The mosquitofish (Gambusia affinis) naturally inhabits freshwater (FW; 1-3‰) and seawater (SW; 28-33‰) ponds in constructed wetland. To explore the physiological status and molecular mechanisms for salinity adaptation of the mosquitofish, cytoprotective responses and osmoregulation were examined. In the field study, activation of protein quality control (PQC) mechanism through upregulation of the abundance of heat shock protein (HSP) 90 and 70 and ubiquitin-conjugated proteins was found in the mosquitofish gills from SW pond compared to the individuals of FW pond. The levels of aggregated proteins in mosquitofish gills had no significant difference between FW and SW ponds. Furthermore, the osmoregulatory responses revealed that the body fluid osmolality and muscle water contents of the mosquitofish from two ponds were maintained within a physiological range while branchial Na+/K+-ATPase (NKA) expression was higher in the individuals from SW than FW ponds. Subsequently, to further clarify whether the cellular stress responses and osmoregulation were mainly induced by hypertonicity, a laboratory salinity acclimation experiment was conducted. The results from the laboratory experiment were similar to the field study. Branchial PQC as well as NKA responses were induced by SW acclimation compared to FW-acclimated individuals. Taken together, induction of gill PQC and NKA responses implied that SW represents an osmotic stress for mosquitofish. Activation of PQC was suggested to provide an osmoprotection to prevent the accumulation of aggregated proteins. Moreover, an increase in branchial NKA responses for osmoregulatory adjustment was required for the physiological homeostasis of body fluid osmolality and muscle water content.
Single-walled carbon nanotubes (SWCNTs) are carbon-based nanomaterials that possess immense industrial potential. Despite accumulating evidence that exposure to SWCNTs might be toxic to humans, our understanding of the mechanisms for cellular toxicity of SWCNTs remain limited. Here, we demonstrated that acute exposure of short (1-3μm) and regular-length (5-30μm) pristine, carboxylated or hydroxylated SWCNTs inhibited cell proliferation in human somatic and human stem cells in a cell type-dependent manner. The toxicity of regular-length pristine SWCNT was most evidenced in NP69>CYT00086>MCF-10A>MRC-5>HaCaT > HEK-293T>HepG2. In contrast, the short pristine SWCNTs were relatively less toxic in most of the cells being tested, except for NP69 which is more sensitive to short pristine SWCNTs as compared to regular-length pristine SWCNTs. Interestingly, carboxylation and hydroxylation of regular-length SWCNTs, but not the short SWCNTs, significantly reduced the cytotoxicity. Exposure of SWCNTs also induced caspase 3 and 9 activities, mitochondrial membrane depolarization, and significant apoptosis and necrosis in MRC-5 embryonic lung fibroblasts. In contrast, SWCNTs inhibited the proliferation of HaCaT human keratinocytes without inducing cell death. Further analyses by gene expression profiling and Connectivity Map analysis showed that SWCNTs induced a gene expression signature characteristic of heat shock protein 90 (HSP90) inhibition in MRC-5 cells, suggesting that SWCNTs may inhibit the HSP90 signaling pathway. Indeed, exposure of MRC-5 cells to SWCNTs results in a dose-dependent decrease in HSP90 client proteins (AKT, CDK4 and BCL2) and a concomitant increase in HSP70 expression. In addition, SWCNTs also significantly inhibited HSP90-dependent protein refolding. Finally, we showed that ectopic expression of HSP90, but not HSP40 or HSP70, completely abrogated the cytotoxic effects of SWCNTs, suggesting that SWCNT-induced cellular toxicity is HSP90 dependent. In summary, our findings suggest that the toxic effects of SWCNTs are mediated through inhibition of HSP90 in human lung fibroblasts and keratinocytes.