Mitochondrial protection by the JNK inhibitor leflunomide rescues mice from acetaminophen-induced liver injury


  • Potential conflict of interest: Nothing to report.


Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses but which can precipitate liver injury at high doses. We have previously found that the antirheumatic drug leflunomide is a potent inhibitor of APAP toxicity in cultured human hepatocytes, protecting them from mitochondria-mediated cell death by inhibiting the mitochondrial permeability transition. The purpose of this study was to explore whether leflunomide protects against APAP hepatotoxicity in vivo and to define the molecular pathways of cytoprotection. Male C57BL/6 mice were treated with a hepatotoxic dose of APAP (750 mg/kg, ip) followed by a single injection of leflunomide (30 mg/kg, ip). Leflunomide (4 hours after APAP dose) afforded significant protection from liver necrosis as assessed by serum ALT activity and histopathology after 8 and 24 hours. The mechanism of protection by leflunomide was not through inhibition of cytochrome P450 (CYP)–catalyzed APAP bioactivation or an apparent suppression of the innate immune system. Instead, leflunomide inhibited APAP-induced activation (phosphorylation) of c-jun NH2-terminal protein kinase (JNK), thus preventing downstream Bcl-2 and Bcl-XL inactivation and protecting from mitochondrial permeabilization and cytochrome c release. Furthermore, leflunomide inhibited the APAP-mediated increased expression of inducible nitric oxide synthase and prevented the formation of peroxynitrite, as judged from the absence of hepatic nitrotyrosine adducts. Even when given 8 hours after APAP dose, leflunomide still protected from massive liver necrosis. Conclusion: Leflunomide afforded protection against APAP-induced hepatotoxicity in mice through inhibition of JNK-mediated activation of mitochondrial permeabilization. (HEPATOLOGY 2007.)

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses. However, when taken at high doses or, rarely in particularly susceptible people at therapeutic doses, APAP can precipitate severe liver injury that can develop into fulminant liver failure.1 The clinical significance of this adverse effect is underscored by APAP being, among all drugs, the single major cause of drug-induced hepatotoxicity in the United States and the United Kingdom.2 The mechanisms underlying APAP-induced liver injury have been studied for several decades, and excellent recent reviews have summarized the cellular and molecular pathways of this toxic response.3–5 Although the initial steps in the sequence of events leading to hepatocyte necrosis (bioactivation of APAP and glutathione depletion) have been well known for many years, the more distal events (signaling pathways that lead to the precipitation of cell death) are less clear. However, recently, the mitochondrial permeability transition (mPT) has been identified as a pivotal mechanism mediating APAP-induced cell death.6, 7 According to this concept, a combination of mitochondrial oxidant stress, increased Ca2+ levels, and other factors may favor the opening of a multiprotein megapore that spans the inner and outer mitochondrial membranes, resulting in the collapse of the transmembrane potential and then osmotic swelling, ultimately causing rupture of the outer mitochondrial membrane and release of cytochrome c and other proapoptotic factors into the cytosol. Among these death proteins are endonuclease G and AIF, which translocate into the nucleus and induce DNA damage.5 Because of the low ATP levels and the highly prooxidant environment in mitochondria, the mode of cell death in mouse liver is caspase-independent oncotic necrosis rather than apoptosis or secondary necrosis.8 Furthermore, it has recently been demonstrated that c-Jun-NH2-terminal protein kinase 2 (JNK2) plays a critical role in mediating APAP hepatotoxicity in mice.9 However, how these processes are related has remained speculative so far.

The treatment of choice after APAP overdose is currently limited to the use of N-acetylcysteine (NAC).10–12 This is based on the mechanistic insight that a small but relevant portion of APAP may be bioactivated by CYP2E1, CYP3A4, and CYP1A2 to reactive N-acetyl-p-benzoquinoneimine (NAPQI), an electrophilic intermediate that rapidly depletes the hepatic GSH pool. This in turn causes oxidant stress that may trigger signaling pathways through mitochondrial toxicity, ultimately leading to lethal cell injury. NAC, a precursor of GSH (cysteine donor) and a nucleophilic antioxidant itself, can effectively antagonize the oxidant stress posed by NAPQI. However, because bioactivation of APAP to NAPQI and the subsequent oxidant stress caused by rapid GSH depletion is an early step in the pathogenesis of APAP-induced liver injury, the antidote has to be given relatively early, before a significant increase in ALT occurs.13 Therefore, the need for alternative, safe, efficacious, and more widely applicable antidotes is warranted.

