Conversion of methylmercury into inorganic mercury via organomercurial lyase (MerB) activates autophagy and aggresome formation.

Methylmercury (MeHg) is converted to inorganic mercury (iHg) in several organs; however, its impact on tissues and cells remains poorly understood. Previously, we established a bacterial organomercury lyase (MerB)-expressing mammalian cell line to overcome the low cell permeability of iHg and investigate its effects. Here, we elucidated the cytotoxic effects of the resultant iHg on autophagy and deciphered their relationship. Treatment of MerB-expressing cells with MeHg significantly increases the mRNA and protein levels of LC3B and p62, which are involved in autophagosome formation and substrate recognition, respectively. Autophagic flux assays using the autophagy inhibitor chloroquine (CQ) revealed that MeHg treatment activates autophagy in MerB-expressing cells but not in wild-type cells. Additionally, MeHg treatment induces the accumulation of ubiquitinated proteins and p62, specifically in MerB-expressing cells. Confocal microscopy revealed that large ubiquitinated protein aggregates (aggresomes) associated with p62 are formed transiently in the perinuclear region of MerB-expressing cells upon MeHg exposure. Meanwhile, inhibition of autophagic flux decreases the MeHg-induced cell viability of MerB-expressing cells. Overall, our results imply that cells regulate aggresome formation and autophagy activation by activating LC3B and p62 to prevent cytotoxicity caused by iHg. These findings provide insights into the role of autophagy against iHg-mediated toxicity.

Inhibition of p38 Mitogen-Activated Protein Kinases Attenuates Methylmercury Toxicity in SH-SY5Y Neuroblastoma Cells.

Methylmercury (MeHg) is a toxic metal that causes irreversible damage to the nervous system, making it a risk factor for neuronal degeneration and diseases. MeHg activates various cell signaling pathways, particularly the mitogen-activated protein kinase (MAPK) cascades, which are believed to be important determinants of stress-induced cell fate. However, little is known about the signaling pathways that mitigate the neurotoxic effects of MeHg. Herein, we showed that pretreatment with a p38 MAPK-specific inhibitor, SB203580, attenuates MeHg toxicity in human neuroblastoma SH-SY5Y cells, whereas pretreatment with the extracellular signaling-regulated kinase inhibitor U0126 and the c-Jun N-terminal kinase inhibitor SP600125 does not. Specifically, we quantified the levels of intracellular mercury (Hg) and found that pretreatment with SB203580 reduced Hg levels compared to MeHg treatment alone. Further analysis showed that pretreatment with SB203580 increased multidrug resistance-associated protein 2 (MRP2) mRNA levels after MeHg treatment. These results indicate that detoxification of MeHg by p38 MAPK inhibitors may involve an efflux function of MeHg by inducing MRP2 expression.

Gadolinium-based contrast agents suppress adipocyte differentiation in 3T3-L1 cells.

Gadolinium-based contrast agents (GBCAs) are widely used in magnetic resonance imaging (MRI) to improve the sensitivity and enhance diagnostic performance. GBCAs are mostly eliminated from the body through the kidney after administration; however small amounts of gadolinium are retained in the brain and other tissues. Although there is increasing concern about the adverse health effects of gadolinium, the cellular effects of GBCAs remains poorly understood. Here, we elucidated the potential cytotoxicity of the GBCAs Omniscan and Gadovist in 12 different cell lines, especially 3T3-L1 adipocyte cell line. Omniscan and Gadovist treatments significantly increased intracellular gadolinium levels in 3T3-L1 cells in a time- and dose-dependent manner. Additionally, Omniscan and Gadovist treatments downregulated the expression of adipocyte differentiation markers, including peroxisome proliferator-activated receptor γ (PPARG), adiponectin (ADIPOQ), and fatty acid-binding protein (FABP4), in 3T3-L1 cells, especially during early differentiation (day 0-2). Moreover, histological analysis using Oil red O staining showed that gadolinium chloride (GdCl3) treatment suppressed lipid droplet accumulation and the expression of adipocyte differentiation markers. Overall, the results showed that Omniscan and Gadovist treatment suppressed adipocyte differentiation in 3T3-L1 cells, contributing to the understanding of the potential toxic effects of GBCA exposure.

Proteasome and p62/SQSTM1 are involved in methylmercury toxicity mitigation in mouse embryonic fibroblast cells.

