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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: Toxicol Lett. 2020 May 24;331:82–91. doi: 10.1016/j.toxlet.2020.05.023

Differential susceptibility of PC12 and BRL cells and the regulatory role of HIF-1α signaling pathway in response to acute methylmercury exposure under normoxia

Tingting Liu a, Qianqian Gao a, Bobo Yang a, Changsheng Yin b,a, Jie Chang a, Hai Qian a, Guangwei Xing a, Suhua Wang a, Fang Li a, Yubin Zhang c, Da Chen d, Jiyang Cai e, Haifeng Shi b, Michael Aschner f, Kwaku Appiah-Kubi g, Dawei He h, Rongzhu Lu a,h,*
PMCID: PMC7366344  NIHMSID: NIHMS1608117  PMID: 32461003

Abstract

Hypoxia-inducible factor 1 (HIF-1) is a critical nuclear transcription factor for adaptation to hypoxia. HIF-1α, the regulatable subunit of HIF-1, is a cytoprotective factor that regulates various physiological activities. We used rat adrenal pheochromocytoma (PC12) cells and the rat hepatocyte cell line BRL to examine the effects of methylmercury (MeHg). Treatment with MeHg led to time- and concentration-dependent toxicity in both lines with statistically cytotoxic effects at 5 μM and 10 μM in PC12 and BRL, respectively, at 0.5 hours. The protein level of HIF-1α was significantly decreased at 2.5 and 5 μM MeHg in PC12 and BRL cells, respectively. Furthermore, MeHg reduced the protein levels of the HIF-1α target proteins glucose transporter-1, vascular endothelial growth factor–A and erythropoietin. Overexpression of HIF-1α significantly attenuated MeHg-induced toxicity in both cell types. Notably, cobalt chloride, a pharmacological inducer of HIF-1α, significantly attenuated MeHg-induced toxicity in BRL cells but not in PC12 cells. An inhibitor of prolyl hydroxylase (PHD), 3, 4-Dihydroxybenzoic acid (DHB), and the proteasomes inhibitor MG132 antagonized the toxicity of MeHg in both cell lines. However, 2-Methoxyestradiol, a HIF-1α inhibitor, significantly increased MeHg-induced toxicity in both cell lines. These data establish that (a) neuronal PC12 cells are more sensitive to MeHg than non-neuronal BRL cells (b) HIF-1α plays a similar role in MeHg-induced toxicity in both cell lines and (c) upregulation of HIF-1α by different methods offers greater cytoprotection against the toxicity of MeHg in PC2 and BRL cell lines.

Keywords: Methylmercury, HIF-1α, PC12, BRL, Susceptibility

1. Introduction

Mercury is a naturally occurring element, found as both inorganic and organic form (Clarkson., 2002;Clarkson., 2006). The primary sources of mercury are man-made emissions from mining, coal burning and other fossil fuel byproducts (Streets et al., 2011). Microorganisms generate organic mercury including methylmercury (MeHg) by promoting the methylation of inorganic mercury, biologically enriching it through the food chain, especially in aquatic fish and mammals. MeHg is the most common form of organic mercury and represents the most considerable human health risk (Clarkson et al., 2006), notably via consumption of MeHg-adulterated fish and seafood (Mahaffey et al. 2004; Sunderland et al., 2018; Tang et al., 2018), especially during pregnancy (Rosa-Silva et al. 2019). Rice is another source of mercury exposure (Gong et al., 2018), especially in China (Zhang et al., 2010). The gastrointestinal tract absorbs approximately 95% of ingested MeHg and widely distributed to liver (López-Berenguer et al., 2019; Ji et al., 2006, Mori et al., 2007; Cuello et al., 2010; Macedo-Júnior et al., 2017), kidneys and the central nervous system (CNS) (Kershaw et al., 1980；Bridges et al., 2010; Khadra et al., 2019). MeHg toxicity in humans can lead reduction of peripheral vision, language impairment, hearing loss, memory loss and motor system disease (Goldberg, 2010; 1994; Grandjean et al., 2011). The mechanisms of the toxicity of MeHg have been linked to induction of oxidative stress (Chen et al., 2020), mitochondrial dysfunction (Qu et al., 2013) and calcium dyshomeostasis (Dreiem et al., 2007; Liu et al., 2019).

