Comparative cytotoxicity of novel mercury species α-mercuri-acetaldehyde and α-mercuri-acetic acid versus methylmercury in SH-SY5Y cells

Abstract

Recently, α-mercuri-acetaldehyde (HgCH₂CHO) and α-mercuri-acetic acid (HgCH₂COOH) have been proposed as potential causative agents of Minamata disease. However, their toxicological profiles remain largely unknown. This study aimed to characterize the cytotoxicity, cellular uptake, and efflux mechanisms of these compounds in SH-SY5Y neuroblastoma cells and to compare these properties with those of methylmercury (MeHg). Cell viability was assessed after 24 hr of exposure to MeHg (1–10 µM), HgCH₂CHO (10–50 µM), or HgCH₂COOH (10–50 µM) using the CCK-8 assay. The roles of L-type amino acid transporter 1 (LAT1) and multidrug resistance-associated proteins (MRPs) were evaluated using the inhibitors JPH203 (1 µM) and MK571 (10 µM), respectively. Intracellular mercury accumulation was quantified after 24 hr of exposure to 3 µM of each compound using thermal decomposition-amalgamation atomic absorption spectrometry. All compounds exhibited dose-dependent cytotoxicity, with a relative toxicity order of MeHg (LC₅₀: 6.4 µM) > HgCH₂CHO (LC₅₀: 14.6 µM) > HgCH₂COOH (LC₅₀: 39.2 µM). LAT1 inhibition had minimal effect on MeHg toxicity but slightly attenuated that of HgCH₂CHO and HgCH₂COOH. Conversely, MRP inhibition markedly enhanced MeHg toxicity, modestly increased that of HgCH₂CHO, and slightly increased that of HgCH₂COOH. Cellular mercury accumulation was consistent with cytotoxicity patterns, showing 10–20-fold lower levels for HgCH₂CHO and HgCH₂COOH than for MeHg. HgCH₂CHO and HgCH₂COOH were approximately 2–5-fold less cytotoxic than MeHg and exhibited substantially lower intracellular mercury levels. Our findings suggest that HgCH₂CHO and HgCH₂COOH are unlikely to have neurotoxic potential comparable to that of MeHg.

INTRODUCTION

Methylmercury (MeHg) is a major environmental toxicant and the causative agent of Minamata disease, causing severe neurological damage that affects both the central and peripheral nervous systems (Eto et al., 2010; Eto and Takeuchi, 1978; Takeuchi et al., 1962). Over the past decades, numerous studies have investigated the mechanisms underlying MeHg-induced neurotoxicity (Antunes dos Santos et al., 2018; Lee et al., 2020; Shinoda et al., 2023). In 2020, James et al. re-examined the Minamata tragedy and argued that α-mercuri-acetaldehyde (HgCH₂CHO) and α-mercuri-acetic acid (HgCH₂COOH) may be novel etiologic candidates for Minamata disease (James et al., 2020). The study integrated high-energy-resolution fluorescence-detected X-ray absorption spectroscopy (HERFD-XAS) of the historical specimen Cat 717 with density functional theory (DFT) calculations. HERFD-XAS revealed sulfur-bound organic mercury as the predominant mercury species, with a minor β-HgS phase. DFT calculations, which modeled acetaldehyde production at the Chisso plant, indicated that the activation barrier for MeHg formation was prohibitively high, making this pathway unlikely to proceed. By contrast, the calculations indicated an energetic preference for forming HgCH₂CHO or HgCH₂COOH, which the authors proposed as stable mercury-containing by-products of the process. However, the toxicological properties of HgCH₂CHO and HgCH₂COOH have not yet been elucidated, highlighting the importance of characterizing their toxicity profiles to better understand their potential health risks.

Two contemporaneous commentaries offered critical perspectives on this reanalysis. Balogh et al. emphasized historical evidence of MeHg in acetaldehyde-production wastes from Chisso and in shellfish from Minamata Bay (Balogh and Tsui, 2020). They also cautioned that long-term formalin storage can alter mercury speciation in tissues, potentially modifying the original mercury speciation profile of Cat 717; they maintained that the broader record remains consistent with MeHg as the etiologic agent. Tohyama criticized the paper for misleading statements and for failing to acknowledge foundational literature that had already established MeHg as the cause of Minamata disease (Tohyama, 2020). He also emphasized that the paper’s conclusion was based only on theoretical assumptions and that merely inferring the presence of HgCH₂CHO and HgCH₂COOH in Cat 717 cannot supplant the established etiology without demonstrating exposure, tissue distribution, accumulation, and dose-dependent toxicity in vivo. These arguments also underscore the need to investigate the toxicity of HgCH₂CHO and HgCH₂COOH.