Methylmercury (MeHg), an environmental pollutant, disrupts and impairs cellular function. MeHg binds to various cellular proteins, causing dysfunction and misfolding, which are considered underlying causes of MeHg toxicity. The p62 protein, also termed SQSTM1, is a ubiquitin-binding protein that targets ubiquitinated substrates to undergo autophagy and plays a key role in ameliorating MeHg toxicity. p62 also delivers ubiquitinated substrates to proteasomes. However, the role of these degradation systems in mitigating MeHg toxicity remains unknown. Herein, we explored the impact of the proteasome inhibitor MG132 on MeHg toxicity and examined the toxicity of co-treatment with MG132 and MeHg in p62KO mouse embryonic fibroblasts (MEFs) by analyzing cell viability, immunoblotting, mRNA levels, immunofluorescence, and the mercury content. The proteasome inhibitor MG132 enhanced MeHg-induced cytotoxicity while reducing intracellular mercury levels in MEFs. Co-treatment with MG132 and MeHg markedly increased levels of p62 and ubiquitinated proteins. Furthermore, co-treatment with MG132 and MeHg reduced p62KO MEF viability compared to that of wild-type MEFs. Our findings suggest that the proteasome participates in mitigating MeHg cytotoxicity, while p62 may play an important role in transporting MeHg-induced ubiquitinated proteins to the proteasome, as well as in autophagy. Collectively, these results imply that p62, and proteasome, and autophagy are vital for cytoprotection against MeHg toxicity.

Methylmercury drives lipid droplet formation and adipokine expression during the late stages of adipocyte differentiation in 3T3-L1 cells.

Chronic exposure to methylmercury (MeHg) is positively associated with obesity and metabolic syndromes. However, the effect of MeHg on adipogenesis has not been thoroughly investigated. This study investigated the effects of continuous exposure to 0.5 µM MeHg on adipocyte differentiation in 3T3-L1 cells. Oil Red O staining and triglycerides (TG) assays demonstrated that MeHg enhanced the TG content in 3T3-L1 cells. MeHg enhanced the mRNA and protein expression of adipocyte differentiation markers including peroxisome proliferator-activated receptor γ, adiponectin, and fatty acid-binding protein, and their expression levels were prominent during the late stages (days 6-8) after the induction of differentiation. In addition, 0.5 µM MeHg promoted the expression of autophagy-related genes, including light chain 3 B-II and p62, after induction of differentiation. Treatment of 3T3-L1 cells with chloroquine (CQ), an autophagy inhibitor, during the early stages (days 0-2) after induction of differentiation inhibited cellular lipid accumulation in the presence of 0.5 µM MeHg. However, treatment with CQ during the late stages (days 6-8) had little effect on the MeHg-induced increase in TG content and the expression of adipocyte differentiation markers. Although the underlying mechanisms in the late stages remain to be completely elucidated, but the present data suggest that autophagy and other mechanisms play critical roles in adipogenesis during MeHg-induced differentiation. Collectively, our results suggest that continuous exposure to MeHg induces TG accumulation and expression of genes related to adipogenesis, especially during the late stages of 3T3-L1 differentiation, which may contribute to an improved understanding of MeHg-induced adipogenesis.

Mesophyll specific expression of a bacterial mercury transporter-based vacuolar sequestration machinery sufficiently enhances mercury tolerance of Arabidopsis.

We aimed to efficiently enhance plant Hg(II) tolerance by the transgenic approach utilizing a bacterial mercury transporter MerC, an Arabidopsis mesophyll specific promoter pRBCS1A, and a vacuolar membrane targeting syntaxin AtVAM3/SYP22. We generated two independent homozygous Arabidopsis pRBCS1A-TCV lines expressing mT-Sapphire-MerC-AtVAM3 under the control of pRBCS1A. Quantitative RT-PCR showed that the transgene was expressed specifically in shoots of pRBCS1A-TCV lines. Confocal analyses further demonstrated the leaf mesophyll specific expression of mT-Sapphire-MerC-AtVAM3. Confocal observation of the protoplast derived from the F1 plants of the pRBCS1A-TCV line and the tonoplast marker line p35S-GFP-δTIP showed the tonoplast colocalization of mT-Sapphire-MerC-AtVAM3 and GFP-δTIP. These results clearly demonstrated that mT-Sapphire-MerC-AtVAM3 expression in Arabidopsis is spatially regulated as designed at the transcript and the membrane trafficking levels. We then examined the Hg(II) tolerance of the pRBCS1A-TCV lines as well as the p35S-driven MerC-AtVAM3 expressing line p35S-CV under the various Hg(II) stress conditions. Short-term (12 d) Hg(II) treatment indicated the enhanced Hg(II) tolerance of both pRBCS1A-TCV and p35S-CV lines. The longer (3 weeks) Hg(II) treatment highlighted the better shoot growth of the transgenic plants compared to the wild-type Col-0 and the pRBCS1A-TCV lines were more tolerant to Hg(II) stress than the p35S-CV line. These results suggest that mesophyll-specific expression of MerC-AtVAM3 is sufficient or even better to enhance the Arabidopsis Hg(II) tolerance. The Hg accumulation in roots and shoots did not differ between the wild-type Col-0 and the MerC-AtVAM3 expressing plants, suggesting that the boosted Hg(II) tolerance of the transgenic lines would be attributed to vacuolar Hg-sequestration by the tonoplast-localized MerC. Further perspectives of the MerC-based plant engineering are also discussed.