Toxic metals, such as cobalt (Lauryn et al., 2013; Bahadori et al., 2019), copper (Martin et al., 2005), nickel (Davidson et al., 2006), aluminum (Al) (Mailloux et al., 2009), and cadmium (Jing et al., 2012; Fujiki et al., 2017) stabilize HIF-1α. Stabilized HIF-1α dimerizes with HIF-1β, which can bind to hypoxia-response elements on target DNA to activate transcription of a series of downstream target genes, including glucose transporter-1 (GLUT-1) (Semenza and Wang., 1992; Hoskin et al., 2003), vascular endothelial growth factor-A (VEGF-A) (Pore et al., 2006; Xu et al.,2018) and erythropoietin (EPO) (Semenza et al., 1991). Upregulation of HIF-1α can significantly antagonize the toxicity of several drugs and toxins by regulating HIF-1α targets (Mailloux et al., 2011; Guo et al., 2016). Yasutake et al., (1997) found that prolonged exposure to MeHg was associated with anemic symptoms in humans, possibly related to the inhibition of EPO, a target of HIF-1α which is reported to play a neuroprotective role on neuronal damage via its anti-apoptotic action (Merelli et al., 2019). Our previous study has proved that the toxicity of MeHg is closely related to the downregulation of HIF-1α (Chang et al., 2019). But the tissue-specific role of HIF-1α in antagonizing toxicity of MeHg remains unclear. The liver and brain are the primary sites of oxygen sensor expression and they also are the most active organs during metabolism and the brain is the first to be protected organs in response to hypoxia (Zhang et al., 2017). To have a complete picture, we thus examined the effect of MeHg on HIF-1α expression and further explored the role of HIF-1α in mediating cytotoxicity and cytoprotection in PC12 and BRL cells.

2. Materials and Methods

2.1. Chemicals and reagents

MeHg, cobalt chloride (CoCl2), 3, 4-Dihydroxybenzoic acid (DHB), MG132, dimethyl sulfoxide (DMSO), and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA), and 2-methoxyestradiol (2-MeOE2) was from Selleck Chemicals (Houston, TX, USA). For Western blot analysis, polyclonal antibodies to HIF-1α were obtained from ImmunoWay (Plano, TX, USA), while those for EPO, GLUT-1 and VEGF-A were obtained from ABclonal (Cambridge, MA, USA). Anti-HIF-1β and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Nerve growth factor (NGF) was purchased from Harlan Laboratories (Houston, TX, USA).

2.2. Cell culture

PC12 is derived from a rat adrenal pheochromocytoma (Greene and Tischler, 1976). After exposure to nerve growth factor (NGF), PC12 cells undergo nerve differentiation (Greene et al., 1976; Parran et al., 2001), so we chose PC12 cells as neuronal cells. In our study, PC12 cells were differentiated in DMEM with 50 ng/ml nerve growth factor and 1 % fetal bovine serum for 9 days. To further assess the toxicity of MeHg to liver and compare the differences with the CNS, we chose normal rat liver cell line BRL cell as non-neuronal cells which is widely used in the study of hepatotoxicity (Liu et al., 2019). PC12 and BRL cells were purchased from the Institute of Cell Biology, Chinese Academy of Science (Shanghai, China). PC12 cells were induced to neuronal differentiation with nerve growth factor. Both cells were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cells were maintained at 37°C in a 5% CO2 environment. The cultured cells were replaced with fresh media twice a week. Cultured cells were used for experiments when they reached 75–85% confluence.

2.3. MTT cell viability assay

Cells were seeded at 5 × 103 cells/well in 96-well plates, incubated for 24 h, then treated with MeHg (at the indicated concentrations) and other agents. PC12 and BRL cells were cultured in 96-well plates and treated with graded concentrations of MeHg (0, 1, 2.5, 5, 10 μM) for 0.5 to 6 h. After pretreatment with CoCl2 (200 μM, 0.5 h), 2-MeOE2 (10 μM, 0.5 h), DHB (1 mM, 6 h) and MG132 (1 μM, 24 h), PC12 or BRL cells were washed twice with phosphate-buffered saline (PBS) and incubated in fresh media with 10 μM MeHg for 0.5 h. MTT solution (0.5 mg/mL) was added to the MeHg-treated cells and incubated for an additional 4 h; the addition of DMSO dissolved dark-blue formazan crystals in viable cells, and the optical density at 490 nm was read with a Bio-Rad 680 Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA).

