Sensitivity of Liver Injury in Heterozygous Sod2 Knockout Mice Treated with Troglitazone or Acetaminophen

Abstract

Recently, it was reported that the intraperitoneal administration of 30 mg/kg/day troglitazone to heterozygous superoxide dismutase 2 gene knockout (Sod2+/−) mice for twenty-eight days caused liver injury, manifested by increased serum ALT activity and hepatic necrosis. Therefore, we evaluated the reproducibility of troglitazone-induced liver injury in Sod2+/− mice, as well as their validity as an animal model with higher sensitivity to mitochondrial toxicity by single-dose treatment with acetaminophen in Sod2+/− mice. Although we conducted a repeated dose toxicity study in Sod2+/− mice treated orally with 300 mg/kg/day troglitazone for twenty-eight days, no hepatocellular necrosis was observed in our study. On the other hand, six hours and twenty-four hours after an administration of 300 mg/kg acetaminophen, plasma ALT activity was significantly increased in Sod2+/− mice, compared to wild-type mice. In particular, six hours after administration, hepatic centrilobular necrosis was observed only in Sod2+/− mice. These results suggest that Sod2+/− mice are valuable as an animal model with higher sensitivity to mitochondrial toxicity. On the other hand, it was suggested that the mitochondrial damage alone might not be the major cause of the troglitazone-induced idiosyncratic liver injury observed in humans.

Introduction

Mitochondria play an important role in ATP production via the Krebs-TCA cycle, the electron transport chain and fatty acid β-oxidation, and apoptosis regulation through cytochrome c release to cytoplasm. Therefore, mitochondria are one of the key organelles in toxicology. Recently, it has been reported that mitochondrial toxicity may be involved in the pathogenesis of idiosyncratic drug-induced liver injury (DILI) (Boelsterli and Lim 2007; Dykens and Will 2007). However, direct evidence of idiosyncratic DILI caused by mitochondrial toxicity has not been observed.

Troglitazone is the first thiazolidinedione developed to treat type 2 diabetes mellitus that works by the activation of peroxisome proliferator–activated receptor γ. However, in spite of acceptable safety profiles in clinical trials, serious cases of idiosyncratic liver injury were reported, and this agent was withdrawn from the market in 2000. In a number of nonclinical safety studies, no symptoms of liver injury were observed with troglitazone exposure. Watanabe et al. (1999) noticed that it was extremely difficult to reproduce liver injury in normal experimental animals. Although animal models with genetic abnormalities were also used in several studies, the authors rarely reproduced the liver injury in these models (Bedoucha et al. 2001; Jia et al. 2000; Watanabe et al. 2000) because of the unknown mechanism of the liver injury by troglitazone. Recently, it was reported that troglitazone induced mitochondrial toxicity, such as mitochondria swelling, decrease in mitochondrial membrane potential, and mitochondrial Ca²⁺ accumulation (Bova et al. 2005; Masubuchi et al. 2006; Tirmenstein et al. 2002). Furthermore, Ong et al. (2007) found increased serum ALT activity and hepatic necrosis in heterozygous superoxide dismutase 2 gene knockout (Sod2+/−) mice administered 30 mg/kg troglitazone intraperitoneally for twenty-eight days.

The Sod2 gene encodes an intramitochondrial antioxidant enzyme that scavenges the superoxide anion radical. Therefore, Sod2+/− mice, which express only 50% of wild-type Sod2 activity, accumulate the consequences of excessive mitochondrial oxidative stress and have a potential to synthesize only 60 % of the amount of ATP of wild-type mice (Ong et al. 2006). It was suggested that these mitochondrial abnormalities result in a decrease in the threshold for liver injury by troglitazone. However, despite the observation of moderate or severe degeneration of hepatocytes in approximately 35% of the Sod2+/− mice administered troglitazone, serum ALT activity was only twice as high as that of Sod2+/− mice administered vehicle and only 1.3 to 1.5 times higher than that of wild-type mice administered troglitazone or vehicle. To reevaluate these observations, we conducted repeated-dose toxicity studies in Sod2+/− mice and wild-type mice treated orally with 300 mg/kg troglitazone daily for twenty-eight days. In addition, acetaminophen has been reported to induce mitochondrial toxicity (Kon et al. 2004; Masubuchi et al. 2005; Reid et al. 2005). Therefore, we hypothesized that Sod2+/− mice are more sensitive to acetaminophen-induced liver injury. To validate the Sod2+/− mouse as an animal model of higher sensitivity to mitochondrial toxicity than the wild-type mouse, we conducted an intraperitoneal single-dose toxicity study in Sod2+/− mice and wild-type mice using acetaminophen.

