Comparative study of susceptibility to methylmercury cytotoxicity in cell types composing rat peripheral nerves: a higher susceptibility of dorsal root ganglion neurons

Abstract

Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.

INTRODUCTION

Methylmercury is an organometallic compound that exhibits neurotoxicity and causes Minamata disease (Eto, 2000). In patients with Minamata disease, the cerebrum, cerebellum, and peripheral nerves are damaged by methylmercury. Sensory impairment of the extremities is an early symptom of Minamata disease, which can be caused by damage to either or both of the postcentral gyrus of the cerebrum and peripheral sensory nerves. It is likely that generalized paresthesia is mainly caused by central neuropathy, whereas paresthesia of the extremities results from peripheral nerve damage.

The characteristic histopathological change in peripheral nerves of Minamata disease patients is marked axonal degeneration of the sensory nerves; such degeneration is not observed in motor nerves (Eto, 2000). We previously observed that methylmercury causes neural degeneration in dorsal root ganglion (DRG) cells and sensory nerve fibers in rats (Shinoda et al., 2019). Among peripheral nerves, methylmercury has a significantly greater damaging effect on sensory nerves than on motor nerves. This selective toxicity for sensory nerves (dorsal root nerves) has been observed in rats administered methylmercury (Jacobs et al., 1977; Su et al., 1997; Cao et al., 2013) but the molecular mechanisms underlying the selective toxicity remain unclear.

Methylmercury is taken up into cells by passive diffusion and by the L-type large neutral amino acid transporter, L-type amino acid transporter (LAT) 1 and LAT2 (Simmons-Willis et al., 2002). Once in the cell, methylmercury reacts covalently with reactive thiols in proteins with toxicological consequences because of its high affinity for sulfhydryl groups (Simpson, 1961). Methylmercury also binds to glutathione by non-enzymatic and possibly enzymatic processes (involving glutathione S-transferases) to form a methylmercury-glutathione conjugate (Rabenstein and Fairhurst, 1975). This conjugate is transported out of cells into the extracellular space by multidrug resistance-associated proteins (MRPs), such as MRP2 (König et al., 1999) to reduce intracellular methylmercury (Toyama et al., 2011). Additionally, MRP2 as well as MRP1 also has the ability to export methylmercury out of the cell (Granitzer et al., 2020). Therefore, the levels of LAT1/2 and MRP1/2 may affect the toxicity of methylmercury through changes in intracellular accumulation.

The purpose of this study was to investigate the mechanisms underlying the high susceptibility of sensory neurons to methylmercury cytotoxicity by comparing differences in the levels of the transporters responsible for uptake and excretion of methylmercury in the three cell types that comprise the peripheral nerves: DRG cells, anterior horn cells (AHC), and Schwann cells.

MATERIALS AND METHODS

Materials

All experimental protocols were evaluated and approved by the Regulations for Animal Research at Tokyo University of Science and Tokyo University of Pharmacy and Life Sciences and were carried out in accordance with the approved protocols. All efforts were made to minimize the number of animals used and their suffering. Three- or four-week-old male Wistar rats (Sankyo Labo Service Corporation Inc., Tokyo, Japan) were purchased and housed in home cages under a 12 hr/12 hr light-dark cycle with ad libitum access to water and food. Methylmercury chloride (MeHgCl, Sigma-Aldrich, Tokyo, Japan) was dissolved in 10 mM L-cysteine solution at a concentration of 10 mM. Poly-L-lysine-coated tissue culture dishes and plates were obtained from Iwaki (Chiba, Japan). Dulbecco’s modified Eagle’s medium (DMEM) and Ca²⁺- and Mg²⁺-free phosphate-buffered saline (CMF-PBS) were obtained from Nissui Pharmaceutical (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Biosera (Kansas, MO, USA). Lipofectamine RNAiMAX and Opti-MEM were obtained from Invitrogen (Carlsbad, CA, USA). QIAzol lysis reagent was obtained from Qiagen (Hilden, Germany). A bicinchoninic acid protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). JPH203 (a LAT1 inhibitor) and MK571 (an MRP inhibitor) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Horseradish peroxidase-conjugated anti-rabbit (#7074) and anti-mouse (#7076) IgG antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). A rabbit polyclonal antibody against LAT1 (KE026) was obtained from Medical Chemistry Pharmaceutical Co. (Hokkaido, Japan). A goat monoclonal antibody against LAT2 (sc-27581), and mouse monoclonal antibodies against MRP1 (sc-18835) and MRP2 (sc-5770) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal antibodies against β-actin and GAPDH were obtained from Fujifilm Wako Pure Chemical Industries (Osaka, Japan). A high-capacity cDNA reverse transcription kit was purchased from Applied Biosystems (Foster City, CA, USA). Gene Ace SYBR qPCR Mix was obtained from Nippon Gene (Tokyo, Japan). Other reagents were obtained from Nacalai Tesque (Kyoto, Japan).

