Exacerbation of gefitinib-induced liver injury by glutathione reduction in mice

Abstract

Gefitinib (GEF) is the first selective tyrosine kinase inhibitor of epidermal growth factor receptor. It is associated with the occurrence of clinical drug-induced liver injury. Although GEF is metabolized to chemically reactive metabolites by cytochrome P450 3A and 1A enzymes and then conjugated to glutathione (GSH), whether these reactive metabolites contribute to GEF-induced toxicity remains unknown. In this study, we investigated whether GSH depletion can sensitize mice to liver injury caused by GEF. Male C57BL/6J mice were intraperitoneally pretreated with L-buthionine (S,R)-sulfoximine (BSO) at 700 mg/kg to inhibit GSH synthesis and then orally administered GEF at 500 mg/kg every 24 hr for 4 consecutive days. The coadministration of BSO and GEF increased plasma alanine aminotransferase (ALT) levels to approximately 700 U/L and 1600 U/L at 72 and 96 hr after the first administration, respectively, whereas the increase in plasma ALT levels in mice receiving GEF at 500 mg/kg alone was limited, suggesting that GSH plays a protective role in GEF-induced liver injury. Histological examination showed nuclear karyorrhexis and sporadic single hepatocyte death in the livers of BSO+GEF coadministered mice. In these mice, the hepatic expression levels of heme oxygenase 1 (Hmox1) and metallothionein 2 (Mt2) mRNA, caspase 3/7 enzymatic activity, and the amounts of 2-thiobarbiuric acid reactive substances were significantly increased, suggesting the presence of oxidative stress, which may be associated with hepatocellular death. Together, these results show that oxidative stress as well as the reactive metabolites of GEF are involved in GEF-induced liver injury in GSH-depleted mice.

INTRODUCTION

Gefitinib (GEF) is a small molecule first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) that was approved in Japan in 2002 and has been widely used for the treatment of non-small cell lung cancer (NSCLC) (Cataldo et al., 2011). EGFR TKIs bind to the ATP-binding site of the tyrosine kinase domain of EGFR, which inhibits the catalytic activity of the kinase, thereby inhibiting downstream signaling pathways responsible for cell proliferation. For the treatment of NSCLC, other EGFR TKIs, erlotinib and afatinib, were also subsequently approved, and their effectiveness in improving progression-free survival has been demonstrated, especially for patients harboring EGFR mutations, such as exon 19 deletions or exon 21 (Leu858Arg) substitution (Lee et al., 2013; Lynch et al., 2004). Despite their effectiveness, EGFR TKIs are associated with adverse events such as skin rashes, diarrhea, interstitial pneumonia, and hepatotoxicity, with which less than 10% of patients discontinue the treatment (Ding et al., 2017).

Most cases of hepatotoxicity associated with GEF present as asymptomatic increases in aminotransferase levels with an incidence of 55% to 70% (Maemondo et al., 2010; Mitsudomi et al., 2010). However, grade 3/4 hepatotoxicity develops in patients (18.5%) who are treated with GEF, which is higher than that in patients treated with erlotinib and afatinib (Takeda et al., 2015; Yang et al., 2017). Hepatocellular necrosis has been observed in the liver biopsy of a patient who developed GEF-induced liver injury (Ho et al., 2005). Thus, among EGFR TKIs, GEF is highly associated with liver injury.

GEF is mainly cleared by metabolism, and the O-demethylated form, O,N-dealkylated form, oxidative defluoridated form and 5 other metabolites are detected in human plasma (McKillop et al., 2004a). O-demethylated GEF is the main metabolite detected in human plasma and is catalyzed by CYP2D6; the rest of the metabolites are catalyzed by CYP3A4 or CYP1A1 (McKillop et al., 2004a). Through metabolism, two quinone imine intermediates, which are chemically unstable and thus can react with nucleophiles such as protein and glutathione (GSH), are formed (Li et al., 2009) (Fig. 1). GSH is an endogenous thiol tripeptide abundant in many tissues where it reacts with free radicals and peroxides to protect cells from oxidative damage (Circu and Aw, 2010; Forman et al., 2009). GSH plays protective roles against acetaminophen (APAP)-induced liver injury by scavenging the reactive metabolite of APAP, N-acetyl-p-benzoquinone imine, in the pathogenesis of APAP-induced liver injury (James et al., 2003). However, the toxicological roles of reactive metabolites of GEF remain unknown.