Materials and Methods

Chemicals

Acetaminophen was purchased from Sigma-Aldrich Japan K. K. (Tokyo, Japan). Troglitazone was synthesized by Sankyo Co., Ltd. (Tokyo, Japan). An amorphous coprecipitate of troglitazone was used for the administration. The administered doses were based on an active drug content of 60.1%. All chemicals and solvents used were of analytical grade.

Animals

The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Sankyo Co., Ltd. B6.129S7-Sod2 ^tm1Leb /J mice (Stock No. 002973) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). This strain had been backcrossed more than five times to C57BL/6 mice. The mice were housed under a specific pathogen-free condition with controlled environmental conditions (23°C ± 1°C, 55% ± 5% relative humidity, twelve-hour dark-light cycle) and free access to water and mouse chow (Certified Rodent Diet 5002, PMI Nutrition International, Inc.). Mice homozygous for the Sod2 ^tm1leb targeted mutation die within twenty-one days of birth (Lebovitz et al. 1996). Therefore, heterozygous mutant (Sod2+/−) mice and littermate wild-type mice, which were produced by breeding Sod2+/− mice to wild-type mice, were used for this study. The genotype was determined by genomic PCR using the mutant allele detectable primer-pair (oIMR0781: 5′-TGT TCT CCT CTT CCT CAT CTC C -3,’ oIMR0782: 5′-ACC CTT TCC AAA TCC TCA GC-3’). In the mutant allele, a 250-bp DNA fragment was amplified by PCR genotyping. The genotype of each individual was accepted when independent PCR genotyping was performed twice and the results were consistent. Male mice were used in all experiments.

Drug Administration and Experimental Design for Troglitazone

The amorphous coprecipitate of troglitazone was suspended in 0.5% carboxymethylcellulose sodium solution (10 mL/kg body weight of 3% troglitazone suspension was given) for oral administration of 300 mg/kg of troglitazone once a day for twenty-eight days. According to New et al. (2007), plasma level (C_max) in mice after a single intraperitoneal administration of 30 mg/kg troglitazone was 9.8 mg/L. On the other hand, it was reported that C_max after a single oral administration of 50 mg/kg and 400 mg/kg troglitazone was 8.78 mg/L and 23.3 mg/L, respectively (Herman et al. 2002). Considering these results, we concluded that the exposure level by a single oral administration of 300 mg/kg troglitazone was higher than that by a single intraperitoneal administration of 30 mg/kg troglitazone. The control animals were given the vehicle (10 mL/kg body weight of 0.5% carboxymethylcellulose sodium solution) alone.

At first, mice that were eight weeks old at the start of drug administration were used in this study. However, Ong et al. (2007) reported that sixteen- to twenty-one-week-old mice were used in their study. Considering the effect of aging, we conducted the second study using thirty-five-week-old mice. For each genotype and each age, two groups were prepared: one was the control group and the other was the troglitazone-treated group. All groups were composed of nine or ten animals. The animals were monitored daily for mortality and clinical signs. After twenty-eight days of treatment, the fed mice were anesthetized with diethyl ether. Blood was collected from the inferior vena cava, and a plasma sample was prepared by centrifuging at 3000 rpm for ten minutes. The liver was quickly excised and weighed. One portion was fixed in 10% neutral buffered formalin for histopathology, and the rest was frozen in liquid nitrogen and stored at −80°C in a deep freezer until use.

