Journal of Hepatology
Volume 42, Issue 1 , Pages 110-116, January 2005

Involvement of mitochondrial permeability transition in acetaminophen-induced liver injury in mice

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan

Received 27 April 2004; received in revised form 16 September 2004; accepted 21 September 2004. published online 12 October 2004.

Article Outline

Background/Aims

Although mitochondria have been demonstrated as primary targets in acetaminophen hepatotoxicity, the mechanism for mitochondria-mediated toxicity has not been defined. We examined the role of mitochondrial permeability transition (MPT) in the acetaminophen-induced liver injury.

Methods

Male CD-1 mice were given intraperitoneally acetaminophen (350mg/kg) without or with cyclosporin A (50mg/kg), a specific inhibitor of MPT. Serum alanine aminotransferase (ALT), a marker of liver injury, and other biochemical parameters were determined.

Results

Acetaminophen-induced ALT leakage was attenuated by co-administration of cyclosporin A. Cyclosporin A did not affect acetaminophen-induced early decrease in hepatic reduced glutathione (GSH) contents, indicating lack of the effect on the metabolic activation. Acetaminophen-induced decrease in mitochondrial GSH and ATP contents, and cytosolic leakage of cytochrome c were attenuated by cyclosporin A, suggesting that mitochondrial oxidative stress and ATP depletion resulting from MPT are principle mechanisms involved in acetaminophen-induced liver injury. Mitochondrial swelling by calcium was exacerbated in the mitochondria isolated from the acetaminophen-treated mice. In vitro exposure of intact mitochondria to N-acetyl-p-benzoquinone imine (NAPQI) with calcium caused mitochondrial swelling.

Conclusions

The present data indicate that the MPT is the principal mechanism in the acetaminophen-induced liver injury and NAPQI is a candidate to open the transition pore.

Keywords: Acetaminophen, Liver injury, Mitochondrial permeability transition, Cyclosporin A, Glutathione, ATP, Cytochrome c, N-Acetyl-p-benzoquinone imine

 

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1. Introduction 

An overdose of the analgesic drug acetaminophen causes liver injury in experimental animals and humans. The toxicity has been shown to be initiated by cytochrome P450 metabolism to N-acetyl-p-benzoquinone imine (NAPQI) [1], [2]. The high reactivity of NAPQI with sulfhydryl groups results in depleting glutathione in hepatocytes, followed by covalent binding to intracellular proteins [3], [4]. Although it has been shown that the relative amount of covalent binding is correlated with the development of the toxicity [3], it is also suggested that the covalent binding is not sufficient for the toxicity [5], [6], [7]. Extensive studies were thus focused on covalent binding to specific protein(s) as a trigger of the toxicity. A number of proteins have been identified as targets of NAPQI by the immunological techniques [8] and recent proteomics [9]. Because development of various mitochondria dysfunctions has been observed with acetaminophen toxicity, which include inhibition of respiration, a decrease in hepatic ATP levels, a decrease in membrane potential, a loss of mitochondrial Ca2+ [10], [11], [12], [13], it was proposed that mitochondria was primary target of the reactive metabolite. Indeed, some of the target proteins were localized in mitochondria fraction including glutamate dehydrogenase, aldehyde dehydrogenase, carbamyl phosphate synthetase-I and ATP synthetase α-subunit [8], [9]. These enzyme activities in the mitochondrial fraction were decreased partially [14], [15], probably as consequences of covalent binding, while it is also estimated that only the loss of the any single enzyme activity could not explain mitochondria-mediated acetaminophen hepatotoxicity. It is thus presumed that the toxicity is accounted for by combination of covalent binding to several functional proteins and/or by secondary event following the covalent binding.

