Long-term in vitro effects of exposing the human HK-2 proximal tubule cell line to 3-monochloropropane-1,2-diol

Abstract

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.

INTRODUCTION

3-Monochloropropane-1,2-diol (3-MCPD) is a food contaminant present in a variety of common foods including infant formula (MacMahon et al., 2013), refined edible oils (Albuquerque et al., 2018), bakery products (Starski et al., 2013), and non-fermented soy sauces (Lee and Khor, 2015). Levels of 3-MCPD in these food categories contain as much as 119 µg/kg in infant formula (Spungen et al., 2018), 24 µg/kg in edible oils (Zelinková et al., 2006), 60 µg/kg in baked goods (Mogol et al., 2014), and 4.86 µg/g in soy sauces (Huang et al., 2005). Free 3-MCPD is generated by at least three known pathways: acid hydrolysis of proteins (Lee and Khor, 2015), heat processing of lipids (Sadowska-Rociek et al., 2018), and hydrolysis-mediated release from 3-MCPD esters in the gastrointestinal tract (Buhrke et al., 2011). As a member of the chloropropanol family, 3-MCPD is essentially a 3-carbon alcohol with a chloride group substituted for a hydroxyl group through a nucleophilic substitution mechanism. Its chemical properties afford its utility in several industrial applications, including use as a freezing point reducer in dynamite (“α-Chlorohydrin | The Merck Index Online,” 2018), an intermediate in the manufacturing of dyes (“α-Chlorohydrin | The Merck Index Online,” 2018), and a potent rodenticide (University of Hertfordshire, 2018; U.S. EPA, 2006).

Many aspects of the toxicity of 3-MCPD are well studied, but its exact mechanisms of action have not been fully elucidated. Its effects have been studied in animal models such as monkeys, rabbits, rats, mice, and birds (Chambers, 2018) and a wide range of pleiotropic effects have been reported. They include hematological disorders (Kirton et al., 1970), non-genotoxic carcinogenicity (Cho et al., 2008b), male reproductive toxicity (Kim et al., 2012), and kidney dysfunction (“3-CHLORO-1,2-PROPANEDIOL (JECFA Food Additives Series 48),” 2001; Cho et al., 2008b; Huang et al., 2018; Onami et al., 2014) in acute and chronic settings. Its presence as a potential food contaminant in U.S. infant formula (Spungen et al., 2018) warrants further investigation of its potential toxicity in long-term settings, since the developing organs of infants may have a heightened vulnerability to 3-MCPD effects if formula is an infant’s primary food source for many consecutive months.

To address the need for further research on the potential toxicity of free 3-MCPD, we focused our investigation on its effects on the renal system since the proximal tubules are a reported target of toxicity in both male and female animal models (Cho et al., 2008a).The kidneys are generally among the most commonly affected organs in clinical toxicity adverse events, as the proximal tubules have the specialized ability to concentrate compounds entering through the glomeruli to levels exceeding those in the blood (Curthoys and Moe, 2014). Harmful compounds can injure cells of the proximal tubules through physical (obstructive) means, such as through the formation of crystals (Bandele et al., 2013, 2014; Ermer et al., 2016; Stine et al., 2014), or biochemical (ischemic, hypoxic, oxidative, or metabolic) mechanisms, such as through drugs, uremic toxins, or heavy metal-containing chemicals that can inhibit fundamental cellular and mitochondrial processes needed to sustain proximal tubule cell vitality and function (Lentini et al., 2017; Nigam et al., 2015; Zalups, 2000). In the case of 3-MCPD, potential mechanisms of renal injury are not well elucidated and comparative data on 3-MCPD levels in tubular fluid and blood plasma are not available. Nevertheless, several in vivo renal effects have been reported, including increased kidney weights, tubular hyperplasia, kidney tubular necrosis, and chronic progressive nephropathy (Cho et al., 2008b, 2008a; Hwang et al., 2009; Sunahara et al., 1993).

