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3-Monochloropropane-1,2-diol (3-MCPD) is a food processing contaminant in some infant formula products and other foods in the United States. Although rodent studies have demonstrated that 3-MCPD and its palmitic esters have the potential to induce nephrotoxicity, our recent human cell culture studies using the human renal proximal tubule cell line HK-2 have not strongly supported this finding. Considering this disparity, we sought to examine whether changes in transporter gene expression on proximal tubule cells could be modulated by these compounds and allow us to glean mechanistic information on a possible indirect path to proximal tubule injury in vivo . If fundamental processes like water and solute transport could be disrupted by 3-MCPD compounds, then a new avenue of toxicity could be further explored in both infant and adult models. In our current study, we used HK-2 cells as an in vitro cellular model of human proximal tubule cells to investigate the effects of low (10 μM) and high (100 μM) 3-MCPD compound exposures to these cells for 24 hours (h) on the expression of 20 transporter genes that are known to be relevant to proximal tubules. Although we detected consistent upregulation of AQP1 expression at the RNA transcript level following HK-2 treatment with both low and high doses of several ester-bound 3-MCPD compounds, these increases were not associated with statistically significant elevations in their protein expression levels. Moreover, we observed a lack of modulation of other members of the AQP protein family that are known to be expressed by human proximal tubule cells. Overall, our study suggests the possibility that 3-MCPD-related nephrotoxicity could be associated with indirect modes of action relating to aquaporin homeostasis, but additional studies with other human-derived models would be pertinent to further explore these findings and to better understand transporter expression differences under different stages of proximal tubule development., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier B.V.)

3-Monochloropropane-1,2-diol (3-MCPD) is a food processing contaminant in the U.S. food supply, detected in infant formula. In vivo rodent model studies have identified a variety of possible adverse outcomes from 3-MCPD exposure including renal effects like increased kidney weights, tubular hyperplasia, kidney tubular necrosis, and chronic progressive nephropathy. Given the lack of available in vivo toxicological assessments of 3-MCPD in humans and the limited availability of in vitro human cell studies, the health effects of 3-MCPD remain unclear. We used in vitro human proximal tubule cells represented by the HK-2 cell line to compare short- and long-term consequences to continuous exposure to this compound. After periodic lengths of exposure (0-100 mM) ranging from 1 to 16 days, we evaluated cell viability, mitochondrial integrity, oxidative stress, and a specific biomarker of proximal tubule injury, Kidney Injury Molecule-1 (KIM-1). Overall, we found that free 3-MCPD was generally more toxic at high concentrations or extended durations of exposure, but that its overall ability to induce cell injury was limited in this in vitro system. Further experiments will be needed to conduct a comprehensive safety assessment in infants who may be exposed to 3-MCPD through consumption of infant formula, as human renal physiology changes significantly during development.

Fatty acid esters of 3-monochloropropane-1,2-diol (3-MCPD) are chemical contaminants found in a wide range of edible oils that are thermally processed during industrial manufacturing of infant formula and other lipid-containing foods in the United States. Rodent studies have unequivocally demonstrated a plethora of in vivo toxicological effects including reproductive, neurological and renal dysfunction. To determine if similar effects are observed in human organ systems, in vitro studies using human cell lines are conducted to assess concordance of the data collected. One limitation to performing such in vitro research is the extremely high hydrophobicity of 3-MCPD esters; dissolving them into aqueous cell culture media is a tremendous challenge. To address this obstacle, we developed a simple protocol to circumvent the immiscibility of 3-MCPD esters and their corresponding free fatty acids into aqueous cell culture media in order to assess the effect of these esters on epithelial cells of kidney origin in vitro ., (© 2019 Published by Elsevier B.V.)

Diglycolic acid (DGA) is present in trace amounts in our food supply and is classified as an indirect food additive linked with the primary GRAS food additive carboxymethyl cellulose (CMC). Carboxymethyl starches are used as a filler/binder excipient in dietary supplement tablets and a thickening ingredient in many other processed foods. We sought to utilize the human proximal tubule HK-2 cell line as an in vitro cellular model system to evaluate its acute nephrotoxicity of DGA. We found that DGA was indeed toxic to HK-2 cells in all in vitro assays in our study, including a highly sensitive Luminex assay that measures levels of an in vitro biomarker of kidney-specific toxicity, Kidney Injury Molecule 1 (KIM-1). Interestingly, in vitro KIM-1 levels also correlated with in vivo KIM-1 levels in urine collected from rats treated with DGA by daily oral gavage. The use of in vitro and in vivo models towards understanding the effectiveness of an established in vitro system to predict in vivo outcomes would be particularly useful in rapidly screening compounds that are suspected to be unsafe to consumers. The merit of the HK-2 cell model in predicting human toxicity and accelerating the process of food toxicant screening would be especially important for regulatory purposes. Overall, our study not only revealed the value of HK-2 in vitro cell model for nephrotoxicity evaluation, but also uncovered some of the mechanistic aspects of the human proximal tubule injury that DGA may cause.