We recently identified leflunomide as a potent cytoprotective agent against APAP toxicity in immortalized human hepatocytes (HC-04 cells).14 Leflunomide is a disease-modifying antirheumatic drug (DMARD) that inhibits T cell proliferation. Because of the drug's inhibitory effects on T cells, leflunomide was subsequently shown to protect from T cell-mediated liver injury in animal models.15 However, our recent studies with hepatocytes have provided evidence that leflunomide also protects from non–T cell-mediated oxidative cell injury. The mechanism of cytoprotection against APAP toxicity in human hepatocytes is an inhibitory step distal to the bioactivation of APAP but proximal to mitochondrial release of cytochrome c.14 We have demonstrated that leflunomide interferes with the signaling pathways involved in the mitochondrial release of death proteins that trigger lethal cell injury, in particular by inhibition of JNK activation. These in vitro data suggest leflunomide could be administered at a relatively late point. However, no in vivo studies of the potential cytoprotective effects of leflunomide in animal models have been reported so far.

The aims of the present work were to explore the protective effects of leflunomide against APAP-induced hepatotoxicity in vivo and to define how long after APAP administration leflunomide still affords mice with protection. We found that leflunomide, a marketed drug used by humans that has a well-defined safety profile, inhibits the JNK-mediated mitochondrial pathway and affords mice with significant protection against APAP-induced liver damage.


APAP, acetaminophen (paracetamol); iNOS, inducible nitric oxide synthase; JNK, c-jun NH2-terminal protein kinase; mPT, mitochondrial permeability transition; NAC, N-acetylcysteine; NAPQI, N-acetyl-p-benzoquinone imine; P-JNK, phosphorylated JNK.

Materials and Methods


All protocols involving animals were in compliance with the Institutional Animal Care and Use Committee and in accordance with the guidelines of the National Advisory Committee for Laboratory Animal Care and Research. Male C57BL/6 mice (Centre for Animal Resources, Lim Chu Kang, Singapore) were kept under controlled environmental conditions (22°C ± 2°C, 75% ± 5% relative humidity, 12-hour dark/12-hour light cycle) and had free access to standard rodent chow (Specialty Feeds Pte Ltd., Glen Forrest, Western Australia) and water. All animals were acclimatized to the housing conditions for at least 1 week and were 12 weeks old at the start of drug treatment.

Drug Administration and Experimental Design.

Acetaminophen (Sigma) was dissolved in Solutol HS-15 (15% in phosphate buffered saline), a nontoxic solvent used for parenteral administration of water-insoluble compounds. Solutol HS-15 (BASF, Germany) is composed of polyglycol monoesters and diesters of 12-hydroxystearic acid and 30% free polyethylene glycol. Fed mice were injected intraperitoneally with APAP or vehicle (10 μl/g) at 9 A.M. Leflunomide (Sigma) was dissolved in solutol HS-15 (7.5%) and injected intraperitoneally (30 mg/kg) at various times after administration of APAP. The dose of leflunomide was not hepatotoxic and was chosen according to previous reports.15, 16 Controls received vehicle alone (10 μl/g). Some mice received the JNK2 inhibitor SP600125 (30 mg/kg, ip, in solutol HS-15). At various time points, the mice were anesthetized with pentobarbital (60 mg/kg). Blood was collected by cardiac puncture, and serum was prepared. The liver was quickly excised and weighed. While one portion was used for histopathology, other pieces were snap-frozen and kept at −80°C or minced and homogenized on ice and immediately used to prepare mitochondrial and cytosolic fractions.

Isolation of Hepatic Mitochondria.

Liver mitochondria were isolated according to standard methods with added protease inhibitor cocktail (Roche Molecular Biochemicals, Basel, Switzerland). Protein content was determined with the Bradford reaction using albumin as the reference protein. Mitochondria were immediately snap-frozen in liquid nitrogen and kept at −80°C until used for analysis, except for measurements of mitochondrial ATP production, for which freshly isolated mitochondria were used.

Serum ALT and Liver Histopathology.

Serum activity of ALT was determined photometrically using a test kit (Catachem, Bridgeport, CT). For histopathological analysis, small pieces of liver were fixed in 10% phosphate-buffered formalin. The fixed tissues were subsequently processed with an automatic tissue processor (Leica TP 1020, Germany) and embedded in paraffin blocks. Tissue sections (5 μm) were stained with hematoxylin-eosin (HE) and analyzed by light microscopy.

