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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.

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.

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.

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.

Mercury accumulation in Arabidopsis shoots is accelerated by endodermis specific expression of fusion proteins of a bacterial mercury transporter MerC and a plant SNARE SYP121 under control of SCARECROW promoter. We previously demonstrated that the CaMV 35S RNA promoter (p35S)-driven ubiquitous expression of a bacterial mercury transporter MerC, fused with SYP121, an Arabidopsis SNARE protein increases mercury accumulation of Arabidopsis. To establish an improved fine-tuned mercury transport system in plants for phytoremediation, the present study generated and characterized transgenic Arabidopsis plants expressing MerC-SYP121 specifically in the root endodermis, which is a crucial cell type for root element uptake. We generated four independent transgenic Arabidopsis lines expressing a transgene encoding mCherry-MerC-SYP121 under the control of the endodermis-specific SCARECROW promoter (hereafter pSCR lines). Quantitative real-time PCR analysis showed that expression levels of the transgene in roots of the pSCR lines were 3-23% of the p35S driven-overexpressing line. Confocal microscopy analysis showed that mCherry-MerC-SYP121 was dominantly expressed in the endodermis of the meristematic zone as well as in the mature zone of the pSCR roots. Mercury accumulation in shoots of the pSCR lines exposed to inorganic mercury was overall higher than the wild-type and comparable to the p35S over-expressing line. These results suggest that endodermis-specific expression of the MerC-SYP121 fusion proteins in plant roots sufficiently enhances mercury uptake and accumulation into shoots, which would be an ideal phenotype for phytoremediation of mercury-contaminated environments.

Methylmercury (MeHg) is a highly toxic pollutant, and is considered hazardous to human health. In our previous study, we found that MeHg induces autophagy and that Atg5-dependent autophagy plays a protective role against MeHg toxicity. To further characterize the role of autophagy in MeHg-induced toxicity, we examined the impact of autophagy on microtubules and nuclei under MeHg exposure using Atg5KO mouse embryonic fibroblasts (MEFs). Low concentrations of MeHg induced a decrease in α-tubulin and acetylated-tubulin in both wild-type and Atg5KO cells. While α-tubulin acetylation was promoted by treatment with tubacin, a selective inhibitor of histone deacetylase 6, MeHg treatment inhibits the increase of tubacin-induced acetylated-tubulin. However, similar effects were observed for treatment with either tubacin or tubacin + MeHg in wild-type and Atg5KO cells. We also found a significant increase in the number of multinuclear cells upon MeHg exposure in Atg5KO MEFs compared to wild-type MEFs. In addition, DNA double strand breaks (DSBs), measured by phosphorylation of the core histone H2A variant (H2AX) on serine 139 (γH2AX), markedly increased in Atg5KO MEFs compared to wild-type MEFs. Our results therefore suggest that autophagy is not a simple elimination pathway of MeHg-induced damaged proteins, but that it also plays a protective role in the context of MeHg-associated DSBs.

Methylmercury (MeHg) is one of the most toxic environmental pollutants, presenting a serious health hazard worldwide. In this study, we examined the potential of derivatives of oleanolic acid (OA), such as OA 3-glucoside, OA 28-glucoside, and OA 3,28-diglucoside, to mitigate MeHg toxicity in vitro and in vivo. We found that OA 3-glucoside suppressed the cellular MeHg uptake by 63.4% compared with that of the control and improved the cell viability from 75.4% to 107.9% upon exposure to cytotoxic MeHg in Caco-2 cells. To verify the anti-MeHg activity of OA 3-glucoside, mice were orally administered MeHg (0, 1.0, or 5.0 mg kg−1·d−1), with or without OA 3-glucoside, and then mercury accumulation was measured in various organs of the mice. The mice co-treated with MeHg and OA 3-glucoside showed significantly lower mercury content in organs such as the cerebrum, cerebellum, liver, kidney, and spleen, with 83.1%, 68.7%, 71.7%, 82.1%, and 18.2% of those in the OA 3-glucoside-untreated group, respectively. This suggested OA 3-glucoside had the potential as an anti-MeHg compound, owing to its ability to suppress the distribution of MeHg into organs. Supporting this hypothesis, the mice treated with MeHg and OA 3-glucoside showed a tendency to survive one day longer than the control mice. Our findings suggest OA 3-glucoside administration alleviates the toxicity of MeHg by suppressing MeHg accumulation in organs.

