Increased cytotoxicity of Pb2+ with co-exposures to a mitochondrial uncoupler and mitochondrial calcium uniporter inhibitor†

Lead (Pb²⁺) 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 Pb²⁺ 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 Pb²⁺ with other chemicals. To test these hypotheses, we exposed HepG2 (human liver) cells to Pb²⁺ alone and in mixtures with other mitochondria-damaging chemicals: carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler that reduces MMP, and Ruthenium Red (RuRed), a dye that inhibits the MCU. After 24 hours, Pb²⁺ alone, the mixture of Pb²⁺ and RuRed, and the mixture of Pb²⁺ 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 Pb²⁺ concentrations. After 48 hours, the co-exposure to elevated Pb²⁺ 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 Pb²⁺ concentrations. We performed ICP-MS analyses on isolated mitochondrial and cytosolic fractions and found no differences in Pb²⁺ 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 Pb²⁺ toxicity in mixtures with mitochondrial toxicants.