Acute Methylmercury Exposure and the Hypoxia-Inducible Factor-1α<math><mrow><mtext>Factor-</mtext><mn>1</mn><mi>α</mi></mrow></math> Signaling Pathway under Normoxic Conditions in the Rat Brain and Astrocytes in Vitro

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 factor-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;factor-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; (HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt;), 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

MeHg induced a significant reduction in HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; 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 HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; expression in response to MeHg toxicity. Moreover, we established that up-regulation of HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; 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.

Primary rat astrocytes were treated with MeHg (0-10μM&lt;math&gt;&lt;mrow&gt;&lt;mn&gt;0&lt;/mn&gt;&lt;mo&gt;-&lt;/mo&gt;&lt;mn&gt;10&lt;/mn&gt;&lt;mspace&gt;&lt;/mspace&gt;&lt;mi&gt;μ&lt;/mi&gt;&lt;mi&gt;M&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt;) for 0.5h&lt;math&gt;&lt;mrow&gt;&lt;mn&gt;0.5&lt;/mn&gt;&lt;mspace&gt;&lt;/mspace&gt;&lt;mi&gt;h&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt;. 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. HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt;, 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 HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; mRNA. Pretreatment with protein synthesis inhibitor (CHX), proteasome inhibitor (MG132), or proline hydroxylase inhibitor (DHB) were applied to explore the possible mechanisms of HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; inhibition by MeHg. To investigate the role of HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; in MeHg-induced neurotoxicity, cobalt chloride (CoCl2&lt;math&gt;&lt;mrow&gt;&lt;mi&gt;CoC&lt;/mi&gt;&lt;msub&gt;&lt;mi&gt;l&lt;/mi&gt;&lt;mi&gt;2&lt;/mi&gt;&lt;/msub&gt;&lt;/mrow&gt;&lt;/math&gt;), 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 HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; levels. The expression of HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; and related downstream proteins was detected in adult rat brain exposed to MeHg (0-10mg/kg&lt;math&gt;&lt;mrow&gt;&lt;mn&gt;0&lt;/mn&gt;&lt;mo&gt;-&lt;/mo&gt;&lt;mn&gt;10&lt;/mn&gt;&lt;mspace&gt;&lt;/mspace&gt;&lt;mi&gt;mg&lt;/mi&gt;&lt;mo&gt;/&lt;/mo&gt;&lt;mi&gt;kg&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt;) for 0.5h&lt;math&gt;&lt;mrow&gt;&lt;mn&gt;0.5&lt;/mn&gt;&lt;mspace&gt;&lt;/mspace&gt;&lt;mi&gt;h&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; 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 10μM&lt;math&gt;&lt;mrow&gt;&lt;mn&gt;10&lt;/mn&gt;&lt;mspace&gt;&lt;/mspace&gt;&lt;mi&gt;μ&lt;/mi&gt;&lt;mi&gt;M&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; MeHg for 0.5h&lt;math&gt;&lt;mrow&gt;&lt;mn&gt;0.5&lt;/mn&gt;&lt;mspace&gt;&lt;/mspace&gt;&lt;mi&gt;h&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; significantly inhibited the expression of astrocytic HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt;, and the downstream genes GLUT-1, EPO, and VEGF-A (p&lt;0.05&lt;math&gt;&lt;mrow&gt;&lt;mi&gt;p&lt;/mi&gt;&lt;mo&gt;&lt;&lt;/mo&gt;&lt;mn&gt;0.05&lt;/mn&gt;&lt;/mrow&gt;&lt;/math&gt;), in the absence of a significant decrease in HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; mRNA levels. When protein synthesis was inhibited by CHX, MeHg promoted the degradation rate of HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt;. MG132 and DHB significantly blocked the MeHg-induced decrease in HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; expression (p&lt;0.05&lt;math&gt;&lt;mrow&gt;&lt;mi&gt;p&lt;/mi&gt;&lt;mo&gt;&lt;&lt;/mo&gt;&lt;mn&gt;0.05&lt;/mn&gt;&lt;/mrow&gt;&lt;/math&gt;). Overexpression of HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; significantly attenuated the decline in MeHg-induced cell proliferation, whereas the inhibition of HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; significantly increased the decline in cell proliferation (p&lt;0.05&lt;math&gt;&lt;mrow&gt;&lt;mi&gt;p&lt;/mi&gt;&lt;mo&gt;&lt;&lt;/mo&gt;&lt;mn&gt;0.05&lt;/mn&gt;&lt;/mrow&gt;&lt;/math&gt;). NAC and Trolox, two established antioxidants, reversed the MeHg-induced decline in HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; protein levels and the decrease in cell proliferation (p&lt;0.05&lt;math&gt;&lt;mrow&gt;&lt;mi&gt;p&lt;/mi&gt;&lt;mo&gt;&lt;&lt;/mo&gt;&lt;mn&gt;0.05&lt;/mn&gt;&lt;/mrow&gt;&lt;/math&gt;). MeHg suppressed the expression of HIF-1α&lt;math&gt;&lt;mrow&gt;&lt;mtext&gt;HIF-&lt;/mtext&gt;&lt;mn&gt;1&lt;/mn&gt;&lt;mi&gt;α&lt;/mi&gt;&lt;/mrow&gt;&lt;/math&gt; and related downstream target proteins in adult rat brain.