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 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 (0–10μM) 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. 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 (0–10mg/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 10μM MeHg for 0.5h 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


Introduction
Methylmercury (MeHg) is a global environmental contaminant targeting the central nervous system (CNS) (Farina and Aschner 2017;Santos et al. 2016). Consumption of MeHg-containing fish products (Canuel et al. 2006;Carrasco et al. 2011;Steuerwald et al. 2000;Stopford and Goldwater 1975) and rice (Rothenberg et al. 2014;Zhang et al. 2010) has been shown to induce neurodegeneration as well as be associated with neurodevelopmental disorders (Belletti et al. 2002;Carocci et al. 2014;Ceccatelli et al. 2010). However, the underlying cellular and molecular mechanisms of MeHg-induced neurotoxicity have yet to be fully elucidated. Meanwhile, there is still a lack of treatment to effectively protect against MeHg-induced brain damage.
Hypoxia-inducible factor-1 (HIF-1), a DNA-binding transcription factor, plays a crucial role in a diverse range of adaptive responses to oxygen tension. HIF-1 is a heterodimer comprising an oxygen-labile a-submit (HIF-1a) and a constitutively expressed b-submit (HIF-1b). Under normal oxygen conditions (>5% O 2 ), prolyl hydroxylase (PHD) has been shown to mediate hydroxylation of HIF-1a (Berra et al. 2003;Ivan et al. 2001). Hydroxylated HIF-1a is rapidly degraded by the ubiquitin proteasome system (UPS), mediated by interaction with the von-Hippel Lindau tumor suppressor gene product (pVHL) (Bruick and McKnight, 2001;Lee et al. 2007). Under hypoxic conditions (<5% O 2 ) or stimulation by other environmental factors, stabilized HIF-1a dimerizes with HIF-1b to transactivate a series of adaptive genes, including vascular endothelial growth factor A (VEGF-A), glucose transporter 1 (GLUT-1), and erythropoietin (EPO) (Semenza and Wang, 1992). Several metals and organic chemicals have been shown to affect HIF-1a expression and activity (Dong et al. 2016;Lee et al. 2009;Liao et al. 2014;Wikenheiser et al. 2013;Wu et al. 2010), whereas increased expression of HIF-1a has been shown to be protective against neurotoxicants (Feng et al. 2014;Lee et al. 2009;Wu et al. 2010). Manganese has also been shown to increase HIF-1a protein levels in Hep2 cells by regulating mitogen-activated protein kinases (MAPKs) (Shin et al. 2010). Clioquinol (a copper and zinc chelator) decreased HIF-1a degradation by inhibiting HIF-1a ubiquitination and hydroxylation in SH-SY5Y cells and HepG2 cells, whereas addition of copper and zinc reversed these effects (Choi et al. 2006). Cadmium was shown to up-regulate HIF-1a expression in human lung epithelial cells via the production of reactive oxygen species (ROS) through the activation of the protein kinase B (Akt), and extracellular signal-regulated protein kinase (ERK) signaling pathways (Jing et al. 2012). Notably, deferoxamine (DFO), an iron chelator, was shown to attenuate neuronal cell death and the generation of ROS caused by MeHg and methylphenyl tetrahydropyridine (MPTP) in in vivo rodent models LeBel et al. 1992). Furthermore, it was shown that by inhibiting the activity of proline hydroxylase (PHD) in mouse hippocampal neurons, DFO decreased degradation of HIF-1a and increased intracellular levels of HIF-1a protein (Hamrick et al. 2005). Recently, several studies have further documented that HIF-1a is neuroprotective because it drives the expression of critical genes that diminish neuronal cell death Sen and Sen 2016). Increased HIF-1a expression and activity have been shown to promote glycolysis and glucose metabolism, thus countering oxidative stress by producing NADH and NADPH to propagate neuroprotective responses (Soucek et al. 2003). HIF-1a has also been shown to improve cerebral blood flow, which could oppose the toxicity of hypoxia (Iyalomhe et al. 2017). Overexpression of HIF-1a and/or HIF-1a target genes, such as VEGF-A and EPO, may be an early adaptation to the oxidative stressors that characterize MeHginduced neuropathology. Thus, we speculate that the molecular events that constitute this early adaptation are likely neuroprotective and might mitigate neuronal injury caused by MeHg.
Previous studies have indicated an association between HIF-1a activity and intracellular ROS production. ROS was reported to promote HIF-1a accumulation by the inhibition of PHD catalytic activity via the oxidization of PHD-bound Fe 2 + (Koivunen et al. 2007;Pan et al. 2007). However, it has been noted that a paradox exists between them, because the HIF-1a-dependent transcriptional program can prevent mitochondrial ROS generation (Jain et al. 2016). It is unclear whether MeHg-induced toxicity is associated with the expression of HIF-1a, whereas the specific relationship between ROS production and HIF-1a also remains unclear. MeHg predominantly accumulates in the CNS, particularly in astrocytes; the latter have been shown to play an important role in mediating MeHg-induced neurotoxicity (Ceccatelli et al. 2010;Shanker et al. 2003), inhibit glutamate uptake, and contribute to secondary excitotoxicity (Aschner et al. 2000;Deng et al. 2014;Liu et al. 2013Liu et al. , 2014. Astrocytes perform diverse important functions in providing support and nutrition (Abbott 2002;Rudge et al. 1994), inducing neuronal differentiation (Barkho et al. 2006) and mediating immune responses (Aschner 1998a(Aschner , 1998bJensen et al. 2013). Therefore, we aimed to investigate the role of HIF-1a in MeHg-induced acute neurotoxicity by using primary rat astrocytes and adult rats, with special emphasis on an ROS mode of toxicity, so as to further identify novel therapeutic targets against MeHg-induced neuronal injury.