Drug Administration and Experimental Design for Acetaminophen

For each group, five or six mice aged ten to seventeen weeks were used in the experiment. Acetaminophen is soluble in saline at 37°C for one hour for intraperitoneal administration. The mice were given 20 mL/kg body weight of 10 mg/mL or 15 mg/mL acetaminophen solution at 200 mg/kg or 300 mg/kg, respectively. The control animals were given 20 mL/kg saline. At one hour, six hours, and twenty-four hours after administration, blood was collected from the inferior vena cava of the anesthetized mice, which were then killed to obtain their livers. Plasma samples were prepared by centrifugation at 3000 rpm for ten minutes and were then used for blood chemical analysis. One portion of the liver was fixed in 10% neutral buffered formalin for histopathology, and the rest was frozen in liquid nitrogen and stored at −80°C in a deep freezer until use.

Blood Chemical and Histopathological Examination

Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bilirubin (T.BIL) were determined using the plasma samples with an autoanalyzer (TBA-200FR, Toshiba Medical Systems Co., Ltd., Tochigi, Japan). Histopathological specimens of the liver were prepared according to routine procedures. These specimens were stained with hematoxylin-eosin and observed under a light microscope.

Hepatic and Mitochondrial GSH Content

Liver homogenates were prepared by homogenization in 50 g/L metaphosphoric acid. The homogenate was centrifuged at 3000 g for ten minutes at 4°C. The clear upper aqueous layer was used for the reduced glutathione (GSH) measurement. Mitochondria from liver tissue were isolated using a Mitochondria Isolation Kit for Tissue (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. A mitochondria pellet was suspended in 50 g/L metaphosphoric acid and homogenized using a polytron homogenizer. The homogenate was centrifuged at 3000 g for ten minutes at 4°C. The clear upper aqueous layer was used in the GSH measurement, and the precipitate was used in the protein measurement. The protein precipitate was dissolved in 1 mL of protein lysis buffer containing 30 mM Tris-HCl, 7 M urea, 2 M thiourea, 5 mM magnesium acetate, and 4% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-l- propane-sulfonate (CHAPS). The protein content was measured using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The GSH content was measured using a BIOXYTECH GSH-400 (OXIS International Inc., Foster City, CA, USA) according to the manufacturer’s instructions.

Cytotoxicity of Acetaminophen in Primary Cultured Hepatocytes

Primary cultured hepatocytes from the Sod2+/− mice and wild-type mice were isolated using a two-step collagenase perfusion method. After the mice were anesthetized with sodium pentobarbital, the liver was perfused in situ with Liver Perfusion Medium (Invitrogen, Carlsbad, CA, USA) for five minutes at 40°C, followed by perfusion with 1 mg/mL collagenase (type I, Wako Pure Chemical Industries, Ltd., Osaka, Japan) in Hank’s buffer for seven minutes at 40°C. The perfusate flow was adjusted to 10 mL/min. After removing nonviable cells by Percoll treatment and washing the viable hepatocytes with Krebs-Henseleit buffer, they were resuspended in modified William’s E medium (Invitrogen) in the presence of 10% FBS, 50 units/mL penicillin, 50 μg/mL streptomycin, 0.1 μM dexamethasone, and ITS+ premix (BD Biosciences, San Jose, CA, USA). The cell viability was determined by trypan blue exclusion. More than 90% of the cells were used in the study. Hepatocytes (2 x 10⁵ cells/well) were plated in a collagen-coated twenty-four-well plate. After six hours of incubation at 37°C in 5% CO₂, cells were overlaid with ice-cold 2.5% Matrigel (BD Biosciences) in William’s E medium at 0.5 mL/well. The cultures were maintained in FBS-free medium for twenty hours, and then 10 mM acetaminophen in modified William’s medium was exposed to the hepatocytes for twenty-four hours. The viability, cellular dehydrogenase activity, and cellular ATP content were assessed by LDH-Cytotoxic Test (Wako Pure Chemical Industries), Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) and CellTiter-Glo Luminescent Cell Viability Assay (Promega K K, Tokyo, Japan), respectively. The viability was measured as 0% and 100% in the treatment with 0.25% Triton X-100 and solvent (DMSO) control, respectively.