Fig. 1

Metabolic pathway of gefitinib (GEF) in humans. GEF undergoes oxidative metabolism to produce reactive intermediates. The reactive intermediates can react with nucleophiles such as protein, DNA and GSH. Note that these metabolic pathways are part of the GEF metabolic pathway. SG, glutathionate. Adapted from Li et al. (2009): Chem. Res. Toxicol., 22,1736-1742.

A better understanding of the pathogenesis of GEF-induced liver injury is needed to guide the development of effective therapies or prevention strategies during therapy or the future development of drugs that have a reduced risk of hepatotoxicity. In this study, we hypothesized that reactive intermediates of GEF could be associated with the pathogenesis of GEF-induced liver injury. First, we administered GEF at a dose that is insufficient to induce liver injury in mice under GSH depletion conditions. We further evaluated the possible mechanism of liver injury by hepatic mRNA expression analysis.

MATERIALS AND METHODS

Animals and ethics statement

Male C57BL/6J mice (8 weeks old) were purchased from Japan SLC (Shizuoka, Japan). The mice were housed in a controlled environment (temperature 23°C ± 2°C, humidity 55% ± 10%, and 12-hr lights on/off cycle) in the institutional animal facility, with ad libitum access to food (CE-2, CLEA Japan, Tokyo, Japan) and water, and they were acclimated to our facility before the experiments began. All animal experiments were approved by the Animal Care Committee of Nagoya University Graduate School of Medicine and were conducted in accordance with the guidelines established by the Institute for Laboratory Animal Research of the Medical School of Nagoya University.

Drug administration

For the GEF dose-escalation study, mice were orally administered GEF (Wako Pure Chemical Industries, Osaka, Japan) suspended in vehicle comprised of dimethylsulfoxide (Wako Pure Chemical Industries)/Cremophor^® EL (Sigma-Aldrich, St. Louis, MO, USA)/water (1/1/8, v/v/v; 10 mL/kg) at dosages of 100, 250, 500, and 750 mg/kg body weight/day every 24 hr for 2 days. Before (0) and 24 hr after the first GEF administration, blood was collected from the jugular vein in the presence of disodium EDTA at a final concentration of 1 mg/mL as an anticoagulant. At 48 hr, blood was collected from the abdominal vein, and the mice were euthanized by exsanguination under isoflurane anesthesia, after which the liver was collected. These dosages were selected based on our preliminary study in which two out of five animals were dead by the single administration of GEF at 1000 mg/kg.

For the combination study of GEF with BSO, mice were intraperitoneally pretreated with L-buthionine (S,R)-sulfoximine (BSO; Sigma-Aldrich) that was dissolved in saline at 70 mg/mL at a dosage of 700 mg/kg body weight/day for 4 days under nonfasting conditions. The 700 mg/kg dose of BSO was selected based on a previous report (Shimizu et al., 2009). One hour after the BSO injection, the mice were orally administered GEF at a dosage of 500 mg/kg/day for 4 days. At 0 (pre), 24, 48, and 72 hr after the first GEF administration, blood was collected from the jugular vein. At 96 hr, blood was collected from the abdominal vein, and the mice were euthanized as described above. Plasma was prepared from these blood samples by centrifugation at 1600 × g at 4°C for 10 min. A piece of each left lateral lobe of the liver was fixed in 10% neutral buffered formalin (Wako Pure Chemical Industries) or in 2.5% glutaraldehyde in PBS (TAAB Laboratories Equipment, Berks, UK) and used for hematoxylin and eosin (H&E) staining and transmission electron microscopy, respectively, as described below. The remaining liver tissue was snap frozen in liquid nitrogen and stored at -80°C until use.

H&E staining

The formalin-fixed liver specimens were embedded in paraffin and sectioned at a 2-µm thickness. The sections were deparaffinized by xylene and rehydrated in a graded series of ethanol and stained with new hematoxylin type M (Muto Pure Chemicals, Tokyo, Japan) for 10 min and eosin Y (Muto Pure Chemicals) for 2 min at room temperature. The H&E-stained sections were observed using an FSX100 inverted microscope (Olympus, Tokyo, Japan).

