Can Galactose Be Converted to Glucose in HepG2 ... - ACS Publications

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Cite This: Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Can Galactose Be Converted to Glucose in HepG2 Cells? Improving the in Vitro Mitochondrial Toxicity Assay for the Assessment of Drug Induced Liver Injury Qiuwei Xu,* Liping Liu, Heather Vu, Matthew Kuhls, Amy G. Aslamkhan, Andy Liaw, Yan Yu, Allen Kaczor, Michael Ruth, Christina Wei, John Imredy, Jose Lebron, Kara Pearson, Raymond Gonzalez, Kaushik Mitra, and Frank D. Sistare Downloaded via UNIV OF SOUTHERN INDIANA on July 20, 2019 at 10:29:10 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Merck & Co. Inc., Kenilworth, New Jersey 07033, United States S Supporting Information *

ABSTRACT: Human hepatocellular carcinoma cells, HepG2, are often used for drug mediated mitochondrial toxicity assessments. Glucose in HepG2 culture media is replaced by galactose to reveal drug-induced mitochondrial toxicity as a marked shift of drug IC50 values for the reduction of cellular ATP. It has been postulated that galactose sensitizes HepG2 mitochondria by the additional ATP consumption demand in the Leloir pathway. However, our NMR metabolomics analysis of HepG2 cells and culture media showed very limited galactose metabolism. To clarify the role of galactose in HepG2 cellular metabolism, U-13C6-galactose or U-13C6glucose was added to HepG2 culture media to help specifically track the metabolism of those two sugars. Conversion to U-13C3-lactate was hardly detected when HepG2 cells were incubated with U-13C6-galactose, while an abundance of U-13C3-lactate was produced when HepG2 cells were incubated with U-13C6-glucose. In the absence of glucose, HepG2 cells increased glutamine consumption as a bioenergetics source. The requirement of additional glutamine almost matched the amount of glucose needed to maintain a similar level of cellular ATP in HepG2 cells. This improved understanding of galactose and glutamine metabolism in HepG2 cells helped optimize the ATP-based mitochondrial toxicity assay. The modified assay showed 96% sensitivity and 97% specificity in correctly discriminating compounds known to cause mitochondrial toxicity from those with prior evidence of not being mitochondrial toxicants. The greatest significance of the modified assay was its improved sensitivity in detecting the inhibition of mitochondrial fatty acid β-oxidation (FAO) when glutamine was withheld. Use of this improved assay for an empirical prediction of the likely contribution of mitochondrial toxicity to human DILI (drug induced liver injury) was attempted. According to testing of 65 DILI positive compounds representing numerous mechanisms of DILI together with 55 DILI negative compounds, the overall prediction of mitochondrial mechanism-related DILI showed 25% sensitivity and 95% specificity.

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INTRODUCTION Mitochondria are important cellular ATP generating organelles that support the function and viability of major organs. Mitochondria produce a steady supply of ATP by efficiently maximizing bioenergetics from substrates such as glucose, fatty acids, and amino acids. Drug induced mitochondrial dysfunction has been implicated in drug induced liver injury (DILI).1,2 However, the implication is usually derived by inference from results extrapolated from in vitro or ex vivo assays due to the lack of sensitive and specific in vivo translational and mechanistic biomarkers in humans. The percentage of human DILI compounds that are attributable to mitochondrial dysfunction is therefore often estimated based on in vitro assay results. Drugs associated with DILI such as amiodarone,4,5 benzbromarone,5,6 nefazodone,7 and troglitazone8−10 are found to impair mitochondria in cells or isolated © XXXX American Chemical Society

mitochondria, and thus mitochondrial impairment is concluded to be a likely contributing factor to DILI for those drugs. Rana et al. surveyed 228 pharmaceutical drugs and provided a reasonable estimate of the percentage of human DILI compounds that are attributable to mitochondrial dysfunction. They show that 11−34% of DILI compounds are associated with mitochondrial toxicity when IC50 values of an in vitro assay, which is based on the oxygen consumption rate (OCR), are less than 1−100 × the maximum total drug plasma concentrations in humans.3 Mitochondria isolated from rat livers following treatment with an equivalent human dose of the antiarrhythmic amiodarone show inhibition of mitochondrial complex I, Received: January 28, 2019 Published: July 4, 2019 A

DOI: 10.1021/acs.chemrestox.9b00033 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology

Figure 1. Leloir pathway, pathways to glycolysis, and oxidation of galactose to galactonate (represented by Gal-6-COOH).

However, there is lack of direct evidence showing the level of this galactose metabolism in HepG2 cells. The Leloir pathway describes the conversion of galactose to glucose with the help of a set of enzymes and two cofactors, ATP and UTP13,14 (Figure 1). Galactose is first phosphorylated to galactose-1-phosphate by galactokinase (step 1 in Figure 1). Galactose-1-phosphate is then activated to UDPgalactose by galactose-1-phosphate uridyltransferase (GALT) at the expense of turning UDP-glucose into glucose-1phosphate (step 2 in Figure 1). Activated UDP-galactose is then epimerized to UDP-glucose by UDP-galactose 4epimerase (step 3 in Figure 1). The net sum of these three reactions is the conversion of galactose to glucose-1-phosphate at the expense of ATP (eq 1). Glucose-1-phosphate can be converted by phosphoglucomutase to glucose-6-phosphate for glycolysis (steps 4 and 5 in Figure 1). In addition, glucose-1phosphate can also be activated to UDP-glucose by UDPglucose pyrophosphorylase (step 6 in Figure 1) to facilitate the Leloir conversion.

uncoupling of oxidative phosphorylation, and modification of membrane phospholipid composition.4 Both amiodarone and the uricosuric benzbromarone cause decreases in mitochondrial membrane potential in isolated rat hepatocytes and lead to reduction of oxidative respiration and uncoupling of oxidative phosphorylation in isolated rat liver mitochondria.5 In HepG2 cells, benzbromarone reduces cellular ATP at a much lower concentration than where cytotoxicity is observed6 and is accompanied by elevations of lactate concentration, reflecting increased glycolysis compensating for impaired ATP production in mitochondria. The antidepressant nefazodone inhibits mitochondrial OCR in isolated rat liver mitochondria and in intact HepG2 cells.7 The antidiabetic troglitazone is shown to decrease mitochondrial membrane potential in HepG2 cells preceding changes in cell permeability and cell count.8 In mitochondria isolated from male CD-1 mice, troglitazone also reduces mitochondrial membrane potential by inducing the opening of the mitochondrial permeability transition pore.9 In human HepaRG hepatoma cells, troglitazone reduces oxygen consumption rates and ATP levels and alters mitochondrial structure.10 To avoid likely compound attrition in later stages of drug development, it may be important, therefore, to screen drug candidate compounds for mitochondrial dysfunction during early discovery and development, and understand the relevance of in vitro toxicity to targeted therapeutic exposures. One of the often cited and commonly used in vitro cellular assays developed for this purpose in the pharmaceutical industry is a cellular ATP IC50 Glc-Gal shift assay in culture media containing either glucose or galactose.11 This assay is often performed in HepG2 cells, an immortal human hepatoma cell line that is derived from liver biopsies of a child with primary hepatoblastoma and hepatocellular carcinoma.12 If a test compound impairs mitochondria, its dose dependent decreases in cellular ATP shift toward significantly lower concentrations of the test compound when media glucose is replaced by galactose. It is presumed that the cost of an additional ATP consumption in the Leloir pathway (Figure 1 and eq 1) renders glycolysis less efficient starting with galactose, and this places greater demands on mitochondrial metabolism. The increased reliance on mitochondrial metabolism is presumed to explain the enhanced sensitivity for detecting inhibition or impairment to mitochondria in HepG2 with galactose.