Determination of Glutathione.

GSH was determined in whole-liver homogenates using monochlorobimane as fluorogen as previously described.17

ATP Biosynthesis Measurement in Isolated Mitochondria.

The rate of ATP biosynthesis was measured in ex vivo–treated isolated mitochondria as previously described,17 except that 50 mM succinate (complex II substrate) and 10 μM rotenone (to inhibit backflow of electrons through complex I) were used instead of glutamate/malate.

Nitrotyrosine Immunohistochemistry.

Deparaffinized histological liver sections were digested with proteinase K at 37°C. Next, tissues were incubated with 3% hydrogen peroxide followed by serum and avidin/biotin (R&D Systems, Minneapolis, MN) to block nonspecific binding. The slides were washed with PBS, incubated with mouse antinitrotyrosine antibody (1:200; Cayman Chemical, Ann Arbor, MI) for 1 hour, washed 3× in PBS, and incubated with the secondary antibody (biotinylated antimouse antibody) and streptavidin-HRP conjugate for 30 minutes. 3,3′-Diaminobenzidine (2.5%) was applied to each section until color developed. Then the slides were washed with deionized water and counterstained with hematoxylin for 30 seconds.

Protein Immunoblotting.

Equal amounts of denatured whole-liver homogenate or mitochondrial protein were loaded per lane, separated on a 10%–15% SDS-PAGE gel under reducing conditions, and subsequently transferred to nitrocellulose membranes. The membranes were blocked in 5% nonfat dry milk prepared with 0.05% PBS-Tween 20 for 1 hour at 25°C. Anti-P-JNK (1:1,000; Calbiochem, San Diego, CA), anti-JNK (1:1,000; Biosource, Camarillo, CA), anti-MKK4 (1:1,000; Santa Cruz Biotech, Santa Cruz, CA), anti-P-MKK4 (1:1,000; Calbiochem), anti-Bcl-2 (1:1,000; Calbiochem), anti-P-Bcl-2 (1:1,000; Calbiochem, San Diego, CA), anti-Bcl-X (1:1,000; Chemicon), anti-P-Bcl-XL (1:1,000; Chemicon), anti-iNOS (1:500; BD Bioscience, San Jose, CA), and anti-murine cytochrome c (1:1,000; Santa Cruz Biotech) were used as primary antibodies. The protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ) after incubation with HRP-conjugated secondary antibody (1:100,000). Anti-β-actin or anti–cytochrome c oxidase (subunit 4) was used to control for protein loading of tissue lysates or mitochondrial fractions, respectively.

Chemokine RT-PCR and Myeloperoxidase Activity.

Total RNA extraction from liver, reverse transcription to cDNA, PCR, and selection of primers were performed as described.18 Myeloperoxidase (MPO) activity was determined in whole-liver homogenates.19

Statistical Evaluation.

The mean ± SD were calculated for each treatment group. For biochemical analyses, all assays were performed in duplicate, and the mean was taken as 1 data point. Significant differences between means were determined by ANOVA and Tukey-Kramer multiple comparison post-tests (InStat, GraphPad software, San Diego, CA). P values of less than 0.05 were considered significant.


Leflunomide Protects from APAP-Induced Acute Liver Injury.

To assess APAP dose response in C57BL/6 mice, we first performed a dose range-finding study because this sensitive strain20 exhibits marked interindividual variability.21 Nonfasted mice developed modest hepatotoxicity 8 hours after and marked hepatic necrosis 24 hours after a single intraperitoneal dose of 750 mg/kg APAP, as judged from increased serum ALT activity and histopathological evaluation (data not shown). This dose was selected as the hepatotoxic standard dose in all subsequent studies.

To explore a possible cytoprotective effect of leflunomide in vivo, groups of mice were treated with APAP or vehicle followed by injection with leflunomide or vehicle. To prevent leflunomide from possibly altering the toxicokinetics and interacting with the uptake or bioactivation of APAP, it was given 4 hours after APAP, at which point the latter would have been fully metabolized in the mice.22 All mice were sacrificed 8 hours after administration of APAP, and the extent of hepatic injury was analyzed. Figure 1 demonstrates that APAP alone increased serum ALT activity dramatically. In contrast, the delayed treatment with leflunomide kept the ALT values at those of the normal controls, indicating that leflunomide prevents APAP-induced parenchymal injury in mice. Control experiments in which leflunomide was directly added to mouse serum ruled out the possibility that the drug might inhibit ALT catalytic activity. Histopathological analysis confirmed the clinical-chemical results; whereas APAP alone caused centrilobular parenchymal necrosis in all mice (Fig. 2), APAP treatment followed by leflunomide (4 hours after APAP dose) afforded complete protection from hepatic necrosis in some mice and attenuated the extent of injury in the others (Fig. 2; Table 1).

