Increased cytotoxicity of Pb with co-exposures to a mitochondrial uncoupler and mitochondrial calcium uniporter inhibitor.

Lead (Pb) is an important developmental toxicant. The mitochondrial calcium uniporter (MCU) imports calcium ions using the mitochondrial membrane potential (MMP), and also appears to mediate the influx of Pb into the mitochondria. Since our environment contains mixtures of toxic agents, it is important to consider multi-chemical exposures. To begin to develop generalizable, predictive models of interactive toxicity, we developed mechanism-based hypotheses about interactive effects of Pb with other chemicals. To test these hypotheses, we exposed HepG2 (human liver) cells to Pb alone and in mixtures with other mitochondria-damaging chemicals: carbonyl cyanide--trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler that reduces MMP, and Ruthenium Red (RuRed), a dye that inhibits the MCU. After 24 hours, Pb alone, the mixture of Pb and RuRed, and the mixture of Pb and FCCP caused no decrease in cell viability. However, the combination of all three exposures led to a significant decrease in cell viability at higher Pb concentrations. After 48 hours, the co-exposure to elevated Pb concentrations and FCCP caused a significant decrease in cell viability, and the mixture of all three showed a clear dose-response curve with significant decreases in cell viability across a range of Pb concentrations. We performed ICP-MS analyses on isolated mitochondrial and cytosolic fractions and found no differences in Pb uptake across exposure groups, ruling out altered cellular uptake as the mechanism for interactive toxicity. We assessed MMP following exposure and observed a decrease in membrane potential that corresponds to loss of cell viability but is likely not sufficient to be the causative mechanistic driver of cell death. This research provides a mechanistically-based framework for understanding Pb toxicity in mixtures with mitochondrial toxicants.