Sodium arsenite and cadmium chloride are two widespread environmental toxicants which have deleterious effects on living organisms. At the cellular level, sodium arsenite and cadmium chloride cause oxidative stress, dysregulation of gene expression, apoptosis, and the unfolding of protein. Furthermore, both chemical stressors individually have the ability to induce heat shock protein (HSP) accumulation. HSPs are molecular chaperones that aid in protein folding, translocation and in preventing stress-induced protein aggregation. Previously, our laboratory demonstrated that treatment of A6 kidney epithelial cells of the frog Xenopus laevis, with either cadmium chloride or sodium arsenite plus a concurrent mild heat shock resulted in an enhanced accumulation of HSPs that was greater than found with the sum of the individual stressors. To the best of our knowledge, no information is available to date on the effect that these two chemical stressors have in combination on HSP accumulation in aquatic organisms. The present study examined the effect of simultaneous sodium arsenite and cadmium chloride treatment on the pattern of HSP30 and HSP70 accumulation in Xenopus A6 cells. Immunoblot analysis revealed that the relative levels of HSP30 and HSP70 accumulation in A6 cells treated concurrently with sodium arsenite and cadmium chloride for 12 h were significantly higher than the sum of HSP30 or HSP70 accumulation from cells subjected to the treatments individually. For instance, the combined 10 µM sodium arsenite plus 100 µM cadmium chloride treatment resulted in a 3.5 fold increase in HSP30 accumulation and a 2.5 fold increase in HSP70 accumulation compared to the sum of the stressors individually. This finding suggested a synergistic action between the two stressors. Pretreatment of cells with KNK437, an HSF1 inhibitor, inhibited the combined sodium arsenite- and cadmium chloride-induced accumulation of HSP30 and HSP70 suggesting that this accumulation of HSPs may be regulated, at least in part, at the level of transcription. Immunocytochemical analysis employing the use of laser scanning confocal microscopy (LSCM) revealed that simultaneous treatment of cells with the two stressors induced HSP30 accumulation primarily in the cytoplasm in a punctate pattern with some dysregulation of F-actin structure. Increased ubiquitinated protein accumulation was observed with combined 10 µM sodium arsenite and 10, 50 or 100 µM cadmium chloride treatment compared to individual stressors suggesting an impairment of the ubiquitin-proteasome degradation system. Finally, while incubation of A6 cells with 1 µM sodium arsenite plus 10 µM cadmium chloride did not induce a detectable accumulation of HSPs, the addition of a 30 °C mild heat shock resulted in a strong accumulation of HSP30 and HSP70. This study has demonstrated that concurrent sodium arsenite and cadmium chloride treatment can enhance HSP accumulation. Since HSP accumulation is triggered by proteotoxic stress, these findings are relevant given the fact that aquatic amphibians in their natural habitat may be exposed to multiple chemical stressors simultaneously.
| Identifer | oai:union.ndltd.org:WATERLOO/oai:uwspace.uwaterloo.ca:10012/7916 |
| Date | 03 September 2013 |
| Creators | Khamis, Imran |
| Source Sets | University of Waterloo Electronic Theses Repository |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation |
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