For mercury phytoextraction, we previously demonstrated in Arabidopsis thaliana that a constitutive and ubiquitous promoter-driven expression of a bacterial mercury transporter MerC fused with SYP121, a plant SNARE for plasma membrane protein trafficking increases plant mercury accumulation. To advance regulation of ectopic expression of the bacterial transporter in the plant system, the present study examined whether merC-SYP121 expression driven by a root epidermis specific promoter (pEpi) is sufficient to enhance mercury accumulation in plant tissues. We generated five independent transgenic Arabidopsis plant lines (hereafter pEpi lines) expressing a transgene encoding MerC-SYP121 N-terminally tagged with a fluorescent protein mTRQ2 under the control of pEpi, a root epidermal promoter. Confocal microscopy analysis of the pEpi lines showed that mTRQ2-MerC-SYP121 was preferentially expressed in lateral root cap in the root meristematic zone and epidermal cells in the elongation zone of the roots. Mercury accumulation in shoots of the pEpi lines exposed to inorganic mercury was overall higher than the wild-type and comparable to the over-expressing line. The results suggest that cell-type specific expression of the bacterial transporter MerC in plant roots sufficiently enhances mercury accumulation in shoots, which could be a useful phenotype for improving efficiency of mercury phytoremediation.

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.

Acute and chronic arsenic (As) toxicity is a global health issue affecting millions of people, which leads to inactivation of over 200 enzymes, particularly those involved in cellular energy pathways and DNA synthesis and repair. The fern Pteris vittata acts as a hyperaccumulator of As and may be useful for phytoremediation to reduce disposal risks by utilizing metal-enriched plant biomass in energy and metal recovery. However, these ferns grow in limited environments and its transplantation and transport can be challenging. Therefore, we generated a transgenic Arabidopsis plant as a seed plant model, capable of accumulating As in their vacuole lumen. This was achieved by transforming the As-resistant bacterial As transporter, ArsB, via fusion with a organelle-targeting signal to the vacuolar membrane, N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs) protein, VAMP711. In this study, we developed the iVenus assay as a method for detecting whether the N- or C-terminus of a membrane protein is located on the cytoplasmic or exoplasmic side, and from the result of the iVenus assay, we generated the transgenic plant introduced N-terminal end of ArsB with VAMP711, localized to the central vacuolar membrane to accumulate As in the shoot and differentiation zone of root.

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.

Cancer cells enhance autophagic activity as a survival measure against metabolic and therapeutic stresses. The inhibition of autophagy may represent a valuable sensitizing target for cancer treatment. Recently, we examined the ability of various cytochalasins to inhibit autophagy and demonstrated the potent inhibitory effect of cytochalasin E (CE) on autophagic flux. The present study was conducted to investigate whether CE inhibited autophagosome-lysosome fusion, and to determine whether CE enhanced chemotherapy-induced cell death. Cell exposure to CE led to the accumulation of microtubule-associated protein light chain 3-II (LC3-II) and sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62) in a dose- and time-dependent manner. Cells treated with CE exhibited distinct formation of p62-positive structures on lysosome-associated membrane protein 2 (LAMP2)-positive lysosomal vesicles. CE treatment following serum starvation robustly reduced cell viability and increased expression levels of LC3-II and p62, in comparison to those of cells treated with CE alone. Furthermore, combination treatment with CE and bortezomib, an inhibitor of the 26S proteasome, showed a synergistic effect in targeting human lung cancer A549 cells. Altogether, our results demonstrated that CE treatment inhibited autophagosome-lysosome fusion, and this activity, in part, augmented bortezomib-induced cell death. Therefore, we concluded that CE may be a potentially effective therapeutic agent against lung cancer, especially in a combination therapy with proteasome inhibitors.