Transmission electron microscopy

The glutaraldehyde-fixed liver tissues were further fixed with 2% osmium tetroxide in PBS (TAAB Laboratories Equipment) at 4°C for 2 hr. The tissues were dehydrated in a graded series of ethanol and 100% propylene oxide (Wako Pure Chemical Industries). Liquid epoxy was prepared by mixing 12.65 mL of epoxy 812 resin (TAAB Laboratories Equipment), 5.575 mL of dodecenylsuccinic anhydride (TAAB Laboratories Equipment), 6.8 mL of methyl nadic anhydride (TAAB Laboratories Equipment), and 0.375 mL of 2,4,6-tris(dimethylaminomethyl)phenol (TAAB Laboratories Equipment). The tissues were immersed in liquid epoxy in inverted embedding capsules (TAAB Laboratories Equipment), and the liquid epoxy was solidified at 60°C for 48 hr and subsequently at room temperature. The tissue within the epoxy was trimmed and submitted to ultrathin sectioning at 60 nm using an ultramicrotome UC7 (Leica Microsystems, Wetzlar, Germany). The sections were stained with uranyl acetate (Sigma-Aldrich) for 10 min and with lead citrate (TAAB Laboratories Equipment) for 3 min and were observed by JEM-1400PLUS transmission electron microscopy (JEOL, Tokyo, Japan).

Plasma biochemical analysis

The ALT and AST levels in the plasma were determined using a Fuji Dri-Chem 4000 V analyzer (Fujifilm, Tokyo, Japan) according to the manufacturer’s protocol.

Real-time reverse transcription (RT)-PCR

Total RNA was extracted from the livers of the mice in the combination study of GEF with BSO using RNAiso PLUS (Takara, Shiga, Japan) according to the manufacturer’s instructions. For mRNA analysis, the total RNA was reverse transcribed using a ReverTra Ace^® qPCR RT kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. In brief, 2 µg of total RNA from pooled groups or individual animals was mixed in 5 × RT buffer, RT-enzyme mix, and 1 pmol of the primer mix in a total volume of 20 µL, and the RT reaction was performed at 37°C for 15 min and at 98°C for 5 min using a thermal cycler Dice^® TP800 (Takara). The expression levels of mRNAs were quantified by real-time PCR. The PCR mixture contained 1 µL of the template cDNA, 5 µL of TB Green Premix Ex Taq solution (Tli RNaseH Plus) (Takara), 0.2 µL of 5 × ROX reference dye, and 4 pmol each of the forward and reverse primers in a total volume of 10 µL. All primers were synthesized at Hokkaido System Science (Hokkaido, Japan; Table S1). Real-time PCR was conducted using an Mx3000P thermocycler (Agilent Technologies, Santa Clara, CA, USA), with an initial incubation at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The relative mRNA expression levels were calculated using the comparative threshold cycle (Ct) method with β-Actin as the reference gene.

Caspase 3/7 enzymatic activity and 2-thiobarbiuric acid reactive substances (TBARS)

The livers (10 mg) from the mice in the combination study of GEF with BSO were homogenized in 100 μL of a buffer solution (250 mM sucrose, 10 mM HEPES [pH 7.4]) on ice. The protein concentration in the liver homogenates was determined using a Bio-Rad Protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Five micrograms of the homogenate in 50 µL was mixed with 50 µL of a Caspase-Glo 3/7 reagent (Promega, Madison, WI, USA) in a 96-well plate and incubated at room temperature for 1 hr. The luminescence was measured using an Infinite^® F200 Pro microplate reader (Tecan, Männedorf, Switzerland). The caspase 3/7 enzymatic activity was normalized to the protein concentration of the homogenates. The amount of TBARS was determined in the liver homogenates (50 µL) from the mice in the combination study of GEF with BSO using a TBARS microplate assay kit (Oxford Biomedical Research, Rochester Hills, MI, USA). The fluorescence in a black 96-well plate was measured at 532 nm excitation and 585 nm emission using a Cytation5 microplate reader (BioTek, Winooski, VT, USA). The amount of TBARS was normalized to the protein concentration of the homogenates.

Statistical analysis

Statistical analyses were conducted using JMP Pro^® software version 14.2.0 (SAS Institute, Cary, NC, USA). Comparisons of multiple groups were performed using Dunnett’s test. A value of P < 0.05 was considered to be statistically significant.