Figure 1.

Serum ALT activity in mice treated with a single intraperitoneal dose of APAP (750 mg/kg) or vehicle (15% solutol HS-15 in PBS), followed by an intraperitoneal dose of leflunomide (30 mg/kg) or vehicle (7.5% solutol HS-15 in PBS) 4 hours after APAP. The animals were sacrificed 8 hours after APAP. Data are mean ± SD (n = 5 mice per group; *P < 0.05).

Figure 2.

Representative liver sections of mice treated with a single intraperitoneal dose of APAP (750 mg/kg) followed by an intraperitoneal dose of (A) vehicle or (B) leflunomide (30 mg/kg) 4 hours after APAP. The animals were sacrificed 8 hours after APAP. The arrow indicates the centrilobular localization of hepatocellular necrosis. H&E staining, original magnification objective ×40.

Table 1. Hepatotoxicity Score of Mice Treated with APAP Alone (750 mg/kg ip) or in Combination with Leflunomide (30 mg/kg ip, 4 hours post-APAP)
TreatmentHistopathology Scores (8 Hours Post-APAP)
  1. Note: Mice were sacrificed 8 hours after treatment with APAP. The criteria for scoring the liver injury were: 0, no injury; 1, minimal injury (only few hepatocytes affected); 2, mild injury (centrilobular necrosis in some lobules, 1–2 rings of necrotic cells); 3, moderate injury (centrilobular necrosis in most lobules, 2–3 rings of necrotic cells); 4, marked injury (centrilobular necrosis in all lobules, 3–4 rings of necrotic cells); 5 severe injury (panlobular confluent necrosis, >5 rings of necrotic cells and hemorrhage). Values are the total number of animals with the indicated score.

Vehicle control (n = 5)500000
APAP (n = 5)003110
APAP + Leflunomide (n = 5)302000
Leflunomide (n = 5)500000

To ascertain whether leflunomide indeed prevents the manifestation of APAP-induced liver injury, not just delays it, the degree of hepatic damage was analyzed 24 hours after administration of APAP (Fig. 3A,B; Table 2). The results clearly demonstrated that leflunomide protected from APAP hepatotoxicity. Whereas APAP alone induced massive confluent necrosis and hemorrhage, leflunomide greatly attenuated the degree of injury. Necrotic cells were confined to small centrilobular rings of a few degenerated hepatocytes without apparent nuclear changes. Furthermore, to more accurately define the length of time that leflunomide might still afford full protection after APAP administration, leflunomide was given 8 hours after APAP dose, and the effect on the extent of liver injury was analyzed after 24 hours. Leflunomide indeed attenuated liver injury, even when given 8 hours post-APAP, and the degree of protection was similar to that obtained 4 hours post-APAP (although some were nonresponders) (Fig. 3C; Table 2). A similar extent of protection was attained with SP600125, a known JNK inhibitor,9 which was used as a positive control (Fig. 3D).

Figure 3.

Representative liver sections of mice all treated with a single intraperitoneal dose of APAP (750 mg/kg), followed by an intraperitoneal injection of (A) vehicle, (B) leflunomide (30 mg/kg) 4 hours after APAP dose, (C) leflunomide (30 mg/kg) 8 hours after APAP dose, or (D) SP600125 (30 mg/kg). All animals were sacrificed 24 hours after APAP. The arrow indicates massive confluent necrosis with hemorrhage. H&E staining, original magnification objective ×20.

Table 2. Hepatotoxicity Score of Mice Treated with APAP Alone (750 mg/kg ip) or in Combination with either Leflunomide (30 mg/kg ip, 4 or 8 Hours Post-APAP) or the JNK Inhibitor SP600125 (30 mg/kg ip, 4 hours post-APAP)
TreatmentHistopathology Scores (24 hours Post-APAP)
  • Note: All mice were sacrificed 24 hours after treatment with APAP. The criteria for scoring the liver injury were the same as those given in Table 1. Values are the total number of animals with the indicated score (*1 mouse died;

  • 2 mice died within 24 hours; not included in score). The JNK inhibitor SP600125 was used as a positive control.