Primary Rat Astrocyte Isolation and Culture
Sprague-Dawley (SD) rats at the 1-2-d postnatal stage were purchased from the Laboratory Animal Center of Jiangsu University.
Primary neonatal rat astrocytes were prepared as previously described (Bai et al. 2015(Bai et al. , 2016Fang et al. 2016;Yang et al. 2018;Yin et al. 2011). The procedures were as follows: Rats were rapidly decapitated, and the cerebral hemispheres were quickly obtained and rinsed with 4°C phosphate buffered solution (PBS). After dissecting away part of the meninges, basal ganglia, midbrain, and blood vessels, the remaining cortical tissues were cut into 1 mm 3 pieces and dissociated with 0.25% trypsin (Gibco) for 15 min at room temperature in the absence of stirring. Cells were grown in Dulbecco's Modified Eagle Medium (DMEM; Hyclone) containing 10% fetal bovine serum (Gibco), and 1% penicillin/streptomycin (Gibco). Culture medium was replaced with fresh medium twice a week, and cultures were maintained in a 5% CO 2 environment at 37°C. Cultured cells were used for experiments when astrocytes reached 75%-85% confluency. Protocols were approved by the Animal Ethics Committee of Jiangsu University of China and were carried out in accordance with the established Guiding Principles for Animal Research.

Cell Proliferation Assay
Cell proliferation was measured in astrocytes cultured in 96-well plates (1 × 10 4 =well) using the MTT assay. At the end of treatment, MTT was added to each well at a final concentration of 0:5 mg=mL and incubated at 37°C for 4 h. Next, the MTT solution was replaced with dimethyl sulfoxide (150 lL=well) for 10 min to dissolve the dark-blue formazan crystals in intact cells, and the absorbance was measured at 490 nm with a Bio-Rad 680 Microplate Reader (Bio-Rad Laboratories).

Lactate Dehydrogenase (LDH) Release Assay
Cytotoxicity was detected using an LDH release assay kit (Jiancheng Bioengineering Institute of Nanjing) according to the manufacturer's protocol. Cells were cultured in 96-well plates (1 × 10 4 =well), and after treatment supernatants were transferred to clean 96-well plates. Absorbance was analyzed at 450 nm using a Bio-Rad 680 Microplate Reader.

Measurement of Intracellular ROS Generation
To measure ROS generation, a fluorometric assay was carried out using the cell membrane permeable dye 2 0 ,7 0 -dichlorofluorescin diacetate (DCFH-DA). In the quantitative experiment of fluorescence intensity, cells cultured in 96-well plate (1 × 10 4 =well) were treated with different concentrations of MeHg (1, 2.5, 5, and 10 lM) for 0:5 h and were then incubated with 10 lmol=L DCFH-DA in serum-free medium for 0:5 h at 37°C. After washing with PBS, the fluorescence intensity was measured with Cytation™ 5 (Biotek), with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. In the imaging experiment, cells cultured in 24-well plates (5 × 10 4 =well) were treated with MeHg (10 lM, 0:5 h) or H 2 O 2 (6 lM, 0:5 h). After washing with PBS, the nuclei were stained with 4 0 ,6-diamidino-2phenylindole (DAPI) for 15 min at room temperature. Then the cells were incubated with 10 lmol=L DCFH-DA in serum-free medium for 0:5 h at 37°C. Finally, the resulting fluorescence was detected with a fluorescence microscope (Zeiss Axio Observer). Cells treated with H 2 O 2 were used as the positive control. The intensity of fluorescence reflects the extent of oxidative stress.