Mitochondrial permeability transition (MPT) is recently focused as a mechanism for drug-induced hepatocyte injury [16], [17], [18]. The MPT represents an abrupt increase in permeability of the mitochondrial inner membrane to allow solutes with a molecular weight less than 1500 [19]. The MPT is promoted by the accumulation of excessive Ca2+ and stimulated by various compounds and conditions. It leads to dissipation of membrane potential (Δψ), uncoupling of oxidative phosphorylation, loss of pre-accumulated Ca2+, and expansion of the matrix volume. MPT causes both apoptotic and necrotic cell death. Acetaminophen could induce hepatocyte apoptosis in vitro as well as necrosis [16], [17], whereas the quantitative determination of cell death after exposure of hepatotoxic dose of acetaminophen in vivo indicated that acetaminophen caused extensively oncotic necrosis rather than apoptosis [20]. Recent papers proposed the possibility of MPT as a mechanism for acetaminophen-induced liver injury [21], [22], but have not been fully supported by experimental data. In the present study, we investigated the possible involvement of MPT in acetaminophen-induced liver injury in male CD-1 mice by using a MPT specific inhibitor, cyclosporin A.

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2. Materials and methods 

2.1. Chemicals 

Acetaminophen and NAPQI were purchased from Sigma-Aldrich (St Louis, MO); cyclosporin A, glutathione, reduced form (GSH) and glutathione disulfide (GSSG) were from the Wako Pure Chemical Ind. (Osaka, Japan); ATP was from Oriental Yeast Co., Ltd (Tokyo, Japan). All other chemicals and solvents were of analytical grade.

2.2. Animals and in vivo treatment 

Male CD-1 mice were purchased from Takasugi Experimental Animals (Saitama, Japan). The mice were used in the experiment at the age of 8–9 weeks. The mice were acclimatized at least 1 week in a climate-controlled room on a 12-hour light-dark cycle and were fed ad libitum. All procedures and care were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory animals. The mice were then fasted for 16h before experiments to sensitize mice to acetaminophen toxicity by decreasing basal levels of liver GSH. Mice received intraperitoneally (i.p.) 10-ml/kg saline or 350mg/kg of acetaminophen. In some experiments, mice received 10-ml/kg i.p. corn oil or cyclosporin A (50mg/kg) in the oil just before saline or acetaminophen. Two, 8 or 24h after the treatment, blood of the mice was collected and the mice were killed to obtain their livers. A portion of the liver was fixed in 10% buffered formalin for histological sections. The blood was allowed to coagulate and the samples were then centrifuged to obtain the serum. In some experiments, the bile duct was cannulated with polyethylene tubing (PE 10) to allow sampling of bile and the bile was collected for 15min.

2.3. Assessment of hepatotoxicity 

Serum alanine aminotransferase (ALT) activities were assayed as a marker of acetaminophen-induced hepatotoxicity. Assays were run on the test kit (Sigma Diagnostics, St Louis, MO). Formalin-fixed tissue sections were embedded in paraffin, mounted onto glass slides, and stained with hematoxylin–eosin (Takasaki Pathologic Center, Gunma, Japan). Hepatotoxicity was also assessed by histological examination of the tissue sections.

2.4. Isolation of liver mitochondria 

Liver mitochondrial fraction was prepared according to the method described by Schneider and Hogeboom [23] with modifications. The livers were isolated and placed in the ice-cold medium containing 250mM sucrose, 10mM HEPES-KOH, pH 7.4, and 0.5mM EGTA. The livers were cut to small cubes with scissors in 50ml of the medium and homogenized five times in a Potter homogenizer. The homogenates were diluted to 100ml per liver and were centrifuged at 770×g for 5min kept at 4°C. The resulting supernatant was decanted and further centrifuged at 9800×g for 10min. The supernatant was discarded, the pellet was suspended in 20ml of the ice-cold isolation medium, and centrifuged at 4500×g for 10min. The final mitochondrial pellet was suspended in 1ml of medium containing 250mM sucrose and 10mM HEPES-KOH, pH 7.4. Protein concentration of the mitochondrial fraction was determined by the method of Lowry et al. [24].