Vehicle control (n = 7)700000
APAP (n = 7)*000124
APAP + leflunomide (4 h) (n = 7)014110
Leflunomide (n = 7)700000
APAP + SP600125 (n = 5)032000
SP600125 (n = 5)500000
APAP + leflunomide (8 h) (n = 5)013010

Leflunomide Does Not Interfere with APAP Bioactivation.

To further ascertain that the cytoprotection afforded by leflunomide was not a result of metabolic inhibition of the specific forms of cytochrome P450 (CYP) that activate APAP to NAPQI, the time course of GSH consumption was measured. Decline in hepatic GSH level is a widely accepted marker for the generation of the reactive intermediate in vitro or in vivo.21 Figure 4 demonstrates that hepatic GSH levels decreased during the first 4 hours and increased again thereafter, indicating that the bulk of APAP was bioactivated in the first few hours. Mice that received leflunomide 4 hours after APAP did not exhibit significantly increased GSH levels compared to mice that received APAP alone. Earlier data had shown that A77 1726, the metabolite into which the prodrug leflunomide is rapidly converted during absorption, did not interfere with CYP2E1 activity (the major APAP-bioactivating form) in mouse liver microsomes.14 Taken together, these data indicate that the mode of cytoprotection by leflunomide is not through inhibition of APAP bioactivation to its mitochondria-reactive metabolite, NAPQI.

Figure 4.

Hepatic GSH levels in mice treated with a single intraperitoneal dose of APAP (750 mg/kg) followed by an intraperitoneal dose of leflunomide (30 mg/kg) (□) or vehicle (▪) 4 hours after APAP. Data are mean ± SD (n = 3 mice per group; n = 5 for 6 and 8 hours; *P < 0.05 versus zero time).

Leflunomide Protects from Mitochondrial Injury and Release of Proapoptotic Proteins.

To determine whether leflunomide affords protection from liver necrosis at mitochondria or at a more distal site, we measured mitochondrial function in intact mitochondria isolated from mice treated with APAP with or without leflunomide. An endpoint was chosen that is affected by several functional changes that all merge in compromised energy production. Determination of the ATP-biosynthesizing capacity in succinate-energized mitochondria ex vivo revealed that APAP treatment had significantly decreased the ability to biosynthesize ATP as compared to that of the vehicle controls (Fig. 5). In contrast, leflunomide largely protected the mitochondria from functional injury and maintained ATP-biosynthesizing capacity at the levels of the normal controls.

Figure 5.

ATP biosynthesis in hepatic mitochondria isolated from mice treated with APAP (750 mg/kg) or APAP plus leflunomide (30 mg/kg, 4 hours after APAP dose). Mitochondria were isolated 6 hours after APAP, and the rate of ATP biosynthesis was assessed in vitro as described in the Materials and Methods section. Control (100% = 112 ± 17 nmol ATP/mg protein/min). Data are mean ± SD (n = 3 mice per group; *P < 0.05).

If leflunomide exerts its cytoprotective activity upstream of mitochondrial signaling, then it should also prevent APAP-induced mitochondrial permeabilization and release of death factors. Therefore, we used Western blotting to determine the levels of one of these death proteins, cytochrome c, in the hepatic mitochondrial fraction of mice treated with APAP with or without leflunomide. Cytochrome c was chosen only as a biomarker of permeabilization, not to imply it plays a key role in APAP-induced liver injury.5 As shown in Fig. 6, APAP treatment resulted in significant loss of cytochrome c from mitochondria (6 hours after APAP dose), which was prevented in those mice treated with APAP and leflunomide given 4 hours after APAP. The results for the cytosolic fraction were less clear, as cytochrome c not only leaks from mitochondria but is also lost from injured cells23 (data not shown). Collectively, these results confirm that the mPT was involved in APAP-mediated liver injury in mice and, more importantly, that leflunomide afforded protection upstream of the mPT.

Figure 6.

Western blot analysis showing the inhibitory effects of leflunomide on APAP-induced mitochondrial release of cytochrome c. Mice were treated with APAP with or without leflunomide 4 hours after APAP dose. The tissues were collected 6 hours after APAP dose, and the mitochondrial fractions were probed with an anti–cytochrome c antibody. Complex IV subunit 4 was used as loading control.