Protective function of the SQSTM1/p62-NEDD4 complex against methylmercury toxicity.

SQSTM1/p62, hereinafter referred to as p62, is a stress-induced cellular protein that interacts with various signaling proteins as well as ubiquitinated proteins to regulate a variety of cellular functions and cell survival. Methylmercury (MeHg) exposure increases the levels of p62, the latter playing a protective role in MeHg-induced toxicity. However, the underlying mechanism by which p62 alleviates MeHg toxicity remains poorly understood. Herein, we report the interaction of p62 with neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4), a HECT E3 ubiquitin ligase. The region of p62 where NEDD4 binds is located at the proline- and arginine (PR)-rich region (amino acids: 102-119), C-terminal extension of the Phox and Bem1 (PB1) domain. To evaluate the importance of the p62-NEDD4 complex, we examined the compensation of deletion mutant (GFP-Δ102-119 p62) for the lack of endogenous p62 in MEFs. GFP-p62/p62KO cells exhibited significantly higher cell viability than GFP-Δ102-119 p62/p62KO cells after treatment with MeHg. Our findings suggest novel mechanisms to alleviate MeHg toxicity through p62-NEDD4 complex formation.

Effects of chemical forms of gadolinium on the spleen in mice after single intravenous administration.

Gadolinium-based contrast agents (GBCAs) are widely used to improve tissue contrast during magnetic resonance imaging. Exposure to GBCAs can result in gadolinium deposition within human tissues and has become a clinical concern because of the potential toxic effects of free gadolinium (Gd3+). Here, we report the impact of a single administration of GBCAs (Omniscan and Gadovist), and Gd3+ on mouse tissues. Five-week-old male BALB/c mice were injected intravenously with GBCAs or Gd3+. Seven days after injection, relatively high levels of gadolinium were detected in the spleen (118.87 nmol/g tissue), liver (83.00 nmol/g tissue), skin (48.56 nmol/g tissue), and kidneys (25.59 nmol/g tissue) of the Gd(NO3)3 (high dose: 0.165 mmol/kg) group; in the bones (11.12 nmol/g tissue), kidneys (7.49 nmol/g tissue), teeth (teeth: 6.18 nmol/g tissue), and skin (2.43 nmol/g tissue) of the Omniscan (high dose: 1.654 mmol/kg) group and in the kidneys (16.36 nmol/g tissue) and skin (4.88 nmol/g tissue) of the Gadovist (high dose: 3.308 mmol/kg) group. Enlargement of the spleen was observed in the Gd3+ group (p < 0.05), but not in the Omniscan or Gadovist groups. Gd3+ caused iron accumulation around the white pulp of the spleen, suggesting that enlargement of the spleen is, at least in part, associated with Gd3+ and/or iron accumulation. Our results may help elucidate the relative risks of different types of gadolinium agents, the mechanisms involved, and even recognition of potential toxic effects of GBCAs.

Phytochelatin-mediated metal detoxification pathway is crucial for an organomercurial phenylmercury tolerance in Arabidopsis.