Cell culture

Primary rat DRG neurons were isolated from 4-week-old male Wistar rats weighing 50–60 g as described by Haratake et al. (2011). The spinal column was dissected from rats and placed in cold Hank’s buffer in an ice bath. The spinal cord was extruded from the spinal column using a syringe and placed in cold DMEM in an ice bath. DRG neurons were isolated as follows. Under a microscope, 30 thoracic and lumbar DRG per rat were dissected out from the spinal column and the spinal roots and peripheral nerve trunks were carefully cut away. Dissected DRG were enzymatically disaggregated at 37°C in Hank’s solution containing 0.05% collagenase for 2 hr and subsequently in Hank’s solution containing 0.05% trypsin plus 0.02% ethylenediaminetetraacetic acid (EDTA) for 1 hr. In addition, AHC was isolated from the spinal cord and then enzymatically disaggregated at 37°C in Hank’s solution containing 0.05% trypsin plus 0.02% EDTA for 1 hr. After washing twice with gentle pipetting in warm DMEM containing 10% FBS, cells were plated onto 35-mm poly-L-lysine-coated dishes or 6-well plates at a density of 0.5~1 × 10⁶ cells/dish. Unless otherwise stated, the cells were cultured in DMEM supplemented with 10% heat-inactivated FBS in a humidified atmosphere of 5% CO₂ in air. Proliferation of non-neurons was suppressed by addition of 10 mM arabinosylcytosine for 24 to 48 hr just after plating, and at later times as required. DRG neurons were subjected to experiments 7–10 days after plating (at 90% confluency). In all experiments, specific DRG and AHC markers were detected before use (Fig. S1). Schwann cells were cultured in Schwann Cell Medium in 60-mm poly-L-lysine-coated dishes at 37°C in a humidified atmosphere of 5% CO₂ in air until confluent. The cells were then seeded at a density of 6 × 10³ cells/cm² in 35-mm culture dishes, 6-well plates, or 96-well plates coated with poly-L-lysine and cultured until 90% confluent for use in the experiments described below.

Cytotoxicity Assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-triphenyl tetrazolium bromide (MTT) assay was used to estimate cell viability, as described previously (Yoshida et al., 2014). Briefly, DRG, AHC, and Schwann cells were pretreated with or without 1 µM JPH203 for 1 hr, transfected with or without LAT1 siRNA for 6 hr, or pretreated with 10 µM MK571 for 1 hr, and then exposed to methylmercury (0.25, 0.5, 1, 3 and 5 µM) for 24 hr and then treated with 5 mg/mL MTT (1/20 volume) for another 4 hr at 37°C. After removing the medium, dimethyl sulfoxide (DMSO) was added to dissolve MTT formazan. Absorbance was measured by a plate reader (EnVision 2104 Multilabel Reader, PerkinElmer, Inc., Waltham, MA, USA) at a wavelength of 540 nm.

Measurement of mercury concentration

Intracellular mercury concentration was determined using a previously described method (Hirooka et al., 2010) with a minor modification. Briefly, DRG, AHCs, and Schwann cells were cultured in 6-well culture plates and exposed to methylmercury. After washing the cell layer twice with CMF-PBS, 1 mL 60% (v/v) nitric acid and 10 µL Au standard solution were added to each well. The solution was transferred to an acid-washed test tube and incubated at 140°C for 30 min in an aluminum heat block, and then the precipitate was re-suspended in 5 mL of 1% (v/v) nitric acid containing 1% (v/v) Au standard solution. Inductively coupled plasma mass spectrometry (using a Nexion 300S, PerkinElmer, Waltham, MA, USA) was then performed on 4.5 mL of each sample to determine mercury content. The remaining cell homogenate (0.5 mL) was analyzed for DNA content by the fluorometric method (Kissane and Robins, 1958) to enable expression of mercury content as pmol/μg DNA.

