Isoniazid-induced cell death is precipitated by underlying mitochondrial complex I dysfunction in mouse hepatocytes

Free Radical Biology and Medicine 65 (2013) 584–594

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Original Contribution

Isoniazid-induced cell death is precipitated by underlying mitochondrial complex I dysfunction in mouse hepatocytes Kang Kwang Lee a, Kazunori Fujimoto a,b, Carmen Zhang a, Christine T. Schwall c, Nathan N. Alder c, Carl A. Pinkert d, Winfried Krueger a, Theodore Rasmussen a, Urs A. Boelsterli a,n a

University of Connecticut, Department of Pharmaceutical Sciences, Storrs, CT 06269, USA Daiichi Sankyo, Medicinal Safety Research Laboratories, Japan c University of Connecticut, Department of Molecular and Cell Biology, Storrs, CT 06029, USA d Auburn University, Department of Pathobiology, Auburn, AL 36849, USA b

art ic l e i nf o

a b s t r a c t

Article history: Received 30 April 2013 Received in revised form 11 July 2013 Accepted 24 July 2013 Available online 30 July 2013

Isoniazid (INH) is an antituberculosis drug that has been associated with idiosyncratic liver injury in susceptible patients. The underlying mechanisms are still unclear, but there is growing evidence that INH and/or its major metabolite, hydrazine, may interfere with mitochondrial function. However, hepatic mitochondria have a large reserve capacity, and minor disruption of energy homeostasis does not necessarily induce cell death. We explored whether pharmacologic or genetic impairment of mitochondrial complex I may amplify mitochondrial dysfunction and precipitate INH-induced hepatocellular injury. We found that INH ( r3000 μM) did not induce cell injury in cultured mouse hepatocytes, although it decreased hepatocellular respiration and ATP levels in a concentration-dependent fashion. However, coexposure of hepatocytes to INH and nontoxic concentrations of the complex I inhibitors rotenone (3 μM) or piericidin A (30 nM) resulted in massive ATP depletion and cell death. Although both rotenone and piericidin A increased MitoSox-reactive fluorescence, Mito-TEMPO or N-acetylcysteine did not attenuate the extent of cytotoxicity. However, preincubation of cells with the acylamidase inhibitor bis-p-nitrophenol phosphate provided protection from hepatocyte injury induced by rotenone/INH (but not rotenone/hydrazine), suggesting that hydrazine was the cell-damaging species. Indeed, we found that hydrazine directly inhibited the activity of solubilized complex II. Hepatocytes isolated from mutant Ndufs4 þ /  mice, although featuring moderately lower protein expression levels of this complex I subunit in liver mitochondria, exhibited unchanged hepatic complex I activity and were therefore not sensitized to INH. These data indicate that underlying inhibition of complex I, which alone is not acutely toxic, can trigger INH-induced hepatocellular injury. & 2013 Elsevier Inc. All rights reserved.

Keywords: Complex I Complex II Drug-induced liver injury Hydrazine Isoniazid Mitochondria Ndufs4 Rotenone

Introduction Isoniazid (INH) is a widely used first-line antituberculosis drug that has been associated with idiosyncratic (host-dependent) drug-induced liver injury (DILI) in susceptible patients [1,2]. The incidence of INH-related hepatic adverse reactions is relatively high, as compared to that induced by other drugs; it has been reported that up to 20% of treated individuals develop increased

Abbreviations: ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; BNPP, Bis-p-nitrophenyl phosphate; DILI, Drug-induced liver injury; ETC, Electron transport chain; HzN, Hydrazine; INH, Isoniazid (isonicotinic acid hydrazide); LDH, Lactate dehydrogenase; OCR, Oxygen consumption rate; OXPHOS, Oxidative phosphorylation; PA, Piericidin A; ROT, Rotenone; TCA, Tricarboxylic acid n Correspondence to: University of Connecticut School of Pharmacy, Department of Pharmaceutical Sciences, 69 N. Eagleville Road Unit 3092, Storrs, CT 06269, USA. Fax: +1 860 486 5792. E-mail address: [email protected] (U.A. Boelsterli). 0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2013.07.038

plasma alanine aminotransferase (ALT) activity and up to 1% of recipients exhibit more severe hepatotoxicity including liver failure [3,4]. The single most important risk factor is age; in fact, there is an excellent positive correlation between the patient's age and the incidence of INH-induced DILI [5–7]. However, other determinants of susceptibility to INH hepatotoxicity have remained largely unclear. For example, establishing positive correlations with genetic variability at several loci involved in INH biotransformation (NAT2, CYP2E1, GSTM1, GSTT1) has remained controversial [8–11]. One reason for the difficulty in identifying susceptibility factors in patients is the lack of a clear understanding of the molecular mechanisms underlying INH-induced liver injury [12]. The concept of novel reactive intermediates, generated by cytochrome P450 (CYP)-dependent or CYP-independent pathways, has recently been revisited [13–15]. Covalent adducts of reactive drug metabolites may lead to an immune response against drug-modified proteins. However, while there is evidence for this concept in some cases [14], this paradigm cannot readily explain all characteristics of