2.4. Lactate dehydrogenase (LDH) release assay

Cell cytotoxicity was determined using an LDH-release assay kit (Jiancheng Bioengineering Institute of Nanjing, Jiangsu, China). Cells (1 × 104/well) were incubated in 24-well plates for 24 hours. Following MeHg treatment, intracellular LDH was released into the culture supernatant by damaged cells. The supernatant was transferred into 96-well plates and measured according to manufacturer’s protocol. The absorbance was detected at 450 nm using a Bio-Rad 680 Microplate Reader. The absorbance value is proportional to the degree of cell damage. Control groups were treated with free media without any agents

2.5. Western blot analysis

To prepare whole protein lysates, the test cells grown in 6-well plates were washed three times with cold PBS, lysed in a radioimmunoprecipitation assay lysis buffer (RIPA) containing 1% phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China), incubated on ice for 10–15 minutes, and collected by centrifugation at 12,000 g at 4°C for 15 minutes. The protein concentration was determined using a BCA protein assay reagent kit (Beyotime, Shanghai, China). Standard Western blotting procedure was as follows: extracted protein was subjected to SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Darmstadt, Germany). Membranes were blocked with 5% nonfat milk and incubated overnight at 4°C with primary antibodies including HIF-1α, GLUT-1, VEGF-A, EPO or β-actin. Membranes were then washed three times in TBST (10 mM Tris–HCl, 120 mM NaCl, 0.1% Tween-20, pH 7.4) buffer before incubation with the secondary antibody. The enhanced chemiluminescence method was used to develop the signals, and membranes were scanned with the MiniChemi Mini Size Chemiluminescent Imaging System (Beijing Sage Creation Science Co. Ltd, Beijing, China) and Image J software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the band density.

2.6. Adenovirus transfection

Adenovirus carrying the HIF-1α gene was obtained from Hanbio Biotechnology Co., Ltd (Shanghai, China). The cells were seeded into 6-well plates with 5 × 104 cells/well and transfected with commercially available HIF-1α or control adenovirus according to MOI (multiplicity of infection=100×108 PFU/mL following the manufacturer’s instructions. Virus solution was diluted in serum-free DMEM medium, and then added to the 6-well plates and incubated at 37°C for 6 hours. The culture media containing the virus was removed and replaced with high-glucose DMEM containing 10% FBS. Experiments were carried out after 48 hours of culture.

2.7. Real-time reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated from the cells using Trizol reagent. RNA samples (2 μg) were reverse-transcribed to generate first-strand cDNA. The primers for rat HIF-1α and β-actin were designed and synthesized by Shanghai Generay Biotech Co., Ltd (Shanghai, China). The following primers were used to amplify HIF-1α mRNA (forward, 5′-TCACAAATCAGCACCAAGCAC-3′; reverse: 5′-AAGGGGAAAGAACAAAACACG-3′) and β-actin mRNA as an internal standard (forward, 5′-CCTAGACTTCGAGCAAGAGA-3′; reverse, 5′-GGAAGGAAGGCTGGAAGA-3′). Fold changes in the expression of the HIF-1α gene were calculated using a comparative threshold cycle (Ct) method using the formula 2−(ΔΔCt).

2.8. Statistical analysis

The results are presented as mean ± standard deviation. Statistical analyses employed a one-way analysis of variance followed by a Dunnett test, a multiple comparison procedure. The assays were repeated at least three times in at least three independently derived PC12 and BRL cell cultures. Significance was taken as p< 0.05.

3. Results

3.1. MeHg reduced cell viability and induced cytotoxicity in PC12 and BRL cells

MeHg caused a time- and concentration-dependent decrease in cell viability in both cell types. The half-maximal inhibitory concentration values of the MTT cell viability reduction varying intervals up to 6 hours in the presence of MeHg at 5 μM or 10 μM in PC12 and BRL cells, respectively. At the 0.5 h incubation, PC12 and BRL cell viability was decreased to 69.5 ± 2.4% and 77.9 ± 3.7% of controls, respectively (p < 0.05) (Fig. 1A). Accordingly, 0.5 h of treatment with 10 μM MeHg was used for subsequent experiments. LDH release was increased in a concentration-dependent manner and was significantly elevated relative to controls (p < 0.05) in PC12 cells treated for 0.5 hours with 2.5 μM MeHg and 10 μM MeHg in BRL cells (Fig. 1B). Accordingly, 0.5 h of treatment with 10 μM MeHg was used for subsequent experiments.