Western blotting

After treatment with methylmercury, DRG, AHC, and Schwann cells were washed twice with ice-cold CMF-PBS. Total cell proteins were prepared by the lysis of cells in SDS sample buffer (50 mM Tris-HCl containing 2% SDS and 10% glycerol, pH 6.8), followed by incubation at 95°C for 10 min. Protein concentration was determined using a bicinchoninic acid (BCA) protein assay reagent kit (Pierce) before 2-mercaptoethanol was added to each sample. Protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane at 2 mA/cm² for 1 hr, according to the method of Kyhse-Andersen (1984). The membranes were blocked with 5% skimmed milk in TTBS (20 mM Tris-HCl containing 150 mM NaCl and 0.1% Tween 20, pH 7.5) and then incubated with anti-LAT1 (1:500), anti-LAT2 (1:500), anti-MRP1 (1:500), anti-MRP2 (1:200), anti-LDHB (1:1000), anti-Islet1/2 (1:1000), anti-GFAP (1:1000), anti-GAPDH (1:5000), or anti-β-actin (1:5000) antibodies at 4°C overnight. After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hr at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence and scanned using an LAS 3000 luminescent image analyzer (Fujifilm, Tokyo, Japan).

LAT1 siRNA transfection

Growing cultures of DRG cells were transfected with LAT1 siRNA at 4°C for 6 hr in serum-free DMEM. Synthetic rat LAT1 siRNA (Slc7a5; ID s132355) was purchased from Thermo Fisher Scientific (Tokyo, Japan). A nonspecific sequence was used as the siRNA negative control (Thermo Fisher Scientific, Tokyo, Japan). Transfection was performed using RNAiMAX Transfection Reagent according to the manufacturer’s protocol. Briefly, each siRNA and RNAiMAX Reagent were dissolved in Opti-MEM and incubated for 20 min at room temperature. After transfection, the medium was replaced with fresh DMEM supplemented with 10% FBS and the cells then incubated at 37°C for 18 hr. The medium was then discarded, and the cells were washed twice with 1% BSA containing DMEM. The cells were treated with or without methylmercury (0.5, 1 and 3 µM) for 24 hr, and the cytotoxicity was evaluated by the MTT assay as described above.

Real time reverse transcription-polymerase chain reaction (RT-qPCR)

Total RNA was extracted using the RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA, USA) and cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster, CA, USA). Real-time PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) with 5 µg cDNA and 0.1 µM primers on a 7500 Real Time PCR system (Applied Biosystems). Thermal cycling parameters were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 sec and 60°C for 1 min. Melting curve analysis and agarose gel electrophoresis with ethidium bromide staining were conducted to ensure a single PCR product of correct length. Levels of LAT1 and β₂-microglobulin mRNA in each RNA sample were quantified by the relative standard curve method. Fold-change for each gene was assessed after normalization of the intensity value to β₂-microglobulin. Sequences of the primers were as follows: rat LAT1 (Slc7a5), 5′-TGAAGGCACCAATCTGGACG-3′ (forward) and 5′-GAAGTAGGCCAGGTTCGTCA-3′ (reverse); rat β₂-microglobulin, 5′-ACCCTGGTCTTTCTGGTGCTTG-3′ (forward) and 5′-GCAGTTCAGTATGTTCGGCTTCC-3′ (reverse).

Statistics

All statistical analyses were performed using Excel software (Microsoft, Redmond, WA, USA) with add-in software, Statcel (OMS, Tokyo, Japan). If not stated otherwise, data are expressed as the mean ± SEM. Data were analyzed for statistical significance by analysis of variance and Bonferroni-type multiple t-test. P values of less than 0.05 were considered to indicate statistically significant differences.

RESULTS

First, we investigated the susceptibility of peripheral nerve cell types, DRG, AHC, and Schwann cells, to methylmercury. As shown in Fig. 1, DRG cell viability was markedly decreased by methylmercury in a concentration-dependent manner, while that of AHC was unchanged. Methylmercury also decreased the viability of Schwann cells; however, the extent of the decrease was minor compared with that of DRG cells.