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INH-induced DILI, including the typical long (4 months median) delay between the onset of treatment and the precipitation of liver injury [5,7]. Unfortunately there are currently no animal models available that recapitulate the clinical picture of INH hepatotoxicity which could elucidate the underlying mechanisms in detail. Even long-term (4-week) administration of INH to mice did not result in clinical or histopathological evidence of liver injury [16]. There is experimental evidence that INH [17] and/or one of its major metabolites, hydrazine [18–21], may interfere with mitochondrial function. However, these mitochondrial changes (mitochondrial oxidant stress) were not sufficient to precipitate overt hepatocyte injury; instead, they may merely indicate a potential subcellular target. For example, being a hydrazine derivative, INH can react with keto acids, which are key intermediates in the TCA cycle, to form hydrazones, and a number of novel hydrazone metabolites have indeed been detected in human urine as revealed by a metabolomics analysis [13]. In addition, there is recent evidence that INH can form adducts with NAD+, not only in bacteria, but also in the host (humans or mice) [22]. Therefore, mitochondrial energy homeostasis and redox balance may be altered; for example, hydrazine has been implicated in decreasing succinate dehydrogenase activity in hepatocytes as an early event [18]. These functional alterations in mitochondria may be without severe consequences in normal cells. However, we hypothesized that underlying abnormal conditions in mitochondria (genetic or acquired) would enhance the sensitivity of hepatocytes to INH and could therefore precipitate INH-mediated cell injury. The aim of this study was to investigate whether pharmacological or genetic inhibition of mitochondrial complex I, which is one of the most frequent sites of mitochondrial abnormalities, would trigger INH-induced hepatocyte injury in a murine model.

Materials and methods

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Hepatocytes were plated in 6- or 48-well plates (6.4  105 or 8.0  104 cells per well) coated with 50 mg/ml rat-tail collagen. Hepatocytes were allowed to attach for 3 h in a humidified atmosphere of 5% CO2, 95% air at 37 1C. Subsequently, hepatocytes were washed once and then incubated in the same medium. Exposure to drugs For all incubations with drugs, serum- and antibiotic-free culture medium was used. Isoniazid was dissolved in culture medium. DMSO was used as a solvent for lipophilic chemicals (final concentrations not exceeding 0.1%). To pharmacologically inhibit mitochondrial respiratory chain complex I or III, rotenone (3 mM), piericidin A (30 nM), or antimycin A (3 mM) was used at concentrations that by themselves did not affect cell viability or decrease energy levels. In some experiments, the cells were pretreated for 2 h with the mitochondria-targeted radical scavenger Mito-TEMPO (3 μM) or the antioxidant N-acetylcysteine (5 mM) prior to exposure to INH. Determination of cell injury Release of lactate dehydrogenase (LDH) into the extracellular medium was taken as an indicator of cytotoxicity. LDH activity was determined with the CytoTox-One Homogeneous Membrane Integrity Assay (Promega, Madison, WI) and expressed as percentage of activity present in the medium as compared to the total intra- and extracellular LDH. Isolation of mitochondria from liver and hepatocytes Mitochondria were isolated from whole mouse liver or heart or from cultured hepatocytes using a Mitochondria Isolation Kit (Pierce Biotechnology, Rockford, IL) and suspended in 2% Chaps/ TBS (25 mM Tris, 0.15 M NaCl; pH 7.2).

Chemicals Isoniazid, hydrazine, rotenone, and piericidin A were purchased from Sigma (St. Louis, MO). Animals and genotyping All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Connecticut. Young adult C57BL/6 J mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and used at age 10–16 weeks. The animals were acclimatized for at least 1 week and kept on a 14/10-h light/dark cycle under controlled environmental conditions. They received mouse chow (Teklad Global Rodent Diet; Harlan Laboratories, Boston, MA) and water ad libitum. Heterozygous Ndufs4 þ /  mutant knock-in mice were obtained (CAP, Auburn University, AL) [23], and a breeding colony was established over five generations before animals were used for experiments. Genotyping of the offspring was performed by tail biopsy prior to weaning as described [23].

Quantitative determination of cellular ATP concentrations and ATP biosynthesis rates in isolated mitochondria ATP content in cultured hepatocytes was determined with the Cell Titer-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). ATP biosynthesis rates were measured in isolated mouse hepatic mitochondria as described previously [25]. Briefly, the assay buffer contained 210 mM mannitol, 70 mM sucrose, 5 mM Hepes (pH 7.4), 5 mM KH2PO4, and 250 μM ADP. To energize mitochondria, 10 mM succinate plus 2 μM rotenone (to prevent electron backflow through complex I) was used. Isoniazid or hydrazine was added at the required concentration immediately before the assay. The reaction was started by the addition of mitochondria (200 μg protein/ml). The samples were incubated for 10 min at 37 1C, and the reaction was terminated by boiling the tubes at 100 1C for 3 min. The supernatants were then frozen at  80 1C until ATP analysis. Chemiluminescence was determined in white 96-well plates. The ATP content was calculated from a standard curve. Isoniazid or hydrazine did not interfere with the luciferin/luciferase reaction.

Hepatocyte isolation and culture Complex I activity measurement Hepatocytes were isolated from 25- to 30-g wild-type or Ndufs4 þ /  mutant male mice by collagenase perfusion through the inferior vena cava, using Percoll purification [24]. Hepatocytes were resuspended in Williams E medium (Life Technologies) containing 10% fetal calf serum, 2 mML-glutamine, 100 U/ ml penicillin, and 100 mg/ml streptomycin. Cell viability was greater than 95% as determined by trypan blue exclusion.