For this current study, our objective was to investigate both short- and progressively long-term effects of free 3-MCPD on human proximal tubule cells in vitro represented by the Human Kidney 2 (HK-2) cell line. HK-2 cells are especially well suited for in vitro toxicology studies, as they retain key features of their primary cell counterparts including anchorage dependence and expression of metabolic energy-related and cytochrome P450 enzymes (Ryan et al., 1994). In comparison to in vivo safety assessments, in vitro cellular models allow for the rapid evaluation of potential toxins at lower costs than in vivo systems typically require. They can also offer helpful insights into their mechanisms of toxicity over different periods of time. Despite these advantages, there is a lack of in vitro studies on the effects of 3-MCPD on proximal tubule cells beyond 48 hr (Buhrke et al., 2018; Liu et al., 2012; Ozcagli et al., 2016; Senyildiz et al., 2017; Sun et al., 2015). As such, we assessed the effects of treating HK-2 cells with varying concentrations of free 3-MCPD for up to 16 days to create an unprecedentedly stringent model of direct and continuous exposure. Potential toxicity was evaluated for cellular and mitochondrial effects, as well as for the biomarker of renal-specific toxicity, Kidney Injury Molecule 1 (KIM-1).

MATERIALS AND METHODS

Cell culture and treatment exposure

HK-2 cells were purchased from ATCC (Manassas, VA, USA) and maintained in Keratinocyte-SFM media containing 10% Fetal Bovine Serum, 50 mg/L Bovine Pituitary Extract and 5 µg/L human recombinant Epidermal Growth Factor (all from Gibco, Waltham, MA, USA) as described previously (Mossoba et al., 2016a, 2016b). In preparation for treatment exposures for the duration of 1, 2, 4, 8, and 16 days, cells were seeded in clear-bottom black- or white-wall (for fluorescence or luminescence assays, respectively), clear-bottom 96-well plates (Greiner, Frickenhausen, Germany) at a density of 2 x 10⁵, 1 x 10⁵, 0.5 x 10⁵, 0.25 x 10⁵, and 0.125 x 10⁵ cells per mL, respectively. Serially diluted concentrations of 3-monochloropropane-1,2-diol (3-MCPD; 0-100 mM), phenylmercuric acetate (PMA; 0-100 µM) (positive control), or valproic acid (VAL; 0-100 mM) (negative control) were prepared in HK-2 media. All treatment compounds were purchased from Sigma (St. Louis, MO, USA) and added to appropriate wells for the intended exposure duration and kept at 37°C in a humidified incubator with 5% CO₂ before performing toxicity assays.

Cell viability assay

Cell viability was evaluated in treated and non-treated HK-2 cells by quantifying the relative levels of ATP using the CellTiter-Glo Cell Viability Assay (Promega, Madison, WI, USA) following the manufacturer’s instructions. White-wall, clear-bottom plates were analyzed using the Omega plate reader (BMG Labtech, Ortenberg, Germany) on luminescence detection mode. Experiments were independently performed at least three times.

Confocal microscopy

HK-2 cells were grown in 8-well Lab-Tek chamber slides from Thermo Fisher Scientific (Waltham, MA, USA) and treated with 0, 0.05, 5 and 50 mM 3-MCPD; 0.05 and 5 mM VAL; 0.05 and 5 µM PMA for different incubation time periods at 37°C/ 5% CO₂. After 1, 2, 4, 8, or 16 days, cells in each chamber slide were fixed using 10% formalin by incubating them for 10 min at room temperature. Cells were then washed twice with PBS and the chamber was removed. Slides were air-dried before 4 µL of fluoroshield 4′,6′-diamidino-2-phenylindole (DAPI) dye (Sigma) were added to each well before covering them with their cover slip. DAPI-stained cells (Ex/Em 358/461 nm) were visualized by confocal fluorescence microscopy using a Leica TCS SP5II microscope (Leica Microsystems, Wetzlar, Germany).

Mitochondrial Membrane Potential assay

Mitochondrial membrane potential (MMP) of treated and non-treated HK-2 populations were evaluated by staining the cells with fluorescent dye JC-10 (Enzo, Farmingdale, NY, USA) for 30 min at 37°C in a humidified incubator with 5% CO₂ before washing them three times with HBSS (Gibco). As described previously (Mossoba et al., 2016a, 2016b), cells plated in black-walled, clear-bottom plates were evaluated for MMP changes by measuring their fluorescence using the Omega plate reader (BMG Labtech) on fluorescence detection mode at excitation wavelength 485 nm and emission wavelengths 520 and 590 nm. Experiments were independently performed at least three times.