Phytochelatins (PCs) are major chelators of toxic elements including inorganic arsenic (As) in plant cells. Their synthesis confers tolerance and influences within-plant mobility. Previous studies had shown that various metal/metalloid ions differentially activate PC synthesis. Here we identified C-terminal parts involved in arsenite- [As(III)] dependent activation of AtPCS1, the primary Arabidopsis PC synthase. The T-DNA insertion in the AtPCS1 mutant cad1-6 causes a truncation in the C-terminal regulatory domain that differentially affects activation by cadmium (Cd) and zinc (Zn). Comparisons of cad1-6 with the AtPCS1 null mutant cad1-3 and the double mutant of tonoplast PC transporters abcc1/2 revealed As(III) hypersensitivity of cad1-6 equal to that of cad1-3. Both cad1-6 and cad1-3 showed increased As distribution to shoots compared with Col-0, whereas Zn accumulation in shoots was equally lower in cad1-6 and cad1-3. Supporting these phenotypes of cad1-6, PC accumulation in the As(III)-exposed plants were at trace level in both cad1-6 and cad1-3, suggesting that the truncated AtPCS1 of cad1-6 is defective in PCS activity in response to As(III). Analysis of a C-terminal deletion series of AtPCS1 using the PCS-deficient mutant of fission yeast suggested important regions within the C-terminal domain for As(III)-dependent PC synthesis, which were different from the regions previously suggested for Cd- or Zn-dependent activation. Interestingly, we identified a truncated variant more strongly activated than the wild-type protein. This variant could potentially be used as a tool to better restrict As mobility in plants.

Bacterial resistance to mercury compounds (mercurials) is mediated by proteins encoded by mercury resistance (mer) operons. Six merE variants with site-directed mutations were constructed to investigate the roles of the cysteine and histidine residues in MerE protein during mercurial transport. By comparison of mercurial uptake by the cell with intact and/or variant MerE, we showed that the cysteine pair in the first transmembrane domain was critical for the transport of both Hg(II) and CH 3Hg(I). Also, the histidine residue located near to the cysteine pair was critical for Hg(II) transport, whereas the histidine residue located on the periplasmic side was critical for CH 3Hg(I) transport. Thus, enhanced mercurial uptake mediated by MerE may be a promising strategy for the design of new biomass for use in the bioremediation of mercurials in the environment.

MerC, encoded by merC in the transposon Tn21 mer operon, is a heavy metal transporter with potential applications for phytoremediation of heavy metals such as mercuric ion and cadmium. In this study, we demonstrate that MerC also acts as a transporter for methylmercury. When MerC was expressed in Escherichia coli XL1-Blue, cells became hypersensitive to CH3Hg(I) and the uptake of CH3Hg(I) by these cells was higher than that by cells of the isogenic strain. Moreover, transgenic Arabidopsis plants expressing bacterial MerC or MerC fused to plant soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) accumulated CH3Hg(I) effectively and their growth was comparable to the wild-type plants. These results demonstrate that when the bacterium-derived merC gene is ectopically introduced in genetically modified plants, MerC expression in the transgenic plants promotes the transport and sequestration of methylmercury. Thus, our results show that the expression of merC in Arabidopsis results in transgenic plants that could be used for the phytoremediation and elimination of toxic methylmercury from the environment.

Some methylmercury (MeHg) is converted to inorganic mercury (Hg2+) after incorporation into human and animal tissues, where it can remain for a long time. To determine the overall toxicity of MeHg in tissues, studies should evaluate low concentrations of Hg2+. Although demethylation is involved, the participating enzymes or underlying mechanisms are unknown; in addition, the low cell membrane permeability of Hg2+ makes these analyses challenging. We established model cell lines to assess toxicities of low concentrations of Hg2+ using bacterial organomercury lyase (MerB). We engineered MerB-expressing HEK293 and HeLa cell lines that catalyze MeHg demethylation. These cells were significantly more sensitive to MeHg exposure compared to the parental cells. MeHg treatment remarkably induced metallothioneins (MTs) and hemeoxygenase-1 (HMOX-1) mRNAs and modest expression of superoxide dismutase 1, whereas catalase and glutathione peroxidase 1 mRNAs were not up-regulated. merB knockdown using small interfering RNA supported the induction of MT and HMOX-1 mRNA by MerB enzymatic activity. Pretreatment with Trolox, a water-soluble vitamin E analog, did not inhibit MeHg-induced elevation of MT-Ix and HMOX-1 mRNAs in MerB-expressing cells, suggesting that Hg2+ works independently of reactive oxygen species generation. Similar results were obtained in cells expressing MerB, suggesting that high MTs and HMOX-1 induction and cytotoxicity are common cellular responses to low intracellular Hg2+ concentrations. This is the first study to establish cell lines that demethylate intracellular MeHg to Hg2+ using bacterial MerB for overcoming the low membrane permeability of Hg2+ and exploring the intracellular responses and toxicities of low Hg2+ concentrations.

