2022 Volume 47 Issue 1 Pages 31-37
Brain susceptibility to methylmercury (MeHg) is developmentally and regionally specific in both humans and rodents, but the mechanism is not well clarified. Reactive sulfur species (RSS) with high nucleophilicity can react with MeHg, leading to the formation of a less toxic metabolite bismethylmercury sulfide, thus exerting cytoprotection. In this study, we assessed the variation of RSS content in the rat brain and evaluated its relevance in sensitivity to MeHg. Analyses of fetal/juvenile rat brains showed low RSS levels in early developmental stages. Site-specific analysis of adult rat brains revealed that cerebellar RSS levels were lower than those of the hippocampus. Microscopically, RSS levels of the granular cell layer were lower than those of the molecular layer in the cerebellum. Thus, low RSS levels corresponded with age and site of the brain that is vulnerable to MeHg. Taken together with the finding that brain RSS were consumed during MeHg exposure, these results indicate that RSS is a factor that defines the specificity of MeHg vulnerability in the brain.
Methylmercury (MeHg), the causative agent of Minamata disease, induces a variety of neurological impairments (Takeuchi, 1982). MeHg can easily pass through the placenta into the fetus via a neutral amino acid carrier (Aschner and Clarkson, 1987), thereby causing adverse effects on fetal brain development (Onishchenko et al., 2007; Montgomery et al., 2008). Human and animal developing fetuses and neonates are considered to be highly susceptible to MeHg (Rice and Barone, 2000; Debes et al., 2016). In fact, children with fetal-type Minamata disease caused by exposure to MeHg in utero were even born to mothers with no symptoms of MeHg poisoning (Harada, 1978). In addition, adult-type Minamata disease, caused by exposure to MeHg during adulthood, shows layer-specific brain lesions in the cerebrum and in the cerebellum (CBM) (Eto et al., 2010). In rodent models of MeHg intoxication, a similar pattern of brain lesions was also observed: neuronal degeneration in the deep layer of the cerebral cortex and in the granular cell layer (GL) of the CBM occurred in adult mice and rats, respectively (Fujimura and Usuki, 2014; Fujimura et al., 2009). In both models, the hippocampus (HPC) showed no pathological changes. Previous studies revealed factors that specifically determine vulnerability to MeHg, such as high MeHg accumulation in the fetal/juvenile brain (Sakamoto et al., 2018) and low expression of antioxidative enzymes in brain regions susceptible to MeHg (Fujimura and Usuki, 2014, 2017), but the mechanisms are not yet fully understood.
MeHg is an electrophile with low electron-deficient moiety that forms adducts by covalently binding to electron-rich nucleophilic substituents such as protein cysteine residues, thereby modulating protein structure and function (Kanda et al., 2014). This modification disrupts the function of redox-regulated proteins, such as thioredoxin reductase (Carvalho et al., 2008), glutaredoxin (Robitaille et al., 2016), glutathione reductase (Carvalho et al., 2008), and superoxide dismutase (Shinyashiki et al., 1996), thereby inducing oxidative stress, which is a critical factor in MeHg toxicity. In contrast, such modifications cause Keap1 (Kelch-like ECH-associated protein 1) and PTEN (phosphatase and tensin homologue) to function as sensor proteins to MeHg and activate electrophilic signals that contribute to cellular defense responses (Kumagai et al., 2013; Unoki et al., 2020, 2016). We previously reported a novel defense mechanism against MeHg via reactive sulfur species (RSS) that are endogenously produced and have high nucleophilicity. While cystathionine γ-lyase (CSE) has been identified as the final trans-sulfuration enzyme essential for cysteine (CysSH) biosynthesis from cystathionine (Steegborn et al., 1999), we reported that CSE can also catalyze the production of cysteine persulfide (CysSSH) from cystine as a substrate (Ida et al., 2014). In turn, CysSSH spontaneously produces other reactive persulfide species, such as glutathione persulfide (GSSH) from glutathione (GSH), and hydrogen sulfide (H2S) as a derivative (Ida et al., 2014; Ono et al., 2014). Thus, CSE acts as an RSS-generating enzyme. Mitochondrial cysteine-tRNA synthetase 2 also catalyzes the formation of CysSSH from CysSH (Akaike et al., 2017). Notably, MeHg can react with RSS, such as H2S and GSSH, leading to the formation of bismethylmercury sulfide [(MeHg)2S], a less toxic metabolite in cells and rat liver (Yoshida et al., 2011; Abiko et al., 2015). Additionally, CSE-deficient mice showed increased susceptibility to MeHg (Akiyama et al., 2019). These findings suggest the importance of RSS-mediated cytoprotection against MeHg. The fact that RSS content fluctuates dynamically during mouse development indicates the necessity to focus on the relationship between RSS content and MeHg sensitivity in vivo (Akiyama et al., 2019). We assessed the variation of RSS content in the brains of MeHg intoxication model rats and evaluated its relevance in sensitivity to MeHg.