Leflunomide Attenuates Peroxynitrite Formation.

A key mechanism by which NAPQI damages mitochondria and causes hepatic necrosis is through the production of peroxynitrite (ONOO).24–26 Peroxynitrite is formed from the reaction of NO with superoxide (mainly generated in mitochondria) and is particularly dangerous because it can be degraded to the ultrareactive hydroxyl radical. After APAP, peroxynitrite is predominantly found in mitochondria.5, 27 To determine whether leflunomide attenuated peroxynitrite formation, we used an immunohistochemical method for nitrotyrosine (a biomarker for peroxynitrite) in the liver of mice treated with APAP with or without leflunomide. Figure 7A,B demonstrates that, as expected, APAP alone induced positive nitrotyrosine immunoreactivity in the centrilobular regions, congruent with the areas of hepatocellular necrosis. Leflunomide greatly prevented APAP-dependent nitrotyrosine immunostaining. This suggests that leflunomide affords protection upstream of mitochondrial peroxynitrite formation, possibly by decreasing the production of either mitochondrial superoxide or NO.

Figure 7.

Immunohistochemical demonstration of nitrotyrosine adducts in liver sections of mice treated with (A) APAP (750 mg/kg) alone or (B) APAP followed by a single dose of leflunomide (30 mg/kg) 4 hours after APAP dose. The animals were sacrificed 8 hours after APAP dose. The brown areas (arrow) indicate the presence of nitrotyrosine, indicative of peroxynitrite formation. (C) Western blot analysis showing hepatic expression of inducible nitric oxide synthase (iNOS) in mice treated with APAP with or without leflunomide 4 hours after APAP dose. The tissues were collected and probed 6 hours after APAP dose. β-Actin was used as loading control.

We previously showed14 that leflunomide does not act as an antioxidant and superoxide scavenger in vitro. However, because leflunomide has been demonstrated to inhibit NO formation in immortalized chondrocytes through inhibition of inducible NO synthase (iNOS),28 we next determined the expression of iNOS in the liver after APAP with or without leflunomide. Western blot analysis revealed that hepatic iNOS was abundantly expressed 6 hours after APAP (Fig. 7C) and that leflunomide attenuated increased iNOS expression. Although the roles of iNOS and NO in the pathogenesis of APAP-induced liver injury are somewhat controversial,5 these data provide a possible explanation for the absence of nitrotyrosine induction in the combined APAP/leflunomide treatment.

Leflunomide Inhibits JNK1/2 Activation and Prevents Bcl-XL and Bcl-2 Phosphorylation.

To further explore possible targets of interaction with leflunomide during activation of the cell death pathways by APAP, we next used protein immunoblotting to determine the expression and activation of JNK. The rationale behind this approach was our previous findings that APAP activates (phosphorylates) JNK1/2 in immortalized human hepatocytes and that leflunomide completely abrogates this effect at early time points.14 Here, APAP greatly increased the levels of P-JNK1/2, as determined 6 hours after administration of a toxic dose (Fig. 8A). Leflunomide administered 4 hours after APAP dose greatly attenuated the expression of P-JNK1/2. These results confirm the in vitro data and suggest that JNK may be a pivotal target of leflunomide. To confirm that the apparently lower levels of P-JNK were not simply a result of lower expression of the nonphosphorylated JNK protein, we used Western blotting to assess expression of JNK. Fig. 8A reveals that JNK expression was not changed significantly either by APAP or by leflunomide.

Figure 8.

Western blot analysis showing the inhibitory effects of leflunomide on APAP-induced increased hepatic levels of (A) activated (phosphorylated) JNK1/2 (P-JNK1/2) or (B) activated (phoshorylated) MKK4 (P-MKK4). Expression of JNK or MKK4 was not affected. Mice were treated with APAP with or without leflunomide 4 hours after APAP dose. The tissues were collected 6 hours after APAP dose, and hepatic lysates were probed with phosphospecific anti-JNK or anti-MKK4 antibody. β-Actin was used as loading control.

To explore whether leflunomide inhibited the stress kinase pathway at a site upstream of JNK, we determined the expression of phosphorylated MKK4, the immediate upstream kinase that activates JNK. Although basal expression of MKK4 remained unchanged in all groups, leflunomide prevented an APAP-induced increase in P-MKK4 (Fig. 8B). These data suggest that leflunomide inhibits the JNK signaling pathway at a more proximal regulatory step, resulting in inhibition of JNK phosphorylation and its downstream effector mechanisms.