Fish consumption has both the risk of methylmercury (MeHg) poisoning and the benefit of obtaining n-3 polyunsaturated fatty acids (n-3 PUFAs), particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). However, the cellular interaction between MeHg and PUFAs remains unknown. Therefore, the aim of this study was to investigate the effects of MeHg and n-3 PUFA exposure on mouse embryonic fibroblasts (MEFs). The results showed that EPA had a negligible effect on MeHg-induced cell death, whereas DHA promoted it. Thiobarbituric acid reactive substance (TBARS) concentrations in cells exposed to DHA and MeHg were higher than in those exposed to EPA and MeHg. Treatment with DHA and MeHg markedly induced the expression of endoplasmic reticulum (ER) stress (CHOP and DNAJB9) and Nrf2 target gene (p62 and HMOX-1) mRNA levels. Unexpectedly, EPA supplementation in addition to DHA and MeHg attenuated DHA- and MeHg-induced cell death and suppressed ER stress and expression of Nrf2 target genes. Our results revealed a differential impact of DHA and EPA on MeHg-induced cell death, and combined treatment with DHA and EPA along with MeHg attenuated MeHg-induced toxicity.

Autophagy is a cell survival process that represents a therapeutic target in cancer treatment. Many types of cytochalasins have been identified and some of them have been reported to interfere with the formation of the autophagosome, although only limited data are available to assess their potential effects. Therefore, in this study, we examined the effects of cytochalasins and structurally related compounds on cell survival and the regulation of autophagy in human lung A549 adenocarcinoma cells. Cytochalasin D (CD) and cytochalasin E (CE) prominently inhibited the growth of A549 cells in a dose-dependent manner. Following treatment with CE, F-actin filaments were disrupted, and the proportion of binucleated cells increased, whereas no such effects were observed with the seven other cytochalasins tested. We found that cytochalasin H (CH), CD, and especially CE could induce the up-regulation of autophagy-related protein (LC3-II) and SQSTM1/p62. Using bafilomycin A1, we demonstrated that CD, CE, and CH inhibited autophagosome turnover, resulting in a dysfunctional autophagic process. The results of this study reveal that CE is the most potent cytochalasin in terms of its ability to induce cell death and inhibit autophagy. CE may therefore be an effective therapeutic agent against lung cancer.

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.

The characteristics of bacteria take up mercury into cells via membrane potential-dependent sequence-divergent members of the mercuric ion (Mer) superfamily, i.e., a periplasmic mercuric ion scavenging protein (MerP) and one or more inner membrane-spanning proteins (MerC, MerE, MerF, and MerT), which transport mercuric ions into the cytoplasm, have been applied in engineering of bioreactor used for mercurial bioremediation. We engineered bacteria to express MerC, MerE, MerF, or MerT with or without MerP to clarify their individual role and potential in transport of mercurial. By immunoblot analysis using specific polyclonal antibody, the proteins encoded by merC, merE, merF, merT or merP, were certainly expressed and identified in the membrane fraction. Bacteria expressing MerC, MerE, MerF or MerT in the absence of MerP transported significantly more C6H5Hg(I) and Hg(II) across bacterial membrane than their isogenic strain. In vivo expression of MerP in the presence of all the transporters did not cause apparent difference to the C6H5Hg(I) transport, but gives an apparently higher Hg(II) transport than that did by MerE, MerF or MerT but not by MerC. Among the four transporters studied, MerC showed more potential to transport Hg(II) across bacterial membrane than MerE, MerF and MerT. Together these findings, we demonstrated for the first time that in addition to MerE and MerT, MerF and MerC are broad-spectrum mercury transporters that mediate both Hg(II) and phenylmercury transport into cells. Our results suggested that MerC is the most efficient tool for designing mercurial bioremediation systems, because MerC is sufficient for mercurial transport into cells.

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/m3in 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.

The feasibility of transgenic tobacco, engineered to express bacterial polyphosphate (polyP), for phytoremediation of mercury pollution was evaluated. T3 progeny of the transgenic tobacco produced a large amount of polyP in leaves and showed a relatively high resistant phenotype to Hg2+ than its wild-type progenitors. These results suggest that the integrated ppk gene, encoding polyphosphate kinase (PPK), a key enzyme for polyP biosynthesis, is stably conserved in tobacco genome, and translated to active PPK which catalyzed biosynthesis of polyP, and suggest that polyP is capable of reducing the cytotoxicity of Hg2+, probably via chelation formation with polyP. The transgenic tobacco expressing polyP accumulated significantly more mercury than its wild-type progenitors from Hg2+-containing agar medium and simulated soils without taxing the tobacco plants suggesting that the transported Hg2+ was accumulated as a less toxic Hg-polyP complex in the tobacco tissues. Based on the results obtained in the present study, the polyP-mediated accumulation of mercury from mercurial-contaminated soils may provide an ecologically compatible approach for phytoremediation of mercury pollution.