Reactive Oxygen Species assay

Reactive oxygen species (ROS) levels present in treated and non-treated HK-2 cells were assayed using ROS-Glo H₂O₂ (Promega), following the manufacturer’s instructions and data were normalized to cell viability as previously described (Mossoba et al., 2016a, 2016b). Cells plated in white-wall, clear-bottom plates were analyzed for luminescence emission using the Omega plate reader (BMG Labtech). Experiments were independently performed at least three times.

KIM-1 assay

Cell culture supernatants of HK-2 cells treated and non-treated with compounds of interest were processed according to manufacturer’s recommendations to quantitate the concentrations of KIM-1 using the R&D systems Human Magnetic Luminex assay (Minneapolis, MN, USA) and the Bio-plex Luminex 200 system (Bio-Rad, Hercules, CA, USA). KIM-1 biomarker levels were normalized to cell viability, as previously described (Mossoba et al., 2016a, 2016b). Experiments were independently performed at least three times.

Statistics

Data was collected and analyzed using Excel (Microsoft, Redmond, WA, USA) and GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Statistical analyses were done by student 2-way ANOVA tests to establish the significance of treatment effect differences, using a P-value of less than 0.05.

RESULTS

Effect of 3-MCPD on cell viability following direct exposure to HK-2 cells in vitro

To understand the basic cytotoxicity profile of 3-MCPD against the human proximal tubule epithelial cells HK-2, we selected a wide range of exposure doses, ranging from 0 to 100 mM. ATP levels were used as a measure of cell viability; they were measured by a luminescence assay in HK-2 cells treated with 3-MCPD for 1, 2, 4, 8, or 16 days at the full range of exposure doses. After just 1 day of exposure, 3-MCPD appeared not to exert a major toxic effect on HK-2 cells until the very high end of the dosing range was applied. As shown in Fig. 1A, a mild reduction in cell viability resulted from exposure to 50 mM of 3-MCPD and this effect became more intense as the maximum tested dose of 100 mM was applied. Compared to the positive control PMA, which reliably induced cell death at very low doses (0.001 mM or 1 µM), and even the negative control VAL, which required a dose of at least 5 mM to exert cytotoxicity, 3-MCPD was only mildly toxic. As the duration of exposure doubled from 1 to 2 days (Fig. 1B), however, the 3-MCPD dose found to induce the lowest observed effect level (LOEL) was reduced by half from 50 to 25 mM. This trend continued as shown in Figs. 1C, D, and E; the LOEL values in the 3-MCPD treatment series continued to drop by roughly half as the exposure time increased by two-fold. To further reflect this result, we calculated median lethal concentration (LC₅₀) values for each treatment and time point. As shown in Table 1, the LC₅₀ values for 3-MCPD were consistently highest among the three compounds tested at every time point selected and actually decreased steadily with every exposure doubling, whereas LC₅₀ values measured for the positive and negative controls decreased sharply as the treatment duration was progressively doubled. Short-term exposure of HK-2 cells to 3-MCPD for 1 and 2 days yielded fairly high LC₅₀ values of 91.4 and 78.6 mM, respectively. Beyond these timeframes, however, the severity of cytotoxicity induced by 3-MCPD increased significantly at 4, 8, and 16 days of exposure, reducing the LC₅₀ values to 29.4, 11.3, and 3.9 mM, respectively. It should be noted that for these experiments, a racemic mixture of 3-MCPD was used, since our preliminary experiments revealed no significant difference in effects of ATP production following the use of its R- or S-enantiomers versus its racemic mixture (data not shown).

Fig. 1

HK-2 cells were treated with 0-100 mM 3-MCPD, 0-100 µM of PMA, or 0-100 mM of VAL for 1, 2, 4, 8, or 16 days (A-E). Cell viability in treated cells was assayed by ATP luminescence and compared to non-treated HK-2 cell viability. Data is representative of three independent experiments. Statistical significance of P < 0.05 for treated vs. non-treated cell populations is indicated by * symbols.

