Analysis of arsenic-modulated expression of hypothalamic estrogen receptor, thyroid receptor, and peroxisome proliferator-activated receptor gamma mRNA and simultaneous mitochondrial morphology and respiration rates in the mouse

Abstract

Arsenic has been identified as an environmental toxicant acting through various mechanisms, including the disruption of endocrine pathways. The present study assessed the ability of a single intraperitoneal injection of arsenic, to modify the mRNA expression levels of estrogen- and thyroid hormone receptors (ERα,β; TRα,β) and peroxisome proliferator-activated receptor gamma (PPARγ) in hypothalamic tissue homogenates of prepubertal mice in vivo. Mitochondrial respiration (MRR) was also measured, and the corresponding mitochondrial ultrastructure was analyzed. Results show that ERα,β, and TRα expression was significantly increased by arsenic, in all concentrations examined. In contrast, TRβ and PPARγ remained unaffected after arsenic injection. Arsenic-induced dose-dependent changes in state 4 mitochondrial respiration (St4). Mitochondrial morphology was affected by arsenic in that the 5 mg dose increased the size but decreased the number of mitochondria in agouti-related protein- (AgRP), while increasing the size without affecting the number of mitochondria in pro-opiomelanocortin (POMC) neurons. Arsenic also increased the size of the mitochondrial matrix per host mitochondrion. Complex analysis of dose-dependent response patterns between receptor mRNA, mitochondrial morphology, and mitochondrial respiration in the neuroendocrine hypothalamus suggests that instant arsenic effects on receptor mRNAs may not be directly reflected in St3-4 values, however, mitochondrial dynamics is affected, which predicts more pronounced effects in hypothalamus-regulated homeostatic processes after long-term arsenic exposure.

Fig 1. The hypothesized mechanisms underlying arsenic-induced endocrine disruption, emphasizing its potential impact on the primary endocrine center.

The melanocortin system within the hypothalamus emerges as a key target of arsenic, with adverse effects anticipated across diverse cellular processes, including mitochondrial respiration and morphology, as well as hormonal signaling pathways. Persistent dysfunction of the melanocortin system is implicated in the etiology of various systemic disorders, such as metabolic syndromes, reproductive dysfunction, mood disorders, and neurodegenerative diseases.

Fig 2. Effects of 3 different doses of a single intraperitoneal injection of sodium (meta)arsenite (arsenic, As) on the expression of hypothalamic nuclear hormone receptors (ERα,β; TRα,β, PPARγ), and hypothalamic mitochondrial state 3–4 respiration.

(A-E): Relative expression level of receptor genes was analyzed by qRT-PCR and normalized to the average of the control gene GAPDH. (F): ADP-dependent (St3) and oligomycin-induced (St4) mitochondrial respiration rates were given in consumed nmol O₂ per ml of final volume in one minute. The data shown here are the mean ± standard deviation (SD) and compared to the non-treated control (ntC). P-values are represented by asterisk (*) where p < 0.05.

Fig 3. The effects of 3 different doses of a single intraperitoneal injection of sodium (meta)arsenite (As) on the mitochondrial microstructure in AgRP and POMC neurons.

(A-B) the size of mitochondria, (C-D) the number of mitochondria is measured per unit section area. (E-F) the proportional mitochondrial matrix (matrix-to-total mitochondrial ratio) area is expressed as a percentage. (G-H) the images of electron micrographs of AgRP and POMC neurons, where arrows indicate examples of POMC-IR and AgRP-IR neurons. The micrographs showcase the effectiveness of the staining technique employed in this study, providing high-quality visualization of the cellular structures of interest. The data presented above (A-F) are expressed as the mean ± standard deviation (SD) and compared to the non-treated control (ntC) group. P-values are represented by asterisks (*,**) where p < 0.05 and p < 0.001, respectively.