Statistical Analyses

All data were analyzed by an F test to evaluate the homogeneity of variance. If the variance was homogeneous, a Student’s t test was applied. If the variance was heterogeneous, an Aspin-Welch’s t test was performed. The value of p < .05 was chosen as an indication of statistical significance. A statistical comparison was performed using statistical software (SAS System Release 8.2, SAS Institute Inc., Cary, NC, USA).

Results

Hepatic Toxicity of Repeated Dosing of Troglitazone to Sod2+/− Mice

No obvious differences in the appearance, body weight, or behavioral activity were detected between Sod2+/− mice and wild-type mice during the administration of 300 mg/kg/day troglitazone (Table 1). The liver weight was slightly, but significantly, increased (111.5%–135.1%) by the administration of troglitazone in all groups, and there were also no differences between Sod2+/− mice and wild-type mice. Plasma ALT activity was also increased (135.9%–156.6%) by the administration of troglitazone in all groups, and there were also no differences between Sod2+/− mice and wild-type mice (Table 2). The administration of troglitazone also contributed to a mild increase in plasma AST and ALP activities in all groups, and again, these liver dysfunction markers were also not different between Sod2+/− mice and wild-type mice. In the histopathological examination, centrilobular hypertrophy of hepatocytes was observed in both Sod2+/− mice and wild-type mice administered troglitazone (Figure 1, Table 3). However, there was no difference in grade between them. Aggregation of lymphocytes, observed in the Sod2+/− mice administered troglitazone and the wild-type mice administered vehicle or troglitazone at thirty-five weeks of age, was assessed to be a change resulting from age. Vacuolation of hepatocytes was observed only in the wild-type mice. No histopathological finding of hepatic necrosis resulting from the administration of troglitazone to Sod2+/−mice was observed.

Hepatic Toxicity of Single Dosing or In Vitro Exposure of Sod2+/− Mice to Acetaminophen

Six hours and twenty-four hours after an administration of 300 mg/kg acetaminophen, plasma ALT activity was significantly increased in Sod2+/− mice, compared to wild-type mice (p < .05 and p < .01, respectively, Figure 2). In particular, six hours after an administration of 300 mg/kg acetaminophen, hepatic centrilobular necrosis was observed only in Sod2+/−mice (Table 4 and Figure 3). Furthermore, twenty-four hours after an administration of 200 mg/kg acetaminophen, a tendency toward increased plasma ALT activity was also observed in Sod2+/− mice. Acetaminophen-induced liver injury is mediated by its reactive metabolite, NAPQI, which is generated by P450 and metabolized by GSH (James et al. 2003). Therefore, we measured the hepatic GSH content one hour and six hours after administration to decide whether the difference in sensitivity of liver injury to acetaminophen depended on the metabolic property of NAPQI. The result showed that the hepatic GSH content was not different between Sod2+/− mice and the wild-type control at the same dosing level, despite a marked reduction by the administration of acetaminophen (Figure 4). This finding suggests that the difference in sensitivity of liver injury by acetaminophen is not a result of a difference in the exposure level of NAPQI. On the other hand, the decrease in mitochondrial GSH level six hours after administration was more pronounced in Sod2+/− mice (51.4%–52.2%, compared to the vehicle control of Sod2+/− mice) than in wild-type mice (75.0%–76.9%, compared to the vehicle control of wild-type mice), despite there being no significance between Sod2+/−mice and wild-type mice administered 300 mg/kg acetaminophen (Figure 5). Considering this result, we supposed that the mitochondria of Sod2+/− mice were exposed to a higher level of oxidative stress.

In primary cultured hepatocytes from Sod2+/− mice, the exposure to 10 mM acetaminophen for twenty-four hours resulted in significantly lower ATP content than that in wild-type mice despite no significant difference in viability and cell activity (Figure 6). Consequently, we speculated that the higher sensitivity to acetaminophen in Sod2+/− mice was a result of ATP depletion by excess oxidative stress.

