2021 Volume 46 Issue 2 Pages 91-97
Methylmercury (MeHg), an environmental electrophile, binds covalently to the cysteine residues of proteins in organs, altering protein function and causing cytotoxicity. MeHg has also been shown to alter the composition of gut microbes. The gut microbiota is a complex community, the disturbance of which has been linked to the development of certain diseases. However, the relationship between MeHg and gut bacteria remains poorly understood. In this study, we showed that MeHg binds covalently to gut bacterial proteins via cysteine residues. We examined the effects of MeHg on the growth of selected Lactobacillus species, namely, L. reuteri, L. gasseri, L. casei, and L. acidophilus, that are frequently either positively or negatively correlated with human diseases. The results revealed that MeHg inhibits the growth of Lactobacillus to varying degrees depending on the species. Furthermore, the growth of L. reuteri, which was inhibited by MeHg exposure, was restored by Na2S2 treatment. By comparing mice with and without gut microbiota colonization, we found that gut bacteria contribute to the production of reactive sulfur species such as hydrogen sulfide and hydrogen persulfide in the gut. We also discovered that the removal of gut bacteria accelerated accumulation of mercury in the cerebellum, liver, and lungs of mice subsequent to MeHg exposure. These results accordingly indicate that MeHg is captured and inactivated by the hydrogen sulfide and hydrogen persulfide produced by intestinal microbes, thereby providing evidence for the role played by gut microbiota in reducing MeHg toxicity.
Methylmercury (MeHg) is an organometal characterized by an electron-deficient moiety that forms covalent bonds with electron-rich nucleophiles (Rabenstein and Saetre, 1977). It is estimated that there are approximately 214,000 cysteine residues in the human genome, 80-90% of which are present in SH groups, S-S bonds, or zinc ligands, whereas the remaining 10-20% are believed to exist as thiolate anions (Jones, 2008). Protein thiolate (deprotonated cysteine residues) readily forms covalent bonds with MeHg in the body, which thereby alters the three-dimensional structure of proteins and ultimately modifies protein function (Kanda et al., 2014; Yang et al., 2020). Indeed, several lines of evidence provide support for theory that MeHg binds to and inhibits the activities of proteins and enzymes involved in combating oxidative stress in cells, including glutaredoxin (Robitaille et al., 2016), glutathione peroxidase (Franco et al., 2009), glutathione reductase, thioredoxin reductase (Carvalho et al., 2008), superoxide dismutase (Shinyashiki et al., 1996), neuronal nitric oxide synthase (Shinyashiki et al., 1998), and Keap1 (Toyama et al., 2007). In addition, MeHg binds to Ca2+ transporters, glutamate transporters, and tubulin, thereby altering their functions (Kanda et al., 2014). Accordingly, exposure to MeHg may have multiple adverse effects on different organs, and particularly brain tissue.
A complex community of approximately 40 trillion bacteria coexist symbiotically within the lumen of the human gut, wherein they produce a diverse range of metabolites (Costea et al., 2018), some of which are transferred to the host and play a range of physiological roles in different organs. In this regard, an important link has been identified between the development of certain diseases and disturbance of the gut microbiota (Sekirov et al., 2010; Fan and Pedersen, 2021). Bacteria within the genus Lactobacillus are important members of the gut microbiota, the activities of which have a wide-ranging influence on both human health and disease (Heeney et al., 2018). The composition of the gut microbiota is influenced by environmental factors such as medications, dietary nutrients, and exposure to environmental chemicals (Costea et al., 2018). Among the latter, humans are constantly exposed to MeHg, even at low concentrations, through the consumption of large fish (Grandjean et al., 2010). However, although some studies have reported that exposure to MeHg can affect the composition of the gut microbiota (Bridges et al., 2018; Lin et al., 2020), the underlying mechanisms are yet to be elucidated. In this study, we sought to investigate whether MeHg affects the growth of Lactobacillus species by binding to their proteins via reactive cysteine residues, as well as to those in host organs.
