Mitochondrial respiratory chain dysfunction mediated by ROS is a primary point of fluoride-induced damage in Hepa1-6 cells

Abstract

To evaluate the mechanism of fluoride (F) mitochondrial toxicity, we cultured Hepa1-6 cells with different F concentrations (0, 1 and 2 mmoL/L) and determined cell pathological morphology, mitochondrial respiratory chain damage and cell cycle change. Results showed that the activities and mRNA expression levels of antioxidant enzymes considerably decreased, whereas the contents of reactive oxygen species (ROS), malondialdehyde (MDA) and nitric oxide (NO) markedly increased. Breakage of mitochondrial cristae and substantial vacuolated mitochondria were observed by transmission electron microscopy. These results indicate the F-induced oxidative damage in Hepa1-6 cells. The enzyme activities of mitochondrial complexes I, II, III and IV were disordered in Hepa1-6 cells treated by excessive F, thereby indicating a remarkable down-regulation. Further research showed that complex subunits also demonstrated the development of disorder, in which the protein expressions levels of NDUFV2 and SDHA were substantially down-regulated, whereas those of CYC1 and COX Ⅳ were markedly up-regulated. Reductions in ATP and mitochondrial membrane potential were detected with the dysfunction of the mitochondrial respiratory chain. The G2/M phase arrest of the cell cycle in Hepa1-6 cells was measured via flow cytometry, and the up-regulated protein expressions of Cyt c, caspase 9, caspase 3 and substantial apoptotic cells were determined. In summary, this study demonstrated that ROS-mediated mitochondrial respiratory chain dysfunction causes F-induced Hepa1-6 cell damage.