Activated JNK, in turn, can phosphorylate (and thus inactivate) Bcl-XL or Bcl-2, two antiapoptotic proteins, thereby shifting the balance of proapoptotic and antiapoptotic members of the Bcl-2 family in favor of the proapoptotic proteins, promoting the mPT.29, 30 Therefore, we measured the expression of phosphorylated Bcl-XL and Bcl-2 ex vivo 6 hours after APAP dose. APAP indeed increased the levels of both P-Bcl-XL and P-Bcl-2, likely as a consequence of increased P-JNK levels (Fig. 9). Leflunomide fully prevented phosphorylation of Bcl-XL or Bcl-2, indicating this may be a relevant mechanism for protection from mPT and cell death. Again, control experiments revealed that this was not caused by differential expression of the nonphosphorylated proteins (Fig. 9).

Figure 9.

Western blot analysis showing the inhibitory effects of leflunomide on APAP-induced increased hepatic levels of phosphorylated Bcl-2 and Bcl-XL (P-Bcl-2 and P-Bcl-XL). Expression of the nonphosphorylated proteins was not affected. Mice were treated with APAP with or without leflunomide 4 hours after APAP treatment. The tissues were collected 6 hours after APAP treatment, and hepatic lysates were probed with phosphospecific antibodies.

Leflunomide Does Not Inhibit APAP-Induced Activation of the Innate Immune System.

As leflunomide is an immunomodulatory drug, we addressed the possibility that its protective effects on APAP hepatotoxicity might be mediated by inhibiting NK or NKT cells, which are abundant in liver and which become highly activated following challenge with APAP.18 Therefore, with RT-PCR we determined the expression of transcripts of KC and MCP-1, two key chemokines that are regulated by activated NK/NKT cells,18 at 8 and 24 hours after APAP dose. Furthermore, we quantified MPO activity as a marker for neutrophil activity in liver. APAP alone markedly increased the expression of transcripts of KC and MCP-1, as well as significantly increased MPO activity, as expected (Fig. 10). However, in the combined APAP + leflunomide group, chemokine expression and MPO activity were not decreased compared to those in APAP alone, indicating that leflunomide at 30 mg/kg did not significantly inhibit the innate immune system activated by APAP.

Figure 10.

Effects of APAP and leflunomide on markers of the innate immune system. (A) Apparent lack of inhibitory effect of leflunomide on the APAP-induced induction of mRNA for the chemokines keratinocyte-derived chemokine (KC) and monocyte chemoattractant protein-1 (MCP-1). Liver tissue was collected from mice treated with APAP with or without leflunomide (4 hours after APAP dose) at 8 or 24 hours after APAP dose. Total RNA was extracted and analyzed by RT-PCR. The data shown are typical of 5 mice per group. (B) Apparent lack of inhibitory effect of leflunomide on the APAP-induced increase in hepatic MPO activity. Myeloperoxidase activity was determined in whole-liver homogenates. Data are mean ± SD (n = 3-5 mice per group; *P < 0.05 versus vehicle control).


The aim of this work was to assess whether the DMARD leflunomide affords protection against APAP-induced liver injury in a murine model of APAP hepatotoxicity. This was based on recent findings indicating that APAP toxicity is mediated by the JNK pathway in mouse liver.9 We had previously shown that leflunomide protects from protoxicant-induced lethal cell injury by preventing the JNK-mediated mitochondrial mPT in immortalized human hepatocytes.14 However, because data on APAP toxicity in vitro cannot always be extrapolated to an in vivo situation, where many physiological factors influence the bioactivation, conjugation, distribution, and elimination of APAP,5 we have performed an in vivo study. In the present study we have demonstrated for the first time that leflunomide, even when given late after the administration of APAP, can provide full protection against a high dose of the hepatotoxicant. We also have shown that the mode of this protection involves both inhibition of JNK1/2-mediated mitochondrial permeabilization and inhibition of iNOS-induced peroxynitrite formation.