2.5. Assay of GSH and GSSG 

GSH and GSSG contents in liver homogenates, mitochondria and bile were determined by an HPLC method according to Keller and Menzel [25] with modifications. The reaction medium (1.0ml) was mixed with 0.5ml solution consisting of 5% metaphosphoric acid/0.1% EDTA (2:7, v/v). The sample was centrifuged (16,000×g, 5min) and the supernatant (0.5ml) mixed with 10ml of 250mM 3-fluorotyrosine as an internal standard for HPLC. The samples were applied after filtration to the HPLC column (Inertsil ODS, GL Sciences Inc., Tokyo). The mobile phase consisting of 0.1% trifluoroacetic acid/methanol (9/1, v/v) was pumped at a flow rate of 1.0ml/min. The effluent from the column was mixed with luminescence reagent consisting of 18.6mM o-phthalaldehyde and 17.1mM 2-mercaptoethanol pumped at a flow rate of 0.2ml/min. The fluorescence intensity at the 355/425nm wavelength pair was monitored.

2.6. Assay of mitochondrial ATP content 

Liver mitochondria were suspended into 0.5ml of 1N HClO4 and disrupted by vortex mixing with the acid. After the neutralization with 2N KOH, the sample was centrifuged (16,000×g, 30s) and supernatant was used for assay for ATP. ATP content was measured with Sigma ATP bioluminescent assay kit based on the luciferin-luciferase method by a chemiluminescence analyzer.

2.7. Detection of cytochrome c in cytosol 

Cytochrome c in hepatic cytosol fraction was detected by immunoblot analysis. Cytosolic proteins (5.0μg) were separated by SDS–PAGE with a 15% polyacrylamide gel. The proteins on the gel were transferred to a polyvinylidine difluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was treated with the anti-cytochrome c antibody (clone 7H8.2C12, Lab Vision Co., Fremont, CA), which was diluted 1:1000 for use. The immunoblots were developed with the enhanced chemiluminescence detection method with reagents from Amersham Pharmacia Biotech (Uppsala, Sweden), according to the manufacturer's instructions.

2.8. Incubation of mitochondria 

Reaction medium containing 1.0mg/ml liver mitochondrial protein, 125mM sucrose, 150mM KCl, 10mM HEPES-KOH, and 2.5μM rotenone was preincubated at 30°C. The liver mitochondria from acetaminophen-treated or control mice were energized by 5mM succinate. The incubation was started by adding 20μM CaCl2 and was performed at 30°C for various time periods. In the experiment to determine in vitro effects of acetaminophen and NAPQI, the liver mitochondria from untreated mice in the above-mentioned mixture with 20μM CaCl2 were energized by 5mM succinate. The incubation was started by adding acetaminophen or NAPQI and was performed at 30°C for various time periods. In some experiments, the mixture included 1μM cyclosporin A.

2.9. Assessment of mitochondrial permeability transition 

Mitochondrial swelling as the indicator of MPT was estimated from the decrease in absorbance at 540nm.

2.10. Assessment of mitochondrial membrane potential 

The electrical transmembrane potential of mitochondria was monitored spectrophotometrically with the cationic dye, rhodamine 123 at the concentration of 0.5μM. After the 2-min preincubation with succinate, incubation was started by the addition of acetaminophen or NAPQI and was performed for 3min. The reaction medium was immediately centrifuged (16,000×g, 30s) and fluorescence intensity of the supernatant was monitored at the 505/535nm wavelength pair. Δψ was calculated by the Nernst equation as described previously [26].

2.11. Statistical analysis 

The experimental groups were compared by analysis of variance, followed by Newman–Keuls multiple comparisons test to determine significant differences between the group means.

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3. Results 

3.1. Protection of mice from acetaminophen-induced liver injury by cyclosporin A 

Administration of acetaminophen (350mg/kg) to male CD-1 mice induced an increase in serum ALT leakage at 8 and 24h after the injection (Table 1). No significant increase in ALT was observed 2h after the treatment (data not shown). The ALT leakage was significantly suppressed by co-administration of cyclosporin A (50mg/kg, i.p.), a typical inhibitor of MPT pore opening, suggesting that MPT is involved in pathogenesis of the acetaminophen-induced liver injury. Cyclosporin A itself was not hepatotoxic under the present condition. Moreover, histological examination of liver tissues 24h after treatment with acetaminophen revealed centrilobular necrosis with hemorrhage, whereas the mice treated together with cyclosporin A showed only minimal hepatic necrosis (Fig. 1).