Gal + ATP ⇆ Glc‐1‐P + ADP

(1)

The significance of the Leloir pathway is its proposed explanation of the utilization of galactose for energy production through glycolysis and energy storage in the form of glycogen, when excess galactose is available. The Leloire pathway is, however, often impaired in rapidly dividing cancer cells.15−18 Both galactose-1-phosphate uridyltransferase and 4-epimerase activities are at a minute fraction of their normal capacities, and such cancer cells can experience growth arrest and death after prolonged replacement of glucose by galactose in culture media. The 4-epimerase is found to be inhibited by NADH or by a pH drift from its optimal pH 8.7 toward slightly lower values such as 7.5−7.0. We have noticed very limited if any galactose consumption in our NMR metabolomics analyses of galactose culture media of HepG2 cells and therefore sought to better understand the metabolism of galactose in HepG2 cells. NMR is a powerful analytical technique enabling simultaneous detection of individual chemicals and quantification without need of individual reference standards. The uniquely uniform response of all nuclei (protons for 1H NMR here) allows direct measurements of chemicals in NMR samples by referencing to one internal or external reference standard prepared at a known concentration. For example, by B


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Figure 2. 1D proton NMR spectra of HepG2 culture media after 24 h incubation with ∼5 mM glucose or 13C6-glucose, 10% dialyzed FBS, and treatment with or without 5 μM oligomycin A (n = 3 for individual conditions) (A) 1H NMR anomeric C1α-H region of glucose. (B) 1H NMR anomeric C1β-H and ring proton region of glucose. (C) 1H NMR methyl proton region of lactate. From top to bottom in each figure: (1) ∼5 mM 13 C6 glucose in starting culture media; (2) ∼5 mM 13C6 glucose and 5 μM oligomycin A in starting culture media; (3) ∼5 mM glucose in starting culture media.

quantitative measurements of metabolites in culture media and inside myotube cells, NMR can show specific mitochondrial toxicities.19 It also enables a direct and quantitative comparison of chemicals in an NMR sample. The molar decrease in glucose by glycolysis can be directly compared to the molar increase in lactate as the end-product under glycolysis. To differentiate products of exogenously added chemicals from

endogenous metabolites, stable isotope labeled chemicals can be added to cell culture media and differentiated from corresponding endogenous chemicals to provide specific tracking of metabolic conversions in vitro or in vivo. Our findings have led to adoption of specific alterations of assay nutrient composition that improves the performance of C


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Figure 3. (A) Measurements of glucose consumption and lactate production in HepG2 cells after 24 h incubation with ∼5 mM glucose (n = 3). From the left to right are concentrations (mM) of glucose and lactate. Teal, 0 h; brown, 24 h. (B) Measurements of 13C6-glucose consumption and 13 C3-lactate production in HepG2 cells after 24 h incubation with ∼5 mM 13C6-glucose and treatment with 5 μM oligomycin A (n = 3). From the left to right are concentrations (mM) of glucose and lactate. Teal, 0 h; brown, 24 h; light green, 24 h treatment with 5 μM oligomycin A. (C) Ratios of ΔLac/ΔGlc calculated from the measurement of 13C6-glucose and 13C3-lactate in HepG2 culture media after 24 h of incubation with ∼5 mM 13 C6-glucose (left) and treatment with 5 μM oligomycin A (right; n = 3). cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC) supplemented with 10% fetal bovine serum (FBS; Gibco, Gaithersburg, MD), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). The cell number initially plated ranged from 50 000 cells/ well in 96-well plates for mitochondrial toxicity screening to 1 or 2 million cells/well in six-well dishes for NMR analyses. The cells were left to adhere to plates in a monolayer by incubation overnight at 37 °C in a humidified incubator with 5% CO2. HepG2 cell cultures were completely confluent at the time of compound treatment. Culture media were completely aspirated, and wells were rinsed once with Dulbecco’s phosphate saline buffer

the HepG2 assay in detecting drug-induced mitochondrial dysfunction.

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EXPERIMENTAL PROCEDURES

Materials. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. The 100% deuterated aqueous buffer (80 mM potassium phosphate at pH = 7.0 with 2 mM DSS-d6) was purchased from Isotec (Miamisburg, OH). Cell Culture. HepG2 cells (HB-8065, ATCC, Manassas, VA) were cultured in collagen coated 96-well plates or six-well dishes. Cells were D


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Chemical Research in Toxicology (ThermoFisher, Waltham, MA) to remove residual media, particularly glucose. Test compounds were prepared as 100 × concentrated stock solutions in dimethyl sulfoxide (DMSO), and the final DMSO concentration in treatment media was 1%. The treatment medium was comprised of Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% dialyzed FBS (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin. Medium pH was adjusted to 7.4 with sodium bicarbonate and 1 N HCl. Treatment media were also supplemented with either glucose (unlabeled or uniformly 13C6-labeled) or galactose (unlabeled or uniformly 13C6labeled) at approximately 5 mM; glutamine was not added to the treatment media unless specified. Cells cultured in media containing test compounds were incubated overnight at 37 °C in a humidified incubator with 5% CO2. There were usually at least three replicate wells for each compound concentration evaluated. At the end of each incubation, culture media were collected for NMR analyses or analyzed for lactate dehydrogenase (LDH) activity using a commercially available assay kit (ThermoFisher). Cellular ATP levels were measured using a Promega CellTiter-GLO luminescence assay (Madison, WI). For the NMR analysis, Oligomycin A was added at 5 nM or 5 μM to HepG2 incubation media containing 13C6-labeled glucose or galactose. While ATP levels decreased in HepG2 cells with increasing concentrations of oligomycin A, the dose−response curve was dramatically impacted by the presence of either glucose or galactose (Supporting Information Figure S1). After 24 h of incubation of HepG2 cells in the absence of glucose but in the presence of 5 mM galactose, little if any ATP was produced at concentrations of oligomycin A above 10 nM. Therefore, 5 nM oligomycin A was appropriate for assessing the metabolism of HepG2 cells in media containing no glucose but 5 mM galactose to avoid excessive cellular toxicity. For NMR analyses, cells were separated from media and washed with phosphate buffered saline (PBS). Cells were then lysed in 80% aqueous methanol on dry ice to quench cellular enzyme activities and precipitate proteins or lipoproteins. Media were also treated with 80% methanol to remove proteins or lipoproteins. Supernatants of cell extract and treated culture media were lyophilized in a refrigerated CentriVap Concentrator (Labconco, Kansas City, MO) connected to a refrigerated Vapor Trap (ThermoFisher). Lyophilized samples were stored at −70 °C prior to NMR analyses. NMR Analysis. Sample Preparation for NMR. To lyophilized samples, 550 μL or 220 μL of 80 mM potassium phosphate (pH 7) containing 2 mM of DSS-d6 (Isotec, Miamisburg, OH) was added, and the samples were thoroughly mixed for complete dissolution and centrifuged at 12 000 rpm at 10 °C for 10 min. Supernatants were transferred to 4-in., 5-mm, or concentric 5−3-mm NMR tubes (New Era or Wilmad, Vineland, NJ). All NMR samples were placed in 8 × 12 NMR tube racks on a SampleJet (Bruker, Bellerica, MA). The sample storage temperature in the SampleJet was set at 10 °C. NMR Instrument Settings. One dimensional proton NMR spectra (1D 1H NMR) were acquired on a Bruker AVANCE III 700 MHz spectrometer equipped with a cold probe or AVANCE II 600 MHz NMR spectrometer with a room temperature probe (Bruker, Bellerica, MA). The probe temperature was set to 25 °C. The 1D 1H NMR spectral width was 8571 (600 MHz) or 10 000 Hz (700 MHz), covering the range from −2.37 ppm to 11.91 ppm. The acquisition time was 3 s, corresponding to a digital resolution of 0.33 Hz. The water signal was suppressed with a WET pulse sequence that was based on the selective excitation of the water peak using sinc shape pulses and dephasing gradients.20 Transients of at least 64 scans were acquired with a relaxation delay of 15 s. NMR Data Processing. Individual NMR FIDs (free induction decay) were apodized with a 0.2 Hz exponential line broadening curve, and extended to 64 000 complex points before the Fourier transformation. NMR spectra were phased properly, and chemical shifts were referenced to the internal DSS-d6 resonance (0 ppm). Spectra were then saved for subsequent metabolite analyses using the dataChord software (One Moon Scientific, Inc., Newark, NJ).