To evaluate the toxicity of the proposed candidate compounds, HgCH₂CHO and HgCH₂COOH, comparison with MeHg is essential because MeHg is the best-characterized benchmark in this context. At the cellular level under physiological conditions, MeHg is efficiently transported as the MeHg–L-cysteine conjugate by L-type large neutral amino acid transporters LAT1 and LAT2 (Simmons-Willis et al., 2002) and forms covalent bonds with protein thiols (Simpson, 1961). MeHg also forms glutathione complexes, and such glutathione conjugates are exported by multidrug resistance–associated proteins (MRPs) (Farina and Aschner, 2019; Granitzer et al., 2020). Consequently, the cellular levels and activities of LAT1/2 and MRP1/2 are expected to modulate intracellular MeHg accumulation and toxicity.

Accordingly, we compared HgCH₂CHO and HgCH₂COOH with MeHg in SH-SY5Y cells, focusing on (i) cytotoxicity, (ii) uptake and efflux pathways, and (iii) intracellular accumulation.

MATERIALS AND METHODS

Compounds and reagents

Methylmercury(II) chloride (MeHg) was purchased from Merck (Rahway, NJ, USA). Mercury acetaldehyde chloride (HgCH₂CHO) and mercury acetate chloride (HgCH₂COOH) were synthesized in the Department of Chemistry, Faculty of Advanced Science and Technology, Kumamoto University, as described in the Supplemental materials. Structures were confirmed as follows: HgCH₂CHO: by ¹H NMR and elemental analysis [¹H NMR (CDCl₃, 500 MHz): δ 3.64 (d sat, J_H-H = 4.2 Hz and J_H-Hg = 139 Hz, 2H), 9.67 (t, J = 4.3 Hz, 1H); Anal. Calcd for C₂H₃ClHgO: C, 8.61; H, 1.08. Found: C, 8.62; H, 1.33.]; HgCH₂COOH: by ¹H NMR and elemental analysis [¹H NMR (D₂O, 400 MHz): δ 2.55 (sat, J_H-Hg = 307 Hz, 2H); Anal. Calcd for C₂H₃ClHgO₂: C, 8.14; H, 1.02. Found: C, 7.99; H, 1.13.]. MeHg, HgCH₂CHO, and HgCH₂COOH were dissolved in sterile water by sonication for 1 hr. For HgCH₂COOH, 1 N NaOH was added to ensure complete dissolution (0.1% v/v 1 N NaOH in the 5 mM stock solution). Stock solutions of MeHg (0.1–1 mM), HgCH₂CHO (1–5 mM) and HgCH₂COOH (1–5 mM) were prepared and diluted 100-fold in culture medium to obtain the working concentrations used in the assays.

Ca²⁺- and Mg²⁺-free phosphate-buffered saline (CMF-PBS) and Dulbecco's Modified Eagle Medium (DMEM, high glucose) were purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan). Fetal bovine serum (FBS) was purchased from Biowest (Nuaille, France) and from Sigma-Aldrich (St. Louis, MO, USA), and was heat-inactivated at 56°C for 30 min. JPH203 (a LAT1 inhibitor) and MK571 (an MRP inhibitor) were purchased from Adooq BioScience LLC (Irvine, CA, USA) and Cayman Chemical (Ann Arbor, MI, USA), respectively. These transporter inhibitors were dissolved in dimethyl sulfoxide (FUJIFILM Wako Pure Chemical Corp.) and added to the cultures at a final DMSO concentration of 0.1% v/v in all conditions. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan).

Cell culture and cytotoxicity assay

The neuroblastoma cell line SH-SY5Y was provided by Dr. Takashi Toyama (Graduate School of Pharmaceutical Sciences, Tohoku University). SH-SY5Y cells were cultured in DMEM supplemented with 10% heat-inactivated FBS in a humidified atmosphere containing 5% CO₂ at 37°C.