KEY MESSAGE: An organomercurial phenylmercury activates AtPCS1, an enzyme known for detoxification of inorganic metal(loid) ions in Arabidopsis and the induced metal-chelating peptides phytochelatins are essential for detoxification of phenylmercury. Small thiol-rich peptides phytochelatins (PCs) and their synthases (PCSs) are crucial for plants to mitigate the stress derived from various metal(loid) ions in their inorganic form including inorganic mercury [Hg(II)]. However, the possible roles of the PC/PCS system in organic mercury detoxification in plants remain elusive. We found that an organomercury phenylmercury (PheHg) induced PC synthesis in Arabidopsis thaliana plants as Hg(II), whereas methylmercury did not. The analyses of AtPCS1 mutant plants and in vitro assays using the AtPCS1-recombinant protein demonstrated that AtPCS1, the major PCS in A. thaliana, was responsible for the PheHg-responsive PC synthesis. AtPCS1 mutants cad1-3 and cad1-6, and the double mutant of PC-metal(loid) complex transporters AtABCC1 and AtABCC2 showed enhanced sensitivity to PheHg as well as to Hg(II). The hypersensitivity of cad1-3 to PheHg stress was complemented by the own-promoter-driven expression of AtPCS1-GFP. The confocal microscopy of the complementation lines showed that the AtPCS1-GFP was preferentially expressed in epidermal cells of the mature and elongation zones, and the outer-most layer of the lateral root cap cells in the meristematic zone. Moreover, in vitro PC-metal binding assay demonstrated that binding affinity between PC and PheHg was comparable to Hg(II). However, plant ionomic profiles, as well as root morphology under PheHg and Hg(II) stress, were divergent. These results suggest that PheHg phytotoxicity is different from Hg(II), but AtPCS1-mediated PC synthesis, complex formation, and vacuolar sequestration by AtABCC1 and AtABCC2 are similarly functional for both PheHg and Hg(II) detoxification in root surficial cell types.

Development of affinity bead-based in vitro metal-ligand binding assay reveals dominant cadmium affinity of thiol-rich small peptides phytochelatins beyond glutathione.

For a better understanding of metal-ligand interaction and its function in cells, we developed an easy, sensitive, and high-throughput method to quantify ligand-metal(loid) binding affinity under physiological conditions by combining ligand-attached affinity beads and inductively coupled plasma-optical emission spectrometry (ICP-OES). Glutathione (GSH) and two phytochelatins (PC2 and PC3, small peptides with different numbers of free thiols) were employed as model ligands and attached to hydrophilic beads. The principle of the assay resembles that of affinity purification of proteins in biochemistry: metals binding to the ligand on the beads and the rest in the buffer are separated by a spin column and quantified by ICP-OES. The binding assay using the GSH-attached beads and various metal(loid)s suggested the different affinity of the metal-GSH interactions, in accordance with the order of the Irving-Williams series and the reported stability constants. The binding assay using PC2 or PC3-attached beads suggested positive binding between PCs and Ni(II), Cu(II), Zn(II), Cd(II), and As(III) in accordance with the number of thiols in PC2 and PC3. We then conducted the competition assay using Cd(II), Mn(II), Fe(II), Cu(II), and Zn(II), and the results suggested a better binding affinity of PC2 with Cd(II) than with the essential metals. Another competition assay using PC2 and GSH suggested a robust binding affinity between PCs and Cd(II) compared to GSH and Cd(II). These results suggested the dominance of PC-Cd complex formation in vitro, supporting the physiological importance of PCs for the detoxification of cadmium in vivo. We also discuss the potential application of the assay.

p62/sequestosome 1 attenuates methylmercury-induced endoplasmic reticulum stress in mouse embryonic fibroblasts.

Methylmercury (MeHg) is a hazardous environmental pollutant that causes serious toxicity in humans and animals, as well as proteotoxic stress. In our previous study, we found that MeHg induces the expression of p62/sequestosome 1 (p62) that selectively targets ubiquitinated proteins for degradation via autophagy, and that p62 might protect cells against MeHg toxicity. To further investigate the role of p62 in MeHg-induced stress responses, we evaluated the role of p62 in MeHg-induced endoplasmic reticulum (ER) stress in p62 knockout (p62KO) mouse embryonic fibroblasts (MEFs). Treatment of wild-type (WT) MEFs were treated with MeHg (1 µM) increased mRNA levels of Chop encoding C/EBP homologous protein, Trib3 encoding Tribbles homolog 3, and Dnajb9 encoding DnaJ heat-shock protein family (Hsp40) member B9 increased, suggesting that ER stress is elicited by MeHg stress. Additionally, p62KO MEFs treated with MeHg showed a higher mRNA expression of Chop and Trib3 relative to that in WT MEFs. Furthermore, knock-in of GFP-p62 to p62KO cells diminished the Chop and Trib3 induction responses to MeHg stress and resulted in a higher cell viability than that of p62KO MEFs. These results suggest that the protective role of p62 against MeHg toxicity is partly mediated by suppressing the ER stress response.

Cadmium transport activity of four mercury transporters (MerC, MerE, MerF and MerT) and effects of the periplasmic mercury-binding protein MerP on Mer-dependent cadmium uptake.