Table 1. LC₅₀ values following HK-2 treatment with 3-MCPD.

	3-MCPD (mM)	PMA (µM)	VAL (mM)
Day 1	91.4	3.2	33.5
2	78.6	1.0	22.4
4	29.4	0.95	2.3
8	11.3	0.19	1.3
16	3.9	1.3x10-6	1.1

To corroborate the use of cellular ATP as a measure of cell viability, we undertook a series of experiments to visualize 3-MCPD effects on cell morphology and death. Using confocal microscopy, standard light transmission and fluorescent apoptosis dye DAPI-stained HK-2 cell images were taken at 1, 2, 4, 8, and 16 days post-treatment with 3-MCPD (Fig. 2A), or control compounds PMA or VAL (Fig. 2B) at select treatment concentrations spanning the range tested in Fig. 1. As shown in Fig. 2, cell density decreased and overall cell morphology changed from spindle to round as treatment concentration or exposure duration increased. Moreover, since the fluorescence intensity of DAPI increases in response to its ability to access A-T rich regions of DNA, its fluorescence intensity is an indicator of reduced cell viability (Cummings et al., 2004; Kapuscinski, 1995). We observed that DAPI intensity increased to its most intense levels at the highest treatment concentrations and exposure time of 16 days.

Fig. 2

HK-2 cells treated with (A) 3-MCPD (0, 0.05, 5 or 50 mM) or (B) control compounds VAL (0.05 and 5 mM) or PMA (0.05 and 5 µM PMA) were by confocal microscopy following staining by DAPI dye. Fluorescence was measured using the excitation and emission wavelengths of 358 and 461 nm, respectively.

3-MCPD induces limited mitochondrial dysfunction and production of reactive oxygen species

Having assessed the potential of 3-MCPD to induce cytotoxicity towards HK-2 cells, we sought to investigate the role of mitochondrial dysfunction in its mechanism of inducing cellular injury. Since mitochondria use their outer and inner membranes to maintain a voltage gradient that is essential for the electron transport chain to produce ATP, we measured the disruption of this membrane potential using the well-established compound JC-10. This fluorescent dye has two emission spectra corresponding to its two conformation states inside the mitochondrial matrix versus the cytosol. As shown in Fig. 3, we measured changes in mitochondrial membrane potential (MMP), as calculated by the ratio of fluorescence emission by the two JC-10 conformations. As expected, after just one day of exposing HK-2 cells to 3-MCPD, a gradual increase in mitochondria having a disrupted membrane potential was found in direct proportion to treatment concentrations between 50 to 100 mM. This increase, however, was not as sharp as that induced by PMA at the very low dose of 2.5 µM. PMA treatment beyond 2.5 µM, failed to induce further elevations in mitochondrial dysfunction. By contrast, VAL treatment after one day of exposure resulted in a gradual increase in mitochondrial dysfunction starting from about 2.5 mM, 1000 times lower than PMA, but at least 20 times higher than 3-MCPD. As show in Figs. 3B-E, increasing the duration of exposure beyond one day led to changes to MMP values at progressively lower treatment concentrations. We also consistently observed a plateau in signal corresponding to the treatment concentrations at which ATP signals had been ultimately low.

Fig. 3

Losses in mitochondrial membrane potential (MMP) were measured in treated HK-2 cells treated for 1, 2, 4, 8, or 16 days (A-E) with 3-MCPD or control compounds by calculated as a ratio of fluorescence emission wavelengths of 520 vs. 590 nm. The graphs shown are representative of three independent experiments. Statistical significance of P < 0.05 for treated vs. non-treated cell populations is indicated by * symbols.

Following up on these findings, our next goal was to establish whether the production of excessive reactive oxygen species (ROS) could be a mechanism of 3-MCPD treatment-induced mitochondrial toxicity; ROS levels are known to be a robust indicator of ‘mitotoxicity’ and cytotoxicity. As show in Fig. 4, we found that mitochondrial injury and compromised cell viability clearly matched up with increases in ROS production. Consistent with the cytotoxicity and mitochondrial dysfunction effects induced by 3-MCPD, relative ROS levels were small compared to both control compounds PMA and VAL (P < 0.05) within each time point assayed and over the 16 days of exposure (Figs. 4A-E).