The CCK-8 assay was used to assess cell viability as described previously (Shinoda et al., 2017). Briefly, cells were seeded in 96-well plates at a density of 2.0 × 10⁴ cells/well and cultured for 24 hr prior to exposure to mercury compounds. Cells were exposed to various concentrations of mercury compounds: MeHg (1–10 µM), HgCH₂CHO (10–50 µM), and HgCH₂COOH (10–50 µM) for 24 hr in a humidified atmosphere containing 5% CO₂ at 37°C. Twenty-four hours after exposure to mercury compounds, CCK-8 solution was mixed with DMEM to the final concentration recommended by the manufacturer, and the mixture was applied to mercury-exposed cells for 1 hr in a humidified atmosphere containing 5% CO₂ at 37°C. After incubation, absorbance was measured at 450 nm using a Varioskan Flash microplate reader (Thermo Scientific, Waltham, MA, USA). For inhibition studies, cells were treated with inhibitors (1 µM JPH203 for 1 hr and 10 µM MK571 for 1 hr) prior to mercury compound exposure. The concentrations of these inhibitors were selected based on our previous study using dorsal root ganglion neurons (Yoshida et al., 2024). Following pretreatment, cells were cotreated with the inhibitor and mercury compounds for an additional 24 hr.

Measurement of intracellular mercury concentration

Intracellular mercury concentration was measured as described in a previous study (Takanezawa et al., 2019). Briefly, cells were seeded in 6-well plates at a density of 6.0 × 10⁵ cells/well and cultured for 24 hr prior to exposure to mercury compounds. Cells were exposed to 3 µM of each mercury compound for 24 hr in a humidified atmosphere containing 5% CO₂ at 37°C. After washing the cell layer once with CMF-PBS, cells were collected with 2 mL of CMF-PBS using a cell scraper and lysed by sonication (Power: 20%, Time set: 20 sec, ON Time: 1.0 sec, OFF Time: 1.0 sec, Auto Tune: ON; VP-050, TAITEC, Saitama, Japan). The intracellular mercury content in the cell lysate was determined using thermal decomposition-amalgamation atomic absorption spectrometry with a direct mercury analyzer MA-3000 (Nippon Instruments Co., Kyoto, Japan). The exposure concentration (3 µM) was selected to enable direct comparison of intracellular mercury accumulation under minimally cytotoxic conditions for all mercury species. It should be noted that this method does not distinguish intracellular mercury from mercury bound to the plasma membrane; thus, the intracellular mercury concentration in this study represents total cell-associated mercury.

Statistical analysis

JMP Pro ver. 18.00 (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses, with the significance level set at 5% (p < 0.05). Data are presented as mean ± standard error of the mean (S.E.M.) from three independent experiments. To evaluate the concentration-dependent toxicity of mercury compounds, Dunnett's test was performed using 0 µM as the control group. The effects of transporter inhibitors were assessed using Welch's t-test with Bonferroni correction.

Use of AI tools

Parts of the manuscript text were edited for English language clarity with assistance from ChatGPT 5.1 (OpenAI, San Francisco, CA, USA). The authors reviewed and are responsible for all content.

RESULTS

First, the dose-dependent cytotoxicity of HgCH₂CHO and HgCH₂COOH in SH-SY5Y cells was evaluated and compared with that of MeHg (Fig. 1). All three mercury species caused a significant, concentration-dependent decrease in cell viability (p < 0.01–0.001 vs. 0 µM control), and the toxicity ranked MeHg > HgCH₂CHO > HgCH₂COOH, with LC₅₀ values of 6.4 µM, 14.6 µM, and 39.2 µM, respectively. Cell viability after exposure to MeHg and HgCH₂CHO decreased rapidly beyond threshold concentrations, whereas that after exposure to HgCH₂COOH decreased more slowly.

Fig. 1

Dose-dependent cytotoxicity of HgCH₂CHO and HgCH₂COOH in SH-SY5Y cells compared with that of MeHg. Cell viability was assessed following exposure to increasing concentrations of each compound. Data are presented as mean ± S.E.M. (n = 9). Statistical significance was determined using Dunnett's test; **p < 0.01, ***p < 0.001 vs. 0 µM control.

