low-dose As could have specific pathophysiologic consequences, because tail metamorphosis is tightly controlled by TH through TR. TH-dependent tail shrinkage was inhibited in a dose-dependent manner by 0.1- 4.0 microM As.

Conclusions: As had similar effects on RAR- and TR-mediated gene regulation as those previously observed for the steroid receptors, suggesting a common mechanism or action. Arsenic also profoundly affected a TR-dependent developmental process in a model animal system at very low concentrations. Because RAR and TH are critical for both normal human development and adult function and their dysregulation is associated with many disease processes, disruption of these hormone receptor-dependent processes by As is also potentially relevant to human developmental problems and disease risk.

Keywords: CYP26A; arsenic (As); deiodinase (DIO1); endocrine; retinoic acid (RA); steroid; thyroid (TH).

Figure 1

Dose–response curves for As, ATRA, and TH in NT2 and GH3 cells calculated using average values for each dose. Data points represent the mean ± SE of data from three separate experiments. (A) Cytotoxicity of As in NT2 cells exposed to As for 24 hr and assessed by colony-forming assay. Data are expressed as colony formation as a percent of the control. (B) Induction of RARE-luc expression in NT2 cells by ATRA. RARE-luc expression is expressed as mean ± SE luciferase (LU) per gram protein. The median effective concentration (EC₅₀) for ATRA induction is approximately 7 nM. (C) Cytotoxicity of As in GH3 cells assessed essentially as described for (A), except that cells were cultured in media plus stripped serum with or without 10 nM T₃. (D) Induction of DIO1 expression by T₃ in GH3 cells assessed essentially as described in (C), except that DIO1 mRNA was assessed by RT-PCR. Data are expressed as a percent of the maximum value. See “Methods” for details.

Figure 2

Effects of As on ATRA induction of RARE-luc expression in NT2 cells. Cells were transfected with the RARE-luc construct 24 hr before treatment with 10 nM ATRA with or without simultaneous treatment with As for 24 hr. See “Methods” for details. Data are expressed as mean ± SE of the values from replicates of experiments. Bars that do not have the same letter are significantly different from each other at p < 0.003 using an unpaired t-test.

Figure 3

Effects of As on ATRA induction of CYP26A mRNA expression in NT2 cells. Cells were treated with 10 nM ATRA with or without simultaneous addition of As for 24 hr; mRNA expression was measured by RT-PCR. See “Methods” for details. Data are expressed as mean ± SE of the values from replicates of experiments. Bars that do not have the same letter are significantly different from each other at p < 0.003 using an unpaired t-test.

Figure 4

Effects of As on T₃ induction of TRE-luc expression in GH3 cells. Cells were transfected with the TRE-luc construct 24 hr before treatment with 2 nM T₃ with or without simultaneous treatment with As for 24 hr. See “Methods” for details. Data are expressed as mean ± SE of the values from replicates of experiments. Bars that do not have the same letter are significantly different from each other at p < 0.003 using an unpaired t-test.

Figure 5

Effects of As on T₃ induction of DIO1 mRNA expression in GH3 cells. Cells were treated with 2 nM T₃ with or without As, and DIO1 mRNA expression was measured 6 hr (A) or 24 hr (B) after treatment. See “Methods” for details. Data are expressed as mean ± SE of the values from replicates of experiments. Bars that do not have the same letter are significantly different from each other at p < 0.01 using pairwise Student’s t-test analysis.

Figure 6

Effects of T₃ on tail fin shrinkage of Xenopus tadpole tails cultured ex vivo as described in “Methods.” Data are expressed as mean ± SE of values from six tails per treatment in three separate experiments.

Figure 7

Effects of As on T₃-mediated tail shrinkage in Xenopus tadpole tails cultured ex vivo shown by representive samples from tail resorption experiments. See “Methods” for details. Morphometric software was used to trace the tail fin area (shown in black), which was used to calculate the differences in area for each tail between day 1 and day 4. Results from these experiments are shown in Figures 6 and 8.

Figure 8

Effects of As on T₃-mediated tail shrinkage in Xenopus tadpole tails cultured ex vivo. See “Methods” for details. Data are expressed as mean + SE of values from 5–6 individual tails per experiment and 4–8 individual experiments per treatment. Bars that do not have the same letter are significantly different from each other at p < 0.01 using pairwise Student’s t-test analysis.