Acute Methylmercury Exposure and the Hypoxia-Inducible Signaling Pathway under Normoxic Conditions in the Rat Brain...

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 $$ ($$), 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.

The aim of this study was to explore the role of $$ 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.

Primary rat astrocytes were treated with MeHg ($$) for $$. 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. $$, and its downstream proteins, glucose transporter 1 (GLUT-1), erythropoietin (EPO), and vascular endothelial growth factor A (VEGF-A) were analyzed by means of Western blotting. Real-time PCR was used to detect the expression of $$ mRNA. Pretreatment with protein synthesis inhibitor (CHX), proteasome inhibitor (MG132), or proline hydroxylase inhibitor (DHB) were applied to explore the possible mechanisms of $$ inhibition by MeHg. To investigate the role of $$ in MeHg-induced neurotoxicity, cobalt chloride ($$), 2-methoxyestradiol (2-MeOE2), small interfering RNA (siRNA) transfection and adenovirus overexpression were used. Pretreatment with N-acetyl-L-cysteine (NAC) and vitamin E (Trolox) were used to investigate the putative role of oxidative stress in MeHg-induced alterations in $$ levels. The expression of $$ and related downstream proteins was detected in adult rat brain exposed to MeHg ($$) for $$ in vivo.

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 $$ MeHg for $$ significantly inhibited the expression of astrocytic $$, and the downstream genes GLUT-1, EPO, and VEGF-A ($$), in the absence of a significant decrease in $$ mRNA levels. When protein synthesis was inhibited by CHX, MeHg promoted the degradation rate of $$. MG132 and DHB significantly blocked the MeHg-induced decrease in $$ expression ($$). Overexpression of $$ significantly attenuated the decline in MeHg-induced cell proliferation, whereas the inhibition of $$ significantly increased the decline in cell proliferation ($$). NAC and Trolox, two established antioxidants, reversed the MeHg-induced decline in $$ protein levels and the decrease in cell proliferation ($$). MeHg suppressed the expression of $$ and related downstream target proteins in adult rat brain.

MeHg induced a significant reduction in $$ protein by activating proline hydroxylase (PHD) and the ubiquitin proteasome system (UPS) in primary rat astrocytes. Additionally, ROS scavenging by antioxidants played a neuroprotective role via increasing $$ expression in response to MeHg toxicity. Moreover, we established that up-regulation of $$ 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.