Individual metabolites were identified based on a proprietary NMR reference spectrum database containing about 700 endogenous metabolites. Metabolite quantification was based on the integration of well resolved peaks, and metabolite concentrations in NMR samples were calculated based on the peak area of the reference DSSd6 of known concentration. The R statistical software (http://www.r-project.org) was used for making box plots.

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RESULTS Mitochondrial Inhibition Increased Glucose Metabolism and Lactate Production in HepG2 Cells. To investigate the suspected increase in glycolysis following mitochondrial inhibition, the turnover of glucose to lactate was monitored using U-13C6-glucose or glucose. HepG2 cells were incubated for 24 h at 37 °C in culture media containing ∼5 mM U-13C6-glucose or unlabeled glucose with or without oligomycin A, an inhibitor of the ATP synthase. Production of U-13C3-lactate and unlabeled lactate could be readily observed and measured on the 1D 1H NMR spectra following the incubation of HepG2 cells (Figure 2). Two anomeric glucose configurations of α and β were visible on NMR spectra. Anomeric H1 of the remaining U-13C6-αglucose appeared as two satellite peaks (5.36 and 5.08 ppm) flanking the position of the doublet H1 peak (5.22 ppm) of unlabeled α-glucose (Figure 2A). Similarly, anomeric H1 of the U-13C6-β-glucose should appear as two satellite peaks, but water suppression by WET suppressed the satellite peak on the left side of the H1 peak (4.64 ppm) of unlabeled β-glucose (Figure 2B), leaving visible only the satellite peak at 4.50 ppm. Lactate could be identified by its C2−H (4.10 ppm) and C3− H3 (1.32 ppm; Figure 2B and Figure 2C). The satellite peaks of 13C2−H (4.22 and 3.98 ppm) were visible but overlapped with Hα of the amino acids in the region (Figure 2B). The two satellite peaks of 13C3−H3 (1.42 and 1.21 ppm) were uniquely distinguishable from any other peaks in the region (Figure 2C). On the basis of the peak integration in Figure 2, glucose decreased from 5.23 mM at 0 h to 4.08 mM at 24 h in the media of HepG2 cells without mitochondrial inhibitor oligomycin A (Figure 3A). Correspondingly, lactate increased from below the limit of detection to 1.94 mM. Similar changes were seen with U-13C6-glucose (decreased from 5.28 mM to 4.16 mM) and U-13C3-lactate (increased from below detection to 1.68 mM; Figure 3B). In the presence of 5 μM oligomycin A, glucose consumption and lactate production increased as seen in U-13C6-labeled glucose and U-13C3-labeled lactate on NMR spectra (Figure 2). Peak integration showed that U-13C6-glucose decreased to 3.28 mM, and U-13C3-lactate more than doubled to 3.51 mM (Figure 3B). In glycolysis, one glucose molecule is converted to two lactate molecules, and no lactate is produced when glucose is completely metabolized to carbon dioxide in mitochondria. The ratio of changes in lactate over changes in glucose (ΔLac/ ΔGlc) for U-13C6-labeled glucose and U-13C3-labeled lactate reveals the extent that glucose is metabolized to lactate in cytosol instead of CO2 in the TCA (tricarboxylic acid) cycle in mitochondria. The ratio ΔLac/ΔGlc is negative due to consumption of glucose and production of lactate in glycolysis. The ΔLac/ΔGlc ratio changed from the average −1.5 in the vehicle control to the average −2.1 in the presence of 5 μM oligomycin A (Figure 3C), confirming that well-established E


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Figure 4. Concentrations of galactose and its metabolites in HepG2 culture media and inside cells after 24 h incubation with increasing concentrations of oligomycin A. The starting culture media contained ∼5 mM galactose. (A) Galactose concentrations (mM) in culture media; (B) amounts of galactose (fmols/cell) in HepG2 cells; (C) amounts of galactonate (fmols/cell) in HepG2 cells; (D) amounts of galactose-1-phosphate (fmols/cell) in HepG2 cells.

mitochondrial inhibitor oligomycin A could increase glycolysis, which was shown experimentally as both increased glucose consumption and lactate production to achieve the stochiometric ΔLac/ΔGlc ratio of −2. Galactose Was Not Much Metabolized to Lactate in HepG2 Cells by the Leloir Pathway and Glycolysis. Because so little consumption of galactose in HepG2 culture media was noted, it prompted us to investigate its level of metabolism. HepG2 cells were incubated for 24 h at 37 °C in the presence of nearly 5 mM galactose with increasing concentrations of oligomycin A (Figure 4). Culture media and cells were collected and processed separately as described in the Experimental Procedures. In the presence of mitochondrial inhibitors such as oligomycin A, galactose levels in culture media of HepG2 cells were slightly higher than those in the absence of oligomycin A (Figure 4A). The slight increase of galactose in media with oligomycin A was matched by its reduced uptake into cells and metabolism to galactonate and galactose-1phosphate (Figure 4B−D). The detectable galactose metabolites were galactonate and galactose-1-phosphate, and they were found only inside HepG2 cells. Both galactonate and galactose-1-phosphate were identified by matching peaks of NMR spectra of

HepG2 cells lysates with the peaks of those compounds in our proprietary NMR spectrum library (Supporting Information Figure S2A and B). To track galactose metabolism and differentiate it from glucose metabolism in HepG2, U-13C6-galactose and U-13C6glucose were separately added to HepG2 culture media. HepG2 cells were incubated for 24 h at 37 °C with starting nutrient concentrations of ∼5 mM U-13C6-glucose or ∼5 mM U-13C6-galactose or ∼5 mM U-13C6-galactose plus ∼5 mM unlabeled glucose. Oligomycin A was added at a concentration of 5 nM or 5 μM; however, 5 μM oligomycin A caused excessive cell toxicity (severe cell loss, not shown) when glucose was absent and replaced by galactose. Figure 5 shows the NMR spectra of culture media after 24 h of incubation of HepG2 cells with five different combinations of U-13C6-glucose, U-13C6-galactose, oligomycin A, and glucose. The anomeric H1α of U-13C6-galactose appeared as two satellite peaks (5.37 and 5.13 ppm, Figure 5A), and it differed from the anomeric H1α of U-13C6-glucose in peak shape and the chemical shifts (5.36 and 5.08 ppm for H1α of U-13C6-glucose). The anomeric H1β of U-13C6-galactose showed only one of its two satellite peaks at 4.46 ppm due to WET water suppression of the downfield peak (Figure 5B). The lactate 13C2−H satellite peak at 4.22 ppm was visible F


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Figure 5. 1D proton NMR spectra of HepG2 cell culture media after 24 h incubation (n = 3 for individual conditions). (A) 1H NMR anomeric C1α-H region of glucose and galactose. (B) 1H NMR anomeric H1β region of glucose and galactose. (C) 1H NMR methyl proton region of lactate. From top to bottom in each figure: (1) 13C6-glucose (∼5 mM) and treatment with 5 μM oligomycin A; (2) 13C6-galactose (∼5 mM); (3) 13C6galactose (∼5 mM) and treatment with 5 nM oligomycin A; (4) 13C6-galactose (∼5 mM) + glucose (∼5 mM); (5) 13C6-galactose (∼5 mM) + glucose (∼5 mM) and treatment with 5 μM oligomycin A.