Mitochondria were suspended in 10 mM Hepes buffer (pH 7.4) containing 250 mM sucrose and 1 mM EDTA. After freeze-thawing (2  ), 50 μg protein/ml was added to the incubation buffer consisting of potassium phosphate (25 mM, pH 7.4), 5 mM MgCl2, and 2.5 mg/ml BSA, 32 μM coenzyme Q1, and 2 μg/ml antimycin A. To determine the rotenone-sensitive portion of complex I activity,

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20 μM rotenone (or DMSO) was added where appropriate. To start the reaction, 100 μM NADH was added, and the change in absorbance at 340 nm (due to oxidation of NADH) was monitored in 96-well microplates using a Safire 2 microplate reader (Tecan, Maennedorf, Switzerland).

Complex II activity measurement Complex II (succinate:ubiquinone oxidoreductase) activity was measured as the succinate-dependent reduction of the artificial electron acceptor, dichlorophenolindophenol (DCPIP) as described [26,27]. Mitochondria were isolated from Saccharomyces cerevisiae (strain D273-10B) [28], incubated in solubilization buffer (50 mM potassium phosphate, pH 7.4, 50 mM KCl, 0.5% (w/v) dodecyl β-D-maltoside) for 25 min at room temperature, and subjected to clarifying centrifugation to sediment nonsolubilized membranes. Supernatants containing complex II were divided into aliquots equaling 0.1 mg of mitochondrial protein. Prior to activity measurements, samples were preincubated with INH or hydrazine for 5 min at room temperature, and then incubated with 10 μM decylubiquinone for an additional 5 min. The reaction was initiated by the addition of sample to assay buffer (50 mM potassium phosphate, pH 7.5, 1 mM EDTA, 2 mM potassium cyanide, 40 μM DCPIP, and 20 mM K-succinate, pH 7.5). The extinction of DCPIP absorbance (ε600 ¼20.7 mM  1 cm  1) was monitored in 1-cm quartz cuvettes in an Amersham Biosciences Ultraspec 2100 pro UV/Vis spectrophotometer over the linear response range (first 40 s). To account for nonenzymatic changes in DCPIP absorbance, measurements were made in the absence of mitochondrial samples and used for background subtraction. Three independent measurements were made for each INH and hydrazine concentration and expressed as the percentage change in complex II activity relative to measurements in the absence of either agent.

Measurement of oxygen consumption rate (OCR) OCR was measured in cultured primary mouse hepatocytes with a XF24 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA) according to the manufacturer's instruction. Freshly isolated mouse hepatocytes were seeded in collagen-coated XF24 microplates at 2  104 cells/100 μl culture medium in each well and incubated in a 5% CO2 humidified atmosphere at 37 1C for 24 h. A XF24 sensor cartridge was placed in a 24-well calibration plate containing 1 ml/ well calibration buffer and left to hydrate at 37 1C. The following day, the culture medium in the wells was aspirated and 675 μl of unbuffered DMEM medium (pH 7.4) supplemented with glucose, glutamine, and sodium pyruvate was added. The cells were then maintained at 37 1C for 30 min to allow for temperature and pH equilibration. Isoniazid and 2-deoxy-D-glucose were directly prepared as 10X stock solutions in nonbuffered DMEM. Rotenone was prepared as 1000X stock solutions in DMSO and diluted to 10X with nonbuffered DMEM. Then, the pH of 2-deoxy-D-glucose in the medium was adjusted to 7.4 with 1 N NaOH and 75 μl of compound in the medium was preloaded into the reagent delivery port A of the XF24 sensor cartridge. The sensor cartridge and the calibration plate were loaded into the XF24 extracellular flux analyzer cartridge calibration. After calibration, the calibration plate was replaced with the cell plate. Three baseline rate measurements of OCR of the mouse hepatocytes were made using a 2-min mix, 3-min hold, and 2-min measure cycle. The compounds were then injected pneumatically by the XF24 analyzer into each well, and four measurements were made using the same conditions.