Fig. 1

Cytotoxicity of methylmercury in rat DRG, AHC, and Schwann cells. Cells were incubated at 37°C for 24 hr in the presence of methylmercury at 0.25, 0.5, 1, 3, or 5 µM. MTT assays were then performed. Values are means ± S.E. of four samples. **Significantly different from the corresponding control, p < 0.01.

We then tested the intracellular accumulation of methylmercury among the three cell types, measured as ²⁰²Hg. As shown in Fig. 2, mercury accumulated markedly within DRG cells in a concentration-dependent manner but mercury accumulation in AHC was minimal and was moderate in Schwann cells.

Fig. 2

Intracellular accumulation of mercury within rat DRG, AHC, and Schwann cells after exposure to methylmercury. Cells were incubated at 37°C for 24 hr in the presence of methylmercury at 0.1, 0.25, 0.5, 1, or 3 µM. Intracellular mercury accumulation was then determined by inductively coupled plasma mass spectrometry. Values are means ± S.E. of six samples. **Significantly different from the corresponding methylmercury concentration in AHCs, p < 0.01. Significantly different from the corresponding methylmercury concentration in Schwann cells, ^#p < 0.05; ^##p < 0.01.

Having observed a clear inverse correlation between cell viability and intracellular accumulation of methylmercury, we next examined the levels of LAT1 and LAT2 (Fig. 3A), which transport methylmercury into cells, and MRP1 and MRP2 (Fig. 3B), which remove methylmercury from cells, in DRG, AHC, and Schwann cells. Constitutive levels of LAT1 were higher in DRG than in AHC and Schwann cells, while constitutive levels of MRP2 in DRG cells were lower than those in AHC and almost the same as those in Schwann cells. Additionally, methylmercury increased MRP2 protein levels in AHC and Schwann cells but not in DRG cells. However, there was no significant difference in the levels of LAT2 and MRP1 among DRG, AHC, and Schwann cells.

Fig. 3

Levels of LAT1/LAT2 and MRP1/MRP2 proteins in rat DRG, AHC, and Schwann cells after exposure to methylmercury. Cells were incubated at 37°C for 24 hr in the presence of methylmercury at 0.5, 1, or 3 µM and LAT1/LAT2 and MRP1/MRP2 protein levels were determined by western blot analysis. [A] LAT1 and LAT2 proteins in DRG, AHC, and Schwann cells. GAPDH was used as an internal control (upper panel). Arrows indicate the positions of LAT1 or LAT2. Quantification of LAT1 band intensities (lower panel). Values are means ± S.E. of three samples. Significantly different from the DRG control, *p < 0.05; **p < 0.01. [B] MRP1 and MRP2 proteins in DRG, AHC, and Schwann cells. β-actin was used as an internal control (upper panel). Arrows indicate the positions of MRP1 or MRP2. Quantification of MRP2 band intensities (lower panel). Values are means ± S.E. of three samples. **Significantly different from the DRG control, p < 0.01. ^#Significantly different from the AHC control, p < 0.05.

Both a selective LAT1 inhibitor, JPH203, (Fig. 4A) and siRNA-mediated suppression of LAT1 (Fig. 4B) attenuated the reduction in cell viability of DRG cells caused by methylmercury. Conversely, a selective MRP2 inhibitor, MK571, increased the reduction in cell viability caused by methylmercury (Fig. 4C).

Fig. 4

Involvement of LAT1 and MRP2 in methylmercury cytotoxicity in DRG cells. Cells were pretreated with 1 µM JPH203 for 1 hr or transfected with LAT1 siRNA for 6 hr or pretreated with 10 µM MK571 for 1 hr, and then incubated at 37°C for 24 hr in the presence of methylmercury at 0.5, 1, or 3 µM. Methylmercury cytotoxicity was evaluated as cell viability determined by the MTT assay. [A] Effect of the LAT1 inhibitor, JPH203, on methylmercury cytotoxicity. Values are means ± S.E. of three samples. **Significantly different from the corresponding “absence of JPH203”, p < 0.01. [B] (Left panel) siRNA-mediated knockdown of LAT1. LAT1 mRNA levels were determined by RT-qPCR. B2M was used as an internal control. Values are means ± S.E. of three samples. **Significantly different from the siControl, p < 0.01. (Right panel) Effect of siRNA-mediated knockdown of LAT1 on methylmercury cytotoxicity. Values are means ± S.E. of two samples. **Significantly different from the corresponding siControl, p < 0.01. [C] Effect of an MRP2 inhibitor, MK571, on methylmercury cytotoxicity. Values are means ± S.E. of three samples. **Significantly different from the corresponding “absence of MK571”, p < 0.01.