when U-13C6-glucose was added to the culture media, but the peak was below detection in the media containing U-13C6galactose. It was equally confirmed in the upfield region of 1.15 to 1.45 ppm where methyl proton peaks of lactate appeared close to threonine methyl peaks (Figure 5C). Incubation of U-13C6-glucose with oligomycin A at 5 μM (Figure 5C) or 5 nM (Supporting Information Figure S3)

produced U-13C3-lactate by showing two satellite peaks (1.42 and 1.21 ppm). The two U-13C3-lactate methyl satellite peaks were hardly detectable when 13C6-galactose was included in four different combinations: (1) U-13C6-galactose alone (Figure 5C); (2) U-13C6-galactose and oligomycin A at 5 nM (Figure 5C) or 5 μM (Supporting Information Figure S3); (3) U-13C6-galactose and glucose (Figure 5C); (4) U-13C6G


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Chemical Research in Toxicology galactose, glucose, and oligomycin A at 5 μM (Figure 5C) or 5 nM (Supporting Information Figure S3). When glucose was included, a prominent doublet peak (1.32 ppm) of unlabeled lactate appeared; however, U-13C3-lactate (1.42 and 1.21 ppm, Figure 5C and Supporting Information Figure S3) was hardly detectable when U-13C6-galactose was included in the incubation regardless of the presence of unlabeled glucose. In addition, U-13C6-glucose was hardly detectable when HepG2 cells were incubated with U-13C6-galactose (Figure 5A and B). The concentration of U-13C6-galactose, unlike U-13C6glucose, after 24 h of incubation in HepG2 appeared to decrease only slightly (Figure 6A). The slight decrease in U-13C6-galactose was observed (i.e., 5.30 ± 0.03 mM to 5.13 ± 0.10 mM) only in the absence of glucose and mitochondrial inhibitors. However, the decrease was statistically insignificant (p = 0.10). In the presence of glucose or oligomycin A or both glucose and oligomycin A, 13C6-galactose concentrations did not seem to decrease. On the contrary, glucose when added together with U-13C6galactose decreased by almost half from 4.27 mM at 0 h to 2.11 mM at 24 h (Figure 6B). Glucose consumption nearly doubled here in comparison to that in the previous section (i.e., 2.16 mM versus 1.15 mM), due to increased cell numbers from 1 million cells to 2 million cells. When 5 μM oligomycin A was added to the incubation media, glucose concentration was reduced further to 0.71 mM at 24 h. Corresponding to the glucose reduction, lactate increased from 0.12 mM at 0 h to 5.49 mM at 24 h when both glucose and U-13C6-galactose were present in the culture media (Figure 6C). The addition of 5 μM oligomycin A resulted in lactate concentration increasing further to 8.96 mM (Figure 6C). Our experiments showed that it was much limited for galactose to be metabolized to glucose and lactate in HepG2 cells; however, glucose could undergo the expected glycolysis to generate lactate in HepG2 cells. HepG2 Cell Metabolism Was Hardly Altered by Galactose. To evaluate the impact of galactose on cellular metabolism in comparison to glucose, HepG2 cells were treated with increasing concentrations of the mitochondrial complex III inhibitor antimycin A in the presence of 5 mM glucose or 5 mM galactose or in the absence of both galactose and glucose for 24 h at 37 °C (Figure 7). The cells were collected and processed as described in the Experimental Procedures. Figure 7 plots the profiles of succinate as an illustration of the changes inside cells with increasing concentrations of antimycin A. Changes of succinate in response to antimycin A treatments in the absence of either sugar resembled those in culture media fed with galactose, and changes in both conditions (no sugars or only galactose) differed from the changes seen in the presence of glucose. Galactose, as shown here, did not noticeably alter cellular metabolism of the HepG2 cell, by itself or in the presence of mitochondrial inhibitors. Glutamine Consumption Increased When Glucose Was Replaced by Galactose in Culture Media. Glutamine is expected to be utilized as a bioenergetics source through conversion to 2-ketoglutarate as anaplerosis to the mitochondrial TCA cycle, particularly in immortal cancer cells. To confirm this, HepG2 cells were fed ∼4 mM glutamine and either ∼5 mM glucose or ∼5 mM galactose. The cells were

Figure 6. (A) Concentrations of 13C6-galactose in HepG2 culture media at 0 h and after 24 h incubation. (B) Concentrations of glucose in HepG2 culture media at 0 h and after 24 h of incubation. (C) Concentrations of lactate in HepG2 culture media at 0 h and after 24 h of incubation. In each figure, from left to right are culture media: (1) 0 h incubation, ∼5 mM 13C6-galactose and ∼5 mM glucose; (2) 24 h incubation starting with ∼5 mM 13C6-galactose; (3) 24 h incubation starting with ∼5 mM 13C6-galactose and ∼5 mM glucose; (4) 24 h incubation starting with ∼5 mM 13C6-galactose and 5 nM oligomycin A; (5) 24 h incubation starting with ∼5 mM 13C6galactose and ∼5 mM glucose and 5 μM oligomycin A.

incubated for 24 h at 37 °C. Glutamine and lactate in culture media were then quantified. When glutamine was added to the culture media, its consumption by HepG2 cells increased when glucose was replaced by galactose (Figure 8). After 24 h of incubation with HepG2, glutamine levels decreased from 3.83 ± 0.15 mM in the presence of glucose to 2.90 ± 0.08 mM in the presence of galactose but the absence of glucose. Increased glutamine consumption in media with galactose was compared to increased lactate production in media with glucose (Figure 8). The compensation by glutamine became evident as glycolysis-driven HepG2 energy metabolism shifted to the glutamine anaplerotic metabolism, feeding the H


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The in vitro mitochondrial toxicity assay in HepG2 cells is based on a relative shift of treatment-related decreases in cellular ATP content (i.e., mitochondria plus cytosol) when glucose in culture media is replaced by galactose (Figure 9A).11 The threshold of ATP IC50 ratios, i.e., IC50glc/IC50gal, can be established by testing the assay with compounds known to inhibit or not to inhibit mitochondria, or to cause direct non-mitochondrial cytotoxicity (Figure 10). In the original assay by Marroquin, glutamine is added to galactose culture media of HepG2,11 but it was instead excluded from our media based on the evidence described in the previous section that glutamine aided anaplerosis, replenishing TCA cycle intermediates, especially when medium glucose was removed and replaced by galactose. By eliminating glutamine from the culture media, we increased the sensitivity of the assay to detect inhibition of mitochondrial fatty acid β-oxidation (FAO, by 4-pentenoic acid or MrkA;21 Figure 9B, top row) or inhibition of mitochondrial complex II (by 3-nitropropionic acid; Figure 9B, top row) or mitochondrial membrane depolarization (by CCCP; Figure 9A); however, the inhibition was not detected or less sensitively detected in the presence of glutamine, exhibiting only negligible shifts of the Glc-Gal ATP IC50 values (Figure 9B bottom row and Supporting Information Figure S6). Importantly, any glucose in FBS could compromise assay sensitivity due to increased cellular ATP production by glycolysis, thus dialyzed FBS was used. The NMR spectrum detected only residual amounts of glucose, lactate, and formate in dialyzed FBS (Supporting Information Figure S4). The amount of glucose detected was approximately 50 μM, instead of the typical concentration of 5 mM seen in undialyzed FBS, enabling a reduction from >500 μM glucose in the conventional assay with 5 mM galactose and 10% FBS to only 5 μM glucose in this modified media. This modified assay was evaluated with 58 compounds (Supporting Information Table S1) reported in peer reviewed publications to be negative or positive for mitochondrial toxicity by investigators from academia or pharmaceutical companies using a variety of in vitro mitochondrial based assays.7,11,21−44 A third category designated as cytotoxic was comprised of compounds known to detrimentally affect cell viability through mechanisms other than mitochondrial toxicity.22−25 Setting a shift in the IC50glc/IC50gal threshold at 2.0 for defining a result as positive, the assay achieved >95% sensitivity and specificity (Figure 10, Table 1). The results also confirmed that increased sensitivity in the culture medium containing galactose was specific to mitochondrial toxicants, and not to general cytotoxicants, and that removal of glutamine and the use of dialyzed FBS enabled good sensitivity in the detection of inhibition of mitochondrial complexes such as FAO and complex II and mitochondrial membrane depolarization. The confidence of the assay was estimated based on a normal probability model of log(IC50glc/IC50gal) values. The confidence for calling positive or negative mitochondrial toxicity based on an IC50glc/IC50gal ratio increased when the measured ratio could be repeated, or the IC50glc/IC50gal ratio significantly differed from the cutoff 2.0 on either side of the threshold, or both above (Supporting Information Figure S5, and Supporting Information Table S2). Prediction of Potential Human DILI Risk Based on in Vitro ATP IC50gal When Testing Positive for Mitochon-