Fig. 1.

Fig. 1

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Effects of methylmercury (MeHg) on cell viability and cytotoxicity in PC12 and BRL cells. PC12 and BRL cells were treated with various concentrations (1–10 μM) of MeHg for 0.5 to 6 h. Cell viability (A) was measured by the MTT assay and cytotoxicity (B) was detected via lactate dehydrogenase (LDH) release. Data show mean ± standard deviation (n = 3).

*p < 0.05, compared with the control group. Statistical analysis was performed by one-way analysis of variance followed by a Dunnett test, a multiple comparison procedure.

3.2. MeHg reduced the HIF-1α protein and its downstream targets, but did not affect the level of HIF-1α mRNA

To investigate whether the toxicity of MeHg was associated with altered the protein level of HIF-1α, PC12 and BRL cells were treated with MeHg (0, 1, 2.5, 5, 10 μM) for 0.5 h. The protein levels of HIF-1α and HIF-1β, the persistently expressed subunit that is also known as aryl hydrocarbon receptor nuclear translocator (ARNT), and the downstream targets of HIF-1α, GLUT-1, VEGF-A and EPO, were evaluated by Western blotting. As shown in Figure 2A, MeHg treatment for 0.5 h significantly reduced the protein level of HIF-1α at 2.5 μM in PC12 cells and 5 μM in BRL cells. With 2.5, 5, 10 μM MeHg, the protein level of HIF-1α decreased to ~12% and ~54% in PC12 and BRL cells, respectively. Measurement of HIF-1α mRNA using RT-PCR did not reveal significantly altered mRNA levels under treatment with MeHg (Fig. 2B). The levels of the HIF-1β did not change significantly after MeHg treatment (Fig. 2C), whereas the HIF-1α-targeted proteins VEGF-A, GLUT-1 and EPO did decrease with increasing MeHg concentration (Fig. 2D). The decrease in HIF-1α was more pronounced in PC12 cells than that in BRL cells, consistent with the heightened sensitivity of PC12 to MeHg. Notably, MeHg decreased the protein levels of GLUT-1 at 1μM in PC12 and 5 μM in BRL cells.

Fig. 2.

Fig. 2

Fig. 2

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Protein levels of HIF-1α and its downstream targets in PC12 and BRL cells after treatment with methylmercury (MeHg). A) Western blotting of HIF-1α in cells treated with different concentrations (1–10 μM) of MeHg for 0.5 h. B) Quantitative RT-PCR was used to measure HIF-1α mRNA expression. C) Western blotting was used to detect the protein level of HIF-1β. D) The downstream proteins of HIF-1α glucose transporter-1 (GLUT-1), vascular endothelial growth factor-A (VEGF-A), and erythropoietin (EPO) after exposure to various concentrations (1–10 μM) of MeHg. β-Actin served as a loading control. Data show mean ± standard deviation (n = 3).

*p < 0.05, compared with the control group. Control groups were treated with free media without any agents. Statistical analysis was performed by one-way analysis of variance followed by a Dunnett test, a multiple comparison procedure.

3.3. Overexpression of recombinant HIF-1α attenuated MeHg-induced cytotoxicity in PC12 and BRL cells

To investigate the role of HIF-1α in MeHg-induced toxicity, we used genetic manipulation to overexpress HIF-1α. At an MOI of 100 × 108 PFU/mL, HIF-1α levels significantly increased compared with control (Fig. 3A). Compared with MeHg alone, pretreatment with adenoviral HIF-1α before treatment with MeHg significantly increased the protein level of HIF-1α and its downstream proteins (Fig. 3B–C). Overexpresssion of HIF-1α also attenuated the cytotoxicity induced by MeHg in both cell lines (Fig. 3D). These results indicated that the cytotoxicity of MeHg was significantly inhibited by the upregulation of HIF-1α in both cell types, establishing that MeHg-induced cytotoxicity can be antagonized by upregulating the protein level HIF-1α.

