Acute Methylmercury Exposure and the Hypoxia-Inducible Factor- 1 α Signaling Pathway under Normoxic Conditions in the Rat Brain and Astrocytes in Vitro

Jie Chang, Bobo Yang, Yun Zhou, Changsheng Yin, Tingting Liu, Hai Qian, Guangwei Xing, Suhua Wang, Fang Li, Yubin Zhang, Da Chen, Michael Aschner, Rongzhu Lu
Environmental Health Perspectives 2019, 127 (12): 127006

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

DISCUSSION: MeHg induced a significant reduction in <mml:math xmlns:mml=""&gt; <mml:mrow> <mml:mtext>HIF-</mml:mtext> <mml:mn>1</mml:mn> <mml:mi>α</mml:mi> </mml:mrow> </mml:math> 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 <mml:math xmlns:mml=""&gt; <mml:mrow> <mml:mtext>HIF-</mml:mtext> <mml:mn>1</mml:mn> <mml:mi>α</mml:mi> </mml:mrow> </mml:math> expression in response to MeHg toxicity. Moreover, we established that up-regulation of <mml:math xmlns:mml=""&gt; <mml:mrow> <mml:mtext>HIF-</mml:mtext> <mml:mn>1</mml:mn> <mml:mi>α</mml:mi> </mml:mrow> </mml:math> might serve to mitigate the acute toxicity of MeHg in astrocytes, affording a novel therapeutic target for future exploration.

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