Table 1. Effect of cyclosporin A on acetaminophen-induced liver injury
ALT8h (IU/l)ALT24h (IU/l)
Control12±116±4
Cyclosporin A17±311±3
Acetaminophen1765±541*2459±548**
Cyclosporin A+acetaminophen396±291#986±260**#

Mice were given acetaminophen (350mg/kg, i.p.) together with or without cyclosporin A (50mg/kg, i.p.), and were killed at 8 or 24h after the treatment. The results are means±SE of 6–12 mice. *P<0.05, **P<0.01, Significantly different from corresponding control. #P<0.05, Significantly different from ‘Acetaminophen’ group.

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  • Fig. 1. 

    Histopathology of liver 24h after administration of acetaminophen. Mice were given acetaminophen (APAP, 350mg/kg, i.p.) together with or without cyclosporin A (CsA, 50mg/kg, i.p.), and were killed 24h after the treatment. Liver sections were subjected to hematoxylin–eosin staining.

Acetaminophen-induced liver injury is mediated by its reactive metabolite, NAPQI. NAPQI binds to GSH, resulting in depleting hepatic GSH, followed by covalently binding to cellular critical targets. Therefore, an early decrease in hepatic GSH is the reflect of NAPQI formation. The hepatic GSH level markedly decreased 2h after the acetaminophen treatment, and the decrease was not affected by the coadministration with cyclosporin A (Fig. 2). Similar results were obtained when measured at an earlier time point (30min; acetaminophen, 2.96±0.71; acetaminophen+cyclosporin A, 3.24±0.38nmol/mg protein). The unchanged depletion rate of GSH suggests that the production of NAPQI is not affected by cyclosporin A at least under these experimental conditions. Cyclosporin A is a substrate of CYP3A [27]. While NAPQI formation is mediated mainly by CYP2E1 in mice [28], several reports indicated that other P450 isozymes including CYP3A also contributed to the metabolism [29], [30]. Thus, it may be possible for cyclosporin A to inhibit CYP3A-dependent NAPQI generation.

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  • Fig. 2. 

    Time course of hepatic GSH and GSSG contents after administration of acetaminophen. Mice were given acetaminophen (APAP, 350mg/kg, i.p.) together with or without cyclosporin A (CsA, 50mg/kg, i.p.), and were killed at various time points. Contents of GSH and GSSG in liver homogenates were determined. The results are means±SE of 3–6 mice. *P<0.05, **P<0.01, Significantly different from corresponding APAP(−) groups.

Similar early decrease was also obtained for GSSG (Fig. 2). Because GSH is oxidized to form GSSG, the decrease in GSSG would be associated with the depletion of GSH. The hepatic GSH level was completely recovered 8h after the injection, whereas hepatic GSSG levels were increased as compared to controls and, furthermore, cyclosporin A potentiated this effect at the later time point (Fig. 2). It is considered that the increases in GSSG is accounted for the oxidative stress secondary to acetaminophen-induced liver injury [21] and by the additional effects of cyclosporin A, which is known to induce oxidative stress [31].

