Acute Methylmercury Exposure and the Hypoxia-Inducible [Formula: see text] Signaling Pathway under Normoxic Conditions in the Rat Brain and Astrocytes in Vitro

BACKGROUND: As a ubiquitous environmental pollutant, methylmercury (MeHg) induces toxic effects in the nervous system, one of its main targets. However, the exact mechanisms of its neurotoxicity have not been fully elucidated. Hypoxia-inducible [Formula: see text] ([Formula: see text]), a transcription factor, plays a crucial role in adaptive and cytoprotective responses in cells and is involved in cell survival, proliferation, apoptosis, inflammation, angiogenesis, glucose metabolism, erythropoiesis, and other physiological activities. OBJECTIVES: The aim of this study was to explore the role of [Formula: see text] in response to acute MeHg exposure in rat brain and primary cultured astrocytes to improve understanding of the mechanisms of MeHg-induced neurotoxicity and the development of effective neuroprotective strategies. METHODS: Primary rat astrocytes were treated with MeHg ([Formula: see text]) for [Formula: see text]. Cell proliferation and cytotoxicity were assessed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl diphenyltetrazolium bromide (MTT) assay and a lactate dehydrogenase (LDH) release assay, respectively. Reactive oxygen species (ROS) levels were analyzed to assess the level of oxidative stress using 2′,7′-dichlorofluorescin diacetate (DCFH-DA) fluorescence. [Formula: see text] , and its downstream proteins, glucose transporter 1 (GLUT-1), erythropoietin (EPO), and vascular endothelial growth factor A (VEGF-A) were analyzed by means of Western blotting. Real-time PCR was used to detect the expression of [Formula: see text] mRNA. Pretreatment with protein synthesis inhibitor (CHX), proteasome inhibitor (MG132), or proline hydroxylase inhibitor (DHB) were applied to explore the possible mechanisms of [Formula: see text] inhibition by MeHg. To investigate the role of [Formula: see text] in MeHg-induced neurotoxicity, cobalt chloride ([Formula: see text]), 2-methoxyestradiol (2-MeOE2), small interfering RNA (siRNA) transfection and adenovirus overexpression were used. Pretreatment with N-acetyl-L-cysteine (NAC) and vitamin E (Trolox) were used to investigate the putative role of oxidative stress in MeHg-induced alterations in [Formula: see text] levels. The expression of [Formula: see text] and related downstream proteins was detected in adult rat brain exposed to MeHg ([Formula: see text]) for [Formula: see text] in vivo. RESULTS: MeHg caused lower cell proliferation and higher cytotoxicity in primary rat astrocytes in a time- and concentration-dependent manner. In comparison with the control cells, exposure to [Formula: see text] MeHg for [Formula: see text] significantly inhibited the expression of astrocytic [Formula: see text] , and the downstream genes GLUT-1, EPO, and VEGF-A ([Formula: see text]), in the absence of a significant decrease in [Formula: see text] mRNA levels. When protein synthesis was inhibited by CHX, MeHg promoted the degradation rate of [Formula: see text]. MG132 and DHB significantly blocked the MeHg-induced decrease in [Formula: see text] expression ([Formula: see text]). Overexpression of [Formula: see text] significantly attenuated the decline in MeHg-induced cell proliferation, whereas the inhibition of [Formula: see text] significantly increased the decline in cell proliferation ([Formula: see text]). NAC and Trolox, two established antioxidants, reversed the MeHg-induced decline in [Formula: see text] protein levels and the decrease in cell proliferation ([Formula: see text]). MeHg suppressed the expression of [Formula: see text] and related downstream target proteins in adult rat brain. DISCUSSION: MeHg induced a significant reduction in [Formula: see text] protein by activating proline hydroxylase (PHD) and the ubiquitin proteasome system (UPS) in primary rat astrocytes. Additionally, ROS scavenging by antioxidants played a neuroprotective role via increasing [Formula: see text] expression in response to MeHg toxicity. Moreover, we established that up-regulation of [Formula: see text] might serve to mitigate the acute toxicity of MeHg in astrocytes, affording a novel therapeutic target for future exploration. https://doi.org/10.1289/EHP5139