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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Toxicol Appl Pharmacol. Author manuscript; available in PMC 2014 January 15.
Published in final edited form as:
PMCID: PMC3534780
NIHMSID: NIHMS425154

Acetaminophen-induced Acute Liver Injury in HCV Transgenic Mice

Abstract

The exact etiology of clinical cases of acute liver failure is difficult to ascertain and it is likely that various co-morbidity factors play a role. For example, epidemiological evidence suggests that coexistent hepatitis C virus (HCV) infection increased the risk of acetaminophen-induced acute liver injury, and was associated with an increased risk of progression to acute liver failure. However, little is known about possible mechanisms of enhanced acetaminophen hepatotoxicity in HCV-infected subjects. In this study, we tested a hypothesis that HCV-Tg mice may be more susceptible to acetaminophen hepatotoxicity, and also evaluated the mechanisms of acetaminophen-induced liver damage in wild type and HCV-Tg mice expressing core, E1 and E2 proteins. Male mice were treated with a single dose of acetaminophen (300 or 500 mg/kg in fed animals; or 200 mg/kg in fasted animals; i.g.) and liver and serum endpoints were evaluated at 4 and 24 hrs after dosing. Our results suggest that in fed mice, liver toxicity in HCV-Tg mice is not markedly exaggerated as compared to the wild-type mice. In fasted mice, greater liver injury was observed in HCV-Tg mice. In fed mice dosed with 300 mg/kg acetaminophen, we observed that liver mitochondria in HCV-Tg mice exhibited signs of dysfunction showing the potential mechanism for increased susceptibility.

Keywords: Hepatitis C virus, acetaminophen, liver, mouse, mitochondria, oxidative stress

Introduction

The prevalence of HCV infection is approximately 2.2–3.0% worldwide (130–170 million people), with the highest infection rates found in the African and the Eastern Mediterranean region (Lavanchy, 2009). After the asymptomatic phase, HCV infection develops into a chronic liver disease in most infected patients and results in liver damage, fibrosis, cirrhosis, liver failure and hepatocellular carcinoma (Tan et al., 2008). Despite major advances in diagnosis and treatment of HCV infection, it remains a growing public health issue in both developed and developing countries (Tsai and Chung, 2010). In the United States, the prevalence of HCV infection in the general population is similar to that worldwide; however, an increase in disease burden over the next decade is expected to occur (Kershenobich et al., 2011).

Acetaminophen (paracetamol, N-acetyl-para-aminophenol) is one of the most widely used pharmaceutical analgesic and antipyretic agents. While it is fairly inexpensive and effective for treatment of common aches and colds, an overdose of acetaminophen can cause acute liver injury (Larson et al., 2005). For example, more than a third of all cases involving acute liver failure in the United States alone are due to overdose of acetaminophen (Lee, 2003); and the majority of these cases are unintentional overdose or involve chronic intake (Kaplowitz, 2005). In addition, it has been estimated that 10% of patients experiencing liver failure due to acetaminophen were taking the drug at recommended doses (Lee, 2007). Even in healthy adult volunteers who received the maximum therapeutic dose for up to 14 days, about one-third exhibited transient, asymptomatic elevations in serum alanine aminotransferase (ALT) levels (Watkins et al., 2006; Harrill et al., 2009). At the same time, several clinical studies and comprehensive reviews of clinical data provide no evidence that acetaminophen, when used as directed, is associated with liver injury (Dart et al., 2010; Lavonas et al., 2010).

Liver injury due to acetaminophen is a complex phenotype, requiring accumulation of its reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalent binding to cellular proteins, oxidative stress, and hepatocellular necrosis, as well as an imbalance between protective and injurious cytokines (Jaeschke and Bajt, 2006). Many factors may facilitate acetaminophen-induced acute liver injury such as excessive alcohol consumption, concomitant use of narcotics, co-treatment with cytochrome P450 2E1 enzyme-inducing xenobiotics, or drugs affecting the glucuronidation pathway (Schmidt et al., 2002; Larson et al., 2005).

A re-analysis of the acetaminophen overdose cases from the Nationwide Inpatient Sample database (1998–2005) in the United States showed that coexistent HCV infection potentiated the hepatotoxicity of acetaminophen (Nguyen et al., 2008). Specifically, HCV significantly increased the risk of acute liver injury, and was also associated with an increased risk of progression to acute liver failure; however, there is no information concerning possible mechanisms of enhanced acetaminophen hepatotoxicity in HCV-infected subjects. Human studies on acetaminophen overdose subjects who are HCV-positive may be difficult because coexistent HCV is present in only a small proportion of patients admitted for acetaminophen toxicity. Thus, HCV transgenic (HCV-Tg) mouse models, in which the HCV core and/or other proteins are expressed (Koike et al., 2010), may present a feasible alternative experimental model.

In this study, we hypothesized that HCV-Tg mice may be more susceptible to acetaminophen hepatotoxicity and that the mechanisms for co-morbidity may be elucidated in an animal model. Male HCV-Tg mice expressing core, E1 and E2 proteins (Lerat et al., 2002) and wild-type C57BL/6J mice were gavaged with a single dose of acetaminophen (300 or 500 mg/kg under fed conditions, or 200 mg/kg under fasted conditions) and liver toxicity was evaluated at 4 and 24 hrs after dosing. While this study shows that liver toxicity in HCV-Tg mice is not markedly exaggerated as compared to the wild-type mice, except when the animals are fasted, we observed that liver mitochondria in HCV-Tg mice exhibited signs of dysfunction in response to acetaminophen treatment.