3.2. Changes in mitochondrial parameters in the mice with acetaminophen-induced liver injury 

Although, the GSH content in liver homogenate was returned to the control level 8h after the acetaminophen treatment (Fig. 2), the content in the mitochondria fraction was lower than control at the same time point. Furthermore, mitochondrial GSSG content markedly increased in the treated mice (Fig. 3). These results indicate mitochondrial oxidative stress, which plays an important role in initiation of the acetaminophen hepatotoxicity [32]. The collapse of the mitochondrial redox balance was partially prevented by cyclosporin A (Fig. 3). Similar results were obtained 24h after the treatment (data not shown). Therefore, increase in tissue GSSG levels observed at 24h after the treatment with cyclosporin A and acetaminophen (Fig. 2) may be derived from outside of mitochondrial compartment. Moreover, biliary excretion of GSSG in this group was higher than other groups (control, 1.85±0.21; cyclosporin A, 2.43±0.46; acetaminophen, 1.97±0.26; cyclosporin A+acetaminophen, 4.67±0.44nmol/min), whereas that of GSH was unchanged (control, 6.26±0.86; cyclosporin A, 6.42±1.19; acetaminophen, 5.60±0.75; cyclosporin A+acetaminophen, 5.39±1.44nmol/min). Because GSSG accumulated in the mitochondrial compartment is not released into bile [33], these data support the idea that the increase in liver GSSG at 24h after the treatment with cyclosporin A and acetaminophen is attributable to the extramitochondrial oxidative stress, which is irrelevant for the liver injury.

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  • Fig. 3. 

    Mitochondrial GSH, GSSG and ATP contents after administration of acetaminophen. Mice were given acetaminophen (APAP, 350mg/kg, i.p.) together with or without cyclosporin A (CsA, 50mg/kg, i.p.), and were killed 8h after the treatment. Contents of GSH, GSSG and ATP in liver mitochondria were determined. The results are means±SE of 3–6 mice. *P<0.05, **P<0.01, Significantly different from corresponding APAP(−) groups. #P<0.05, ##P<0.01, Significantly different from corresponding CsA(−) groups.

Depletion of mitochondrial ATP, which is likely more critical event for the toxicity, was observed in the acetaminophen-treated mice and was also partially prevented in the mice treated with cyclosporin A (Fig. 3). Because mitochondrial oxidative stress and the depletion of ATP has been implicated with MPT [34], the beneficial effects of cyclosporin A support our conclusion that MPT is a key step in the acetaminophen-induced liver injury. Leakage of cytochrome c into cytosol, which is correlated with MPT and a signal for apoptotic cell death, was detected in the acetaminophen-treated mice and was slightly attenuated by the coadministration of cyclosporin A (Fig. 4). This result also demonstrates the opening of the cyclosporin-sensitive MPT pore in the acetaminophen hepatotoxicity. MPT is characterized by a progressive permeabilization of the inner mitochondrial membrane dependent on the excessive amount of intramitochondrial Ca2+ and results in mitochondrial swelling [16], [17]. Energized mitochondria from control mice tolerated Ca2+ at the concentration of 20μM without undergoing the MPT as assessed by mitochondrial swelling (Fig. 5). By contrast, mitochondria from acetaminophen-treated mice were much more sensitive to the MPT induction. A large-amplitude swelling was observed with mitochondria from the treated mice and was prevented by the addition of cyclosporin A (1μM) into the reaction medium (Fig. 5).

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  • Fig. 4. 

    Leakage of cytochrome c to cytosol after administration of acetaminophen. Mice were given acetaminophen (350mg/kg, i.p.) together with or without cyclosporin A (50mg/kg, i.p.), and were killed 24h after the treatment. Hepatic cytosol fractions from the mice were analyzed by Western blot analysis with antibody against cytochrome c. Each lane represents a sample from a single mouse. The results are representative blots from 3 to 5 mice.

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  • Fig. 5. 

    Swelling of liver mitochondria from mice after administration of acetaminophen. Mice were given acetaminophen (350mg/kg, i.p.) and were killed 8h after the treatment along with control mice. Mitochondria (1mg/ml) of the mice were incubated in the reaction medium containing 125mM sucrose, 150mM KCl, 10mM HEPES-KOH, 2.5μM rotenone, 20μM CaCl2, and was energized by 5mM succinate. Absorbance at 540nm was monitored after adding CaCl2. The dotted line shows the incubation of mitochondria from acetaminophen-treated mice with 1μM cyclosporin A. The results are representatives from 3 to 5 experiments.