MeHg (Tokyo Chemical Industry, Tokyo, Japan) was mixed with GSH (Nacalai Tesque, Kyoto, Japan) in equal molar ratios in water to prepare a 1000-ppm solution as an MeHg-GSH adduct so that it remained stable in water. Then the solution was diluted to 5 or 20 ppm for use as drinking water. Drinking water containing the same amount of GSH was used as a control. Female and male Wister rats were purchased from Clea Japan (Tokyo, Japan), followed by 1-week acclimation before commencing the treatments described below. For the embryonic/juvenile brain study, 11-week-old rats were mated and pregnancies were confirmed. From the first day of pregnancy, 5 ppm MeHg was administered to maternal rats via their drinking water throughout gestation and lactational periods. Embryonic and juvenile brains were collected during embryonic day (E) 14 to postnatal day (P) 55 at the indicated points (three independent samples for each point). For the adult brain study, 8-week-old male rats were divided into five groups of six rats. MeHg-containing water (20 ppm) was administered to rats in drinking water for 1, 2, 3, and 4 weeks exposure to MeHg. The rats were then deeply anesthetized by isoflurane inhalation and transcardially perfused with saline. Brains were isolated and divided into each brain region. All experiments were carried out in accordance with the guide for the care and use of laboratory animals, issued by the National Institute for Minamata Disease.
Acute cerebellar slice preparationCBM was mounted with cyanoacrylate adhesive on the specimen disk of a vibratome DTK-3000 (Dosaka EM, Kyoto, Japan). The CBM was covered with ice-cold saline and then sliced in 200-μm steps with vibratome settings as follows: frequency, 50%; speed, 10 mm/min; width, 15 mm. The slices were transferred to a plastic dish filled with ice-cold saline and separated into GL and molecular layer (ML) using spring scissors under a stereomicroscope Stemi 305 cam (Carl Zeiss, Oberkochen, Germany). Isolation of each layer was confirmed by Western blotting with antibodies for layer specific proteins, anti-calbindin (Abcam, Cambridge, UK) and anti-NeuN (Merck Millipore, Burlington, MA, USA). Anti-GAPDH (Abcam) was used as a control.
Sulfur nucleophile detectionLiquid chromatography tandem mass spectrometry (LC/MS/MS) analysis with β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) probe was used to determine the levels of sulfur nucleophiles, including persulfides, in the rat brain as previously reported (Akiyama et al., 2019; Akaike et al., 2017). The brain samples prepared above were sonicated in lysis buffer [50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, and 1% protease inhibitor cocktail] as 100 mg of sample/mL. The lysates were centrifuged (9,000 ×g, 10 min, 4°C) to remove insoluble material. The supernatants were filtered through an ultrafiltration membrane, Amicon Ultra-0.5 3K MWCO (Merck Millipore), to obtain low-molecular-weight (LMW) fractions. The LMW fractions were incubated with 5 mM HPE-IAM (Molecular Biosciences, Boulder, CO, USA) at 37°C for 30 min to yield HPE-AM adducts of sulfur nucleophiles. Aliquots containing HPE-AM adducts were diluted two- to four-fold with 0.1% formic acid containing known amounts of isotope-labeled internal standards, which were then analyzed by LC/MS/MS for sulfur nucleophile determination. A triple quadrupole mass spectrometer, EVOQ Qube (Bruker, Billerica, MA, USA), coupled to an Advance ultra-high-performance liquid chromatography (UHPLC) system (Bruker) was used to perform LC/MS/MS. Sulfane sulfur-derived HPE-AM adducts were separated by Advance UHPLC with a Triart C18 column (50 × 2.0 mm id; YMC, Kyoto, Japan) at 40°C. Mobile phases A (0.1% formic acid) and B (0.1% formic acid in methanol) at a flow rate of 0.2 mL/min were linearly mixed by using a gradient system as follows: 3% B for 3 min; linear increase over 12 min to 95% B; maintaining at 95% B for 1 min before returning linearly to 3% B. A heated electrospray ionization source was used to obtain MS spectra and the ion source settings were as follows: spray voltage, 4000 V; cone temperature, 350°C; heated probe temperature, 250°C; cone gas flow, 25 psi; probe gas flow, 50 psi; nebulizer gas flow, 50 psi.