Fig. 3.

Fig. 3

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Effects of adenoviral overexpression of HIF-1α on methylmercury (MeHg)-induced acute cell injury in PC12 and BRL cells. A) Western blotting of HIF-1α protein in cells after 48 hours of transfection with commercially available HIF-1α or control adenovirus. B, C) Transfected cells were treated with MeHg (10 μM for 0.5 hours), followed by western blot analysis of HIF-1β and downstream proteins glucose transporter-1 (GLUT-1), vascular endothelial growth factor-A (VEGF-A), and erythropoietin (EPO). D) Cell viability was measured by MTT assay. *p < 0.05, compared with the control group; #p < 0.05, compared with null+MeHg group. Control groups were treated with free media without any agents. Statistical analysis was performed by one-way analysis of variance followed by a Dunnett test, a multiple comparison procedure.

3.4. Pharmacological induction of HIF-1α and its targets by CoCl2 protected BRL but not PC12 cells against MeHg-induced acute cytotoxicity

PC12 and BRL cells were treated with 10 μM MeHg for 0.5 hours after pretreatment with CoCl2 (200 μM for 0.5 hours). CoCl2 is commonly used to induce hypoxic conditions, which leads to the induction of HIF-1α protein (Tripathi et al., 2019; Yu et al., 2019). As shown in Figure 4A–B, the levels of HIF-1α, GLUT-1, VEGF-A and EPO significantly increased (p < 0.05) after pretreatment with CoCl2 compared with MeHg alone. The viability of BRL cells upon pretreatment with CoCl2 before MeHg was significantly increased (p < 0.05) compared with that under MeHg treatment alone (Fig. 4C). However, in PC12 cells, CoCl2 pretreatment did not significantly enhance cell viability. These results indicated that CoCl2 affords protection by inducing upregulation of HIF-1α in non-neuronal cells, but not neuronal cells which are more sensitive to oxygen-induced cytotoxicity.

Fig. 4.

Fig. 4

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Effects of cobalt chloride (CoCl2) on methylmercury (MeHg)-induced acute cell injury. PC12 and BRL cells were grown with or without pretreatment with CoCl2 (200 μM for 0.5 h) followed by treatment with MeHg (10 μM). A, B) Protein expression of HIF-1α and downstream targets glucose transporter-1 (GLUT-1), vascular endothelial growth factor-A (VEGF-A), and erythropoietin (EPO) was analyzed by western blotting. β-Actin served as the loading control. C). Cell viability was measured by MTT assay. Data show mean ± standard deviation (n = 3). *p < 0.05, compared with the control group; #p < 0.05, compared with the MeHg group. n, no significance compared with the MeHg group. Control groups were treated with free media without any agents. Statistical analysis was performed by one-way analysis of variance followed by a Dunnett test, a multiple comparison procedure.

3.5. Pharmacological induction of HIF-1α and its targets by DHB attenuated MeHg-induced acute cytotoxicity in PC12 and BRL cells

DHB was reported to act as an iron chelater and its inhibitory actions occur via the displacement of 2-oxoglutarate and ascorbate (Majamaa et al., 1986; Siddiq et al., 2005). Iron, 2-oxoglutarate and ascorbate are the important cofactor of PHD, so DHB is widely used as a PHD inhibitor in the study of upregulation of HIF-1α. Cells were pretreated with 1 mM 3, 4-Dihydroxybenzoic acid (DHB) for 6 hours, then treated with MeHg (10 μM, 0.5 hours). As shown in Figure 5A–B, compared with the control group, DHB alone significantly induced the protein levels of HIF-1α and its targets in both PC12 and BRL cells (p <0.05), while the MeHg-treated group showed significant inhibition the protein levels of HIF-1α and its targets. Upon pretreatment with DHB, the levels of HIF-1α and its downstream targets GLUT-1, VEGF-A and EPO, were significantly higher than those in the MeHg-alone groups, both in PC12 and BRL cells. DHB also mitigated the MeHg-induced decrease in cell viability in PC12. An analogous response was also noted in BRL cells (Fig. 5C). DHB increased cell viability by ~ 15% and ~ 10% in MeHg-treated PC12 and BRL cells, respectively. These results supported the notion that the protective effects of DHB against MeHg-induced cytotoxicity were due to the activation of HIF-1α pathway.