3.3. In vitro effects of acetaminophen and NAPQI on isolated mitochondria 

Incubation of energized mitochondria in the presence of Ca2+ (20μM) and NAPQI (5–50μM) induced a large-amplitude swelling (Fig. 6). Addition of cyclosporin A (1μM) prevented the mitochondrial swelling induced by NAPQI. On the other hand, acetaminophen did not induce swelling at the acetaminophen concentration up to 5mM (data not shown). The transmembrane potential of mitochondria energized with succinate was assessed in the presence of Ca2+ by using a cationic dye, rhodamine 123 as an indicator. Incubation of mitochondria with NAPQI (50μM) caused a decrease in membrane potential in the presence of Ca2+ and the mitochondrial depolarization was also prevented by the addition of cyclosporin A (Fig. 6 insert).

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  • Fig. 6. 

    Swelling and depolarization of liver mitochondria from mice in vitro treated with NAPQI. Mitochondria (1mg/ml) of untreated mice were incubated in the reaction medium containing 125mM sucrose, 150mM KCl, 10mM HEPES-KOH, 2.5μM rotenone, 20μM CaCl2, 5–50μM NAPQI and were energized by 5mM succinate. Absorbance at 540nm was monitored after adding NAPQI. Numbers in the figure are concentrations (μM) of NAPQI. The dotted line shows the incubation of mitochondria with 50μM NAPQI and 1μM cyclosporin A. The plots inserted are Δψ values obtained from the fluorescence intensity at the 505/535nm wavelength pair after incubation of the same mixture with 0.5mM rhodamine 123. The addition of chemicals (C, no addition; N, 50μM NAPQI; CsA, 50μM NAPQI+1μM cyclosporin A) are shown in the bottom. The results are representatives of 3 experiments.

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4. Discussion 

The present study demonstrates that in vivo treatment of male CD-1 mice with acetaminophen results in MPT and suggests that the MPT is involved in pathogenesis of acetaminophen-induced liver injury. Although a number of mitochondrial proteins have been identified as targets of covalent binding of NAPQI, a reactive metabolite of acetaminophen [8], [9], the pathogenic role of the covalent binding in the toxicity has not been fully elucidated. On the other hand, MPT was recently proposed as a mechanism of the acetaminophen hepatotoxicity associated with the covalent binding [21], [22]. Indeed, the other reports presented protective effect of cyclosporin A against the acetaminophen toxicity [35], [36]. However, one of them observed the protective effect of combination of cyclosporin A, fructose and trifluoperazine against acetaminophen hepatotoxicity [35], and thus the contribution of blocking MPT to the overall protection remains unknown. Another assessed the systemic toxicity (LD50) but not liver injury as in vivo toxicity of acetaminophen [36]. Therefore, the present data indicating that cyclosporin A protected mice against acetaminophen-induced liver injury is the first report to demonstrate the in vivo pathogenic role of MPT in the hepatotoxicity of acetaminophen.

Several mechanisms may underlie acetaminophen-induced, probably NAPQI-mediated MPT. MPT pore constitutes a complex assembly of voltage-dependent anion channel in the outer membrane, adenine nucleotide translocase in the inner membrane and cyclophilin D in the matrix [34]. None of these proteins has reported to be a direct target of the covalent binding of NAPQI, allowing us to postulate the oxidative damage of the mitochondrial membrane protein(s) by NAPQI, because it possesses oxidant characteristics [37]. Indeed, it has been reported that mitochondrial NADPH and protein thiols were oxidized by NAPQI [38]. If the membrane thiol is oxidized, essentially thiol cross-linkage is formed, the conformation is changed, followed by opening of the pore. Adenine translocator has been proposed as a protein to form cross-linkage [39]. It was reported that late treatment of acetaminophen-treated mouse hepatocytes with dithiothreitol, a thiol-reducing agent, but not N-acetylcysteine prevented the liver injury [40], suggesting that reversal of an oxidation state, which increases the probability of the open state of the pore, is effective for the hepatoprotection. On the other hand, the voltage-dependent regulation of the MPT pore has been characterized by depolarization of the membrane ant resultant increase in probability of the pore opening. An important factor determining the voltage gating potential of the MPT pore is also redox state of vicinal cysteine thiols, which are closely associated with the voltage-sensing element of the MPT pore [41]. These findings suggest the mitochondrial oxidative stress is responsible for occurrence of MPT. Furthermore, it was reported that NAPQI was able to raise cell calcium probably by inhibition of plasma membrane Ca2+-ATPase through its depleting effect on GSH and protein-bound SH groups [10]. The increase in cellular Ca2+ in vivo should sensitize liver mitochondria to NAPQI-induced MPT pore opening.