DISCUSSION

Methylmercury-induced DRG cell injury in Minamata disease model rats causes progressive axonal degeneration (Cao et al., 2013). Methylmercury accumulates to high levels in peripheral DRG nerves of rats continuously administered methylmercury by subcutaneous injection (Somjen et al., 1973). In the present study, we revealed that DRG cells are more susceptible to methylmercury cytotoxicity compared with AHC and Schwann cells. Our findings indicate that the mechanisms underlying this susceptibility of peripheral sensory nerves include high levels of methylmercury accumulation within the DRG cells, caused by high constitutive levels of LAT1 and low constitutive levels of MRP2. The susceptibility of DRG cells to methylmercury is consistent with it causing selective injury to sensory nerves rather than motor nerves.

According to Somjen et al. (1973), methylmercury accumulates to higher levels in the sensory ganglia than in the spinal cord, spinal roots, and peripheral nerves of rats exposed to methylmercury; no difference in the mercury content of dorsal and ventral roots was observed. Based on these results, the authors postulated that spinal ganglia are the primary targets of poisoning by methylmercury, and that sensory axons degenerate because their parent cell bodies are damaged. In other words, they considered that methylmercury-induced sensory nerve-specific axonal degeneration is not a direct injury to nerve fibers by methylmercury, but a secondary injury resulting from apoptotic cell death of DRG neurons caused by excessive accumulation of methylmercury. Therefore, as they suggested, it is likely that the selective degeneration of peripheral sensory nerve axons may result from damage to DRG cells caused by high levels of accumulated methylmercury. Eleven types of sensory neuron have been identified by transcriptome analysis (Usoskin et al., 2015); therefore, it is possible that one or more of these sensory neuron type(s) are susceptible to methylmercury; this susceptibility may be involved in the sensory disturbance experienced by Minamata disease patients.

Mechanisms underlying differences in expression levels of LAT1 and MRP2 among DRG, AHC, and Schwann cells remain to be clarified. LAT1 levels can be upregulated by intracellular signaling pathways mediated by HIF-1α, ATF4, and possibly other signaling molecules (Zhang et al., 2020). Methylmercury downregulates the levels of HIF-1α by inhibiting proline hydroxylase (Chang et al., 2019). MRP2 is regulated by the NRF2/KEAP1 system (Vollrath et al., 2006) and methylmercury activates the transcription factor NRF2 to upregulate MRP2 (Toyama et al., 2007). Our preliminary experiments indicated that methylmercury activated NRF2 more strongly in DRG than in AHC (data not shown). It is likely that constitutive levels of LAT1 and MRP2 are high and low, respectively, in DRG cells may be due to a strong influence of signaling molecules required for regulation of LAT1 and MRP2 expression. In other words, the regulation of LAT1 and MRP2 expression is more sensitive to such signal molecules in DRG than in AHC and Schwann cells.

The present data provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients. LAT1 and MRP2 may be involved in the high susceptibility of DRG cells to methylmercury cytotoxicity. Specifically, high constitutive LAT1 levels and low MRP2 levels in DRG cells are involved in the susceptibility of sensory nerves to methylmercury cytotoxicity through the intracellular accumulation of high levels of methylmercury.

ACKNOWLEDGMENTS

This study was supported by the Study on the Health Effects of Heavy Metals organized by the Ministry of the Environment, Japan (T.K. and Y.S), by JSPS KAKENHI Grant Number JP19K19417 (E.Y.), and by the Kato Memorial Bioscience Foundation (E.Y.). We are grateful to Professor Morio Nakayama and Dr. Sakura Yoshida, Graduate School of Biomedical Sciences, Nagasaki University, for their kind advice concerning the isolation of rat DRG neurons. We also thank Jeremy Allen, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Conflict of interest

The authors declare that there is no conflict of interest.

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