To prevent environmental mercury poisoning incidents, an effective technology for treating mercury-polluted environments is urgent. Recently, with advances in biotechnology, bioremediation utilizing microorganisms to remove mercurials from contaminated sites has become one of the most rapidly developing fields of environmental restoration. A number of bioremediation strategies, including biotransformation, biosorption, and bioprecipitation of mercurials, have been developed to treat mercurial-polluted environments and mercury-containing waste. To construct bacteria that are capable of specifically accumulating mercury, we have genetically engineered Escherichia coli to express a mercury transport system and organomercurial lyase enzyme simultaneously, and overexpress polyphosphate, a strong chelator of essential divalent metals. The mercury accumulation system was designed so that overexpressed polyphosphate would serve as a mercury accumulator; the mercury transport system would make the bacterial cell specifically accumulate mercury; and the intracellular accumulation process would allow the bioaccumulation system to be less sensitive to ambient conditions. The applicability of the new engineered bacteria in the environmental remediation of mercurials is evaluated and discussed in this review.

A new whole-cell bacterial sensor for the detection of low concentration of mercury in environment was constructed by gene fusion between a mercury resistance (mer) operon from pMR26 of Pseudomonas strain K-62 and a promoterless luxAB gene from Vibrio harveyi. The luminescence-based biosensor was evaluated for the selectivity and sensitivity of the detection of mercury. Cadmium, lead, chromium and zinc ions did not interfere with the assay even at same concentration compared to Hg2+. Methylmercury, phenylmercury and mercuric sulfide also did not affect the biosensor. These results reveal that the specificity of the construct is restricted to bioavailable Hg2+. The sensitivity of the biosensor was improved by decreasing the cell density in the bioassay in addition to genetically expressing an Hg2+ transport system which was expected to increase the amount of mer operon-inducing mercury in the cytoplasm. In optimized assay conditions, the lowest detectable concentration of Hg2+ was 2 pM with 1 ml sample. This detection limit is enough to detect this compound in many contaminated and some pristine environmental samples.

Feasibility of biological mercury removal from wastewater was examined by using alginate-immobilized cells of Escherichia coli carrying mer-ppk fusion plasmid pMKB18. Immobilized cells engineered to express mercury-transport system, organomercurial lyase and polyphosphate efficiently removed organic and inorganic mercury from contaminated wastewater over a wide concentration range of mercurials, probably via intracellular accumulation mediated by ppk-specified polyphosphate. Bioaccumulation of mercury was selective compared to other metals such as Cd(2+), Pb(2+) and Cr(6+). The immobilized cells could be used repeatedly (at least three times) without large loss of mercury removal activity. From these results, it is concluded that the mer-ppk fusion plasmid and the immobilized cells are useful for simultaneous removal of organic and inorganic mercury from contaminated wastewater.

To investigate the role of aromatic amino acids of MerT protein in phenylmercury transport, two merT variants (pMRTm1P and pMRTm2P) with specific site-directed mutations were constructed and examined their effects on C6H5Hg+-transport. Substitution of Phe-36 and Trp-40 residues located on the periplasmic loop of MerT in turn with Ala and Val, respectively, did not affect the Hg2+-uptake, but caused a significant reduction in the C6H5Hg+-uptake by bacterial cells with intact merT gene. Introduction of specific mutations changing Phe-108, 114, 115 to Ala, and Tyr-110, 116 to Ser in the C-terminal region of the third transmembrane of MerT also caused large reduction in the uptake of C6H5Hg+, but had no effect on the Hg2+-uptake. In addition, both mutations caused a significant reduction in the hypersensitivity to C6H5Hg+, but without affecting the Hg2+-hypersensitive phenotype. Together these results suggest that the aromatic amino acids of MerT protein may play an important role in the transport of C6H5Hg+ across the cell membrane.