In addition, recent evidence has indicated that reactive sulfur species (e.g., hydrogen sulfide and reactive per/polysulfide) act as regulators of MeHg toxicity via the formation of sulfur adducts that are less electrophilic, such as bismethylmercury sulfide [(MeHg)2S] (Yoshida et al., 2011). We previously reported that MeHg reacts with hydrogen sulfide and the persulfide GSSH to yield (MeHg)2S, which has been identified as a detoxified metabolite of MeHg in cells and rat liver (Yoshida et al., 2011; Abiko et al., 2015). Furthermore, a deficiency in cystathionine γ-lyase (CSE), a reactive sulfur species-producing enzyme, has been demonstrated to increase the susceptibility to MeHg (Akiyama et al., 2019), thereby indicating that a reduction in the amounts of reactive sulfur species in the body enhances the detrimental effects of MeHg exposure on health. Given that the gut is a sulfur-rich environment, which is primarily attributable to the production of hydrogen sulfide by sulfate-reducing bacteria (Rey et al., 2013), it is conjectured that MeHg is captured in the gut by reactive sulfur species produced by gut bacteria. In the present study, we confirmed the production of reactive sulfur species by gut bacteria and investigated the role of the gut microbiota in reducing the health risks associated with exposure to MeHg.
MeHg and 1,2-NQ were purchased from Sigma-Aldrich (St. Louis, MO, USA). β-(4-Hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) was obtained from Molecular Biosciences (Boulder, CO, USA), and sodium sulfide (Na2S), sodium disulfide (Na2S2), and biotin-PEAC-maleimide (BPM) were purchased from Dojindo (Kumamoto, Japan). Horseradish peroxidase (HRP)-conjugated anti-biotin antibodies and anti-rabbit IgG secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). A polyclonal antibody against 1,2-NQ was prepared as reported previously (Miura and Kumagai, 2010). PD mini Trap™ G-25 was purchased from GE Healthcare (Madison, WI, USA). All other reagents and chemicals used were of the highest grade available.
Female specific pathogen-free (SPF) C57BL/6 mice, male MCH mice, and male germ-free (GF) MCH mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). The male GF MCH mice were maintained in vinyl isolators, whereas the other mice were housed in plastic cages and maintained in a climate-controlled animal room (temperature, 24°C ± 1°C; humidity, 55% ± 5%) under a 12-hr light/dark cycle (lights on from 07:00 to 19:00). MeHg dissolved in water was administered via oral intubation. C57BL/6 mice were administered either drinking water or antibiotics (1 mg/mL ampicillin and 0.5 mg/mL vancomycin) in drinking water for 14 days, and then administered MeHg (5 mg/kg). All experiments were conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments issued by the Science Council of Japan.
Protein samples were separated using SDS-PAGE and electro-transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA) at 2 mA/cm2 for 1 hr. The membranes were blocked in 5% skim milk at 25 °C for 1-hr, incubated overnight with primary antibodies at 4°C, and then incubated with secondary antibodies at room temperature for 2-hr. The stained protein bands were detected using an enhanced chemiluminescence system (Nacalai Tesque, Kyoto, Japan) using an LAS 3000 imager (Fujifilm, Tokyo, Japan).
A BPM-labeling assay was performed to determine the availability of protein thiols following incubation with electrophiles. In this procedure, proteins are allowed to react with electrophiles, followed by incubation with BPM. A reduction in BPM-binding reflects the amount of modified thiols. The BPM-labeling assay was performed as described previously. Briefly, the feces of SPF or GF mice were homogenized in 100 mM HEPES buffer (pH 7.5), and the resulting homogenates were centrifuged (9,000 × g, 10 min, 4°C) to remove insoluble material. The supernatants thus obtained were filtered through a PD mini Trap™ G-25 column to yield high molecular weight (HMW) fractions, which were incubated with MeHg or 1,2-NQ at 37°C for 5 min, after which they were incubated with BPM at 37°C for 30 min. The samples were subsequently mixed with a half volume of SDS-PAGE loading buffer [62.5 mM Tris-HCl (pH 6.8), 8% glycerol (v/v), 2% SDS (w/v), and 0.005% bromophenol blue (w/v)] containing 50 mM tris(2-carboxyethyl)phosphine, incubated at 95°C for 5 min, and analyzed by using western blotting with an HRP-conjugated anti-biotin antibody or anti-1,2-NQ antibody.