Mercury superfamily proteins, i.e. inner membrane-spanning proteins (MerC, MerE, MerF and MerT) and a periplasmic mercury-binding protein (MerP), transport mercury into the cytoplasm. A previous study demonstrated that a Mer transporter homolog exhibits cadmium transport activity; based on this, the present study aimed to evaluate the cadmium transport activity of MerC, MerE, MerF and MerT and the effects of MerP co-expression in Escherichia coli. Bacteria expressing MerC, MerE, MerF or MerT without MerP were more sensitive to cadmium and significantly absorbed more cadmium than did the control strain. Expression of MerP in combination with MerC, MerE, MerF or MerT increased the bacterial sensitivity to cadmium and cadmium accumulation compared to a single expression of MerC, MerE, MerF or MerT. Cadmium uptake mediated by MerC, MerE, MerF or MerT was inhibited under cold or acidic conditions. These findings suggest that MerC, MerE, MerF and MerT are broad-spectrum heavy metal transporters that mediate both mercury and cadmium transport into cells and that MerP accelerates the cadmium transport ability of MerC, MerE, MerF and MerT.

Selection of Agar Reagents for Medium Solidification Is a Critical Factor for Metal(loid) Sensitivity and Ionomic Profiles of Arabidopsis thaliana.

For researchers in the plant metal field, the agar reagent used for the solid plate medium is a problematic factor because application of different agar types and even a different lot of the same agar type can mask the plant metal-related phenotypes and impair the reproducibility. In this study, we systematically assessed effects of different agar reagents on metal(loid) sensitivity and element accumulation of the Arabidopsis metal sensitive mutants. Three established mutants (cad1-3, cad1-6, and abcc1/2), and three different types of purified agar reagents (Type A, Type E, and Nacalai) with two independent batches for each reagent were subjected to the analyses. First, we found that element concentrations in the agar reagents largely varied among the agar types. Then the effects of agar reagents on the mutant metal(loid)-sensitivity were examined under As(III), Hg(II), Cd(II), and excess Zn(II) conditions. A significant variation of the mutant metal(loid)-sensitivity was observed among the different agar plates but the variation depended on the combination of metal(loid) stress and agar reagents. Briefly, the type-dependent variation was more evident under As(III) and Hg(II) than Cd(II) or excess Zn(II) conditions. A lot-dependent variation was also observed for Type A and Type E but not for Nacalai: hypersensitive phenotypes of cad1-3, cad1-6, and abcc1/2 under As(III) or Hg(II) treatments were diminished when different batches of the Type A or Type E agar types were used. We also found a significant variation of As and Hg accumulation in the wild-type and cad1-3. Plant As and Hg concentrations were remarkably higher and the difference between the genotypes was more evident when grown with Type A agar plates. We finally analyzed ionomic profiles in the plants exposed to As(III) stress. Agar-type specific ionomic changes in cad1-3 were more observed with the Type A plates than with the Nacalai plates. The presented results overall suggest that suitability of agar reagents for metal(loid)-related phenotyping depends on the experimental design, and an inappropriate selection of agar reagents can mask even very clear phenotypes of the established mutant like cad1-3. We also discuss perspectives on the agar problem in the plant metal study.

Significant contribution of autophagy in mitigating cytotoxicity of gadolinium ions.

Gadolinium-based contrast agents (GBCAs) are widely used in clinical magnetic resonance imaging (MRI). Free gadolinium ions (Gd3+) released from GBCAs potentially increase the risk of GBCA-related toxicity. However, the cellular responses to Gd3+ and the underlying mechanisms responsible for protection against Gd3+ remain poorly understood. Recently, autophagy has been considered a cell survival mechanism against various toxic metals. Here, we investigated the relationship between Gd3+ and autophagy, as well as the effect of autophagy inhibition on the survival of cells exposed to Gd3+. We found that the increased expression of microtubule-associated protein 1 light chain 3 (LC3)-II, a marker protein of autophagy, in Gd3+-exposed human embryonic kidney 293 (HEK293) cells. Moreover, we found a greater accumulation of LC3-II after exposure to an autophagy inhibitor, chloroquine (CQ), combined with Gd3+ than that after exposure to CQ alone, suggesting that Gd3+ activated autophagy in HEK293 cells. Furthermore, we found that Gd3+ reduced cell viability, which was more pronounced after CQ treatment. Our findings indicated that autophagy exerted a cytoprotective effect against Gd3+ toxicity, suggesting a potential link between autophagy and GBCA-associated adverse events.