RESULTS

Dose-dependent changes in GEF-induced liver injury in mice

To determine the toxic dose of GEF alone, we performed a GEF dose-escalation study. The administration of GEF at 750 mg/kg once daily for two days increased plasma ALT levels to approximately 1000 U/L at 24 hr and 7000 U/L at 48 hr (Fig. 2A). However, the administration of GEF at 100, 250 or 500 mg/kg did not increase plasma ALT levels. Histopathological examination of the H&E-stained livers of mice receiving GEF (750 mg/kg) showed hepatocellular death with nuclear condensation or karyorrhexis (Fig. 2B) whereas no abnormalities were observed in the livers of mice receiving 100, 250, and 500 mg/kg of GEF. Hence, the maximum nontoxic dose of 500 mg/kg was used in the subsequent study to investigate the involvement of reactive metabolites in GEF-induced liver injury under GSH-reduced conditions.

Fig. 2

Dose-dependent changes in plasma ALT levels in mice. Mice were orally administered GEF at 100, 250, 500, or 750 mg/kg at 0 and 24 hr, and the livers were dissected at 48 hr. (A) Plasma ALT levels. Data are shown as the mean ± SEM (n = 4-6). (B) A representative H&E-stained liver section of mice 48 hr after the first GEF administration (750 mg/kg). Arrowheads in the H&E-stained section denote apoptotic bodies. Scale bar: 100 µm. **P < 0.01 vs. the 0-hr group (Dunnett test).

BSO pretreatment exacerbates GEF-induced liver injury in mice

To investigate the toxic response to GEF under GSH-reduced conditions, we performed a combination study of GEF with BSO in mice. Plasma ALT and AST levels were significantly increased at 72 and 96 hr in mice receiving both BSO and GEF (BSO+GEF) once daily for 4 days compared to the vehicle control, although the administration of neither BSO nor GEF alone significantly increased their levels at these time points (Fig. 3A). However, plasma ALT levels in mice receiving GEF alone at 24 and 48 hr were statistically higher than those of vehicle control and tended to gradually increase over time. Gross examination showed that the livers of mice receiving GEF alone were slightly pale and those of BSO+GEF mice were even paler than those of mice receiving GEF alone (Fig. 3B). The livers of mice receiving BSO were slightly darker than those of the vehicle-treated control. H&E-stained liver sections from mice receiving BSO+GEF showed sporadic single hepatocellular death with karyorrhexis but the damage is not confined to either the centrilobular or periportal region (Fig. 3C). Transmission electron micrographs showed the disappearance of microvilli (Fig. 3D, upper) and mitochondrial swelling (Fig. 3D, lower) in the coadministered mice. No abnormalities were observed in the histopathological examinations of any of the vehicle control groups or in mice receiving BSO or GEF.

Fig. 3

Effects of BSO pretreatment on GEF-induced liver injury in mice. Mice were intraperitoneally pretreated with BSO at 700 mg/kg, and after 1 hr, GEF was administered at 500 mg/kg every 24 hr for 4 consecutive days. (A) Plasma ALT and AST levels. Data are shown as the mean ± SEM (n = 4-8 mice). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the vehicle group (Dunnett test). (B-D) Macroscopic photos (B), H&E-stained sections (C), and transmission electron micrographs (D) of the livers. Representative photos are shown. Arrowheads: disappearance of microvilli; arrows: mitochondrial swelling. Scale bars: 100 µm (C), 2 µm (D, upper), and 1 µm (D, lower).

Alteration of mRNA levels, caspase 3/7 enzymatic activity, and TBARS levels in the liver

The mechanism of GEF hepatotoxicity under GSH-reduced conditions was investigated using the liver samples from the mice in the combination study. The mRNA expression levels of 37 genes associated with the apoptosis pathway, endoplasmic reticulum stress, oxidative stress response, inflammatory reaction, and fatty acid and cytokine levels were first determined using pooled hepatic RNA from each treatment group at the time of necropsy (Fig. 4A). The profile indicated that many genes were differentially expressed in the mice receiving both BSO and GEF with induction of oxidative stress markers, Hmox1 and Mt2, and pro-apoptotic genes, Cidec, Cdkn1a, and perforin, and down-regulation of an anti-apoptotic gene Bcl6 and a pro-apoptotic cytokine IFNγ. Since Cdkn1a, Hmox1, and Mt2 levels appeared to be differentially regulated in only BSO+GEF-treated mice, these genes were further examined in individual mice (Fig. 4B). The mRNA levels of Cdkn1a, Hmox1, and Mt2 were upregulated in only mice receiving both BSO and GEF, suggesting that oxidative stress and cell cycle arrest may be associated with liver injury. The hepatic caspase 3/7 enzymatic activity was significantly increased in mice receiving both BSO and GEF compared to that in mice receiving vehicle (Fig. 4C). The amount of TBARS, which are degeneration byproducts, including malondialdehyde, of lipid peroxidation and are considered an oxidative stress marker, was also significantly increased in mice receiving both BSO and GEF compared to that in mice receiving vehicle (Fig. 4D). Thus, oxidative stress and an apoptosis pathway is considered to be involved in liver injury in mice receiving BSO and GEF.