Mercury detectionBrain samples prepared above were lysed in 5 N NaOH at 60°C for 20 min and neutralized with an equal amount of 5 N HCl. Total Hg concentrations in the samples were determined by the oxygen combustion-gold amalgamation method using an MA2000 Hg analyzer (Nippon Instruments, Tokyo, Japan) as described previously (Fujimura et al., 2012).
Western blottingCerebellar layer samples were lysed in T-PER tissue protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA) with gentle sonication. Protein concentration in the samples was measured by a protein assay bicinchoninic kit (Nacalai Tesque). Samples were heated in sample buffer [50 mM Tris-HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, 0.003% bromophenol blue] at 95°C for 5 min before application to SDS-PAGE. Proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked and then incubated with primary antibody at room temperature for 1 hr. The membrane was washed, then incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hr. Immunoreactive bands were visualized using chemiluminescence and scanned using a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA). Band intensities were quantified using Multi Gauge software (Fujifilm, Tokyo, Japan).
Statistical analysisThe statistical significance of differences between the results was assessed by Tukey’s post hoc test or unpaired t-test, using GraphPad Prism 8 (Graphpad Software, San Diego, CA, USA). p < 0.05 was considered to indicate a significant difference.
For sulfur nucleophile detection, we employed LC/MS/MS analysis combined with an alkylation agent HPE-IAM as previously reported (Akaike et al., 2017; Akiyama et al., 2019). In this system, sulfur nucleophiles could be quantified as their HPE-AM adducts (e.g., CysS-HPE-AM, CysSS-HPE-AM and Bis-S-HPE-AM for CysSH, CysSSH and H2S). We first measured the sulfur nucleophile content in the rat brain during development from E14 to P55. CysSH, its persulfide CysSSH, and H2S were significantly increased in an age-dependent manner compared with E14, while GSH and its persulfide GSSH did not show such an increase (Fig. 1A). This analysis was also performed at E20, P1, and P20 in offspring brains from mothers exposed to MeHg (5 ppm) via drinking water, and CysSSH and H2S contents were significantly decreased at E20 and P1, but not P20, compared with control (Fig. 1A). In this exposure condition, Hg accumulates in the offspring brain during the fetal period and declines after birth (Fujimura et al., 2012); thus, Hg content was significantly increased at E20 and P1 compared with control, but negligible at P20 (Fig. 1B). These results suggest that the brain content of RSS, such as CysSSH and H2S, which increases markedly after birth, is consumed under MeHg exposure in vivo.
Sulfur nucleophile contents and their alterations by MeHg exposure in the developing rat brain. (A) Maternal rats were exposed to MeHg (5 ppm) via drinking water throughout gestation and lactation periods. Brains of offspring were collected at the indicated embryonic day (E) and postnatal day (P), and sulfur nucleophile contents were determined by LC/MS/MS. As a control, offspring brains from unexposed maternal rats were used. Each value is the mean ± standard error for three independent experiments. *p < 0.05, **p < 0.01, compared with E14 control. †p < 0.05, compared with control at the same age. (B) Hg contents in brain samples prepared as in (A). Each value is the mean ± standard error for three independent experiments. **p < 0.01, compared with control at each time point.