Fig. 5.

Fig. 5

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Effects of 3, 4-Dihydroxybenzoic acid (DHB) on methylmercury (MeHg)-induced acute cell injury. PC12 and BRL cells were pretreated with DHB (1 mM for 6 h), followed by treatment with MeHg. A, B) Protein expression of HIF-1α and downstream targets glucose transporter-1 (GLUT-1), vascular endothelial growth factor-A (VEGF-A), and erythropoietin (EPO) was analyzed by western blotting. β-Actin served as the loading control. C) Cell viability was measured by MTT assay. *p < 0.05, compared with the control group; #p < 0.05, compared with the MeHg group. Control groups were treated with free media without any agents. Statistical analysis was performed by one-way analysis of variance followed by a Dunnett test, a multiple comparison procedure.

3.6. Pharmacological induction of HIF-1α and its targets by MG132 attenuated MeHg-induced acute cytotoxicity in PC12 and BRL cells

The 26s proteasome finally degrades PHD-hydroxylated HIF-1α after interaction with the von Hippel Lindau tumor suppressor protein (Schofield et al., 2004; Domene and Illingworth 2012). A blocker of the proteasome, MG132 (Wang et al., 2019), was used to investigate the protective role of HIF-1α in the toxicity of MeHg. MG132 was protects against the degradation of the HIF-1 complex in cells transferred from hypoxia to normoxia and is able to induce HIF-1 complex formation when added to normoxic cells (Salceda and Caro 1997). Cells were pretreated with MG132 at a concentration of 1 μM for 24 hours, and then treated with MeHg (10 μM, 0.5 hours). Western blotting analysis showed that, compared with the MeHg-treated group, MG132 significantly increased the protein levels of HIF-1α and its downstream targets GLUT-1, VEGF-A and EPO (Fig. 6A–B). Cell viability experiments showed that, compared with the control group, the viability in the MeHg group was significantly decreased. Compared with the MeHg treatment-alone group, HIF-1α overexpression by MG132 prevented MeHg-induced cell toxicity (Fig. 6C). MG132 increased cell viability by ~ 16% and ~ 8% in MeHg-treated PC12 and BRL cells, respectively.

Fig. 6.

Fig. 6

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Effects of MG132 on methylmercury (MeHg)-induced acute cell injury in PC12 and BRL cells. Cells were pretreated with or without MG132 (1 μM for 24 h), followed by treatment with MeHg. A, B) Protein expression of HIF-1α and downstream targets glucose transporter-1 (GLUT-1), vascular endothelial growth factor-A (VEGF-A), and erythropoietin (EPO) was analyzed by western blotting. β-Actin served as the loading control. C) Cell viability was measured by MTT assay. *p < 0.05, compared with the control group; #p < 0.05, compared with the MeHg group. Control groups were treated with free media without any agents. Statistical analysis was performed by one-way analysis of variance followed by a Dunnett test, a multiple comparison procedure.

3.7. 2-MeOE2 decreased HIF-1α protein levels and its targets enhanced MeHg-induced cytotoxicity in both PC12 and BRL cells

To further investigate the role of HIF-1α in MeHg-induced toxicity, PC12 and BRL cells were pretreated with 2-MeOE2 (10 μM for 0.5 hours), an inhibitor of HIF-1α (Docherty et al., 2019), followed by treatment with MeHg (10 μM for 0.5 hours). The protein levels of HIF-1α and its downstream targets significantly decreased upon pretreatment with 2-MeOE2 (p < 0.05) compared with cells treated with MeHg alone (Fig. 7A–B). The viability of PC12 and BRL cells pretreated with 2-MeOE2 before MeHg was also significantly decreased (p < 0.05) compared with cells treated with MeHg alone (Fig. 7C). These results indicated that the inhibition of HIF-1α exaggerated MeHg-induced cytotoxicity in PC12 and BRL cells, further corroborating the cytoprotective role of HIF-1α.

Fig. 7.