These conclusions were derived from a number of direct and indirect observations. First, leflunomide did not inhibit the initiating events of APAP bioactivation because leflunomide was injected 4-8 hours after APAP, a point at which formation of NAPQI had been completed. Second, leflunomide apparently did not suppress neutrophil function and NK and NKT cells, which are all critically involved in APAP toxicity.18 Third, leflunomide exerted its protective effects proximal to the mitochondrial release of death proteins, as the drug fully prevented the APAP-induced collapse of ATP biosynthesis in mitochondria. Thus, combined with the in vitro data obtained in hepatocytes31 and other cell types,32 our results indicate that leflunomide inhibits either the mPT directly or interferes with a regulatory step upstream of the mPT.14

JNK is a pivotal regulator of mitochondrial permeabilization.33–35 It is well known that oxidant stress (e.g., derived from APAP bioactivation) can directly activate the JNK pathway.36 Another possibility is that ROS may inactivate inhibitors that normally suppress JNK activation (e.g., MAP kinase phosphatases) by oxidizing critical cysteine residues,37, 38 the outcome of which would be the same. Activated (phosphorylated) JNK in turn phosphorylates members of the Bcl-2 family (e.g., Bcl-2 and Bcl-xL) and thereby blocks their antiapoptotic function.39 Recent data also indicate that JNK phosphorylates (and thereby activates) the proapoptotic protein Bax.9, 40 Because it is the balance between proapoptotic and antiapoptotic factors that determines the fate of a cell, this imbalance can lead to cell demise. Our data indeed provide evidence that leflunomide suppresses APAP-induced phosphorylation of JNK1/2 and, hence, of Bcl-2 and Bcl-XL. This prevents the mPT, release of death proteins and antioxidants from mitochondria, loss of ATP production, and ultimately protects from cell death and necrosis (Fig. 11). However, the exact upstream site where leflunomide interferes with the JNK cascade is still unclear.

Figure 11.

Proposed pathways and mechanisms of the protective effects of leflunomide on APAP-induced mPT and liver injury. APAP alone produces the reactive metabolite NAPQI, which induces oxidant stress (peroxynitrite) and activates the JNK pathway. Activation (phosphorylation) of JNK1/2 (P-JNK) in turn phosphorylates (inactivates) the antiapoptotic proteins Bcl-2 and Bcl-XL as well as phosphorylates (activates) the proapoptotic protein Bax. This leads to a shift in the overall equilibrium of the Bcl-2 family members toward induction of the mPT and precipitation of cell death. JNK also up-regulates iNOS, which further enhances peroxynitrite production. Leflunomide blocks the JNK pathway and, hence, all downstream events.

A second mechanism by which leflunomide might protect from APAP is the attenuation of peroxynitrite formation, a pivotal regulator of APAP toxicity.5, 25 The absence of nitrotyrosine staining after leflunomide points to inhibition of either superoxide or nitric oxide. As APAP up-regulated iNOS in our mouse model and because leflunomide greatly inhibits iNOS expression, it is likely that decreased mitochondrial NO formation contributes to this protection. Although the role of iNOS after APAP has remained controversial, iNOS-null mice were protected from APAP toxicity,41 and iNOS induction enhanced APAP hepatotoxicity,42 supporting the role of iNOS. Also, a potent iNOS inhibitor (ONO-1714) protected from APAP-induced liver injury.43 The mechanism of iNOS inhibition by leflunomide is not clear but could be a consequence of JNK inhibition, as JNK is an established regulator of iNOS44, 45 (Fig. 11). In turn, peroxynitrite has been shown to be a signaling molecule in JNK activation.46

In our mouse model, we show that leflunomide is still hepatoprotective as late as 8 hours after APAP administration. This is a clear advantage over the conventional use of NAC, which blocks more proximal events. Other cytoprotective compounds in addition to NAC have been evaluated for their potential to protect from lethal cell injury. For example, a nonpeptide mimetic of superoxide dismutase has been successfully used in mice,47 underscoring again that superoxide (reacting with NO to peroxynitrite) plays a critical role in the pathogenesis of APAP hepatotoxicity. However, such SOD mimetics have not yet been sufficiently investigated for potential use in patients. In contrast, leflunomide is an approved drug with a known safety profile that could be utilized therapeutically. One potential advantage of leflunomide over NAC and other antioxidants is that it acts more downstream of the signaling cascade, suggesting that it could be given relatively late.

In conclusion, we demonstrate that the DMARD leflunomide protects from acute APAP-induced liver injury in mice through inhibition of the JNK2/Bcl-2 protein family–mediated pathway of mitochondrial permeabilization and prevention of peroxynitrite formation. Because leflunomide is an approved drug, its possible clinical application needs to be explored.