Assessment of mitochondrial transmembrane potential and mitochondrial superoxide generation Mitochondrial transmembrane potential (ΔΨm) was measured with tetramethylrhodamine methylester (TMRM, 100 nM, Molecular Probes/Invitrogen), a cationic dye that selectively accumulates within the mitochondrial matrix. Changes in fluorescence were recorded with an Olympus Bx51 fluorescence microscope. Mitochondrial superoxide was determined with the cell-permeable fluorogenic probe, Mito-HE (MitoSOX Red; Invitrogen) as described [29]. The drug-pretreated cells were loaded with MitoSox (25 nM) for 10 min at 37 1C and washed with phenol red-free Williams E medium containing 2 mML-glutamine, and the mitochondrial superoxidederived MitoSox fluorescence was determined at 396 nm (excitation) and 580 nm (emission). Blue native gel electrophoresis Native polyacrylamide gel electrophoresis (PAGE) was carried out as described [23] using Mini-PROTEAN precast gels (Bio-Rad, Hercules, CA). Briefly, 120 μg mitochondrial protein was suspended in 2% Chaps/TBS, mixed with an equal volume of Native Sample Buffer (Bio-Rad) followed by incubation on ice for 30 min, and loaded into individual lanes of 7.5% Mini-PROTEAN TGX Gel. The gels were electrophoresed at 150 V (constant) for 1 h. SDS-PAGE and Western blotting Individual lanes of the native gel were cut into strips and equilibrated separately in 1% SDS/2.5% β-ME/1X TGS buffer at room temperature for 10 min. Each strip was loaded on one Any kD IPG Mini-PROTEAN TGX Gel. The wells of the IPG gels were filled with 0.5% low-melt ReadyPrep Overlay Agarose (Bio-Rad) to ensure good contact between the strip and gel. Precision Plus Protein Dual Color Standards (Bio-Rad) were loaded at the bottom side of a strip. Each gel was electrophoresed at 20 mA constant for approximately 1 h until the molecular standards dye front reached the bottom black line of a gel unit. Next, proteins were transferred from the acrylamide gels to nitrocellulose membranes for Western blotting. The membranes were blocked in 5% nonfat dry milk in 0.2% Tween 20/PBS at room temperature for 1 h. Anti-Ndufs4 antibody (2C7CD4AG3, Abcam, Cambridge, MA) was used at 1:1000 dilution and incubated with the membranes at 4 1C overnight. Goat anti-mouse IgG secondary antibody conjugated with HRP was used at 1:10,000 dilution at room temperature for 1 h. Enhanced chemiluminescence (ECL) detection (Pierce) was used for imaging. Statistical analysis All data were expressed as mean 7SD or SE as indicated. If there was normal distribution, a standard analysis of variance was used, followed by Dunnett's test for multiple comparisons versus the control group. When normality failed, a Kruskal-Wallis oneway analysis of variance on ranks was used followed by Dunn's test for multiple comparison versus the control group. A P value o0.05 was considered significant. Results Isoniazid impairs hepatocellular energy production without causing hepatocellular injury To study the effects of INH on hepatocellular energy production, we first used primary mouse hepatocytes. The parent INH

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(up to 3000 μM) was not cytotoxic and did not significantly increase LDH release into the extracellular medium as compared to solvent controls over an exposure period of 48 h (data not shown). These data confirm previous studies [30,31] and indicate that, in murine hepatocytes, INH alone does not cause acute cell injury over a wide concentration range. However, because previous studies had suggested that INH may affect mitochondrial function (by mechanisms that were not defined) [17,19], we next determined the effects of INH on mitochondrial respiration and energy production. We found that acute exposure of hepatocytes to INH inhibited the OCR in a concentrationdependent manner, causing an approximately 25% decrease in the OCR at 3000 μM (Fig. 1A). Although the effect was mild and became significant at high concentrations only, these data identify the mitochondrion as a potential target. In line with these results, we also found that INH decreased the hepatocellular net ATP content, with an approximately 20% loss of ATP steady-state levels at 24 h exposure to>300 μM INH (Fig. 1B). Although such a moderate decrease in energy levels is not acutely cytotoxic to hepatocytes, it demonstrates that INH may cause either increased ATP utilization or inhibition of its biosynthesis. To determine the mode of interference with energy homeostasis, we next determined the rate of ATP biosynthesis, using succinateenergized mitochondria isolated from mouse liver. We found that INH (up to a concentration of 3000 μM) did not inhibit ATP biosynthesis. In contrast, exposure of isolated mitochondria to hydrazine caused a concentration-dependent decrease in succinate-driven ATP synthesis (Fig. 2A), suggesting that hydrazine, rather than the parent INH, may interfere with energy homeostasis. To further explore this hypothesis, we utilized isolated mitochondria from S. cerevisiae (yeast cells do not have complex I that would potentially interfere with the assay). We found that hydrazine, but not the parent INH, concentration dependently inhibited complex II activity (Fig. 2B). Complex II activity was measured in detergent-solubilized samples to avoid nonspecific effects of the inhibitor on the membrane bilayer; therefore, the data suggest that hydrazine exerted a direct effect on the protein complex. These data confirm and extend earlier studies demonstrating that hydrazine has the potential to inhibit succinate dehydrogenase activity in cells as an early event [13,18]. While these earlier studies had suggested that the inhibition of mitochondrial energy production could be related to the reaction of hydrazine with keto acids that are involved in electron transport and the TCA cycle, we found that the addition of excess quantities of substrate (succinate) did not attenuate the inhibitory

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effect of hydrazine on complex II (data not shown), thus minimizing the possibility of a nonspecific effect on substrate or cosubstrate depletion.

Fig. 2. Effect of isoniazid (INH) or hydrazine (HzN) on rates of ATP biosynthesis or mitochondrial complex II activity. (A) Isolated mouse hepatic mitochondria were energized with succinate (in the presence of rotenone, to prevent backflow of electrons through complex I), and the ADP-stimulated biosynthesis rates were determined over 10 min (linear phase). Data are mean 7SD of triplicate incubations using two independent mitochondrial preparations (n¼ 8). * Po 0.05 vs solvent control. (B) Solubilized complex II isolated from S. cerevisiae mitochondria were energized with succinate and exposed to INH or HzN. Data are mean7 SE of three independent mitochondrial preparations. * P o0.05 vs solvent control (100%).