Western Blotting Analysis
To prepare the whole protein lysate, cells were washed three times with cold PBS, lysed in lysis buffer (RIPA lysis buffer containing 1% PMSF, obtained from Beyotime), incubated on ice for 15 min, and collected after 12,000 × g centrifugation at 4°C for 15 min. The protein concentration was determined by a BCA protein assay reagent kit (Beyotime). A total of 60 lg of cell total protein was separated using 10% or 12% SDS-PAGE, then transferred to a PVDF membrane (Millipore). After being blocked with 5% skim milk in TBST (10 mM Tris-HCl, 120 mM NaCl, 0.1% Tween ® 20, pH 7.4) for 2 h at room temperature, the membranes were incubated with specific primary antibodies overnight at 4°C (Table 1). On the second day, the membranes were washed three times with TBST buffer and further incubated with HRP-labeled secondary antibodies at room temperature for 1 h, and then washed three times in TBST. Finally, signal density of the immunoblots was performed using the MiniChemi Mini Size Chemiluminescent Imaging System (Beijing Sage Creation Science Co., Ltd.) and analyzed with Image J software (National Institutes of Health).

Adenovirus-Mediated HIF-1a Overexpression
Adenovirus carrying the HIF-1a gene was obtained from Hanbio Biotechnology Co., Ltd. Astrocytes were seeded in 6-well plates at a 60% confluence, and cells were transfected with pHBAd-EF1-MCS-GFP according to multiplicity of infection ðMOIÞ = 30 × 10 8 PFU=mL 8 h later, cells were supplemented with fresh medium and continuously cultured for an additional 48 h to observe transfection efficiency by means of Western blotting.

siRNA Transfection
The small interfering RNAs (siRNAs) for HIF-1a were designed and purchased from Guangzhou Ribobio Co., Ltd. The sequences of the three siRNAs were as follows: Astrocytes were plated in 6-well plates at 60% confluence, transfected with HIF-1a siRNA (50 nM), or a negative control (NC, a scrambled sequence) siRNA (50 nM) with Lipofectamine ® 2000 in Opti-MEM medium (Invitrogen), according to the manufacturer's protocol. The transfected cells were incubated at 37°C for an additional 48 h and validated the silencing efficacy via Western blotting analysis.

Total RNA Extraction and Quantitative Real-Time PCR (RT-PCR)
Total RNA was extracted from astrocytes using the TRIzol reagent (Invitrogen). Five hundred nanograms of total RNA were reverse-transcribed to cDNA using an RT reagent kit (TaKaRa), which was then amplified with SYBR green dye on a CFX96 Real-Time PCR Detection System (Bio-Rad). The relative quantification of mRNA levels was calculated using the standard 2 -DDCt relative quantification method.

Animal Model
Male SD rats (8 wk old) weighing 200 ± 20 g were obtained from the Laboratory Animal Center of Jiangsu University (Accreditation Number: SCXK [SU] 2018-0012). The rats were housed in a temperature-and humidity-controlled room (22 ± 2 C and 50 ± 10% relative humidity) with a 12 h:12 h light:dark cycle (dark phase from 1900 to 0700) and were maintained on standard laboratory chow with ad libitum access to water and food (n = 6 per cage). The rats were acclimatized for at least 7 d prior to MeHg exposure. Rats were divided randomly into six groups (n = 6): Group I: Normal saline was injected intraperitoneally for 0:5 h Group II: MeHg [2 mg=kg body weight (BW)] was injected intraperitoneally for 0:5 h Group III: MeHg (4 mg=kg BW) was injected intraperitoneally for 0:5 h Group IV: MeHg (6 mg=kg BW) was injected intraperitoneally for 0:5 h Group V: MeHg (8 mg=kg BW) was injected intraperitoneally for 0:5 h Group VI: MeHg (10 mg=kg BW) was injected intraperitoneally for 0:5 h At the end of the exposure period, the rats were anesthetized by intraperitoneal (IP) injection of pentobarbital to alleviate any potential discomfort and pain. Next, the brain cortices were rapidly frozen in liquid nitrogen and transferred to a -80 C freezer. All experimental procedures were conducted following a protocol approved by the Animal Care and Use Committee and Animal Ethics Committee of Jiangsu University (Accreditation Number: SYXK [SU] 2013-0036). All animals used in this study were treated humanely according to the institutional guidelines, with full consideration for the alleviation of distress and discomfort.