Figure 7. Changes of the succinate level inside HepG2 cells after 24 h of incubation with increasing concentrations of antimycin A. The starting culture media contained 5 mM glucose or 5 mM galactose or no sugars.

Figure 8. Concentrations of glutamine and lactate in HepG2 culture media after 24 h incubation in the presence of either ∼5 mM glucose or ∼5 mM galactose and ∼4 mM glutamine at 0 h. From left to right are concentrations of glutamine and lactate. Light blue, media in the presence of glucose; brown, media in the presence of galactose.

mitochondrial TCA cycle via the glutamate to 2-keto-glutarate pathway. Concentrations of both glutamine and lactate were higher in glucose media than those in galactose media. A high glutamine concentration indicated reduced consumption of glutamine in glucose media, and high lactate concentrations indicated increased production of lactate. Glutamine consumption increased by 0.9 mM, while lactate production was decreased by 6.7 mM when glucose was replaced by galactose in culture media. Since each glucose produces two lactates in glycolysis, the ratio of glutamine/glucose for the anaplerotic metabolism of glutamine is 0.9:(6.7/2) = 1:3.7. Therefore, to maintain similar cellular bioenergetics, much less glutamine (i.e., one glutamine molecule) was needed to compensate for missing glucose (i.e., 3.7 glucose molecules). Improvement of the in Vitro Mitochondrial Toxicity Assay in HepG2 Cells by Nutrient Selection. The elimination of glutamine and use of dialyzed 10% FBS to avoid residual glucose was designed to improve the ATP IC50 Glc-Gal shift assay of drug-induced mitochondrial toxicity in HepG2 cells. The cell cultures were incubated starting with 5 mM glucose or 5 mM galactose for 24 h at 37 °C. I


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Figure 9. (A) ATP% in HepG2 cells in response to increased concentrations of antimycin A, CCCP, or Etomoxir in 2 different nutrient conditions (i.e., 5 mM galactose or 5 mM glucose, 10% dialyzed FBS, and no glutamine at 0 h) after 24 h of incubation. (B) ATP% in HepG2 cells in response to increased concentrations of pentenoic acid, MrkA, or 3-nitropropionic acid in four different nutrient conditions (i.e., 5 mM galactose or 5 mM glucose, 10% dialyzed FBS, and in the absence or presence of 4 mM glutamine at 0 h) after 24 h incubation. Top row: no added glutamine. Bottom row: in the presence of 4 mM glutamine. Gal-Gln: media with 5 mM galactose but no glutamine. Glc-Gln: media with 5 mM glucose but no glutamine. Gal+Gln: media with 5 mM galactose and 4 mM glutamine. Glc+Gln: media with 5 mM glucose and 4 mM glutamine. ATP% was normalized with respect to the level in vehicle control 1% DMSO.

drial Toxicity in HepG2 Cells. There exists a large confidence gap from defining mitochondrial toxicity above certain concentrations in vitro to using the data as an explanation and proof of toxicity observed in vivo in animals, let alone for human DILI. However, we attempted to establish evidence for the practical utility of an empirical relationship that might interpret potential DILI risk for a compound exhibiting mitochondrial toxicity in vitro with respect to potent exposures in DILI. We related the IC50gal to the estimated maximum total drug concentrations in a human portal vein (Cinlet) achieved at maximal recommended therapeutic daily doses. Both DILI positive and negative compounds were selected based on information in the NIH LiverTox database (https://livertox.nih.gov/), FDA DILIrank Data set, product labels, publication, and drug registrations.45−60 DILI positive compounds included marketed or marketed but withdrawn drugs reported to cause acute liver failure or lactic acidosis in

humans and drugs discontinued by pharmaceutical companies at clinical stages of drug development due to strong liver safety transaminase signals but without any documented instances of acute liver failure (manuscript in preparation by Monroe et al.). DILI negative compounds were not associated with documented instances of acute liver failure but might have some reports of observed transaminase increases. The selected set of 120 compounds (65 DILI positives, and 55 DILI negatives) was tested using the ATP IC50 Glc-Gal shift assay in HepG2 cells modified as described here to eliminate glutamine and trace glucose by using 10% dialyzed FBS (Supporting Information Tables S3 and S4). Among 65 DILI positive compounds, 18 were tested positive by the in vitro mitochondrial toxicity assay, and among 55 DILI negative compounds, 8 were tested positive by the in vitro mitochondrial toxicity assay. For 26 compounds that tested positive by the in vitro mitochondrial toxicity assay, in vitro J


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Figure 10. ATP IC50Glc/IC50Gal ratios of 58 compounds (categorized as cytotoxic, mitochondrial toxicity negative, and mitochondrial toxicity positive, Supporting Information Table S1) tested in HepG2 after 24 h incubation in culture media containing either 5 mM glucose or 5 mM galactose at 0 h (each with 10% dialyzed serum, and no glutamine).

Figure 11. A plot of log10(IC50gal/Cinlet) versus IC50gal of 26 drugs that were found to impair mitochondria in HepG2 in vitro (Supporting Information Table S3). Eighteen drugs were DILI positive, and eight were DILI negative. A horizontal line sets a threshold of IC50gal/Cinlet ≤ 3. If a compound is tested positive by the in vitro mitochondrial ATP IC50 Glc-Gal shift assay and IC50 gal ≤ 3 × Cinlet, it may be likely to pose a concern for human DILI.

ATP IC50gal values determined in 10% FBS with 24 h of incubation were compared with estimated Cinlet.27,61,62 Figure 11 illustrates a plot of log10 of IC50gal/Cinlet versus IC50gal. We explored a threshold of the concentration ratio of IC50gal/Cinlet to link a compound found to have in vitro mitochondrial toxicity to DILI in humans with estimated Cinlet exposures. A threshold of IC50gal/Cinlet = 3 was selected to best separate a majority of DILI positive drugs (represented by solid red circles) from DILI negative drugs (represented by open green circles) in Figure 11. The empirical interpretation is that when a compound is tested positive (i.e., IC50glc/ IC50gal > 2) in HepG2 cells for mitochondrial toxicity and the determined IC50gal is within 3 × Cinlet, it may be likely to pose a concern for human DILI. Conversely, if drug IC50gal for in vitro mitochondrial toxicity exceeds 3 × Cinlet, the human dose level is less likely to cause mitochondria related human DILI, or human DILI is likely caused by underlying mechanisms other than mitochondrial toxicity. The sensitivity and specificity of the empirical prediction demonstrated 25% sensitivity and 95% specificity among the 120 tested compounds (Table 2). The positive predictivity of this modified HepG2 ATP IC50 Glc-Gal shift assay for a druginduced mitochondrial inhibition mechanism of human DILI across this 120-compound test set was 84%, and negative predictivity was 47%.