Next, to compare how each mercury compound accumulates in and is exported from cells with MeHg, we examined the effects of transporter inhibitors. Figure 2 shows the effects of transporter inhibitors on cell viability in SH-SY5Y cells after exposure to HgCH₂CHO and HgCH₂COOH, compared with that after exposure to MeHg. Cotreatment with the 1 µM LAT1 inhibitor did not attenuate MeHg cytotoxicity (Fig. 2A, p > 0.05; change in viability relative to no-inhibitor controls: +1.9, −3.4, −0.1, +1.8, and −2.7% at 1–10 µM MeHg, respectively). In contrast, the LAT1 inhibitor significantly but only modestly attenuated the cytotoxicity of HgCH₂CHO (p < 0.001; +15.1% at 20 µM) and HgCH₂COOH (p < 0.05–0.001; +11.2, +17.3, and +15.5% at 30–50 µM, respectively). Cotreatment with the 10 µM MRP inhibitor enhanced MeHg cytotoxicity across a broad range (Fig. 2B, p < 0.01–0.001; −8.8, −17.7, and −36.5% at 2–5 µM) and enhanced the cytotoxicity of HgCH₂CHO at 10 µM only (p < 0.001; −42.3%), whereas the inhibitor significantly but only slightly enhanced the cytotoxicity of HgCH₂COOH over a broad concentration range (p < 0.05–0.01; −14.0, −13.4, and −10.5% at 20–40 µM). These findings indicate that LAT1 may not contribute to the uptake of MeHg, HgCH₂CHO, and HgCH₂COOH in SH-SY5Y cells. MeHg and HgCH₂CHO are likely to be partly effluxed via MRP, whereas the efflux of HgCH₂COOH may depend on alternative transport mechanisms.

Fig. 2

Effects of transporter inhibitors on cell viability in SH-SY5Y cells after exposure to HgCH₂CHO and HgCH₂COOH, compared with that after exposure to MeHg. Cells were cotreated with MeHg, HgCH₂CHO, or HgCH₂COOH and specific transporter inhibitors. Data are shown as mean ± S.E.M. (n = 9). Statistical significance was assessed using Welch’s t-test with Bonferroni correction; *p < 0.05, **p < 0.01, ***p < 0.001 vs. the absence of each respective inhibitor. JPH203: LAT1 inhibitor (1 µM); MK571: MRP inhibitor (10 µM).

To investigate intracellular mercury accumulation in cells treated with each mercury compound, we compared intracellular mercury levels at non-cytotoxic concentrations. Figure 3 shows the intracellular mercury levels in SH-SY5Y cells exposed to 3 µM HgCH₂CHO and HgCH₂COOH, compared with those in cells exposed to 3 µM MeHg. The intracellular mercury levels in cells exposed to HgCH₂CHO (mean ± S.E.M., p < 0.001; 144.6 ± 7.3 ng/mg protein), and HgCH₂COOH (p < 0.001; 79.5 ± 5.5 ng/mg protein) were significantly lower than those in the MeHg (1,565.9 ± 90.9 ng/mg protein). These results are consistent with the toxicity ranking observed in Figs. 1 and 2.

Fig. 3

Intracellular mercury levels in SH-SY5Y cells exposed to 3 µM HgCH₂CHO and HgCH₂COOH, compared with those in cells exposed to 3 µM MeHg. Mercury accumulation was quantified in cells treated with each compound. Data are presented as mean ± S.E.M. (n = 9). Statistical significance was evaluated using the Tukey–Kramer test; ***p < 0.001.

DISCUSSION

The cytotoxicity of HgCH₂CHO and HgCH₂COOH was lower than that of MeHg; however, our results demonstrate that both compounds possess cytotoxic properties in SH-SY5Y cells. LAT1 may not contribute to the uptake of HgCH₂CHO and HgCH₂COOH in SH-SY5Y cells, suggesting that cellular entry of these mercury species likely involves other transporters. HgCH₂CHO may be exported via MRPs, whereas the efflux of HgCH₂COOH likely involves other transporters in SH-SY5Y cells. At the same exposure concentration, the intracellular mercury levels in cells exposed to HgCH₂CHO and HgCH₂COOH were significantly lower than those in cells exposed to MeHg, and these findings were consistent with the toxicity ranking.

HgCH₂CHO and HgCH₂COOH were approximately 2–5-fold less cytotoxic than MeHg, while remaining within the same order of magnitude. Additionally, their intracellular mercury levels aligned with this toxicity rank, being 10–20-fold lower than those for MeHg under non-cytotoxic conditions. A previous study reported that the intracellular mercury levels in cells exposed to 1 µM MeHg for 24 hr were approximately 600–700 ng/mg protein (Takanezawa et al., 2023). This previous study and our results suggest that intracellular mercury levels are proportional to the exposure dose under certain conditions. HgCH₂CHO and HgCH₂COOH may require higher extracellular concentrations to elicit toxicity because their uptake efficiency is low, and the resulting intracellular mercury accumulation is limited. Nevertheless, we cannot fully exclude the possibility that the toxicity per unit of intracellular mercury may be relatively higher under specific conditions, which warrants further investigation.