Selected Lactobacillus species (L. reuteri, L. gasseri, L. casei, and L. acidophilus) were grown aerobically at 37°C on Lactobacilli MRS agar plates and broth (BD, Franklin lakes, NJ, USA). Overnight bacterial cultures were seeded at 1% under different test conditions. Aliquots of cultures (200 µL) were added to the wells of 96-well microtiter plates, which were incubated under aerobic conditions at 37°C, and bacterial growth was assessed at 1-hr intervals. Optical density at an absorbance of 600 nm was recorded using a SpectraMax iD3 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Using an ultrasonic disruptor (Tomy, Tokyo, Japan), 100 mg of SPF MCH or GF MCH mouse feces was homogenized in 1 mL methanol, and the homogenate thus obtained was centrifuged at 9,000 × g for 10 min at 4°C. HPE-IAM (5 mM) was added to the resulting supernatant and left to stand for 30 min at 37°C, after which an equal volume of 0.1% formic acid was added. Aliquots containing HPE-AM adducts were diluted twofold with 0.1% formic acid containing known amounts of isotope-labeled internal standards (Akiyama et al., 2019), which were then analyzed using LC-ESI-MS/MS for determination of sulfur nucleophiles. An EVOQ Qube™ triple quadrupole mass spectrometer (Bruker Daltonics, Billerica, MA, USA), coupled to an Advance™ UHPLC system (Bruker Daltonics), was used for LC-ESI-MS/MS. Sulfur nucleophile-derived HPE-AM adducts were separated by Advance™ UHPLC using a YMC-Triart C18 column (50 × 2.0 mm internal diameter) under the following elution conditions: mobile phase A (0.1% formic acid) with a linear gradient of mobile phase B (0.1% formic acid in methanol) from 5% to 90% for 15 min at a flow rate of 0.2 mL/min at 40°C. A heated electrospray ionization source was used to obtain the MS spectra with the following ion source settings: spray voltage, 4000 V; cone temperature, 350°C; heated probe temperature, 250°C; cone gas flow, 25 psi; probe gas flow, 50 psi; and nebulizer gas flow, 50 psi.
The concentrations of mercury within mouse organs were measured using an atomic absorption mercury detector (model MD-A or MA-2/BC-1; Nippon Instruments, Osaka, Japan).
Statistical significance was assessed based on analyses of variance (ANOVA), with correction for multiple comparisons in post hoc analysis. All statistical analyses were performed using GraphPad Prism (Graphpad Software, San Diego, CA, USA), and a p-value < 0.05 was considered significant.
We initially examined whether gut bacterial proteins have reactive cysteine residues that react with MeHg. The presence of reactive cysteine residues in proteins extracted from the feces of specific pathogen-free (SPF) mice with intact gut microbiota and germ-free (GF) mice without gut microbiota were assessed using a BPM assay. We accordingly detected reactive cysteine residues in the fecal protein of SPF mice, whereas very few were found in the feces of GF mice (Fig. 1A), thereby indicating that the majority of reactive cysteine residues originating from proteins were derived from gut bacteria. Furthermore, in the presence of MeHg, we observed a concentration-dependent reduction in the number of microbial reactive cysteine residues (Fig. 1B), thereby indicating that the gut microbiota-derived proteins were modified by MeHg via the reactive cysteine residues. Additionally, we also demonstrated that amounts of the reactive cysteine residues of gut bacterial proteins were reduced by exposure to 1,2-NQ, another electrophile (Kumagai and Abiko, 2017), as shown in Fig. 1C, and further confirmed the modification of gut microbial proteins by 1,2-NQ via immunoblotting using a 1,2-NQ antibody (Fig. 1D). Collectively, these results indicate that MeHg adversely affects the gut microbiota via a modification of bacterial and host proteins.
Covalent modification of gut bacterial proteins by electrophiles. (A) High molecular weight (HMW) fractions of proteins derived from the feces of specific pathogen-free (SPF) or germ-free (GF) mice were incubated with biotin-PEAC-maleimide (BPM), and the reaction mixtures were subjected to western blot analysis. (B and C) HMW fractions derived from the feces of SPF mice were incubated with (B) methylmercury (MeHg) or (C) 1,2-NQ, and then reacted with BPM. The reaction mixtures were thereafter subjected to western blot analysis. (D) Covalent modification of HMW fractions from the feces of SPF mice treated with 1,2-NQ, as detected using western blotting.
We subsequently examined the growth rates of selected Lactobacillus species (L. reuteri, L. gasseri, L. casei, and L. acidophilus) following exposure to different concentrations of MeHg, and accordingly observed that MeHg inhibited the growth of L. reuteri, L. casei, and L. acidophilus in a concentration-dependent manner (Fig. 2A-C). In contrast, MeHg was found to have minimal effect on the growth of L. gasseri (Fig. 2D), thereby indicating that sensitivity to MeHg may differ among Lactobacillus species. The mechanisms underlying differences in the sensitivity of gut bacteria to MeHg need to be clarified in future studies.