Next, we assessed the brain region-specific content of sulfur nucleophiles and the effects of MeHg exposure on these in the adult rat brain. Adult rats were exposed to MeHg (20 ppm) via drinking water, and whole brain (WB), HPC, and CBM were sampled weekly up to 4 weeks. In this exposure condition, Hg accumulations in both HPC and CBM increased in a time-dependent manner but were not significantly different compared with WB, suggesting that MeHg exposure level was equal in all brain parts at each time point (Fig. 2B). As MeHg exposure progressed, there was a significant decrease in CysSSH and H2S in WB compared with its unexposed control (0 week), and this tendency was also observed in both HPC and CBM (Fig. 2A). However, RSS content in CBM was significantly lower than that of HPC in some cases: CysSSH at all weeks, H2S at 2 and 3 weeks, and GSSH at 1–4 weeks (Fig. 2A). Microscopically, we assessed the layer-specific sulfur nucleophile contents in CBM exposed to MeHg under identical conditions. Under a stereomicroscope, ML and GL of an acute cerebellar slice could be distinguished by their textural differences (Fig. 3A); the layer-specific sample preparations were confirmed by layer-specific marker proteins—calbindin and NeuN for ML and GL, respectively (Fig. 3B). Similar to the results for CBM in Fig. 2A, CysSSH and H2S decreased along with the progression of MeHg exposure in the sample without layer separation (Whole), and this tendency was also observed in the GL (Fig. 3C). However, ML only showed a significant decrease of CysSSH at the 3-weeks exposure point (Fig. 3C). Furthermore, RSS content in GL was significantly lower than that of ML in some cases: CysSSH at 2 and 3 weeks, H2S at 3 and 4 weeks, and GSSH at all weeks (Fig. 3C). In adult rats, CBM and especially its granule cells were vulnerable to MeHg, while HPC was robust (Fujimura and Usuki, 2014). Therefore, these results suggest that brain sites with low RSS content are vulnerable to MeHg.
Region-specific alteration of sulfur nucleophiles during MeHg exposure in adult rat brain. Adult rats were exposed to 20 ppm MeHg via drinking water for up to 4 weeks. Whole brain (WB), hippocampus (HPC) and cerebellum (CBM) were collected at the indicated points. (A) Brain region-specific sulfur nucleophile contents determined by LC/MS/MS. Each value is the mean ± standard error for three independent experiments. ap < 0.05, bp < 0.05 and cp < 0.05 compared with WB, HPC and CBM at 0 weeks, respectively. *p < 0.05, HPC compared with CBM at the same week. (B) Hg contents in the brain samples prepared as in (A). Each value is the mean ± standard error for three independent experiments.
Layer-specific sulfur nucleophile contents in adult rat cerebellum (CBM) exposed to MeHg. (A and B) Layer-specific isolation of rat CBM. Representative images of acute cerebellar slice (A) and Western blots of layer-specific samples isolated from acute cerebellar slice (B) are shown. Band intensities are presented as fold relative to whole samples. Each value is the mean ± standard error for three independent experiments. ML, molecular layer; GL, granular cell layer. Scale bars: 200 μm. (C) Cerebellar layer-specific sulfur nucleophile contents determined by LC/MS/MS. Adult rats were exposed to 20 ppm MeHg via drinking water for up to 4 weeks. Whole, GL, and ML were collected as in (A) at the indicated points. Each value is the mean ± standard error for three independent experiments. ap < 0.05, bp < 0.05, and cp < 0.05 compared with whole, HPC, and CBM at 0 weeks, respectively. *p < 0.05, GL compared with ML at the same week.
We previously reported that RSS is essential for the repression of MeHg toxicity in vivo, using mice deficient RSS-producing enzyme CSE (Akiyama et al., 2019, 2020). We also showed that CysSSH is increased in the mouse brain in a developmental stage-dependent manner (Akiyama et al., 2019). In the present study, we demonstrated the same phenomenon in the rat brain, and further expanded the dynamics of RSS distribution to site-specificity. In these analyses, we found that RSS was reduced by MeHg exposure, but not thiols such as CysSH, confirming that RSS is dominantly responsive to MeHg in a physiological state, presumably due to low pKa value (e.g., CysSH, 8.29; CysSSH, 4.34) (Cuevasanta et al., 2015). Such RSS consumption by MeHg suggests that RSS contributes to the maintenance of redox homeostasis in the brain through converting MeHg to the less toxic metabolite (MeHg)2S (Yoshida et al., 2011; Abiko et al., 2015) and preventing the disruption of electrophilic signaling (Ihara et al., 2017). Therefore, this study proposes RSS as one of the factors that define the specificity of MeHg vulnerability in the brain.
This work was supported by JSPS KAKENHI Grant Numbers JP19K16368 and JP21K06572 to T. U. and JP18H05293 to Y. K. We are grateful to Michiko Fuchigami and Shiori Fukuhama for their excellent technical assistance. We thank Gillian Campbell, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.
Conflict of interestThe authors declare that there is no conflict of interest.