The present data suggest that LAT1 may not contribute to the uptake of HgCH₂CHO and HgCH₂COOH, and HgCH₂CHO may be exported via MRPs, whereas the efflux of HgCH₂COOH likely involves other transporters in SH-SY5Y cells. A previous study reported that MeHg is efficiently transported as the MeHg–L-cysteine conjugate by LAT1 and LAT2 (Simmons-Willis et al., 2002). MeHg also forms glutathione complexes, and these conjugates are exported by MRPs (Farina and Aschner, 2019; Granitzer et al., 2020). However, under the conditions of this study, we did not observe a contribution of LAT1 to MeHg uptake, which is likely attributable to differences in cell type. HgCH₂CHO and HgCH₂COOH correspond to an aldehyde and a carboxylic acid, respectively. They are expected to have low membrane permeability by passive diffusion, particularly the carboxylate under physiological pH (Moser et al., 2025), which could limit cellular entry when active transport is minimal. Even when net cellular entry is limited, inorganic and organic mercury compounds may still disrupt cellular function through direct modulation of membrane proteins (e.g., Ca²⁺ channels) and/or interactions with vulnerable thiol groups within the plasma membrane that impair transport processes (Albrecht et al., 1993; Szücs et al., 1997; Wang and Horisberger, 1996). The results of MRP inhibitor experiments suggest that HgCH₂CHO may form glutathione complexes that are partly exported by MRPs, whereas HgCH₂COOH may have a low propensity to form glutathione complexes. However, the speciation of the mercury complexes under physiological conditions as well as their physicochemical parameters remain unclear and warrant further investigation.

James et al. argued that HgCH₂CHO and HgCH₂COOH are likely to persist as stable mercury-containing by-products of the acetaldehyde production process (James et al., 2020). By contrast, Balogh et al. and Tohyama emphasized the historical record repeatedly identifying MeHg in various sources, rendering James’s conclusion difficult to reconcile with these observations (Balogh and Tsui, 2020; Tohyama, 2020). Balogh et al. further argued that if HgCH₂CHO and HgCH₂COOH had been abundant, independent analytical studies would have reported them; instead, MeHg was consistently detected (Balogh and Tsui, 2020). However, it is possible that the extraction, pretreatment, distillation, and recrystallization methods available at the time, which were optimized for MeHg, did not efficiently recover these low-solubility mercury species. It is also possible that HgCH₂CHO and HgCH₂COOH were converted to MeHg under physiological or environmental conditions. Thus, the detection of MeHg in historical samples does not necessarily rule out the presence of these species.

This study has several limitations. First, we primarily investigated the cytotoxicity of HgCH₂CHO and HgCH₂ COOH and did not provide definitive evidence that HgCH₂CHO and HgCH₂COOH were present in historical factory effluents. Second, toxicity assessments were only performed in SH-SY5Y neuroblastoma cells; those in other cell types or primary neuronal cultures remain unexamined. Third, the mechanisms of cellular uptake/efflux and cell death of these species are insufficiently resolved, limiting causal interpretation of exposure–response relationships. Our findings do not challenge MeHg as the principal etiologic agent of Minamata disease; nevertheless, it remains possible that HgCH₂CHO and HgCH₂COOH formed as intermediates during MeHg production in the Chisso factory and could be present in factory effluents. Additionally, historical materials such as sediment and Cat 717 may be reexamined to explore the presence of HgCH₂CHO and HgCH₂COOH and to evaluate their bioaccumulation potential. Collectively, these examinations would not negate MeHg’s role in Minamata disease but could deepen our mechanistic understanding of the disease.

ACKNOWLEDGMENTS

The authors would like to express their sincere gratitude to Dr. Toyama for kindly providing the SH-SY5Y cell line used in this study.

Funding

No funding was received for the work.

Conflict of interest

The authors declare no competing interests.

Data availability

Contact the corresponding authors directly to request the underlying data.

Author contributions

Conceptualization: Yo Shinoda

Formal analysis: Kaito Yamashiro

Investigation: Kaito Yamashiro, Shun Kono, Takumi Katsuzawa, Sachie Arae, Ryo Irie, Yuuki Fujimoto

Supervision: Toshiyuki Kaji, and Yo Shinoda

Visualization: Kaito Yamashiro, Shun Kono, Takumi Katsuzawa

Writing – original draft: Kaito Yamashiro, Sachie Arae, Ryo Irie

Writing – review & editing: Shun Kono, Takumi Katsuzawa, Sachie Arae, Ryo Irie, Yuuki Fujimoto, Tsutomu Takahashi, Yasuyuki Fujiwara, Yasuhiro Shinkai, Toshiyuki Kaji, and Yo Shinoda

Ethical approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

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