Various changes caused by MPT and associated with toxicity are presented, which include membrane depolarization, uncoupling of oxidative phosphorylation, and release intramitochondrial ions and metabolic intermediates [10]. Among them, MPT as a source of superoxide was proposed to be an important event in acetaminophen hepatotoxicity [21], [22]. The generation of superoxide supports the theory that oxidative stress in addition to covalent binding is involved in the acetaminophen hepatotoxicity. In the present study, acetaminophen-induced mitochondrial oxidative stress was suppressed by cyclosporin A (Fig. 3). It is thus suggested that the mitochondrial oxidative damage is the result of MPT, as well as the cause of MPT as described above. Furthermore, depletion of mitochondrial ATP in the acetaminophen-treated mice was also attenuated by cyclosporin A (Fig. 3), which occurs as a result of the MPT [34]. Recent studies have suggested that nitric oxide (NO) as well as superoxide is involved in the acetaminophen-induced liver injury [42], [43]. NO is known to trap superoxide and generate peroxynitrite, which is highly reactive and toxic. Because peroxynitrite also induces MPT [44], the MPT may provide the mechanism how NO participates in the acetaminophen hepatotoxicity, although we have not investigated NO-dependent pathways in the present study.

We detected leakage of cytochrome c into cytosol in the acetaminophen-treated mice, which is correlated with MPT pore opening (Fig. 4). The leakage were slightly attenuated by cyclosporin A, supporting our conclusion that the protection against acetaminophen hepatotoxicity is accounted for by the protection against MPT. Furthermore, leakage of cytochrome c implies induction of apoptosis because cytochrome c is known to be a signal of apoptosis cascade by activating caspases such as caspase 9 and then caspase 3 [45]. Acetaminophen could induce hepatocyte apoptosis in vitro as well as necrosis, whereas the quantitative determination of cell death after exposure of hepatotoxic dose of acetaminophen in vivo indicated that acetaminophen caused extensively oncotic necrosis rather than apoptosis [20]. Thus, it is reasonable to postulate that necrotic cell death is mainly induced also in the present study, although we have not determined the type of cell death. It is considered that depletion of ATP induced by the MPT and by other events prevents the apoptotic cell death, which requires ATP [46], in acetaminophen-induced liver injury. On the other hand, it was recently reported that although caspases and apoptosis were important in initiating the acetaminophen-induced liver injury, the apoptotic pathway was only incompletely activated in response to acetaminophen treatment, and instead it degenerated to induce premature secondary necrosis [47].

In conclusion, MPT can explain the mechanism for acetaminophen-induced liver injury in vivo in mice, which is based on the consensus that mitochondria are the important target in the acetaminophen hepatotoxicity. NAPQI is considered to play an important role in the MPT. Suppression of the MPT resulted in protection not only from acetaminophen-induced liver injury but also from the known events closely related to the pathogenesis in the hepatotoxicity. Because the MPT is down-stream of covalent binding of NAPQI to the hepatocellular targets and linked with the secondary oxidative stress, it is suggested that blocking MPT should be a strategy for development of therapeutic agent for the acetaminophen overdose, which can be administrated later than N-acetylcysteine, which contributes to detoxify NAPQI.

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Acknowledgements 

This study was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan.

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PII: S0168-8278(04)00429-5

doi:10.1016/j.jhep.2004.09.015

Journal of Hepatology
Volume 42, Issue 1 , Pages 110-116, January 2005

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