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Abstract 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.

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.

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

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.

Abstract Methylmercury (MeHg) is a widely distributed environmental pollutant that causes a series of cytotoxic effects. However, molecular mechanisms underlying MeHg toxicity are not fully understood. Here, we report that sequestosome1/p62 protects mouse embryonic fibroblasts (MEFs) against low-dose MeHg cytotoxicity via clearance of MeHg-induced ubiquitinated proteins. p62 mRNA and protein expression in MEFs were temporally induced by MeHg exposure p62-deficient MEFs exhibited higher sensitivity to MeHg exposure compared to their wild-type (WT) counterparts. An earlier and higher level of accumulation of ubiquitinated proteins was detected in p62-deficient cells compared with WT MEFs. Confocal microscopy revealed that p62 and ubiquitinated proteins co-localized in the perinuclear region of MEFs following MeHg treatment. Further analysis of MEFs revealed that ubiquitinated proteins co-localized with LC3-positive puncta upon co-treatment with MeHg and chloroquine, an autophagy inhibitor. In contrast, there was minimal co-localization in p62-deficient MEFs. The present study, for the first time, examined the expression and distribution of p62 and ubiquitinated proteins in cells exposed to low-dose MeHg. Our findings suggest that p62 is crucial for cytoprotection against MeHg-induced toxicity and is required for MeHg-induced ubiquitinated protein clearance.

A chemical factory, using a production technology of acetaldehyde with mercury catalysis, was located southeast of Qingzhen City in Guizhou Province, China. Previous research showed heavy mercury pollution through an extensive downstream area. A current investigation of the mercury distribution in ambient air, soils, and plants suggests that mobile mercury species in soils created elevated mercury concentrations in ambient air and vegetation. Mercury concentrations of up to 600 ng/m3 in air over the contaminated area provided evidence of the mercury transformation to volatile Hg(0). Mercury analysis of soil and plant samples demonstrated that the mercury concentrations in soil with vaporized and plant-absorbable forms were higher in the southern area, which was closer to the factory. Our results suggest that air monitoring using a portable mercury analyzer can be a convenient and useful method for the rapid detection and mapping of mercury pollution in advanced field surveys.

There is increasing demand for solutions against cadmium pollution to secure food safety, and phytoremediation is one of the potential tools. We previously found that a bacterial mercury transporter MerC possesses cadmium uptake activity and its overexpression as a fusion protein with a plasma-membrane resident SNARE protein, SYP121 enhances the cadmium uptake ability of Arabidopsis plants. In this study, we examined whether two different root cell-type specific expression systems of MerC-SYP121 fusion protein could efficiently enhance cadmium accumulation of Arabidopsis plants, compared to the p35Sdriven ubiquitous expression system. Representative transgenic lines expressing MerC-SYP121 in root surface cells (pEpi lines) or root endodermal cells (pSCR lines), established in our previous studies, were subjected to different cadmium treatments along with the p35S line. A vertical agar plate assay showed that root surface-specific line pEpi, as well as the p35S line, showed about 15% higher cadmium accumulation in shoots after one-day 10 µM cadmium treatment, compared to the wild-type Col-0. On the other hand, the endodermis-specific line pSCR accumulated 30% less cadmium in its shoots. A similar cadmium accumulation pattern in shoots was observed under the environmentally relevant much lower cadmium treatment of 0.1 µM for 4 d, using the hydroponic culture system. To further examine the potential of the MerC-SYP121 expression system for cadmium phytoremediation, the transgenic plants were hydroponically exposed to 0.1 µM for 4 weeks. The cadmium accumulation after the 4 weeks of treatment was again 16% higher in the pEpi shoots compared to that of Col-0, whereas the p35S line only showed 6% higher Cd concentration. Shoots of the pSCR line accumulated slightly less cadmium compared to Col-0. Ionomic profiles in these plants were analyzed, however, pSCR-specific patterns were not evident. Nevertheless, in our previous studies, pEpi and pSCR lines both efficiently accumulated more mercury in shoots than in the wild-type. The presented results suggest that the effects of cell-typespecific MerC-SYP121 expression differ by the target metals and its expression in root surface cells rather than that in endodermis is suitable for enhancing root cadmium uptake and subsequent shoot accumulation. [ABSTRACT FROM AUTHOR]

Phytochelatin (PC) synthesis has been well demonstrated as a major metal tolerance mechanism in Arabidopsis thaliana, whereas its contribution to long-distance element transport especially in monocots remains elusive. Using rice as a cereal model, we examined physiological roles of Oryza sativa phytochelatin synthase 1 (OsPCS1) in the distribution and detoxification of arsenic (As) and cadmium (Cd), two toxic elements associated with major food safety concerns. First, we isolated four different transcript variants of OsPCS1 as well as one from OsPCS2. Quantitative real-time reverse transcription–PCR (RT-PCR) of each OsPCS transcript in rice seedlings suggested that expression of OsPCS1full, the longest OsPCS1 variant, was most abundant, followed by OsPCS2. Heterologous expression of OsPCS variants in PCSdeficient mutants of Schizosaccharomyces pombe and A. thaliana suggested that OsPCS1full possessed PCS activity in response to As(III) and Cd while the activity of other PCS variants was very low. To address physiological functions in toxic element tolerance and accumulation, two independent OsPCS1 mutant rice lines (a T-DNA and a Tos17 insertion line) were identified. The OsPCS1 mutants exhibited increased sensitivity to As(III) and Cd in hydroponic experiments, showing the importance of OsPCS1-dependent PC synthesis for rice As(III) and Cd tolerance. Elemental analyses of rice plants grown in soil with environmentally relevant As and Cd concentrations showed increased As accumulation and decreased Cd accumulation in grains of the T-DNA line. The Tos17 mutant also exhibited the reduced Cd accumulation phenotype. These contrasting effects on As and Cd distribution to grains suggest the existence of at least partially distinct PC-dependent pathways for As and Cd. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]