Table 2. Summary of the Performance of Predicting DILI by the Modified in Vitro Mitochondrial Toxicity ATP Glc-Gal Shift Assay in HepG2 Cells with Media Containing 10% Dialyzed FBS and No Glutamine in vitro mitochondrial toxicity and IC50gal within 3 × Cinlet mito toxicity and DILI

+

−

+

16

49

sensitivity 16/65 = 25%

−

3

52

specificity 52/55 = 95%

positive predictivity 16/19 = 84%

negative predictivity 49/101 = 47%

DILI

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DISCUSSION HepG2 cells retain certain biosynthetic pathways common to normal liver parenchymal cells; however, the capacity of their cellular metabolism is altered.63 In the presence of 10 mM glucose, ATP in HepG2 cells remains steady under aerobic conditions, and lactate production by HepG2 under aerobic

Table 1. Summary of ATP IC50 Glc-Gal Shift Assay Performance in HepG2 Cells with Media Containing 10% Dialyzed FBS and No Glutamine Glc-Gal shift assay outcomes +

−

+

26 true positive

1 false negative

sensitivity 26/27 = 96%

−

1 false positive

30 true negative

specificity 30/31 = 97%

positive predictivity 26/27 = 96%

negative predictivity 30/31 = 97%

in vitro mitochondrial toxicity reported

compound categories

K


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The increased aerobic glycolysis of HepG2 cells following treatment with oligomycin A is similar to anaerobic glycolysis.63 The increased lactate production under either mitochondrial inhibition or anaerobic glycolysis can be attributed to the equivalency of the interruption of the electron transfer chain (ETC) by mitochondrial complex inhibition or deprived oxygen supply. Deprived oxygen supply disables complex IV by preventing the availability of oxygen from harvested electrons through the ETC prior to complex IV. When galactose was provided to culture media, galactonate and galactose-1-phosphate were produced inside HepG2 cells as a consequence of galactose uptake (Figures 4B, C, and D and Supporting Information Figure S2A and B). Their appearance inside HepG2 cells was consistent with the measured marginal decrease in galactose concentration in culture media (Figures 4A and 6A) after 24 h of incubation with the control vehicle of 1% DMSO. In the presence of mitochondrial inhibitors such as oligomycin A, both galactose uptake into HepG2 cells and its feeble metabolism were almost completely interrupted (Figures 4 and 6A). The detection of galactonate inside HepG2 cells indicated galactose oxidation as previously reported in the liver67,68 (step 7 in Figure 1). Hard to detect 13C-labeled glucose and 13Clactate in HepG2 culture media when U-13C6-galactose was added supports the reported deficiency of galactose bioenergetics metabolism in hepatoma cells;15−18 i.e., galactose is not readily converted to glucose and lactate in HepG2 cells. Cancer cell lines, including HeLa and L cells, Ehrlich ascites tumor, and mammary carcinoma, are reported to be unable to grow in galactose and are shown to be defective in galactose metabolism.15 Both enzymes galactose-1-phosphate uridyltransferase and UDP-galactose 4-epimerase are reported to operate at a minute fraction of their normal capacities if at all. Limited activity of galactose-1-phosphate uridyltransferase in hepatoma cells explains the accumulation of galactose-1phosphate inside HepG2 cells (Figure 4D, and Supporting Information Figure S2B). This deficiency in metabolism of galactose-1-phosphate in HepG2 cells explains the scant presence of U-13C6-glucose and U-13C3-lactate on the proton NMR spectra (Figure 5) of culture media when U-13C6-galactose, instead of U-13C6glucose, was added to HepG2 culture media. Even the presence of unlabeled glucose could not assist too much the generation of U-13C6-glucose and U-13C3-lactate from U-13C6galactose. It suggests that ATP generated by unlabeled glucose was not the limiting factor in the Leloir galactose→glucose conversion, as outlined in Figure 1. U-13C3-lactate was clearly visible as satellite peaks at 4.22, 1.42, and 1.21 ppm in culture media containing U-13C6-glucose. Like HeLa cells, HepG2 hepatoma cells likely have deficient activities in galactose-1phosphate uridyltransferase or UDP-galactose 4-epimerase or both uridyltransferase and 4-epimerase. Therefore, ATP production by HepG2 cells from galactose is largely paralyzed by the disabled galactose→glucose conversion. This resulted in reduced cell division of HepG2 (data not shown) and rapid decreases of cellular ATP and elevation of cellular leakage proteins such as LDH (data not shown) at lower concentrations of mitochondrial inhibitors in media containing galactose as compared to glucose. HepG2 cells were much more vulnerable toward drug-induced mitochondrial toxicity in galactose, and they were far more resistant to mitochondrial inhibition in the presence of glucose by being able to rely on

glycolysis is almost 5 times as high as in primary human hepatocytes.63 The high lactate production under aerobic conditions suggests HepG2 has a high baseline capacity for glycolysis in comparison to primary hepatocytes. Lactate production at the expense of glucose could be observed on the NMR spectra of HepG2 cells fed with either unlabeled glucose or U-13C6-glucose (Figure 2). The amount of lactate produced (i.e., 1.94 mM) by HepG2 cells fed unlabeled glucose was slightly higher than by cells fed U-13C6glucose (i.e., 1.68 mM). In the absence of glucose, HepG2 cells can still produce a small amount of lactate,63 suggesting that lactate can be produced from nutrients other than glucose. The production of U-13C3-lactate could only derive from U-13C6glucose, thus explaining why the amount of U-13C3-lactate produced was less than that of unlabeled lactate. Cellular ATP can be generated in mitochondria by a steady and efficient TCA cycle, or more quickly in cytosol by the less efficient biochemical pathway of glycolysis. The TCA cycle extracts a maximum amount of ATP by metabolizing intermediate metabolites completely and efficiently to CO2. ATP generation by glycolysis requires continuous supply of NAD+ that is converted to NADH when glucose is metabolized to pyruvate (Figure 12). The necessity of the

Figure 12. Illustration of the equivalency of glucose and glutamine in supplying and maintaining cellular ATP in HepG2 cells where galactose is hardly metabolized for the gain of ATP. Similar levels of intracellular ATP can be achieved by 3.7 glucose aerobic glycolysis (left) or anaplerotic metabolism of one glutamine in the absence of glucose but the presence of galactose in HepG2 cells (right).

NAD+ regeneration from NADH drives the conversion of pyruvate to lactate, and consequently, a large amount of lactate is generated by glycolysis. Relying on aerobic glycolysis to generate ATP despite the presence of oxygen is a hallmark of immortal cancer cells such as HepG2, and the process is wellknown and termed the Warburg Effect.64,65 In cultured HeLa cells, as many as 80% of glucose molecules are reported to be metabolized by glycolysis to produce lactate.66 In HepG2, the percentage could be calculated based on U-13C3-lactate production and U-13C6-glucose consumption. In the absence of glutamine, the ratio (ΔLac/ΔGlc) was −1.5. The theoretical maximum value is −2.0 since one sixcarbon glucose can at most produce two three-carbon lactates. Therefore, similar to the percentage in HeLa cells, 75% (i.e., 1.5/2.0) of U-13C6-glucose was metabolized by glycolysis to produce U-13C3-lactate in HepG2 cells. In the presence of 5 μM oligomycin A (ATP synthase inhibitor), U-13C6-glucose shifted almost completely to glycolysis to produce U-13C3lactate. Increased lactate production indicates the need for cells to maintain cellular ATP levels with the help of increased glycolysis when mitochondria are inhibited. L