Fig. 4

HK-2 cells were exposed to 0 to 100 mM of 3-MCPD, 0-100 µM of PMA, or 0-100 mM of VAL and quantitatively assayed for reactive oxygen species (ROS) levels 1, 2, 4, 8, or 16 days (A-E) post-treatment. Data shown is representative of three independent experiments. Statistical significance of P < 0.05 for treated vs. non-treated cell populations is indicated by * symbols.

Renal injury biomarker KIM-1 is not strongly elevated by 3-MCPD exposure

To investigate the specificity of 3-MCPD effects towards proximal tubule cells, we measured levels of KIM-1 in the cell culture supernatants of HK-2 cells treated with 3-MCPD or control compounds for 1, 2, 4, 8, or 16 days. KIM-1 is an FDA-qualified biomarker of renal injury that has been shown to be a reliable biomarker of proximal tubule cell injury in vitro. As shown in Fig. 5, relative KIM-1 concentrations resulting from 3-MCPD exposure were below the limit of quantitation until high enough treatment concentrations were applied. Even when detectable, these measurements were significantly below those of the negative control VAL as well as the positive control PMA exposures (P < 0.05). By contrast, KIM-1 production was readily detectable following one day of control compound exposure in proportion to the treatment concentration, beginning at 5 µM of PMA or 75 mM of VAL (Fig. 5A). Although two days of direct 3-MCPD exposure did not yield appreciable levels of KIM-1 production relative to control compounds, relatively small concentrations of this biomarker were measured after 4 days of continuous exposure starting at the 75 mM treatment dose (Figs. 5B & C). Extended durations of HK-2 cell exposure to 3-MCPD similarly only yielded low levels of KIM-1 when 5 to 10 mM treatment doses were used (Figs. 5D & E).

Fig. 5

Kidney injury biomarker signature of KIM-1 expression was measured following 1, 2, 4, 8, or 16 days (A-E) by Luminex xMAP technology. Data is representative of three independent experiments. Statistical significance of P < 0.05 for treated vs. non-treated cell populations is indicated by * symbols.

DISCUSSION

A plethora of in vivo studies done mostly in rat models have demonstrated the potential for 3-MCPD to induce a variety of renal and other effects in acute and chronic settings (“3-CHLORO-1,2-PROPANEDIOL (JECFA Food Additives Series 48),” 2001; Cho et al., 2008b; Huang et al., 2018; Onami et al., 2014). In vitro renal studies, however, have only been done in rat NRK-52E and human HEK or TK cell models to confirm the short-term effects of 24 or 48 hour exposures (Buhrke et al., 2018; Liu et al., 2012; Ozcagli et al., 2016; Senyildiz et al., 2017; Sun et al., 2015). Since the kidneys are among the list of organs targeted by 3-MCPD, we sought to investigate both the short- and long-term effects of directly exposing this compound to human proximal tubule cells in vitro. At least one pharmacokinetic study has revealed that whole kidney and blood levels of 3-MCPD are similar (Abraham et al., 2013). The ability of the proximal tubule portion of the renal system to concentrate 3-MCPD present in glomerular filtrate, however, does not exclude the possibility of 3-MCPD levels reaching values higher than those found in the blood. Any resulting increase in exposure dose relative to other organs may thus injure the proximal tubules relatively profoundly. Whether this concentrating effect is partly responsible for the kidneys being a target of 3-MCPD toxicity is unknown, but we sought to investigate its effects in a rigorous in vitro model of directly exposing this food contaminant to human proximal tubule cells for up to 16 days continuously.