To investigate the biochemical mechanism responsible for NO-induced neurotoxicity, the effect of sodium nitroprusside(SNP), a NO-generating agent, on PC12 cells was studied. The cell density was dose-dependently inhibited by SNP. Neuronally differentiated PC12 cells showed a higher resistance to SNP than the undifferentiated cells. The inhibitory effect was enhanced by 8-Br-cGMP, and reduced by methylene blue. However, 8-Br-cGMP alone had no significant cytotoxicity. SNP also inhibited [3H]-thymidine incorporation into the cells in a dose-dependent manner. The dose response curves for reducing cell density and for inhibiting thymidine incorporation; were found to be virtually superimposable. These results suggested that cytotoxieity elicited by NO seemed to be due to inhibition of DNA synthesis in PC12 cells.

The bacterial merE gene derived from the Tn21 mer operon encodes a broad-spectrum mercury transporter that governs the transport of methylmercury and mercuric ions across bacterial cytoplasmic membranes, and this gene is a potential molecular tool for improving the efficiency of methylmercury phytoremediation. A transgenic Arabidopsis engineered to express MerE was constructed and the impact of expression of MerE on methylmercury accumulation was evaluated. The subcellular localization of transiently expressed GFP-tagged MerE was examined in Arabidopsis suspension-cultured cells. The GFP-MerE was found to localize to the plasma membrane and cytosol. The transgenic Arabidopsis expressing MerE accumulated significantly more methymercury and mercuric ions into plants than the wild-type Arabidopsis did. The transgenic plants expressing MerE was significantly more resistant to mercuric ions, but only showed more resistant to methylmercury compared with the wild type Arabidopsis. These results demonstrated that expression of the bacterial mercury transporter MerE promoted the transport and accumulation of methylmercury in transgenic Arabidopsis, which may be a useful method for improving plants to facilitate the phytoremediation of methylmercury pollution.

Although it has been shown that exposure to diesel exhaust (DE) is linked to the induction or exacerbation of respiratory disorders, the major components responsible have not been fully identified. We examined the effects of airway exposure to nanoparticle-rich DE (NR-DE) or DE without particles on allergic pulmonary inflammation in mice. We also investigated the cellular responses to intratracheal instillation of NR-DE particles (NR-DEP). ICR mice inhaled one of four different mixtures (control air, low-concentration DE, high-concentration DE, and high-concentration DE without particles) for 8 weeks in the presence or absence of repeated intratracheal administration of ovalbumin (OVA). In a separate study, NR-DEP and/or OVA were repeatedly administrated intratracheally to mice. High-concentration NR-DE or DE without particles substantially exacerbated OVA-induced eosinophilic airway inflammation. This exacerbation was concomitant with increases in lung levels of Th2 cytokines such as interleukin (IL)-4, IL-5, and IL-13 and of chemokines such as monocyte chemotactic protein-1. Furthermore, in the presence of allergen, both DE without particles and high-concentration NR-DE strongly enhanced the production and release of myeloperoxidase into the alveolar spaces. Repeated administration of NR-DEP did not substantially affect the allergic asthma. These results strongly suggest that gaseous compounds in NR-DE aggravate murine allergic airway inflammation, mainly via amplification of the Th2 response.

To study the physiological role of the red pigments in soil strain Pseudomonas K-62, we isolated a red pigment-deficient white mutant from the soil strain by treatment with mitomycin C and compared the phenotypic properties of the mutant and parent strain. The red pigments, which were classified as one of carotenoids based on their physicochemical properties, were separated into two groups, designated pigment A and B respectively on NH-Chromatorex HPLC.The crude pigments and pigment B which could react with Hg2+ in the wild-type Pseudomonas K-62 and its mercury-resistant plasmid-deficient strain were enhanced by the addition of Hg2+. The white mutant thus obtained showed a greater sensitivity to Hg2+ than the wild-type reddish strain despite containing the resistant plasmids. The major component in pigment B was identified by mass spectrometric analysis as 1-hydroxy-1-methoxy-1,2, 1′,2′,7′,8′-hexahydro-ψ,ψ-caroten-4-one, a carotenoid monoketone. These results suggested that red pigments, especially pigment B, may account, at least partially, for defense against Hg2+ in the bacterial environments.

To clarify whether the merT and merP genes play a role in the transport of methylmercury, we constructed a deletion plasmid, pMRD141 which lacked the genes conferring the organomercurial lyase and the mercuric reductase from plasmid, pMRA17 containing the entire broad-spectrum mercury resistance determinants of the 26 kb-plasmid from Pseudomonas K-62. Plasmid, pMRD141 showed hypersensitivity to Hg2+ but still expressed a normal sensitivity to methylmercury. The mercury-induced hypersensitive cells carrying pMRD141 took up appreciably more 203Hg2+ than the induced resistant cells with pMRA17 and the sensitive cells with cloning vector, Bluescript II SK(+) but no difference in the uptake of CH3(203)Hg2+ among these three strains was found. These results suggested that the merT and merP are only involved in the Hg2+ transport but do not participate in the transport of methylmercury.

