Mechanisms of Apoptotic Cell Death by Stainless Steel Nanoparticle Through Reactive Oxygen Species and Caspase-3 Activities on Human Liver Cells

Background: Nanoparticles are widely used in pharmaceutical, agriculture, and food processing industries and in many other fields. However, the effect of stainless steel nanoparticles (SSNPs) remains unclear. So in this study, we evaluate the effect of SSNPs’ toxicity on human liver (CHANG and HuH-7) cell lines over 24 and 48 h. Methods: We have analyzed the quality, shape, and size of SSNPs using x-ray diffraction (XRD), energy dispersive x-ray (EDX) scanning electron microscope (SEM), and transmission electron microscope (TEM). The cytotoxicity and cell growth were determined by using the MTT and wound healing tests. The oxidative stress parameters were determined by measuring ROS generation and antioxidant enzymes, such as glutathione (GSH) and superoxide dismutase (SOD), due to SSNP exposure on human liver cell lines over 24 and 48 h. The confirmation of the apoptotic effect of SSNPs on livers cells was determined by the Western blot analysis for the expression of apoptotic proteins, such as Bax, bcl2, and p53, and real-time PCR for the expression of apoptotic genes, such as Bax, bcl2, caspase-3, and p53. Results: We have observed the dose- and time-dependent cytotoxicity and apoptosis of SSNPs on both cells. The results showed that SSNPs induced cell toxicity, inhibited cell growth, GSH, and increased generation of intracellular ROS and SOD levels at higher concentrations of exposure in both cells. SSNPs showed an apoptotic activity with upregulation of Bax, caspase-3, and p53 and downregulation of the bcl2 gene expression in CHANG and HuH-7 cell lines. Moreover, the immunoblotting assay confirmed the apoptotic activity of SSNPs in cells. Conclusion: In conclusion, these findings demonstrated that SSNPs showed toxic effects on human liver cells via activating the caspase-3 activity and they induced more toxicity in HuH-7 cells than in CHANG cells.