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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

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]