MeHg affects the growth of Lactobacillus. (A-D) Growth curves for (A) Lactobacillus reuteri, (B) L. casei, (C) L. acidophilus, and (D) L. gasseri in the absence or presence of different concentrations of MeHg for 24 hr. (E and F) Growth curve of L. reuteri in the absence or presence of 10 µM MeHg and (E) 100 µM Na2S2 or (F) 100 µM Na2S.
MeHg is inactivated by reactive sulfur species, including hydrogen sulfide and reactive persulfide, via the formation of sulfur adducts such as (MeHg)2S (Abiko et al., 2015). These sulfur adducts are weakly electrophilic and less toxic than MeHg (Yoshida et al., 2011), and therefore, using Na2S and Na2S2 as models for hydrogen sulfide and reactive persulfide, respectively, we investigated whether reactive sulfur species provide a protective effect against the suppression of Lactobacillus growth caused by exposure to MeHg (Yu et al., 2018; Agné et al., 2015; Bogdándi et al., 2019). We accordingly found that the MeHg-induced inhibition of L. reuteri growth was restored by Na2S2 treatment (Fig. 2E), whereas in contrast, Na2S had little effect (Fig. 2F). These results thus indicate that Na2S2 inactivates MeHg, thereby preventing the deleterious effects of MeHg on the growth of Lactobacillus via protein modification. The differences observed in the protective effects of Na2S2 and Na2S could be attributed to the lower pKa value of Na2S2 (~5), relative to that of Na2S (~7) (Kawagoe et al., 2017; Gun et al., 2004). In this regard, species of Lactobacillus are known to produce lactic and acetic acids, and when cultured, typically reduce the pH of the medium to approximately 4 to 5. Consistently, in the present study, the measured Lactobacillus culture medium pH values were between 4 and 6 (data not shown). Thus, unlike Na2S2, Na2S may be unable to deprotonate and react with MeHg under the low pH conditions generated by lactic acid bacteria.
To confirm the presence of gut microbiota-derived hydrogen sulfide and hydrogen persulfide, we went on to compare the levels of reactive sulfur species in the feces of SPF and GF mice. We accordingly found that the levels of these two sulfides were reduced in the feces of GF mice compared with those in SPF mouse feces (Fig. 3A and B), thereby indicating that gut microbiota contribute not only to the production of hydrogen sulfide but also to that of hydrogen persulfide.
The gut microbiota generates hydrogen sulfide and hydrogen persulfide, and reduces mercury in organs. (A and B) Liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) analysis was employed to determine the levels of (A) hydrogen sulfide and (B) hydrogen persulfide in low molecular weight fractions derived from the feces of specific pathogen-free (SPF) or germ-free (GF) mice. Data are shown as the means ± SEM. **p < 0.01 and ****p < 0.001, compared with control mice, as determined using t-tests. H2S, hydrogen sulfide; H2S2, hydrogen persulfide. (C-F) C57BL/6 mice initially received either drinking water (control) or antibiotics (1 mg/mL ampicillin and 0.5 mg/mL vancomycin) in drinking water for 14 days, after which they were orally administered MeHg (5 mg/kg). After 3 days, we determined the accumulation of mercury in the (C) cerebellum, (D) lungs, (E) liver, and (F) kidneys. Data are shown as the means ± SEM. *p < 0.05, ***p < 0.005, compared with control mice, as determined using t-tests.
In our previous study using CSE KO female mice (C57BL/6J background), we reported that the accumulation of mercury in organs is promoted by a reduction in MeHg inactivation via sulfur adduct formation, as a consequence of a reduction in reactive sulfur in vivo (Akiyama et al., 2020). In the present study, we investigated the role of the gut microbiota in mercury accumulation subsequent to MeHg exposure. In antibiotics-treated C57BL/6J female mice, we observed an increased accumulation of mercury in the cerebellum, lungs, and liver, although not in the kidneys, following exposure to MeHg, compared with those in the control mice harboring gut microbiota (Fig. 3C-F). These results accordingly provide evidence that MeHg is captured and inactivated by hydrogen sulfide and hydrogen persulfide in the gut, thereby indicating that gut bacteria possibly contribute to reducing the health risks associated with exposure to MeHg.
This work was supported by Grants-in-Aid (JP20H03490 and JP 20K21530 to Y.-G.K., JP18H05293 to Y. K., and JP18K14895 to M. A.) for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and AMED (JP18gm6010004h0003 to Y.-G.K.). We would like to thank Editage (www.editage.com) for English language editing.
The authors declare that there is no conflict of interest.