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Removing glutamine from culture media containing galactose reduced the ATP IC50gal value of the FAO inhibitor Etomoxir73 by at least 20 times, from 44 μM (Supporting Information Figure S6) to less than 2 μM (Figure 9A). Similarly, 13- and 63-fold reductions of ATP IC50 gal values were observed for FAO inhibitors 4-pentenoic acid and MrkA, respectively (Figure 9B);21,74,75 a 4-fold reduction of ATP IC50gal was seen for the complex II inhibitor 3-nitropropionic acid (Figure 9B), and a more than 6-fold reduction of ATP IC50gal was achieved for CCCP (Figure 9A and Supporting Information Figure S6). Both fatty acid β-oxidation and mitochondria complex II are two different branches off the main mitochondrial ETC (i.e., complexes I→III→IV), and mitochondrial membrane depolarization by uncouplers is independent of mitochondrial ETC. The metabolic pathway of glutamine→glutamate→2-ketoglutarate could circumvent the inhibition of FAO and complex II and membrane depolarization by anaplerosis to the mitochondrial TCA cycle. This is a likely explanation for the improved sensitivity and specificity of the ATP IC50 Glc-Gal shift assay in the absence of glutamine. The exclusion of glutamine and reduction of glucose in dialyzed FBS helped enlarge the gap of ATP IC50 values in media containing glucose or galactose, thereby enhancing assay sensitivity, and thus the ratio of IC50glc/IC50gal exceeded the threshold cutoff for Etomoxir, MrkA, 4-pentenoic acid, and 3nitropropionic acid. The improvement of the assay from detecting these four mitochondrial toxicants helped increase the assay sensitivity from 81% (22/27) to 96% (26/27; Table 1). The ATP IC50 Glc-Gal shift assay is an indirect method for detecting mitochondrial inhibition or impairment; therefore, the prediction could be influenced by selecting a threshold cutoff and the likelihood of measuring an IC50glc/IC50gal ratio. The threshold of 2.0 was based on a maximum separation of mitochondrial positive compounds from negative and cytotoxic compounds (Figure 10). If the IC50glc/IC50gal ratio was close to the threshold (e.g., 1.8−2.2), repeated measurements helped confirm the category of a test compound. The confidence level of a measured ratio of, for example, 2.2 increased from 69% to 82% if the ratio could be repeatedly measured thrice (Supporting Information Table S2). If an IC50glc/IC50gal ratio was >3.0, the confidence was >99% in categorizing the test compound mitochondrial inhibition or impairment with just a single replicate (Supporting Information Figure S5). The gap between in vitro assay detection of drug mitochondrial toxicity and a conclusion of in vivo manifestation of likely mitochondria-related DILI arises from the complications of drug absorption, distribution, metabolism, and excretion in vivo. Most drugs also bind significantly to proteins, and drug protein binding alters free drug concentration. The presence of serum protein maintained in the in vitro ATP IC50 Glc-Gal shift assay was 1/10th that of in vivo. We analyzed the relationship of in vitro ATP IC50gal values of total added drug concentrations of 26 drugs (tested positive by the ATP IC50 Glc-Gal shift assay in HepG2) with their estimated in vivo total drug concentration in a liver portal vein Cinlet. In consideration of human DILI, the calculated liver exposure concentration Cinlet is likely much more relevant than systemic exposure Cmax, although peripheral systemic Cmax is often used to predict DILI based on in vitro mitochondrial toxicity assays.3,76 A plot of log10 of IC50gal/Cinlet versus IC50gal was constructed to select an in vitro cutoff threshold for an optimal

cytosolic ATP production through glycolysis (Figures 9 and 12). Galactose contributes little to protection of cells from inhibitors of mitochondrial activity. In terms of bioenergetics and mitochondrial toxicity, the presence of galactose was almost equivalent to the absence of all sugars and did not appear to alter HepG2 cellular metabolisms to a noticeable level (Figure 7). It is reported that galactose undergoes oxidation inside livers to produce galactonate.68 The oxidation occurs mainly in the liver microsome fraction, and oxidation in the mitochondrial fraction shows only one-half of the activity. Its formation in livers is attributed to a galactose dehydrogenase or an alcohol dehydrogenase. Galactonate is detected in patients with galactosemia.67 The defect in galactosemia is usually considered an absence of the activity of galactose-1-phosphate uridyltransferase,69,70 which is needed for the Leloir pathway to convert galactose to glucose. As a consequence of galactosemia, galactose-1-phosphate is seen to accumulate inside cells in vitro,69 or in vivo in galactosemia patients.71 The detection of both galactose-1-phosphate and galactonate inside HepG2 cells matches the reported biochemistry of galactosemia. Glutamine can contribute significantly to bioenergetics in immortal HeLa cells.66 At least 65% of glutamine contributes to HeLa bioenergetics in the presence of glucose. Likewise, glutamine is taken up by HepG2 and contributes to bioenergetics.72 When HepG2 cell culture media were switched from glucose to galactose with glutamine present, one additional glutamine was consumed and 7.4 (6.7/0.9) fewer lactates were produced (Figure 8). As many as 3.7 (i.e., 7.4/2) glucose molecules were likely thus to be compensated by one glutamine to support HepG2 bioenergetics when glucose was removed and replaced by galactose. Anaplerosis by each molecule of glutamine to the TCA cycle can generate at least nine molecules of ATP.66 The net ATP generation by glycolysis is two. Therefore, 7.4 (i.e., 2 × 3.7) ATP molecules lost from glycolysis due to glucose replacement by galactose could be adequately compensated by nine ATP generated in mitochondria by consumption of one additional glutamine. If a small fraction of 3.7 molecules of glucose was metabolized in mitochondria in the absence of mitochondrial inhibitors, more than 7.4 ATP molecules could conceivably be generated, likely bringing the number of ATP molecules close to nine as generated by one glutamine. It confirms the efficient ATP production in mitochondria compared to the aerobic glycolysis. Therefore, consumption of a small amount of glutamine could compensate for a large amount of glucose consumption in aerobic glycolysis to presumably maintain ATP levels in hepatoma HepG2 cells when glucose was replaced by galactose (Figure 12). Therefore, in order to improve the sensitivity and specificity of the ATP IC50 Glc-Gal shift assay in HepG2, the culture media were formulated (1) to exclude glutamine and (2) to use dialyzed FBS to reduce glucose from 500 μM to 5 μM in galactose media. The improved in vitro assay, without glutamine and with even further reduction of glucose, enabled the detection of mitochondrial toxicity by inhibitors of FAO or complex II with IC50glc/IC50gal ratios well exceeding the set threshold of 2.0 (Figure 9B, Supporting Information Table S1). It also improved the sensitivity in the detection of mitochondrial membrane depolarization (CCCP in Figure 9A and Supporting Information Figure S6). M


