Acute Methylmercury Exposure and the Hypoxia-Inducible Factor-1α 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 $\text{factor-}1\alpha$ ($\text{HIF-}1\alpha$), 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 $\text{HIF-}1\alpha$ 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 ($0-10\mu M$) for $0.5h$. 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. $\text{HIF-}1\alpha$, 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 $\text{HIF-}1\alpha$ mRNA. Pretreatment with protein synthesis inhibitor (CHX), Proteasome Inhibitor (MG132), or proline hydroxylase inhibitor (DHB) were applied to explore the possible mechanisms of $\text{HIF-}1\alpha$ inhibition by MeHg. To investigate the role of $\text{HIF-}1\alpha$ in MeHg-induced neurotoxicity, cobalt chloride ($\mathrm{CoC}{l}_2$), 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 $\text{HIF-}1\alpha$ levels. The expression of $\text{HIF-}1\alpha$ and related downstream proteins was detected in adult rat brain exposed to MeHg ($0-10\mathrm{mg}/\mathrm{kg}$) for $0.5h$ 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 $10\mu M$ MeHg for $0.5h$ significantly inhibited the expression of astrocytic $\text{HIF-}1\alpha$, and the downstream genes GLUT-1, EPO, and VEGF-A ($p<0.05$), in the absence of a significant decrease in $\text{HIF-}1\alpha$ mRNA levels. When protein synthesis was inhibited by CHX, MeHg promoted the degradation rate of $\text{HIF-}1\alpha$. MG132 and DHB significantly blocked the MeHg-induced decrease in $\text{HIF-}1\alpha$ expression ($p<0.05$). Overexpression of $\text{HIF-}1\alpha$ significantly attenuated the decline in MeHg-induced cell proliferation, whereas the inhibition of $\text{HIF-}1\alpha$ significantly increased the decline in cell proliferation ($p<0.05$). NAC and Trolox, two established antioxidants, reversed the MeHg-induced decline in $\text{HIF-}1\alpha$ protein levels and the decrease in cell proliferation ($p<0.05$). MeHg suppressed the expression of $\text{HIF-}1\alpha$ and related downstream target proteins in adult rat brain.

Discussion: MeHg induced a significant reduction in $\text{HIF-}1\alpha$ 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 $\text{HIF-}1\alpha$ expression in response to MeHg toxicity. Moreover, we established that up-regulation of $\text{HIF-}1\alpha$ 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.