Discussion

In our study, no symptoms of liver injury were observed in Sod2+/− mice administered 300 mg/kg/day orally for twenty-eight days, which differed from the report of Ong et al. (2007). Mild increase in the liver weight and plasma ALT activity and hypertrophy of the hepatocytes were thought to be caused by the administration of troglitazone, not by the heterozygous disruption of the Sod2 gene, because symptoms of the same degree were observed in both genotypes. Increase in liver weight and hypertrophy of the hepatocytes resulting from the administration of troglitazone (≥ 400 mg/kg/day for thirteen weeks, p.o.) to B6C3F1 mice were also observed in previous reports (McGuire et al. 1997). Even though plasma ALT or ALP was slightly elevated, the histopathological examination demonstrated that oral administration of 300 mg/kg/day troglitazone to Sod2+/− mice for twenty-eight days did not induce hepatic necrosis.

We do not have a reasonable interpretation as to why our results were different from the report of Ong et al. (2007). The differences between our experimental design and the author’s report are summarized as follows. (1) The administration method in this study was an oral treatment because this route is used in a clinical regimen, whereas Ong et al. selected the intraperitoneal route. (2) The test article used in this study was an amorphous coprecipitate of troglitazone, whereas Ong et al. used a crystalline form. (3) The solvent used in this study was carboxymethylcellulose sodium solution, whereas Ong et al. used Solutol HS-15 composed of polyglycol mono- and diesters of 12-hydroxystearic acid and 30% free polyethylene glycol. (4) The ages of the mice used in this study were eight and thirty-five weeks, whereas Ong et al. used sixteen- to twenty-one-week-old mice. (5) The troglitazone dose in this study was 300 mg/kg/day, whereas Ong et al. administered 30 mg/kg/day troglitazone to the animals. It was speculated that some of these factors might be a cause of the difference of the results, although it was irrelevant to the essential mechanism of troglitazone-induced toxicity. We considered that an oral administration of 300 mg/kg/day troglitazone would result in a larger AUC and higher C_max than an intraper-itoneal administration of 30 mg/kg/day troglitazone, based on the reports of Herman et al. (2002) and New et al. (2007), as well as our earlier studies. In fact, we observed an increase in the liver weight and hepatocellular hypertrophy, which was caused by the administration of troglitazone, although Ong et al. (2007) did not mention this in their report. Considering this, we concluded that troglitazone-induced liver injury was not reproduced.

On the other hand, in the acetaminophen 300 mg/kg intra-peritoneal single dose study at six hours, we observed an increase in plasma ALT activity and centrilobular hepatocellular necrosis in Sod2+/− mice, but not in wild-type mice. Correlating with these lesions, the administration of acetaminophen to Sod2+/− mice showed excessive oxidative stress in mitochondria, manifested by a decrease in mitochondrial GSH content. Furthermore, acetaminophen exposure led to a significant decrease in the ATP content of primary cultured hepatocytes from Sod2+/− mice. Consequently, we concluded that Sod2+/− mice were more sensitive to liver injury by acetaminophen. Although Ong et al. (2007) speculated that Sod2+/− mice underwent liver injury resulting from the administration of even a clinical dose for a long period (> twenty-eight days), we succeeded in causing liver injury by the single administration of an overdose of acetaminophen. If it is possible to evaluate the potential for mitochondrial toxicity or DILI by a single administration of an excessive dose to Sod2+/−mice, it will be useful in the toxicological screening of new drug candidates. Now, we are conducting a single-dose toxicity study in Sod2+/− mice treated with drugs that have been reported to have a potential of mitochondrial toxicity or idiosyncratic DILI.

In summary, we concluded that Sod2+/− mice are useful as an animal model with a decreased threshold of liver injury or mitochondrial toxicity through a single-dose toxicity study of acetaminophen by intraperitoneal administration. However, increased plasma ALT activity and hepatic necrosis were not observed in Sod2+/− mice administered 300 mg/kg/day troglitazone orally for twenty-eight days. Therefore, the present study suggested that the mitochondrial damage alone is not the major cause of troglitazone-induced idiosyncratic liver damage observed in humans.