Article

Chemical Research in Toxicology prediction of in vivo human DILI. A threshold of IC50gal/Cinlet = 3 was deemed optimum to define the bottom half in Figure 11 where compounds were positive for in vitro mitochondrial toxicity and 84% (16/19) of those compounds were human DILI positive. According to the criteria, it marked a risk concern of DILI when IC50gal/Cinlet < 3. However, it was not predictable for mitochondrial toxicity-related DILI when IC50gal/Cinlet ≥ 3 since there were more DILI negatives (71% = 5/7) than positives (29% = 2/7; Figure 11). In the bottom half of Figure 11, where IC50gal/Cinlet < 3, 16 compounds are DILI positive as indicated as solid red circles (Supporting Information Table S3). The DILI negative compounds (open green circles) that were indistinguishable here from the DILI positives are sertraline, canagliflozin, and entacapone. Sertraline, a selective serotonin reuptake inhibitor (SSRI), was approved in 1991 in the United States and remains in wide use with almost 40 million prescriptions yearly. However, 1% of patients on Sertraline have demonstrated modest liver enzyme elevations. Acute liver failure due to Sertraline has been reported but is very rare.77 Canagliflozin is indicated for type 2 diabetes mellitus (T2DM) and works by inhibiting sodium-coupled glucose cotransporters in renal proximal tubule epithelial cells.78 No DILI has been reported, but a FDA Drug Safety Communication warns for the risk of acute kidney injury.79 A recent in vitro study of Canagliflozin in human renal proximal tubule epithelial cells shows off-target activity as dual inhibition of mitochondrial glutamate dehydrogenase and complex I.80 Entacapone, like Tolcapone, is used for the treatment of patients with Parkinson syndrome as a catechol-O-methyltransferase (COMT) inhibitor. Both Tolcapone and Entacapone uncouple mitochondrial oxidative phosphorylation and inhibit mitochondrial enzyme complexes; however, Tolcapone is a more potent mitochondrial inhibitor than Entacapone in causing hepatotoxicity.81 There have been no reports of hepatotoxicity for Entacapone. Efficient metabolism of Entacapone to a glucuronide metabolite may be important for limiting DILI,82−85 and phase II biotransformation of Entacapone would be deficient in HepG2 cells. In the top half of Figure 11 (IC50gal/Cinlet > 3), where in vitro mitochondrial toxicity may not be responsible for DILI, there are two DILI positive compounds: cyproterone acetate and ximelagatran. Cyproterone acetate is a steroidal antiandrogen for the treatment of androgen-dependent conditions. Although specific mechanisms of cyproterone related DILI are unknown, the acute liver injury is believed to be an idiosyncratic reaction to one of its metabolites.45 HepG2 cells are often poorly capable of drug metabolism. Ximelagatran is an antithrombotic agent via direct thrombin inhibition. After long-term (>35 days) treatments, increased hepatic enzymes are often accompanied by symptoms such as fever and rash, and a pharmacogenomics study shows that transaminase increases observed clinically with ximelagatran treatment are associated with major histocompatibility complex (MHC) alleles.51 Investigations of other possible underlying mechanisms indicate mitochondrial involvement to be unlikely. On the basis of the results from a set of 65 DILI positive compounds and 55 DILI negative compounds (Supporting Information Tables S3 and S4), the empirical prediction of

human DILI using IC50gal/Cinlet < 3 showed good specificity (95%) and positive predictivity (84%) using the in vitro mitochondrial toxicity assay described in this paper (Table 2). Since numerous and diverse mechanisms can account for DILI, negative predictivity (47%) of the mitochondrial toxicity is expected to be low. The sensitivity of 25% is very close to the 26% reported in a recently published DILI prediction model when 10× drug systemic Cmax in humans is used as a cutoff to compare with IC50s of in vitro mitochondrial toxicity assays.3 The sensitivity of the empirical but data-driven prediction of human DILI presented in Table 2 also suggests that the percentage of compounds causing mitochondria-related human DILI may be approximately 20−30% of all compounds that present with DILI. The modified ATP IC50 Glc-Gal shift assay described here is expected to improve the data-driven empirical model prediction with its more sensitive detection of mitochondrial inhibition to include, for example, FAO and complex II mechanisms. The sensitivity value of potential human DILI prediction would be affected by the number of the included DILI compounds that could inhibit FAO and complex II. We have not evaluated the same test set of 120 pharmaceutical compounds using the unmodified ATP IC50 Glc-Gal shift assay with glutamine and undialyzed FBS because of its poor performance in identifying and categorizing mitochondrial inhibition (Figure 9B and Supporting Information Figure S6). This drug-induced mitochondrial inhibition model is intended for internal drug development consideration when, for example, a set of candidate compounds can be stack ranked. This model, however, requires continued performance testing and evaluation. In summary, biochemical understanding of nutrients important to HepG2 bioenergetics helps to improve the in vitro assay for mitochondrial toxicity screening and enables the assay to assist the selection of drug molecules by mitigating mitochondrial toxicity according to maximum therapeutic exposure levels in the liver. In our study of HepG2 cells for mitochondrial toxicity screening, it was observed that galactose was hardly converted to glucose via the Leloir pathway in HepG2 cells. Therefore, galactose did not significantly alter HepG2 cellular bioenergetics. Increased glutamine consumption by HepG2 cells when glucose was replaced by galactose helped maintain cellular ATP levels at an equivalence of one glutamine versus 3.7 glucoses. By utilizing dialyzed FBS and excluding glutamine, our ATP IC50 Glc-Gal shift assay in HepG2 cells was better able to detect mitochondrial toxicity, especially those inhibitors of mitochondrial FAO and complex II. We established an empirical prediction of probable concerns of human DILI for a compound testing positive by the modified and improved in vitro ATP IC50 Glc-Gal shift assay in HepG2 when its IC50gal was within 3-fold of the calculated total maximum concentrations (Cinlet) in a liver portal vein. Additional work is underway to pursue translational biomarkers to assess and monitor for mitochondrial toxicity in animal models for compounds testing positive by in vitro mitochondrial toxicity assays.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.9b00033. N



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Figures S1−S6, Tables S1−S4, references (PDF)

Article

REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Address: Merck & Co., Inc., 770 Sumneytown Pike, WP81205 West Point, PA 19486. Phone: 215-652-6004. Fax: 215 993-7537. E-mail [email protected]. ORCID

Qiuwei Xu: 0000-0002-8042-6049 Author Contributions

Q.X. was responsible for the manuscript outline and generation of figures and tables. Q.X., J.L., and F.D.S. were responsible for conceiving the overall strategy of assessing mitochondrial toxicity and interpretation of experimental data. L.L., H.V., M.K., A.K., and M.R. were responsible for the generation of experimental data and data analysis. Q.X., A.A., A.K., J.L., and F.S. were responsible for creation and critical review of the manuscript. J.I. and C.W. were responsible for the conception and experiments of in vitro galactose metabolisms in various types of cells. A.L. was responsible for the statistical analysis of the experimental data. A.A. and Y.Y. were responsible for annotating drugs and compounds of the in vitro mitochondrial toxicity assay and calculation of a Cinlet of DILI drugs. K.P. and R.G. were responsible for the biomarker development of mitochondrial toxicity. R.G. and K.M. provided critical review of the relevance of in vitro mitochondrial toxicity to in vivo manifestation. Funding

The work in this paper was funded internally by Safety Assessment and Laboratory Animals Resources at Merck & Co., Inc. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Sharon O’Brien for her creation of the graph of the Table of Contents.

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ABBREVIATIONS U-13Cn, n uniformly 13C-labeled carbons; DILI, drug induced liver injury; NMR, nuclear magnetic resonance; FID, free induction decay; WET, water suppression enhanced through T1 effects; OCR, oxygen consumption rate; DSS-d6, 2,2dimethyl-2-silapentane-5-sulfonate sodium or sodium 3(trimethylsilyl)-1-propanesulfonate; DMSO, dimethyl sulfoxide; ATP, adenosine triphosphate; ADP, adenosine diphosphate; NADH, reduced nicotinamide adenine dinucleotide; NAD+, oxidized nicotinamide adenine dinucleotide; Gal, galactose; Glc, glucose; UDP, uridine diphosphate; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; FBS, fetal bovine serum; DMEM, Dulbecco’s Modified Eagle’s Medium; EMEM, Eagle’s Minimum Essential Medium; PBS, phosphate buffered saline; TCA, tricarboxylic acid; ETC, electron transfer chain; LDH, lactate dehydrogenase; GALT, galactose-1-phosphate uridyltransferase; CPT-1, carnitine palmitoyltransferase-1; Cinlet, maximum concentration of a drug in the liver portal vein; Cmax, maximum concentration of a drug in human blood; SSRI, selective serotonin reuptake inhibitor; T2DM, type 2 diabetes mellitus; COMT, catechol-O-methyltransferase; MHC, major histocompatibility complex O


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