Cadmium elicits alterations in mitochondrial morphology and functionality in C3H10T1/2Cl8 mouse embryonic fibroblasts

Abstract

Background: Cadmium is a widespread contaminant and a recognized carcinogen. We previously showed that the administration of low cadmium doses for 24 hours treatment to healthy C3H10T1/2Cl8 cells at the beginning of Cell Transformation Assay (CTA), up regulates genes involved in metal scavenging and antioxidant defense, like metallothioneines, Glutathione S transferases and heat shock proteins. Still, although most cells thrive normally in the following weeks, malignancy is triggered by cadmium and leads to foci of transformed cells appearance at the end of the CTA. In this work we aim at elucidating the early metabolic deregulation induced by cadmium, underlying healthy cell transformation into malignant cells.Methods: Respiratory metabolism was investigated through Seahorse Agilent assays in different conditions, while oxidative stress level was assessed through fluorescent probes; DNA damage was evaluated by Comet assay and mitochondrial morphology was analyzed in confocal microscopy.Results: Results show that, although initial response to cadmium is effective in balancing oxidative stress, through mitochondria rearrangement, SOD1 activity is inhibited, leading to increased O 2 level, which in turn causes DNA strand breaks. From the metabolic point of view, cells increase their glycolytic flux, although all extra NADH produced is still efficiently reoxidized by mitochondria.Conclusions: Our results confirm previously shown response against cadmium toxicity; new data about glycolytic increase and mitochondrial rearrangements suggest pathways leading to cell transformation.General significance: In this work we exploit the widely used, well known CTA, which allows following healthy cells transformation into a malignant phenotype, to understand early events in cadmiuminduced carcinogenesis.

Insights into cadmium-induced carcinogenesis through an in-vitro study using C3H10T1/2Cl8 cells: the multifaceted role of mitochondria

“…This is likely accomplished through a mitochondrial reorganization in a tightly crowded distribution around the nucleus, as shown by confocal microscopy analysis. A similar picture was previously observed in C3H cells treated with CdCl 2 for 24 h (Oldani et al 2020b). However, all the NADH produced by Cd-treated C3H10T1/2Cl8 cells was e ciently reoxidized on the electron transport chain.…”

“…Finally, no autophagy activation was detected in the two foci, suggesting that the autophagy observed after cadmium administration for 24 h was part of the early mechanism of defense against this metal. Comparing data reported in this paper with those previously obtained on healthy C3H10T1/2Cl8 cells treated with CdCl 2 for 24 h (Oldani et al 2020b), we observe that each focus maintains some but not all the alterations observed in C3H10T1/2Cl8. This nding suggests that the different alterations did not originally occur in all treated cells, but that different cells reacted to CdCl 2 exposure in different ways.…”

“…9, together with images from healthy C3H cells, already described in a previous paper. As previously reported (Oldani et al 2020b), mitochondria in C3H cells were found to be evenly distributed through the cytoplasm, extending from the nucleus to the cell periphery in a regular network (Fig. 9a); their morphology was thin and lamentous (Fig.…”

“…Previous data obtained in our laboratory (Oldani et al 2020b) showed that SOD1 activity is impaired by 24 h treatment with CdCl 2 , leading to O 2 level increase; however, toxicogenomic data showed that SOD1 coding gene is not dysregulated by CdCl 2 treatment neither in C3H cells (Callegaro et al 2018), nor in human SH-SY5Y neuroblastoma cells (Forcella et al 2020). In foci, we could not detect any impairment in SOD1 activity, suggesting that this enzyme inhibition is an early event caused by CdCl 2 administration, far before the 4 to 6 weeks needed for the in vitro cell transformation.…”

“…Autophagy activation was also investigated, since previous data, obtained in our laboratory, showed that a 24 h treatment with CdCl 2 can induce autophagy in C3H10T1/2Cl8 mouse embryonic broblasts (Oldani et al 2020b); however, neither F1 nor F3 focus showed autophagy activation, as reported in Fig. 10, showing no difference in AMPK phosphorylation.…”