Fig. 1. Effect of isoniazid (INH) on mitochondrial respiration and ATP homeostasis in primary cultures of mouse hepatocytes. (A) Oxygen consumption rates were determined in a XF24 extracellular flux analyzer (Seahorse). After equilibration of the system, INH was added at different concentrations (arrow). Data are mean 7 SD of 12 wells including three independent cell preparations. * Po 0.05 vs solvent control. (B) Time course of intracellular ATP concentrations following exposure of hepatocytes to different concentrations of INH. Data are mean 7SD of quadruplicate wells using two independent hepatocyte preparations (n ¼8). * P o0.05 vs solvent control.

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Underlying complex I dysfunction triggers INH-mediated lethal cell injury in hepatocytes In normal mitochondria, the moderate decrease in OXPHOS caused by hydrazine-induced complex II inhibition can likely be compensated for by the normal activity of complex I and has little consequences for the cell's energy metabolism. However, we hypothesized that under conditions of compromised complex I function, INH may cause more severe mitochondrial dysfunction and amplify the toxic response. To test this hypothesis, we used both a pharmacological and a genetic approach to alter complex I function. First, we used nontoxic concentrations of ROT, which is a potent inhibitor of the ubiquinone binding site of complex I. Control experiments revealed that ROT indeed inhibited complex I activity in mitochondria isolated from hepatocyte cultures (by 60% at 3 μM) (Fig. 3A) which, consequently, led to a dissipation of the mitochondrial inner transmembrane potential (ΔΨm) (Fig. 3B). In contrast, INH, even at high concentrations (3000 μM), neither impaired complex I activity nor decreased the ΔΨm. However, we found that combined exposure of hepatocytes to INH and ROT (each at nontoxic concentrations) caused massive acute cell injury as determined by greatly increased LDH release and extensive ATP depletion (Fig. 4A and B). Pretreatment (for 30 min) with the pancytochrome P450 (CYP) inhibitor 1-aminobenzotriazole (100 μM) did not alter the extent of combined INH/ROT-induced cell injury (data not shown), suggesting that it was not a CYP-mediated reactive intermediate that was involved in the toxic response. However, the acyl amidase inhibitor, bis-p-nitrophenol phosphate (BNPP) protected cells from the combined ROT/INH-induced cytotoxicity in a concentration-dependent manner (Fig. 4C), suggesting that the toxic species might be hydrazine (or acetylhydrazine) rather than the parent INH. Furthermore, these data indicate that it is not ROT that kills the cells, but an amidase-mediated hydrolytic cleavage product of INH. If this cleavage product

(hydrazine) was the toxic species, then one would expect that the combination of hydrazine and ROT would be similarly toxic to INH/ROT, but that BNPP would not afford protection against cell injury. Indeed, we found that hydrazine/ROT cotreatment caused a rapid loss of the cellular ATP content resulting in lethal cell injury that was insensitive to BNPP (Fig. 5). To minimize the possibility that a ROT-specific effect not related to complex I inhibition was responsible for the observed potentiating effects on INH-associated cell injury, we used piericidin A, which is another complex I inhibitor that features the same mechanism of binding to complex I as ROT [32]. Piericidin A at concentrations that were nontoxic if given alone (30 nM) equally potentiated the effects of INH, massively increasing LDH release and dramatically decreasing net ATP levels, thus confirming the crucial role of intact complex I function in INH tolerability (Fig. 6A and B). Next, we sought to identify the major mechanisms underlying the potentiating effects of ROT on INH toxicity. Because one of the consequences of complex I inhibition is an increased release of superoxide anion [32], we first studied the role of increased mitochondrial oxidant stress on INH toxicity. Control experiments revealed that, in intact hepatocytes, both ROT (3 μM) and piericidin A (30 nM) at these nontoxic concentrations only moderately enhanced the fluorescence of mitochondria-targeted hydroethidine (MitoSox Red), which is indicative of (but not specific for) hydroxyethidium generated by reaction of the probe with mitochondrial superoxide (Fig. 7A). In contrast, INH (1000 μM) alone or combined treatment with ROT/INH or PA/INH significantly increased MitoSox Red-derived fluorescence at 3 and 6 h (later time points were not considered due to the increasing cytotoxicity of the combined treatment). The reason why INH alone acutely causes increases in mitochondrial superoxide is not clear, but it has been suggested that not only hydrazine but also hydrazides (e.g., INH) can spontaneously reduce O2 to superoxide by

Fig. 3. Effect of isoniazid (INH) or rotenone (ROT) on complex I activity and the mitochondrial inner transmembrane potential (ΔΨm). (A) Isolated mouse hepatic mitochondria were used to determine complex I activity in the presence or absence of INH or ROT. Data are mean 7SD of three independent mitochondrial preparations using duplicate determinations. * Po 0.05 vs solvent control. (B) Cells were loaded with TMRM, washed, and exposed to INH or ROT for 2 h. The photomicrographs show results from one cell preparation typical of 3 independent experiments. The bright (punctate) fluorescence indicates the mitochondrial accumulation of TMRM.

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Fig. 4. Potentiating effects of nontoxic concentrations of rotenone (ROT) on INH-induced cell injury (A) and intracellular ATP content (B). Hepatocytes were coexposed to 1000 μM INH and/or 3 μM ROT for the indicated time periods. INH alone did not significantly alter LDH release or ATP content as compared to solvent controls (not shown). Data are mean 7 SD of quadruplicate wells using two independent cell preparations. * P o0.05 vs ROT alone. (C) Protective effects of the acyl amidase inhibitor, bis-pnitrophenol phosphate (BNPP), on combined INH/ROT-induced cell injury. Data are mean 7 SD of triplicate wells using two independent cell preparations. * P o0.05 vs INH/ ROT alone.