It has been shown that pulmonary exposure to diesel exhaust particles (DEP) disrupt immune systems, presenting as exacerbating effects on allergic manifestations (e.g., allergic asthma). To date, the impact of nano-level DEP on health has not been fully elucidated. Our institute (the National Institute for Environmental Studies) established an 'environmental nanoparticle exposure system applied in animals' in 2005 and, since then, the health effects of exposures to these types of agents have been explored. The present study was designed to investigate the in vitro effects of nanoparticle-rich DEP (NRDEP) on primary splenocytes from atopy-prone hosts. NC/Nga mouse-derived splenic mononuclear cells were co-cultured with NRDEP (0-50 µg/ml); thereafter, cell viability/proliferation was evaluated via a WST-1 assay, production/release of interleukin (IL)-17A in the culture supernatants by ELISA, and expression of RORγt (retinoic acid-related orphan receptor-γt) in cell lysates by Western blot analyses. The results indicated that NRDEP reduced cell viability/proliferation in a dose-related manner-significantly so at a level of 50 µg/ml NRDEP. In contrast, up to 10 µg NRDEP/ml increased RORγt expression in the splenocytes and subsequent IL-17A production/release by the cells in a dose-dependent manner with an overall trend (with significance vs 1 µg NRDEP/ml and 10 µg NRDEP/ml for IL-17A); 50 µg NRDEP/ml tended to inhibit the transcription factor expression and cytokine production/release. These results suggest that NRDEP can activate naive splenic mononuclear cells from atopy-prone animals in terms of RORγt and IL-17A induction (T(H)17 response).

It has been shown that pulmonary exposure to diesel exhaust particles (DEP) disrupts the immune system, presenting as exacerbating effects on allergic manifestations (e.g., allergic asthma). However, since a model inhalation system has not been developed, the impact of nano-level DEP on health has not been satisfactorily investigated. Our institute (the National Institute for Environmental Studies) established an "environmental nanoparticle exposure system applied in animals" in 2005 and since then, we have explored the health effects of exposure to these types of agent. The present study was conducted to investigate the in vitro effects of nanoparticle-rich DEP (NRDEP) on primary splenocytes from atopy-prone hosts. NC/Nga mouse-derived splenic mononuclear cells were co-cultured with NRDEP (0-50 µg/ml); thereafter, the production/release of interleukin (IL)-18 in the culture supernatants was evaluated by means of ELISA. NRDEP increased IL-18 production/release by splenocytes in a dose-dependent manner with an overall trend (with significance vs. 10 µg/ml of NRDEP). In contrast, 50 µg/ml of NRDEP inhibited production/release. These results suggest that NRDEP can activate naive splenic mononuclear cells from atopy-prone animals in terms of IL-18 induction.

The bacterial merC gene from the Tn21-encoded mer operon is a potential molecular tool for improving the efficiency of metal phytoremediation. Arabidopsis SNARE molecules, including SYP111, SYP121, and AtVAM3 (SYP22), were attached to the C-terminus of MerC to target the protein to various organelles. The subcellular localization of transiently expressed GFP-fused MerC-SYP111, MerC-SYP121, and MerC-AtVAM3 was examined in Arabidopsis suspension-cultured cells. We found that GFP-MerC-SYP111 and GFP-MerC-SYP121 localized to the plasma membrane, whereas GFP-AtVAM3 localized to the vacuolar membranes. These results demonstrate that SYP111/SYP121 and AtVAM3 target foreign molecules to the plasma membrane and vacuolar membrane, respectively. To enhance the efficiency and potential of plants to sequester and accumulate cadmium from contaminated sites, transgenic Arabidopsis plants expressing MerC, MerC-SYP111, MerC-SYP121, or MerC-AtVAM3 were generated. The transgenic plants that expressed MerC, MerC-SYP121, or MerC-AtVAM3 appeared to be normal, whereas the transgenic that expressed MerC-SYP111 exhibited severe growth defects. The transgenic plants expressing merC-SYP121 were more resistant to cadmium than the wild type and accumulated significantly more cadmium. Thus, the expression of MerC-SYP121 in the plant plasma membrane may provide an ecologically compatible approach for the phytoremediation of cadmium pollution.