Fig. 7

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Effects of 2-methoxyestradiol (2-MeOE2) on methylmercury (MeHg)-induced acute cell injury. PC12 and BRL cells were pretreated with 2-MeOE2 (10 μM for 0.5 hours), followed by treatment with MeHg. A, B) Protein expression of HIF-1α and downstream targets glucose transporter-1 (GLUT-1), vascular endothelial growth factor-A (VEGF-A), and erythropoietin (EPO) was analyzed by western blotting. β-Actin served as the loading control. C) Cell viability was measured by MTT assay. *p < 0.05, compared with the control group; #p < 0.05, compared with the MeHg group. Control groups were treated with free media without any agents. Statistical analysis was performed by one-way analysis of variance followed by a Dunnett test, a multiple comparison procedure.

4. Discussion

The present study shows that MeHg induced a time- and concentration-dependent decrease in cell viability in both PC12 and BRL cells. MeHg was toxic at lower concentrations in PC12 versus BRL cells. MeHg also decreased the protein levels of HIF-1α and its targets in a concentration-dependent manner. To further elucidate the relationship between MeHg toxicity and HIF-1α, we used both pharmacological (CoCl2, DHB, MG132, 2MeOE2) and genetic interventions to upregulate or downregulate HIF-1α, and showed efficacy in antagonizing and exacerbating the toxicity of MeHg in both cell lines. Additionally, HIF-1α mRNA was detected with RT-PCR, establishing the inability of MeHg to affect transcription and suggesting that its effects are secondary to the promotion of HIF-1α degradation via enhanced activity of the PHD and the proteasomal function. These results demonstrate that HIF-1α plays an essential protective role in MeHg-induced cytotoxicity, and that the protective role of HIF-1α is not cell-specific, although it is more pronounced in neuronal cells.

Over the past several decades, the mechanism(s) associated with MeHg toxicity have been continuously addressed but are yet to be fully understood. Overproduction of ROS and inactivation of the antioxidant defense system represents a key mechanism of MeHg-induced cytotoxicity (Ali et al., 1992; Chung et al., 2019; Xin et al., 2019). Recent reports have also established a role for HIF-1α in regulating ROS production (Li et al., 2019). Activation of HIF-1α may inhibit ROS production (Wang et al., 2018; Sun et al., 2019). And loss of HIF-1α has been reported to induce ROS production (Saito et al., 2015).

MeHg as a well-known pollutant poses a severe threat to human health (Driscoll et al., 2013; Chung et al., 2019). The central nervous system (CNS) is particularly vulnerable to MeHg (Grandjean et al., 2011; López-Berenguer et al., 2019) which may be related to accumulating large quantities of mercury compared with other organs (Gonzalez et al., 2005). Cell proliferation in the present study showed that PC12 was more sensitive to MeHg induced toxicity than BRL cells (Fig.1A–B). Miura., et al (1999) also use PC12 cells as neuronal cells and HeLa cells as non-neuronal cells, firstly proved that neuronal cells is more sensitively towards the toxicity of MeHg. As an essential detoxification organ (Spry et al., 1991; Goldstein et al., 1996), Maulvault et al., (2016) reported that liver has the highest rate of elimination of MeHg compared with brain may via promoting the demethylation of MeHg (Ung et al., 2010; Evans et al., 2016).

We also showed that MeHg led to the decreased of the protein levels of HIF-1α (Fig. 2A) and its targets (Fig. 2D–G) in both cells, and that the protein of HIF-1α was significantly decreased at a lower concentration of MeHg in PC12 cells, consistent with their heightened sensitivity to O2 concentrations (Conrad et al., 2001). The lower protein levels of HIF-1α in PC12 cells after treatment with MeHg likely accounts for their heightened sensitivity to MeHg. The downregulation of HIF-1α upon exposure to MeHg may be multifactorial. MeHg-induced damage to the CNS and liver is associated with altered mitochondrial function (Lee et al., 2016; Tofighi et al., 2011; Mori et al., 2007) which leads to a pseudo-normoxic state with decreased consumption of O2, and increased O2 may cause the downregulation of HIF-1α (Ferlazzo et al., 2019). Second, damaged mitochondrial function leads to increased intracellular ROS levels, in turn altering HIF-1α protein levels (li et al., 2014; Movafagh et al., 2015; sun et al., 2019; Liu et al., 2020). Thirdly, impairment in mitochondrial function may lead to disordered glucose utilization, in turn downregulating HIF-1α levels by increasing 2-oxoglutarate, the cofactor of PHD (Dehne et al., 2010; Bergström et al., 2013).

