Study of cadmium-induced cytotoxicity using two-photon excitation endogenous fluorescence microscopy

We demonstrate that using time-resolved two-photon excitation endogenous fluorescence microscopy, the cadmium (Cd)-induced cellular toxic level can be assessed by the free-to proteinbound reduced nicotinamide adenine dinucleotide (free/bound NADH) ratio in a living cell. NADH fluorescence excited at 730 nm is captured at different times following exposure to cadmium at a variety of concentrations. The temporal characteristics of NADH fluorescence from mitochondrial and nuclear compartments are analyzed, respectively. The results show that cadmium induces a significant increase of the free/bound NADH ratio in mitochondria and nucleus, caused by the inhibition effect on the electron transport chain (ETC) and the stimulating effect on the glycolysis pathway, respectively. It is found that induction of metallothionein (MT) in cells occurs after 4h of exposure to a sublethal concentration of Cd and reaches a peak at 6h. More importantly, the increase in MT level can effectively suppress the elevation of the free/bound NADH ratio caused by a subsequent exposure to a higher concentration of Cd, indicating that MT plays a key role in protecting cells from Cdinduced toxicity. Our findings show that the free/bound NADH ratio can potentially be used as a sensitive indicator of toxic and carcinogenic actions induced by Cd.