Statistical Analysis
All data are presented as the mean ± standard deviation (SD). All assays were repeated at least three times with at least three independently derived astrocyte cultures. Statistical significance was assessed by analysis of variance (ANOVA) with correction for multiple comparisons in post hoc analysis. CHX treatment assay was analyzed by a two-way ANOVA with Dunnett's post hoc test. Other multiple comparisons were analyzed by one-way ANOVA with Dunnett's or Tukey's post hoc test. All statistical analyses were performed using GraphPad Prism software. A p value of <0:05 was considered to be statistically significant.

Effects of Methylmercury on Astrocyte Proliferation, Cytotoxicity, and Production of ROS
To investigate the effects of MeHg on cell proliferation and cytotoxicity in astrocytes, we incubated astrocytes with this organometal (0-10 lM) for 0:5 h. The MTT assay and LDH leakage were subsequently assessed. As shown in Figure 1A, compared with control cells, those treated with MeHg exhibited lower cell proliferation, which appeared to be concentration-dependent. MeHg treatment significantly increased LDH release in astrocytes, especially at concentrations of 5 and 10 lM ( Figure 1B). Exposure to MeHg (5 and 10 lM, 0:5 h) induced a ∼1.6-fold increase in astrocytic LDH release compared with controls (p < 0:05). Next, we analyzed the effects of MeHg on ROS production by 2 0 ,7 0 -dichlorofluorescin diacetate (DCFH-DA) fluorescence. As is shown in Figure 1C, MeHg at 5 and 10 lM caused a significant increase in ROS formation. Additionally, fluorescence images also showed that the fluorescence intensity of ROS was obviously enhanced in MeHg (10 lM, 0:5 h) group ( Figure 1D).

Effects of Methylmercury on the Expression of HIF-1a-Related Proteins in Astrocytes
We incubated astrocytes in the absence or presence of MeHg (1-10 lM) for 0:5 h and assessed HIF-1a and HIF-1b expression by Western blotting. MeHg-treated astrocytes exhibited a significant reduction in HIF-1a expression in a concentrationdependent manner (Figure 2A). In contrast, HIF-1b remained unaffected irrespective of the MeHg concentration ( Figure 2B). Next, we analyzed the expression of HIF-1a targets (GLUT-1, EPO, and VEGF-A) in astrocytes treated with various concentrations of MeHg. A significant decrease in the expression of GLUT-1, EPO, and VEGF-A proteins was observed in astrocytes treated with 10 lM MeHg for 0:5 h (p < 0:05) ( Figure  2C). These results are consistent with the ability of MeHg to decrease astrocytic HIF-1a levels, followed by a decrease in, GLUT-1, EPO, and VEGF-A expression.

