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Chloropropanols are chemical contaminants that can be formed during industrial processing of foods, such as lipids used in commercially available infant and toddler formula in the USA. Many researchers have studied the most common chloropropanol contaminant, 3-monochloropropane-1,2-diol (3-MCPD), as well as its lipid ester derivatives. A plethora of toxicological outcomes have been described in vivo, including effects on the heart, nervous system, reproductive organs, and kidneys. To better understand the concordance of some of these effects to in vitro outcomes, we focused our research on using an in vitro cellular model to investigate whether the proximal tubule cells of the kidney would be vulnerable to the effects of free 3-MCPD and nine of its common esters in commercial formula. Using the established human kidney proximal tubule cell line, HK-2, we performed 24-h treatments using 3-MCPD and nine mono- or di-esters derived from palmitate, oleate, and linoleate. By directly exposing HK-2 cells at treatment doses ranging from 0 to 100 μM, we could evaluate their effects on cell viability, mitochondrial health, reactive oxygen species (ROS) production, and other endpoints of toxicity. Since chloropropanols reportedly inhibit cellular metabolism through interference with glycolysis, we also tested the extent of this mechanism. Overall, we found mild but statistically significant evidence of cytotoxicity at the highest tested treatment concentrations, which were also associated with mitochondrial dysfunction and transient perturbations in cellular metabolism. Based on these findings, further studies will be required to better understand the effects of these compounds under conditions that are more physiologically relevant to human infant and toddler proximal tubules in order to mimic their exposure to chloropropanol-containing foods.

Classic toxicology studies often utilize in vivo animal models. Newer approaches employing in vitro organ-specific cellular models have been developed in recent years to help accelerate the speed and reduce the cost of traditional toxicology testing. Toward the goal of supporting in vitro cellular model research with a regulatory application in mind, we have developed a 'designer' human kidney cell line called HK2-Vi that can fluorescently measure the cytotoxicity of potential toxins on proximal tubule cell viability in a direct exposure in vitro model. HK2-Vi was designed to be a reagent-less kinetic assay that can yield data on short- or long-term cell viability after toxin exposure. To generate HK2-Vi, we used monocistronic lentiviral transduction methods to genetically engineer a human kidney cell line called HK-2 to stably co-express two transgenes. The first is Perceval HR, which encodes a fluorescent biosensor of both cytosolic ATP and ADP and the second is pHRed, which encodes a biosensor of cytosolic pH. Relative levels of cellular ATP and ADP effectively serve as a reliable and robust indicator of cell viability. Because the fluorescence Perceval HR is pH-dependent, we co-expressed the pHRed genetic biosensor to correct for variations in pH if necessary. Heterogenous populations of transduced renal cells were enriched by flow cytometry before monoclonal cellular populations were isolated by cell culture methods. A single clonal population of co-transduced cells expressing both Perceval HR and pHRed was selected to be HK2-Vi. This established cell line can now serve as a tool for in vitro toxicology testing and the methods described herein serve as a model for developing designer cell lines derived from other organs.