Fig. 5. Potentiating effects of nontoxic concentrations of rotenone (ROT) on hydrazine (HzN)-induced cellular ATP content. Hepatocytes were exposed to HzN (1 mM) and/or ROT (3 μM) for the indicated time periods (A), or to the indicated concentrations of HzN alone or in combination with ROT (3 μM) for 24 h in the presence or absence of bis-pnitrophenol phosphate (BNPP, 3 mM) (B). Data are mean7 SD of triplicate wells using two independent cell preparations. * P o0.05 vs HzN alone.

autooxidation [33]. Thus, if increased superoxide were the major factor in triggering INH toxicity, it would be expected that cellpermeable antioxidants would attenuate the degree of INH/ROT toxicity. We therefore preloaded hepatocytes with the mitochondria-targeting superoxide scavenger, Mito-TEMPO or with the antioxidant N-acetylcysteine (NAC). However, we found that neither Mito-TEMPO (up to 3 μM) nor NAC (up to 5 mM) attenuated combined INH/ROT-induced cell injury (Fig. 7B). Taken together, these data suggest that effects other than increased mitochondrial

oxidant stress signaling may be implicated in the potentiating effects on INH toxicity in the presence of complex I inhibitors. Finally, to further explore the mechanisms of the combined exposure of hepatocytes to ROT and INH-induced toxicity, we measured mitochondrial respiration in cultured mouse hepatocytes. As shown in Fig. 8, we found that ROT treatment alone resulted in an approximately 60% inhibition of cellular respiration. This effect was enhanced in the presence of INH. Collectively, these data suggest that extensive inhibition of OXPHOS

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Fig. 6. Potentiating effects of piericidin A (PA) on INH-induced cell injury (A) and intracellular ATP content (B). Hepatocytes were coexposed to 1000 μM INH and/or 30 nM PA for 24 h. INH alone did not alter LDH release or ATP content as compared to solvent controls ( P > 0.05; not shown). Data are mean 7 SD of quadruplicate wells using two independent cell preparations. * Po 0.05 vs PA alone.

Fig. 7. Time course of drug-induced hepatocellular superoxide steady-state levels and effects of antioxidants on INH/ROT-induced cell injury. (A) Hepatocytes were exposed to INH (1000 μM), ROT (3 μM), PA (30 nM), or combinations thereof as indicated. The cells were washed and loaded with the fluorogenic probe, dihydroethidine coupled to a mitochondria-targeted moiety (MitoSox Red). Fluorescence was determined in situ with a plate reader. Data were corrected for background fluorescence and are mean 7 SD of quadruplicate wells using two independent cell preparations. * Po 0.05 vs solvent. (B) Hepatocytes were exposed to combined INH/ROT in the presence or absence of the mitochondrially targeted radical scavenger Mito-TEMPO or the antioxidant N-acetylcysteine (NAC). Neither Mito-TEMPO nor NAC alone increased LDH release as compared to solvent controls ( P > 0.05). Data are mean7 SD of quadruplicate wells using two independent cell preparations.

by complex I dysfunction, combined with the INH-mediated inhibition of complex II activity, might lead to an energy crisis resulting in acute cell loss. Hepatocytes from Ndufs4 þ /  mice are resistant to INH In addition to the pharmacologic model, we also used a genetic model to assess the effects of altered complex I function. We used a mutant mouse that was generated by knocking-in a truncated form of Ndufs4, which is a nuclear-encoded subunit of complex I [23], and which generally has been considered essential for the

Fig. 8. Effect of combined rotenone (ROT) and isoniazid (INH) exposure on mitochondrial respiration in primary cultures of mouse hepatocytes. Oxygen consumption rates were determined in a XF24 extracellular flux analyzer (Seahorse). After equilibration of the system, compounds were added at the indicated concentrations (arrow). Data are mean7 SD of 12 wells including three independent cell preparations. *P o 0.05 vs solvent control. 2-DG, 2-deoxyglucose (inhibitor of glycolysis).

assembly of the large protein complex. Because homozygous mutant mice are not viable, we used heterozygous Ndufs4 þ /  mice; these mice have been shown to exhibit a 25–30% decrease in complex I activity in heart, brain, and skeletal muscle, concomitant with a 2- to 3-fold increase in lactate levels in these organs [23]. Because no quantitative information on the protein expression levels of wild-type Ndufs4 in the liver was available, we first performed a blue native polyacrylamide gel electrophoresis (BNPAGE) analysis (two-dimensional) of hepatic mitochondrial proteins to detect the protein. Western immunoblotting revealed that the native Ndufs4 subunit was present in the heterozygous mice at a level of approximately 80% of that in wild-type mice (Fig. 9A). However, we found that hepatic mitochondrial complex I activity was not significantly different between the two genotypes (Fig. 9B), suggesting that there is a threshold effect for complex I in mouse liver. In line with this, hepatocytes isolated from Ndufs4 þ /  mice were not more sensitive to INH than wild-type mice and did not exhibit apparent signs of cell injury up to an INH concentration of 3000 μM (data not shown). Although basal levels of hepatic lactate were increased in Ndufs4 þ /  mice by 66% as

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Fig. 9. Characterization of hepatic mitochondrial function in Ndufs4 þ /  and wild-type mice. (A) Western immunoblot following blue native gel electrophoresis showing the Ndufs4 subunit (circled). The arrow indicates the cross-reactive band used to normalize for equal loading [53]. The blot shows one experiment typical of three. (B) Complex I activity in isolated hepatic mitochondria from untreated mutant or wild-type mice. (C) Hepatocellular lactate concentrations in mutant or wild-type mice. Data are mean 7 SD of triplicate determinations using 3 mice each.