Effects of Protein Synthesis, Proteasome, and PHD Inhibitors on Protein Expression of HIF-1a and Downstream Effectors in Methylmercury-Treated Astrocytes
To determine the means by which MeHg regulated HIF-1a expression, we analyzed the expression of the HIF-1a gene with RT-PCR. As shown in Figure 3A, MeHg pretreatment did not affect HIF-1a mRNA levels. Therefore, we further explored the potential causes of the significant reduction in HIF-1a levels caused by MeHg in astrocytes. CHX is a broad-spectrum and nonspecific protein synthesis inhibitor, and astrocytes were harvested after CHX pretreatment (400 lM) for 0.5, 1, and 2 h with and without MeHg treatment (10 lM, 0:5 h). In addition, MeHg treatment alone group (10 lM for 0.5, 1, and 2 h) was also established. Then, HIF-1a protein expression was analyzed ( Figure  3B). In comparison with the control group, the protein level of HIF-1a in CHX alone group decreased in a time-dependent manner. Notably, when compared with the corresponding time point of CHX treatment alone group, the level of HIF-1a protein was found to be significantly lower in the CHX + MeHg group (p < 0:05, Figure 3B). We pretreated astrocytes with or without MG132 (1 lM, 24 h), a proteasome inhibitor, followed by 10 lM MeHg, and incubated the cells for an additional 0:5 h. The cells were harvested at the end of the treatment, and the levels of HIF-1a and its targets were measured via Western blotting. As shown in Figure 3C, MG132 treatment inhibited the significant MeHg-induced reduction in HIF-1a and its target proteins in astrocytes. Previous research has suggested that HIF-1a degradation is attributable to the activation of proline hydroxylase (PHD) Figure 2. Effects of MeHg on the expression of HIF-1a-related proteins in astrocytes. (A) Western blotting for HIF-1a in astrocytes following MeHg (0, 1, 2.5, 5 or 10 lM, 0:5 h) exposure. (B) Western blotting for HIF-1b in astrocytes following MeHg (0, 1, 2.5, 5, or 10 lM, 0:5 h) exposure. (C) Effect of MeHg (0, 1, 2.5, 5, or 10 lM, 0:5 h) exposure on the expression of the downstream proteins of HIF-1a, including GLUT-1, EPO, and VEGF-A. b-actin was used as the internal control. Note: Data are presented as mean ± SD from three independent experiments (n = 3). con, control (culture medium treatment without MeHg); EPO, erythropoietin; GLUT-1, glucose transporter 1; HIF-1a, Hypoxia-inducible factor-1a; HIF-1b, Hypoxia-inducible factor-1b; MeHg, methylmercury; VEGF-A, vascular endothelial growth factor A. * p < 0:05 vs. control by one-way ANOVA with Dunnett's post hoc test. (Marxsen et al. 2004). To define the potential contribution of PHD-mediated inhibition of HIF-1a in response to MeHg treatment, we treated astrocytes with 1 mM DHB [a small-molecule prolyl hydroxylase (PHD) inhibitor] (Siddiq et al. 2005) for 6 h and exposed astrocytes to 10 lM MeHg for 0:5 h. Notably, compared with cells treated with MeHg alone, astrocytic HIF-1a expression in cells treated with MeHg and either the proteasome inhibitor ( Figure 3C) or the PHD inhibitor ( Figure 3E) was significantly higher. In addition, both the proteasome inhibitor and PHD inhibitor restored MeHg-inhibited cell proliferation ( Figure 3D and 3F).

HIF-1a Protein Expression and Cell Proliferation after Pretreatment with Antioxidants in Methylmercury-Treated Astrocytes
We investigated whether ROS production were associated with decreased HIF-1a expression in MeHg-exposed astrocytes. Astrocytes were pretreated with NAC (2:5 mM, 4 h) and Trolox (2:5 mM, 4 h) followed by 10 lM MeHg for 0:5 h. NAC and Trolox are antioxidants that are commonly used to mitigate ROS (Aremu et al. 2008;Bai et al. 2015;Kaur et al. 2010). As shown in Figures 5A and 5C, cells pretreated with either NAC or Trolox and then treated with MeHg exhibited higher HIF-1a protein levels, similar to those of control cells, than those treated with MeHg alone. Pretreated cells exhibited higher levels (as much as ∼ 30%) of cell proliferation than those treated with MeHg alone (p < 0:05) ( Figure 5B and 5D).

Protein Expression of HIF-1a and Downstream Effectors following MeHg Administration in in Vivo Rat Models
Finally, to evaluate the in vivo effects of MeHg on HIF-1a protein expression, we established a SD rat model. Animals were administered with MeHg (0, 2, 4, 6, 8, and 10 mg=kg, each group with 6 rats) by intraperitoneal injection and sacrificed 0:5 h later. The expression of HIF-1a and its target proteins in brain tissues was measured by Western blotting. As shown in Figure 6, MeHg treatment significantly decreased the expression of HIF-1a and its targets, especially in rats exposed to 8 mg=kg and 10 mg=kg MeHg (p < 0:05).