Fig. 4

Hepatic mRNA levels, caspase 3/7 enzymatic activity, and amount of TBARS. Mice were intraperitoneally pretreated with BSO at 700 mg/kg, and after 1 hr, GEF was administered at 500 mg/kg every 24 hr for 4 consecutive days. (A-B) mRNA expression levels in pooled hepatic RNA of each group (A) and of individual mice (B) at 96 hr after the first administration were determined by RT-qPCR. Each mRNA expression level was normalized to that of β-actin. (C and D) Caspase 3/7 enzymatic activity and amount of TBARS in hepatic homogenates from individual animals were determined by a Caspase-Glo 3/7 assay kit and a TBARS microplate assay kit, respectively. Data are shown as the mean ± SEM (n = 4-8). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the vehicle group (Dunnett test). Cdkn1a, cyclin-dependent kinase 1a; Hmox1, heme oxygenase 1; Mt2, metallothionein 2.

DISCUSSION

GEF is the first-approved EGFR TKI that is used for the treatment of NSCLC patients with EGFR mutations, such as exon 19 deletions or exon 21 (Leu858Arg) substitutions. GEF is associated with a high frequency of asymptomatic ALT elevation and liver injury that occasionally leads to the discontinuation of the use of GEF. Although GEF is metabolized to reactive metabolites (Fig. 1), whether the formation of reactive metabolites is related to GEF-induced liver injury in vivo remains unknown. In this study, we employed mice with GSH reduced by BSO to test this hypothesis.

BSO is an inhibitor of GSH synthesis that acts by irreversibly inhibiting γ-glutamyl-cysteine synthase (γ-GCS), the rate-limiting enzyme for GSH synthesis (Griffith, 1982). BSO decreases cellular GSH content in many organs, including not only the liver but also the kidney, heart, intestine, and skeletal muscle (Watanabe et al., 2003); therefore, GSH-reduced animals or cells are expected to be a good model for detecting potential toxicity caused by xenobiotics. In this study, we demonstrated that the administration of GEF (500 mg/kg) to mice in combination with BSO for 4 consecutive days induced liver injury, whereas the administration of GEF (500 mg/kg) alone did not (Fig. 3), suggesting that a decrease in GSH content is a key factor for GEF-induced liver injury. Reportedly, BSO pretreatment sensitizes rodents to liver injury caused by acetaminophen (Masubuchi et al., 2011), amodiaquine (Shimizu et al., 2009), ticlopidine (Shimizu et al., 2011), and methimazole (Akai et al., 2016; Kobayashi et al., 2012). Furthermore, BSO pretreatment also sensitizes human hepatoma-derived HepaRG cells to cell death when they are cotreated with drugs, such as flutamide and ketoconazole (Xu et al., 2018). Since most of these drugs are known to be metabolized to chemically reactive metabolites (Stepan et al., 2011), it can be deduced that reactive metabolites of GEF are responsible for liver injury.

The metabolism of GEF has been extensively studied both in vitro and in vivo (McKillop et al., 2004a, 2004b, 2004c, 2005; Li et al., 2007). Liu et al. identified 34 metabolites and adducts of GEF in human liver microsomes, most of which were also produced in mouse liver microsomes (Liu et al., 2015). Among the metabolites, two quinone imine intermediates, which are trapped by GSH, were formed (Liu et al., 2015). The formation of quinone imine intermediates from GEF is catalyzed by CYP3A4 and CYP1A1 in humans (Fig. 1), and it is inhibited in Cyp3a-null mice and human liver microsomes treated with ketoconazole, a CYP3A inhibitor (Li et al., 2009). Since quinone imine is highly reactive, a decrease in GSH content by BSO treatment possibly allows the binding of GEF-reactive metabolites to endogenous macromolecules such as proteins, thereby leading to cellular and organ toxicity. This study observed plasma ALT elevation and karyorrhexis, mitochondrial swelling, and hepatocyte microvillus loss in mice receiving both BSO and GEF, supporting the hypothesis that reactive intermediates are associated with hepatotoxicity.

