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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.

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.

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.

Methylmercury (MeHg) is a widespread environmental pollutant and causes a serious hazard to health worldwide. However, molecular mechanisms underlying MeHg toxicity remain elusive. We show that MeHg reduced mouse embryonic fibroblast (MEF) viability in a dose-dependent manner. Furthermore, MeHg treatment increased levels of autophagy markers LC3-II and p62, possibly by acting on the MAPKs signaling pathway in several cell types. MeHg exposure elevated the number of LC3 puncta in stable GFP-LC3 MEFs and the number of autophagic vacuoles. The accumulation of LC3-II and p62 increased further when complementing MeHg with autophagy inhibitor, chloroquine. Moreover, we found that autophagy-related gene 5-deficient (Atg5-/-) MEFs exhibited higher sensitivity and higher levels of p62 compared to their wild-type counterparts following MeHg exposure. This suggested that p62 was upregulated at the transcription level by MeHg and degraded by Atg5-dependent autophagy. Our data demonstrate that MeHg exposure promotes autophagy, and Atg5-dependent autophagy serves to protect cells from MeHg cytotoxicity.

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.