Discussion
In the present study, we report that, compared with the vehicle control, treatment with 5 or 10 lM MeHg for 0:5 h resulted in lower cell proliferation and higher ROS generation in primary rat astrocytes, and these results were associated with lower expression of HIF-1a. To define the mechanism(s) of the MeHginduced alteration in HIF-1a expression, first we detected the mRNA level of HIF-1a after MeHg treatment. The results showed that MeHg exposure resulted in a significant reduction in HIF-1a levels without affecting HIF-1a transcription. Then, we speculated that whether MeHg affected the degradation of HIF-1a protein. The results showed that treating cells with CHX + MeHg results in lower protein expression of HIF-1a than does treating cells with CHX alone in three different time points. Because CHX blocks protein synthesis, it would stand to reason that the effect of MeHg in these cells is associated with protein degradation. Additionally, we also demonstrated that the proteasome inhibitor (MG132) and the PHD inhibitor (DHB), both of which block protein degradation of HIF-1a, partially restored the inhibitory effect of MeHg on HIF-1a protein, further suggesting that the MeHg-induced decrease in HIF-1a protein is due to the promoted degradation. Furthermore, taking advantage of genetic and pharmacological methods to alter inherent levels of the HIF-1a protein, we provide evidence directly linking MeHg toxicity with HIF-1a inhibition. It is noteworthy that lower HIF-1a protein expression was related to the production of ROS, as demonstrated by NAC and Trolox. Taken together, our Data are presented as the mean ± SD from three independent experiments (n = 3). con, control (culture medium treatment without MeHg); HIF-1a, Hypoxia-inducible factor-1a; MeHg, methylmercury; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl diphenyltetrazolium bromide; NAC, N-acetyl-cysteine, Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. * p < 0:05 vs. control, # p < 0:05 vs. MeHg-only group. novel observations suggest that ROS act as an important regulator of HIF-1a inhibition in MeHg-treated astrocytes and that overexpression of HIF-1a may afford a novel neuroprotective effect in the treatment of MeHg-induced neurotoxicity. Moreover, we also found that MeHg inhibited the expression of HIF-1a in the SD rat brain, which better revealed the relationship between MeHg and the HIF-1a signaling pathway found in the in vitro and in vivo experiments.
MeHg is an established neurotoxicant (Aschner and Syversen 2005;Clarkson and Magos 2006;Culbreth and Aschner, 2016;Farina et al. 2011aFarina et al. , 2011b, whose mechanism(s) of toxicity have yet to be fully understood; however, oxidative stress seems to play a role (Aschner et al. 2007;Santos et al. 2018;Ishihara et al. 2016). Oxidative stress is one of the known mechanisms for the induction of HIF-1a. Previous studies have consistently shown that metals, such as lead (Das et al. 2015), aluminum (Mailloux and Appanna 2007), manganese (Han et al. 2009), and zinc (Pan et al. 2013;Park et al. 2018), as well as other xenobiotics, such as isoflurane (Cao et al. 2018;Sun et al. 2013), can increase HIF-1a expression. In contrast, concentration-dependent inhibition of HIF-1a in Hep3B cells has been reported in response to cadmium and inorganic mercury (Hg) treatment (Horiguchi et al. 2000).
We speculated that two factors contribute to the decreased level of HIF-1a protein in response to MeHg treatment. First, MeHginduced mitochondrial damage might cause a reduction in oxygen consumption and increase relative oxygen content, leading to redistribution of oxygen and binding to PHD and an increase in PHD activity (Wu et al. 2010), whereas other metals, including inorganic mercury, mimicked a hypoxic state and induced pseudohypoxia to promote the translocation of HIF-1a (Mailloux and Appanna 2007). MeHg did not change the level of HIF-1a in brain microvascular pericytes (Hirooka et al. 2013). Second, MeHg might increase 2oxoglutarate (2-OG) levels, a cofactor of PHD. 2-OG has been shown to increase PHD activity, resulting in increased degradation of HIF-1a and a net decrease in HIF-1a protein (Dehne et al. 2010;Mailloux et al. 2009;Matsumoto et al. 2006). As shown in Figure  3, MeHg appeared to have activated PHD and UPS, thus promoting HIF-1a degradation.