Fig. 10. Effects of long-term treatment with isoniazid (INH) or hydrazine (HzN) on serum markers of hepatocellular injury in mice. Groups of 4 mice each were treated with 100 mg/kg/day INH or 50 or 100 mg/kg/day HzN by oral gavage for 4 weeks. Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) activities were determined in sera. Data are mean 7SD of 4 mice/treatment group.

compared to wild-type mice (Fig. 9C), the data suggest that minor changes in the abundance of wild-type complex I are not critical for potentiating INH toxicity as long as overall complex I function is not compromised in hepatocytes.

confirm and extend earlier studies and suggest that INH is not hepatotoxic to mice; instead, hydrazine, a major metabolite of INH, may contribute to the hepatic toxicity of INH.

Hydrazine, but not INH, is hepatotoxic in normal mice

Discussion

To mimic the clinical situation, where delayed onset rather than acute hepatic injury is typically observed in DILI patients receiving INH, we performed a subchronic mouse study. Healthy male C57BL/6J mice received daily oral treatment (gavage) with 100 mg/kg INH, for 4 weeks, and the hepatic effects of INH were assessed. As described by others [34], we found that the currently used markers of hepatocellular injury (serum ALT and AST) were not significantly increased as compared to vehicle controls (Fig. 10A); in fact, ALT was even decreased, likely due to an interaction of the drug with pyridoxal phosphate, a cofactor for ALT and AST [34,35]. Histopathological evaluation also did not reveal any signs of overt hepatic injury (data not shown). In contrast, daily oral treatment of mice with hydrazine (50 or 100 mg/kg) for 4 weeks caused increased serum ALT and AST activity in some (but not all) mice (Fig. 10B). The reason for this interindividual variability is not known. Histopathological analysis revealed hepatocyte swelling and multifocal necrosis in one of four mice exposed to the high dose of hydrazine. These data

Although INH among all drugs causing DILI has a relatively high incidence of hepatocellular injury in susceptible patients, conventional models involving normal hepatocytes or healthy laboratory animals have proven unsuccessful in reproducing the clinical picture of liver injury, even when high doses of INH were used. Here, we demonstrate that altering mitochondrial function with small nontoxic doses of chemical inhibitors of complex I can trigger massive hepatocellular energy depletion and cell injury by an otherwise nontoxic dose of INH. The mechanisms of potentiation of INH-associated hepatocyte injury by the complex I inhibitors are not fully understood. Although rotenone, piericidin A, and other inhibitors cause increased intramitochondrial release of superoxide due to a partially diverted electron flow from the electron transport chain to molecular oxygen [36], this increased oxidant stress and its downstream signaling are unlikely to be the major cause of cell death. This conclusion was based on the observations that two mitochondria-targeted radical scavengers and antioxidants, Mito-

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TEMPO and NAC, which normally are effective in protecting complex I-associated increased superoxide formation [37,38], did not attenuate the cell injury induced by combined ROT/INH treatment. Furthermore, antimycin A, a complex III inhibitor, which also causes increased release of superoxide, did not potentiate INH-induced cell injury (unpublished data). Instead, it is likely that the lethal cell injury was caused by massive ATP depletion, dropping to levels below a critical threshold. While cells with partially compromised complex I function (due to chemical or genetic inhibitors) still have complex II to transfer electrons from substrates to ubiquinone, this alternative electron supply may be severely limited if cells are exposed to INH (or its hydrolytic metabolite, hydrazine) which impairs complex II function. Indeed, we have found that the precipitation of hepatocyte injury was dependent on acyl amidase-mediated hydrolytic cleavage of the parent INH to hydrazine (or acetyl hydrazine), because inhibition of acyl amidase largely prevented the development of cell injury. This suggests that hydrazine (or acetyl hydrazine), rather than the parent INH, may be the cell-damaging species in this model. Indeed, hydrazine hepatotoxicity has been well documented in rats, though less well in mice. For example, hydrazine can cause decreases in hepatocellular ATP levels by interfering with ATP production [39,40]. Metabonomics analyses have revealed that hydrazine induces a number of metabolic perturbations based on markers of mitochondrial dysfunction. These include increases in lactate levels, increased citrulline concentrations, and decreases in succinate and other TCA cycle intermediates [41,42]. High concentrations (mM range) of hydrazine have also been shown to induce cytotoxicity in freshly isolated rat hepatocytes in suspension [20] or short-term culture [21]. Exposure of rat hepatocytes to hydrazine caused a timedependent decrease in succinate dehydrogenase activity that clearly preceded the collapse of cell viability [18]. Here we demonstrate a direct acute effect of hydrazine on complex II activity on solubilized complex II isolated from yeast mitochondria. The concentrations of hydrazine required to obtain an inhibitory effect on complex II are high; however, hydrazine appears to be behaving in this regard like another well-characterized complex II inhibitor, 3-nitropropionic acid (3-NP), which binds to the active site of complex II SdhA flavoprotein unit [43]. The mode of inhibition by 3-NP is not competitive inhibition of succinate; rather, it is a suicide inactivator of the complex, exerting its effects slowly over time, featuring a high Ki ( 0.2 mM), not unlike what we see with hydrazine. The major reason why we chose to investigate complex I dysfunction, as opposed to other mitochondrial abnormalities, as a potential underlying determinant of INH hepatotoxicity is its known susceptibility to genetic changes impacting its function. In fact, complex I deficiency is the most frequently diagnosed form of disturbances in the OXPHOS system [44]. The clinical presentations of complex I deficiency exhibit great variability [44]; while for many known mutations in complex I the clinical consequences are obvious and severe, there is increasing evidence that there may be phenotypically silent mutations that would only become relevant if the cell is challenged with an additional stress (e.g., drugs targeting mitochondria). Among all five complexes of the electron transport chain, complex I is the most vulnerable [45]. Because complex I has the largest number of subunits (at least 45), seven of which are encoded by mtDNA, it is also statistically more likely to be affected by mutations than other ETC complexes. For example, the most frequent ones include the 4977-bp “common deletion” and mutations in tRNA or rRNA genes [45]. The most severe mutations are probably eliminated out of the female germline during oogenesis, but many mutations are only mild to moderately deleterious [46]. In addition, the majority of complex I mutations are in the nuclear encoded genes, not in the mtDNA encoded genes [44]. Finally, apart from these genetic changes, there are age-related acquired changes that

