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Cadmium Toxicity Towards Testicular Cell Lines

Reproduction, sexual development, environmental compound exposure

Date : 15/12/2016

Author Information

Biola

Uploaded by : Biola
Uploaded on : 15/12/2016
Subject : Medicine

Cadmium exposure is a risk factor for male infertility. I examined the effects of cadmium chloride on the viability, proliferation, morphology and migration of mouse reproductive cell lines. Both cell lines exhibited time and/or concentration dependent reductions in cell viability in response to cadmium as determined by series of assays including MTT reduction, lactate dehydrogenase release, trypan blue dye exclusion and live/dead assays. Analysis of haematoxylin and eosin (H & E) and Coomassie Brilliant Blue stained cell monolayers indicated that exposure to 1 M and 12 M concentrations of cadmium for 4 h induced morphological changes consistent with reduced formation of the leading edge and an increased population of rounded cells in both cell lines. Analysis of fixed cell monolayers by indirect immunofluorescence using antibodies against cytoskeletal proteins revealed disruption of the microfilament and microtubule networks in cells treated with both concentrations of CdCl2 cells, but no obvious effect on intermediate filament organisation. Cell migration was inhibited in both cell lines by 1 and 12 M CdCl2 treatments as determined by live cell imaging in a 24 h scratch assay.

Western blotting analysis suggested that CdCl2 induced a gradual reduction in the levels of actin, although 1 M CdCl2 had no significant effect in Sertoli cells. However, a transient rise and fall in the levels of cofilin and p-cofilin (suggesting increased actin binding activity), respectively, were observed after 4 h exposure to this concentration of Cd2+, followed by a decrease in both (i.e. decreased activity) after 24 h in Sertoli cells. Leydig cells showed a significant decrease in the level of actin only with 12 M Cd2+at the two time points. However, there was a transient fall and rise in the levels of cofilin and p-cofilin, respectively after 24 h. Further analysis of Western blots of 1 M CdCl2 treated Sertoli cells suggested slight decreases in the levels of tubulin and stathmin 1, the latter showing increased levels of phosphorylation (suggesting overall decreased tubulin binding activity) over 24 h. In contrast, tubulin levels were significantly increased by both Cd2+ concentrations after 4 h and by 1 M at 24 h in Leydig cells. Data indicate that stathmin 1 levels were significantly decreased and increased by 12 M Cd2+ at 4 h and 24 h respectively, with a significant increase and decrease in phosphorylation state under the same conditions respectively in the same cell line. This indicates that Cd2+ interferes with the ability of stathmin to destabilise MTs.

Further analysis of Western blots from CdCl2 treated cell lysates indicated that the two concentrations of CdCl2 caused significant increases in HSP70 from 4 h to 24 h and in HSP90 at 24 h in Sertoli cells. HSP60, however, showed no significant changes under all conditions tested in this cell line. In contrast, in Leydig cells, HSP60, HSP70, and HSP90 were significantly increased by exposure to 12 M Cd2+ at both time points. Whilst there was no change in the HSP90 with 1 M Cd2+, HSP60 and HSP70 were significantly increased by exposure to the same M Cd2+ concentration at 4 h and 24 h, respectively.

Although there were changes in the levels of both total and phosphorylated forms of MAPKs examined, evaluation of phosphorylation ratios showed increased phosphorylation and activation of p38 under all conditions tested in both cell lines except with 12 M Cd2+ at 24 h, which showed no significant effect on p38 phosphorylation in Leydig cells. While MEK and ERK increased activation was observed with 12 M Cd2+ at 4 h and 24 h in Sertoli cells, their phosphorylation states were differentially affected under the same conditions in Leydig cells Interestingly, JNK phosphorylation was significantly reduced with 12 M CdCl2 at all time points and with 1 M Cd2+ at 24 h in Sertoli cells. Similarly, it was significantly reduced by both CdCl2 concentrations at 4 h and 24 h in Leydig cells. This indicates that Cd2+ exposure disrupts key signalling pathways involved in the function and survival of testicular cell lines

Proteomic analysis of 1 M CdCl2 treated Sertoli cell lysates showed that at least 14 proteins were differentially expressed after either 4 or 24 h exposure, four of which were tentatively identified, one of which is important in spermatogenesis. In conclusion, the two testicular cell lines exhibit similar levels of sensitivity to CdCl2 but differ in their precise molecular and morphological responses to the cytotoxic effects of this heavy metal.

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