GEF frequently induces liver injury in patients with NSCLC who were previously treated with chemotherapy, such as cisplatin (Sugiura et al., 2013). For the treatment of NSCLC, cisplatin or carboplatin in combination with docetaxel, gemcitabine, irinotecan, paclitaxel, or vinorelbine are used. Because cisplatin and paclitaxel induce CYP3A4 expression (Masuyama et al., 2005; Harmsen et al., 2009), it is inferred that the formation of reactive metabolites produced by CYP3A4 may be increased in humans who were previously treated with these agents. Thus, the induction of CYP3A4 may be a clinical risk factor for GEF-induced liver injury. Furthermore, the induction of CYP1A1 increases GEF-induced apoptosis via intracellular ROS accumulation and mitochondrial dysfunction (Wang et al., 2017). Since CYP1A1 expression is induced by cigarette smoke and the proton pump inhibitor omeprazole (Elsherbiny and Brocks, 2011; Yoshinari et al., 2008), exposure to these agents may increase the risk of GEF-induced liver injury. Although further studies are required, the elucidation of risk factors affecting GEF-induced toxicity could guide development of prevention strategies during drug therapy.

GEF is associated with skin rashes, interstitial pneumonia, and hepatotoxicity. A recent report indicates that EGFR TKI-induced skin rash is due to EGFR/MEK (MAPK/ERK kinase) inhibition and the subsequent increase in Krüppel-like transcription factor 4 (KLF4) and IL-36γ by parental compounds (Satoh et al., 2020). In contrast, GEF-induced hepatotoxicity appears not to be explained by EGFR inhibition because mice lacking EGFR in hepatocytes (conditional liver-specific EGFR-knockout mice) do not display any abnormalities in the liver (Natarajan et al., 2007). Thus, it has been suggested that EGFR inhibition in healthy normal livers does not contribute to liver damage. Our data suggest that oxidative stress is involved in the pathogenesis of GEF-induced liver injury because it is accompanied by the increased hepatic expression of Hmox1 and Mt2. The Hmox1 and Mt2 genes are under the control of the Nrf2 transcription factor, and their expression induction indicates the presence of oxidative stress. In response to oxidative stress, Nrf2 induces the expression of its target genes to protect cells from oxidative stress (Klaassen and Reisman, 2010). In fact, treatment of HepG2 cells with GEF generates malondialdehyde, which is a toxic lipid peroxidation product (Chen et al., 2018). Thus, under reduced hepatic GSH conditions, excessive reactive oxygen species may damage hepatic organelles, including mitochondria. An increase in Cdkn1a mRNA was also observed in the livers of mice receiving BSO and GEF. Cdkn1a is a cyclin-dependent kinase inhibitor that initiates cell cycle arrest between G₁- and S-phase (Dutto et al., 2015). In APAP-induced liver injury, Cdkn1a expression levels are also increased after the development of liver injury (Bhushan and Apte, 2019; Bird et al., 2018). Importantly, the knockdown of Cdkn1a results in the acceleration of liver regeneration without altering the severity of liver injury, suggesting that Cdkn1a impairs liver regeneration after liver injury.

A limitation of this study is that the reactive metabolites responsible for GEF-induced liver injury were not identified. As mentioned above, a number of GEF metabolites are detected in human and mouse liver microsomes; however, reactive intermediates of GEF have not been detected in rat plasma (McKillop et al., 2004c). Further studies using Cyp3a-null mice could answer whether reactive metabolites of GEF are responsible for GEF-induced liver injury.

In conclusion, the present study demonstrated that GEF induces liver injury in GSH-reduced mice. Our data suggest that reactive metabolites cause oxidative stress and that subsequent mitochondrial swelling and hepatocyte apoptosis are possible mechanisms underlying GEF-induced liver injury.

ACKNOWLEDGMENTS

The authors wish to thank Koji Itakura from the Division for Medical Research Engineering, Nagoya University Graduate School of Medicine for the transmission electron microscopy. This work was supported by a Grant-in-Aid for Scientific Research (C) (No. 19K07099) from the Japan Society for the Promotion of Science.

Conflict of interest

The authors declare that there is no conflict of interest.

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