may alter the function of complex I. For example, not only does complex I function decrease with age, but the inhibitory effects of rotenone on complex I respiration become more pronounced with age [47]. This increased vulnerability of complex I during aging should be considered in view of the observed striking age-dependent susceptibility to INH in patients. In this study, rotenone was selected because it is a widely used pharmacological tool with a well-defined mechanism of interaction with complex I [32]. Thus, chemical inhibition of complex I was used as an alternative to using a genetic approach, and not to model potential environmental exposure to this chemical in humans. Although rotenone has been used as a pesticide in the management of fish populations, it is unlikely that environmental exposure to rotenoids is a significant susceptibility factor to DILI, mostly because of rotenone's short half-life in the environment, its limited use, and its low absolute bioavailability [48]. Although identifying a hazard, the cellular model described in this study does not exactly mirror clinical DILI with respect to three key elements. First, the INH concentrations in this and other studies are high and clearly greater than the peak plasma concentrations observed in patients following INH therapy. Typically, in patients, the Cmax for INH can attain 50–100 μM [49], but the portal concentrations and the drug concentrations in the hepatic sinusoids may be considerably higher. Similarly, the hydrazine concentrations used were higher than those generated in situ and measured in the plasma of patients with INH hepatotoxicity [50]. Second, the toxic response induced in this experimental study reflects an acute lethal cell injury, whereas one of the clinical hallmarks of INH-induced DILI is the delayed onset of the disease, typically highest after several months of long-term therapy [5,7]. Third, INH is usually combined with other antituberculosis drugs, including rifampicin; therefore, the observed clinical hepatotoxicity could be mediated, at least in part, by the comedication. For example, a recent study with PXR-humanized mice has revealed that rifampicin causes hepatic accumulation of protoporphyrin IX as a direct consequence of PXR activation [51]. Nevertheless, our model identifies mitochondria as a target of INH and hydrazine toxicity, and it is compatible with the concept of lowlevel exposure for an extended period of time. It is possible that gradual mitochondrial stress brought about by INH and/or hydrazine, if superimposed on underlying genetic dysfunction of electron transport, will gradually damage mtDNA and increase the extent of heteroplasmy in a hepatocyte containing a mixture of wild-type and damaged mitochondria, until a threshold is reached that results in cell injury (or organ damage) [52]. Thus, our model, although demonstrating an acute hazard, is still compatible with gradual damage of the mitochondriome in hepatocytes. It would also explain the striking positive correlation of the incidence of INH-associated DILI with increasing age, as mitochondrial oxidant stress-related mtDNA mutations are increasing as a function of time. Because currently there are no validated mouse models of hepatic complex I activity available, it is difficult to translate the results obtained from this in vitro study into an animal model. The Ndufs4 þ /  knock-in model [23] did not exhibit decreased hepatic complex I activity, despite moderately decreased expression of the Ndufs4 protein subunit. Accordingly, treatment of these mutant mice with INH, or exposure of hepatocytes isolated from these mice to INH, did not result in enhanced sensitivity as the energy homeostasis in the liver apparently was not compromised. In conclusion, we have demonstrated that nontoxic concentrations of mitochondrial complex I inhibitors can precipitate and greatly potentiate the toxicity of otherwise harmless concentrations of INH in hepatocytes, and that this is likely mediated through its acylamidase-mediated release of the hydrazine moiety, which inhibits complex II. Thus, this is a side-by-side influence on both complexes. Our model is compatible with the concept that underlying abnormalities in mitochondrial function, although

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clinically silent, may be a determinant of susceptibility to DILI [52], including INH-associated liver injury.

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