Acute methylmercury exposure and the hypoxia-inducible factor-1a 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

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Abstract

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 factor-1α (HIF-1α), 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 HIF-1α 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 lM) for 0:5 h. 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 20,70-dichlorofluorescin diacetate (DCFH-DA) fluorescence. HIF-1α, 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 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 inhibition by MeHg. To investigate the role of HIF-1α in MeHg-induced neurotoxicity, cobalt chloride (CoCl2 ), 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 levels. The expression of HIF-1α and related downstream proteins was detected in adult rat brain exposed to MeHg (0–10 mg=kg) for 0:5 hin 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 lM MeHg for 0:5 h significantly inhibited the expression of astrocytic HIF-1α, and the downstream genes GLUT-1, EPO, and VEGF-A (p <0:05), in the absence of a significant decrease in HIF-1α mRNA levels. When protein synthesis was inhibited by CHX, MeHg promoted the degradation rate of HIF-1α. MG132 and DHB significantly blocked the MeHg-induced decrease in HIF-1α expression (p <0:05). Overexpression of HIF-1α significantly attenuated the decline in MeHg-induced cell proliferation, whereas the inhibition of HIF-1α significantly increased the decline in cell proliferation (p <0:05). NAC and Trolox, two established antioxidants, reversed the MeHg-induced decline in HIF-1α protein levels and the decrease in cell proliferation (p <0:05). MeHg suppressed the expression of HIF-1α and related downstream target proteins in adult rat brain. DISCUSSION: MeHg induced a significant reduction in HIF-1α 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 expression in response to MeHg toxicity. Moreover, we established that up-regulation of HIF-1α 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.

Original languageEnglish (US)
Article number127006
JournalEnvironmental health perspectives
Volume127
Issue number12
DOIs
StatePublished - Jan 1 2019

ASJC Scopus subject areas

  • Public Health, Environmental and Occupational Health
  • Health, Toxicology and Mutagenesis

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