HIF-1a regulates the expression of a large number of genes that are involved in glucose metabolism, energy production, angiogenesis, migration, and other cellular processes Semenza 2002). Thus, reduced availability of HIF-1a and the subsequent reduction in expression of its target genes may cause cellular dysfunction. VEGF-A, GLUT-1, and EPO are typical downstream target proteins of HIF-1a (Ashok et al. 2017;Cramer et al. 2003;Mense et al. 2006;Semenza 2002). VEGF-induced angiogenesis has previously been shown to improve the blood supply to neurons and slow the clinical deterioration in Alzheimer's disease (Bogaert et al. 2006). VEGF and EPO have been shown to improve tissue oxygen content by promoting erythropoiesis and angiogenesis, which may stimulate genes involved in glucose transport and metabolism (Ashok et al. 2017;Semenza 2002). It is noteworthy that HIF-1a complexes can regulate the biological functions of various genes, such as those involved in vascular growth, oxygen transport, and energy metabolism (Czibik 2010). Thus, we suggest that the mechanism of MeHg neurotoxicity involves, at least in part, a reduction in HIF-1a and its downstream proteins (GLUT-1, EPO, and VEGF-A), leading to secondary alterations in blood flow, tissue oxygenation, and ATP production. In addition, studies on the regulation of PHD have generated interest as investigational drugs for anemia and neurodegenerative diseases. The use of a small molecule of HIF-1a PHD inhibitor (FG-4592), has been promoted to clinical trials for treating anemia by increasing erythropoiesis through HIF-1a mediated transcription (Besarab et al. 2016;Chen et al. 2017;Haase 2017), as well as for treating Parkinson's disease by HIF-1a-targeted positive regulation of redox biology and mitochondrial function ). In the present study, the suppression of MeHg toxicity by both CoCl 2 and adenovirustargeted HIF-1a overexpression suggested that activation of the HIF-1a pathway can reverse inhibition of cell proliferation by MeHg ( Figure 4). Thus, we suggest that HIF-1a can be pursued as a novel target for MeHg poisoning.
MeHg-induced toxicity is mediated, at least in part, by oxidative stress (Polunas et al. 2011;Yee and Choi 1996). In addition, rapid degradation of HIF-1a may result from ROS generation (Beattie et al. 2005;Cairo et al. 1996;Henkel and Krebs 2004;Jaeckel et al. 2005;Li et al. 2006;Sato and Kondoh 2002). We initially considered that ROS generation could play a role in the altered HIF-1a protein expression to mediate MeHg toxicity. Treatment of rat astrocytes with oxygen radical scavengers (NAC and Trolox) resulted in higher HIF-1a protein expression and increased cell proliferation (Figure 5), suggesting that detoxification of ROS by HIF-1a is associated with its neuroprotective effects. Reductions in HIF-1a expression in MeHg-treated astrocytes and rats suggest that the up-regulation of HIF-1a affords neuroprotective effects. Taking into account the limitations of the pharmacological and genetic regulations of clinical toxicity and the ability of antioxidants to promote HIF-1a expression, the use of antioxidants should be considered for clinical detoxification. However, MeHg can affect many cellular and molecular functions if the MeHg exposure concentrations and duration are sufficient. Figure 6. Protein expression of HIF-1a and downstream effectors following MeHg administration in in vivo rat models. Adult Sprague-Dawley rats were administered with MeHg (0, 2, 4, 6, 8, 10 mg=kg) by intraperitoneal injection for 0:5 h. Whole brain lysates were analyzed by Western blotting for the indicated proteins. Note: Data are presented as mean ± SD (n = 6 rats=group). con, control (intraperitoneal injection of saline); EPO, erythropoietin; GLUT-1, glucose transporter 1; HIF-1a, Hypoxia-inducible factor-1a; MeHg, methylmercury; VEGF-A, vascular endothelial growth factor A. * p < 0:05 vs. control group by one-way ANOVA with Dunnett's post hoc test.
Thus, additional research on the toxicological significance of HIF-1a suppression by acute exposure to MeHg is warranted, in addition to a more precise delineation of its protective mechanisms.
In conclusion, we demonstrated that MeHg led to astrocytic neurotoxicity in vitro by reducing levels of HIF-1a and its downstream proteins, which may result from increasing PHD activity, which in turn promoted HIF-1a degradation. Furthermore, the up-regulation of HIF-1a imparted protection against MeHg neurotoxicity. Taken together, these findings-from both in vivo and in vitro experiments-suggest that HIF-1a may be as of yet an unrecognized and important mediator or regulator of MeHginduced neurotoxicity, and this means of increasing HIF-1a activation in astrocytes may afford a therapeutic strategy for mitigating MeHg poisoning.