The results from our study revealed that both overall cytotoxicity and mitochondrial dysfunction went hand-in-hand and that higher treatment concentrations yielding reductions in ATP levels matched up well with surges in MMP changes and ROS production levels. Within each tested duration of exposure, as 3-MCPD treatment concentrations were increased towards the maximum tested dose of 100 mM, measured ATP levels decreased proportionally. Increasing 3-MCPD treatment doses also led to significantly greater MMP loss and ROS production. As the duration of exposure was increased towards the maximum tested timepoint of 16 days, the trend between 3-MCPD dose and changes in cell viability, MMP loss, and ROS production endpoints was well conserved, however the severity of these effects increased over treatment duration, as reflected by the LD₅₀ values shifting to lower and lower treatment doses. The relationships among these effects can be explained based on the hypothesis that 3-MCPD may act as a biochemical toxin that interferes with key mitochondrial pathways. As 3-MCPD perturbs one or more mitochondrial functions, then the resulting stress on oxidative phosphorylation would not only yield bursts of ROS production, but also lead to overall cell death. Indeed, research by others demonstrated that 3-MCPD could trigger caspase-dependent apoptosis in human embryonic kidney HEK293FT cells in vitro in a dose-dependent manner within cellular respiration and that 3-MCPD-associated apoptosis could also be measured in rat testicles in vivo and Leydig cells in vitro (Barocelli et al., 2011; Peng et al., 2016; Sun et al., 2013).

Although the cytotoxicity profiling performed in this study revealed that 3-MCPD compromised cell viability in a dose- and duration-dependent manner, its toxic effects only became statistically significant at the high end of the tested dose range (~50 to 100 mM) for short exposures (1-2 days). Even when exposures lasted as long as 16 days, the 1 mM treatment dose of 3-MCPD had no effect on cell viability or any of the other endpoints. These observations are underscored by the additional finding that 3-MCPD was less toxic than our negative control valproic acid at all time points tested. Valproic acid has served as an effective non-toxin or negative control in other renal in vitro and in vivo studies and in this current study, the potential toxicity of valproic acid towards proximal tubules was observed starting at doses greater than 5 mM for 1 day, but this in vitro cellular dose is considerably greater than the LOEL values shown in other publications (Brar et al., 2014; Van Beneden et al., 2011; Xi et al., 2018).

The relative mildness of 3-MCPD is also supported by our finding that KIM-1 levels neither appreciably rose in response to 3-MCPD treatment concentration nor to exposure duration. Although KIM-1 is an FDA-qualified biomarker of renal injury in vivo (Bonventre et al., 2010), it has also been demonstrated by multiple research groups to be a valuable in vitro cellular biomarker of proximal tubule cell injury (Luo et al., 2016; Mossoba et al., 2017; Veach and Wilson, 2018). KIM-1 expression is triggered in proximal tubule cells in response to a wide variety of toxic insults and has been shown to be part of a mitogen-activated protein kinase (MAPK)-mediated cell regenerative mechanism to repair injured cells (Song et al., 2019; Zhang and Cai, 2016). Although 3-MCPD clearly affected cell viability, MMP loss, and ROS production in our study, its effects led to only relatively low levels of KIM-1 expression that were not consistently elevated as the duration of 3-MCPD exposure increased in time. Although it is not clear why a robust KIM-1 signal was not detected on a consistent basis in parallel with the trends observed for the other endpoints, one possible explanation could be that 3-MCPD inhibits the MAPK pathway such that the ability of HK-2 cells to undergo a KIM-1-mediated cell repair process is compromised. This hypothesis is supported by other publications showing that other chloropropanols can indeed modulate MAPK pathways (Liu et al., 2016b; Xiao et al., 2018). Notably, as we measured KIM-1 expression following exposure to the control compounds PMA and VAL in our current study, its levels did rise as expected at low doses of PMA and high doses of VAL, confirming that the assay itself performed as expected.

PMA was used throughout this study as a useful positive control toxin, as organomercury compounds readily enter proximal tubule cells through organic ion and other transporters and induce toxicological activity through molecular interactions that occur at critical nucleophilic sites in (and around) target cells, including interacting with intracellular glutathione metabolism, thus offsetting the regulation of critical cellular events ranging from gene expression to antioxidant defense and nutrient metabolism (Lentini et al., 2017; Nigam et al., 2015; Wu et al., 2004; Zalups, 2000). Organomercury compounds have a well-studied ability to induce toxicity towards proximal tubule cells both in vivo animal models (Friberg et al., 1957; Hirano et al., 1986; White et al., 1961) as well as in cellular in vitro models (Choi et al., 2016; Zalups et al., 2004) and induce toxicity biomarkers like ROS production and KIM-1 expression reliably in such systems (Liu et al., 2016a; Mossoba et al., 2019; Shi et al., 2011; Zhang et al., 2017; Zhou et al., 2008).