Previous studies have established that PHD inhibitors afford neuroprotection against ischemic and anoxic stress in an HIF-1α dependent manner (Hyvarinen et al., 2010; Davis et al., 2018; Li et al., 2018). DHB, as an inhibitor of PHD, can reverse 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration, secondary to the upregulation of HIF-dependent proteins, HO-1 and MnSOD (Lee et al., 2009). In our study, DHB attenuated the toxicity of MeHg in both cell lines, which might also be related to HIF-1α and its downstream proteins (Fig. 4A–B). Cobalt chloride oxidizes iron and deactivates enzymes by destroying hydroxylase responsible for the degradation of HIF-1a, replacing iron at the active site of hydroxylase (Leggett 2008). Salnikow et al., (2004) suggest that oxidation/depletion of ascorbic acid also an important mechanism of cobalt chloride to affect the PHD enzymes. As a hypoxia mimic, CoCl2 not only inducing the protein level of HIF-1α, but also inducing mitochondrial damage (López-Hernández et al., 2015), ATP depletion, and ROS production (Jones et al., 2013; Lauryn et al., 2013;). PC12 cells, representing a neuronal cell line, are more sensitive to decreased O2 concentrations (Conrad et al., 2001) and require higher production of ATP. This concept as mentioned above might be the underlying mechanism why CoCl2 showed high efficacy in attenuating MeHg-induced toxicity in BRL cells but failed to do so in PC12 cells (Fig. 3C). HIF-1α is hydroxylated by PHD, resulting in its ubiquitination by the von Hippel Lindau E3 ubiquitin ligase and subsequent degradation by the proteasome (Harten et al., 2010). MG132 as an inhibitor of the proteasome could inhibit chymotrypsin-like (ChTL) activity which is a novel approach to the treatment of stroke therapy and other diseases of the central nervous system (Wojcik et al., 2004; Chen et al., 2015). In our study, MG132 also significantly prevented the toxicity of MeHg by blocked MeHg-induced HIF-1α depletion, which further proves the cytoprotective effect of MG132. Upregulation of HIF-1α significantly antagonized the toxic effects of MeHg but not completely abated the toxic effects, suggesting that other mechanisms are involved in the toxicity of MeHg which should be further explored.

5. Conclusion

MeHg had a differential toxic effect on neuron-like and non-neuronal cells that may be related to the extent of downregulation of HIF-1α. HIF-1α overexpression may play a similar protective role against MeHg induced toxicity to different organs. But more specific indicators such as dopamine in the nervous system and transaminase in the liver system are needed to explore the different sensibility towards the toxicity of MeHg in different cell lines. And additional cell lines and animal models are needed to determine if HIF-1α is a novel and general target for therapeutic intervention in response to MeHg-induced toxicity.

Highlights.

Neuronal PC12 cells were more sensitive than BRL cells to the toxicity of MeHg

MeHg decreased HIF-1α in PC12 cells with a lower concentration than that in BRL cells

Up-regulation of HIF-1α could antagonize the toxicity of MeHg in both cell lines

Acknowledgments

We thank Dr. Peter Spencer at Oregon Health & Science University for critical comments. This work was supported in part by the Natural Science Foundation of China (No. 30872139, 81273124, 31100964) and R01 ES07331, R01 ES10563, and ES020852 from the National Institute of Environmental Health Sciences (NIEHS).

Abbreviations

2-MeOE2

2-Methoxyestradiol

CoCl2

Cobalt chloride

DHB

3, 4-Dihydroxybenzoic acid

DMSO

Dimethyl sulfoxide

EPO

Erythropoietin

GLUT-1

Glucose transporter 1

h

Hour(s)

HIF-1α

Hypoxia-inducible factor-1α

HIF-1β

Hypoxia-inducible factor-1β

LDH

Lactate dehydrogenase

MeHg

Methylmercury

MG132

Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (Proteasome inhibitor)

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl diphenyltetrazolium bromide

PHD

prolyl hydroxylase

ROS

Reactive oxygen species

VEGF-A

Vascular endothelial growth factor A

CNS

Central nervous system

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