It has been shown that pulmonary exposure to diesel exhaust particles (DEP) disrupt immune systems, presenting as exacerbating effects on allergic manifestations (i.e., allergic asthma). However, since inhalation system could not be developed, impact of nano-level DEP on health has not been satisfactorily elucidated. Our institute (National Institute for Environmental Studies) established the "environmental nanoparticle exposure system applied in animals" in 2005, and, since then, we have explored the health effects of the exposure. As part of our ongoing research, the present study was aimed to investigate the effects of nanoparticle-rich DEP (NRDEP) on the characterization of primary atopy-prone splenocytes in vitro. NC/Nga mouse-derived splenic mononuclear cells were co-cultured with NRDEP (0-50 µg/ml); thereafter, the surface expression of CD11c, CD80, CD86, CD69, and CD40L was evaluated by means of flow cytometry. NRDEP increased the surface expression of these molecules on the splenocytes in a dose-dependent manner with an overall trend (with significance vs. 50 µg/ml of NRDEP). These results suggest that NRDEP can activate naive splenic mononuclear cells from atopy-prone animals.

An Escherichia coli strain was generated by fusion of a merA-deleted broad-spectrum mer operon from Pseudomonas K-62 with a bacterial polyphosphate kinase gene (ppk) from Klebsiella aerogenes in vector pUC119. A large amount of the ppk-specified polyphosphate was identified in the mercury-induced bacterium with the fusion plasmid designated pMKB18 but not in the cells without mercury induction. These results suggest that the synthesis of polyphosphate as well as the expression of the mer genes is mercury-inducible and regulated by merR. The E. coli strain with pMKB18 was more resistant to both Hg2+ and C6H5Hg+ than its isogenic strain with cloning vector pUC119. The recombinant strain accumulated more mercury from Hg2+- and C6H5Hg+-contaminated medium. Hg2+ transported into the cytoplasm appeared to be bound by chelation with the polyphosphate produced by the recombinant cells. The transported phenylmercury was degraded to Hg2+ before the chelation since polyphosphate did not directly chelate with C6H5Hg+. These results indicate that polyphosphate is capable of reducing the cytotoxicity of the transported Hg2+ probably via chelation between polyphosphate and Hg2+.

To evaluate the utility of polyphosphate kinase gene (ppk)-specified polyphosphate in mercury remediation, a fusion plasmid, pMK27, with ppk from Klebsiella aerogenes and mercury transport genes, merT and merP, from Pseudomonas K-62, was constructed. The transcription and translation of ppk, merT and merP were found to be mercury-inducible. The ppk-specified polyphosphate was identified in cells preinduced by Hg2+, but not in cells without mercury induction, suggesting that the synthesis of polyphosphate is regulated by merR. The hyper-sensitive phenotype to Hg2+, shown by bacteria with pMRD141, which contains merT and merP, was almost completely restored to its original levels when the ppk was introduced into the plasmid, suggesting that the Hg2+-toxicity was reduced by the polyphosphate, probably via chelation formation. Bacteria with pMK27 accumulated approximately 6-fold more mercury than the bacteria with cloning vector, pUC119. These results clearly demonstrate that the polyphosphate is capable of retaining mercury in the cells without taxing the cells. Based on the results obtained in the present study, the fusion plasmid pMK27 may serve as a strategy for mercury remediation.

pMRA17 cloned from Pseudomonas K-62 plasmid pMR26 specified the resistance to both organic and inorganic mercurials. DNA sequence of this broad-spectrum resistant mer operon was determined. The 5504-bp sequence includes six open reading frames (ORFs), five of which were identified as merR , merT , merP , merA and merB in order by analysis of deletion mutants and by comparison with the DNA and amino acid (aa) sequences of previously sequenced mer operons. The merB encoding organomercurial lyase showed a less identity than the other mer genes with those from other broad-spectrum resistance operons. The remaining ORF named merE , located between merA and merB , had no significant homology with the published mer genes and seemed to be a new gene which may involve in phenylmercury resistance. Induction experiments and maxicell analyses of the mer -polypeptides revealed that pMRA17 mer operon expressed mercurial-inducible phenotype and the merB and merE as well as the merA were under the control of MerR which could activate not only by mercuric ion but also by organomercurials. © 1997 Elsevier Science B.V. All rights reserved.