Taken together, the data presented in this study are in agreement with the short-term (up to 48 hour) exposure studies performed in vitro with other cell lines (Buhrke et al., 2018; Liu et al., 2012; Ozcagli et al., 2016; Senyildiz et al., 2017; Sun et al., 2015) and further support the notion that 3-MCPD may be a mild toxin towards human proximal tubule cells. Moreover, no adverse kidney (or other) events among humans have been attributed to 3-MCPD-associated food consumption or occupational exposure (“3-CHLORO-1,2-PROPANEDIOL (JECFA Food Additives Series 48),” 2001) and a thorough toxicological evaluation in a non-human primate model showed hematological problems instead of any renal effects (Kirton et al., 1970). It is interesting that some in vivo rodent model studies have demonstrated the potential for 3-MCPD exposure to induce nephropathic changes that include increased kidney weight, tubular necrosis, hyperplasia, cysts, or tumors (Cho et al., 2008b, 2008a; Hwang et al., 2009; Sunahara et al., 1993), while a two-year rodent study reported no significant toxic renal effects (Jeong et al., 2010). Our present cellular in vitro study, however, was neither designed to measure the potential for 3-MCPD to differentiate between necrotic vs. apoptotic types of HK-2 cell death, nor to induce neoplastic events that could lead to in vivo hyperplasia or other aberrant growths. Nevertheless, our findings clearly point to the mildness of 3-MCPD toxicity towards proximal tubule cells in our in vitro cellular HK-2 model.

To address the apparent discrepancy between reports of only mild or no significant adverse renal effects and published 3-MCPD-associated renal injuries described in rodent models, there are three main considerations to note. First, rat studies have shown that 3-MCPD ingestion is associated with the deregulation of the endogenous antioxidant protein DJ-1 (Sawada et al., 2016), whose function relates to mitochondrial protection (Parrado-Fernández et al., 2018; Salazar et al., 2018; Wang et al., 2018). Its loss of function may not be directly mediated by 3-MCPD, but rather indirectly through DJ-1 oxidation by reactive oxygen species (Buhrke et al., 2018). At this time, it remains unknown whether this reported mode of toxicity by 3-MCPD is predominantly present in rats or in all mammals, since DJ-1 is also present in humans.

Second, since 3-MCPD can undergo conversion through multiple metabolic pathways (Lynch et al., 1998), it is possible that its metabolite identities and levels differ between species and each metabolite possesses varying levels of organ-specific toxicity. Indeed the initial oxidation of 3-MCPD to β-chlorolactaldehyde acid and later conversion to β-chlorolactic acid and oxalic acid have been attributed to renal toxicity in rats, at least in part through the inhibition of glycolysis (Bakhiya et al., 2011; Buhrke et al., 2011; Ermer et al., 2016). The presence of 3-MCPD-derived metabolites have not been studied in non-rodent mammals, leaving open the possibility that their damaging effects may be avoided in humans altogether if they are not even readily produced.

Finally, it should be noted that the apparent lack of overt toxicity by 3-MCPD towards human proximal tubule cells in vitro may in part be simply due to HK-2 cells not having the appropriate transporter proteins expressed at optimal levels that would be required for efficient passage of this compound through the cell membrane. Identifying which transporter proteins facilitate cellular entry is being examined in a separate study. If certain transporters are found to be important for 3-MCPD entry into human proximal tubule cells, then evaluating their expression levels in proximal tubule cells of infant kidneys may offer unique insight into the implications of leaving this food contaminant unregulated through infant formula. Ultimately, the health effects of 3-MCPD at low dose levels, but for chronic exposures will require studies in bonafide infant models, as their immature renal physiology will likely play a role in the toxicity profile of 3-MCPD.

ACKNOWLEDGMENTS

This work was supported by internal funding from the U.S. Food and Drug Administration.

Conflict of interest

The authors declare that there is no conflict of interest.

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