Materials and Methods

Animals and experimental design

Male HCV-Tg mice (SL-139 strain, 15–30 weeks old) containing the structural genes (core, E1, E2, and p7, nucleotides 342–2771) of HCV genotype 1b, strain N, under the control of the murine albumin promoter/enhancer on C57BL/6J background (Korenaga et al., 2005) were used in the present study. Transgenic animals were identified after weaning by E2 gene polymerase chain reaction (PCR) on ear DNA as reported previously (Korenaga et al., 2005). Wild type C57BL/6J mice of the matching age were obtained from the Jackson Laboratory (Bar Harbor, ME). All animals were housed in polycarbonate cages on Sani-Chips irradiated hardwood bedding (P. J. Murphy Forest Products, Montville, NJ) and allowed free access to NTP-2000 diet (Zeigler Brothers, Gardners, PA) and water under a 12-h light-dark cycle. All studies were approved by the UNC Institutional Animal Care and Use Committee.

Mice were administered a single dose of acetaminophen (300 mg/kg; 99% pure; Sigma-Aldrich, St Louis, MO; n=8–10 per group) or vehicle (10 ml/kg; 0.5% methyl 2-hydroxyethyl cellulose; Sigma-Aldrich; n=5–7 per group) by oral gavage at approximately 9 a.m. without an overnight fast. Animals were sacrificed 4 or 24 hrs after treatment via exsanguination by cutting both the abdominal aorta and vena cava under anesthesia (Nembutal®; 100 mg/kg, i.p.; Abbott Laboratories, Chicago, IL). Blood samples were collected from the vena cava. The liver was quickly removed following exsanguination, and sections of the left lateral and medial lobes were placed in 10% phosphate-buffered formalin for histopathology. Liver mitochondria were isolated from 100 mg of fresh tissue, using a Mitochondria Isolation Kit for Tissue (Pierce, Rockford, IL) as previously described (Jeannot et al., 2012). Remaining liver tissue was snap frozen in liquid nitrogen and stored at −80°C until used.

Separate groups of male mice (HCV-Tg or C57BL/6J, 20–25 weeks old) were treated with acetaminophen (300 mg/kg, no overnight fast) or vehicle as detailed above. In one experiment, [U-13C]glucose (500 mg/kg, 2.78 mmol/kg; Cambridge Isotopes, Andover, MA) was injected i.p. 3 hrs and 15 min after treatment to evaluate glucose metabolism in the liver following acetaminophen treatment using nuclear magnetic resonance (NMR) spectroscopy as detailed by (Saito et al., 2010) with modifications as detailed below. Animals were sacrificed 4 hrs after dosing with acetaminophen or vehicle, and blood and liver samples were collected as detailed above.

In another experiment, mice were injected with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO, Alexis Biochemicals, San Diego, CA) i.p. in two doses of 1 g/kg (diluted in sterile phosphate-buffered saline, 15 ml/kg) at 2 and 1 hrs before sacrifice at 24 hrs after a single dose of acetaminophen (300 mg/kg, no overnight fast) or vehicle. Serum ALT and aspartate aminotransferase levels were determined using Vitro350 analyzer (Ortho-Clinical Diagnostic, Rochester, NY).

A separate experiment was conducted to evaluate the role of fasting. Groups of male mice (HCV-Tg or C57BL/6J, 8–10 weeks old) were treated with acetaminophen (200 mg/kg in mice fasted overnight before treatment, or 500 mg/kg in fed animals) or vehicle as detailed above. Animals were sacrificed 24 hrs after dosing with acetaminophen or vehicle, and blood and liver samples were collected as detailed above.

Mitochondria glutathione measurements

Levels of total, reduced (GSH) and oxidized (GSSG) glutathione were assessed in mitochondria (4 hrs samples only) using high-performance liquid chromatography with coulometric electrochemical detection as described previously (James et al., 2009).

Liver histopathology and immunohistochemistry protocol

Liver tissues were stored in formalin solution for 72 hrs and then transferred to 70% ethanol. Formalin-fixed liver tissue was then embedded in paraffin, and 5 μm sections were mounted on glass slides. Sections were stained with hematoxylin and eosin, and examined under light microscopy. Extent of liver necrosis was quantified by a point counting technique (Bammler et al., 2005). For immunohistochemistry, formalin fixed, paraffin-embedded liver sections were deparaffinized, rehydrated, hydrolyzed in 4N HCl at 37°C for 20 min, incubated in pepsin solution (Dako Cytomation, Carpinteria, CA) at 37°C for 20 min, and Peroxidase Blocking Reagent (Dako) for 5 min at 37°C. Dako EnVision System HRP kit (all antibodies were diluted by saline containing 1 % bovine serum albumin) was used for the detection of 4-hydroxynonenal (rabbit anti-4-hydroxynonenal (HNE, Alpha Diagnostics, San Antonio, TX, 1:200, 30 min) or F4/80 (rat anti-mouse F4/80 antibody, Serotec, Raleigh, NC, 1:100 dilution, 60 min). The reaction products were visualized using diaminobenzidine, sections were counter-stained with hematoxylin, dehydrated and mounted. The number of F4/80-positive cells per 200× centrilobular field was determined under light microscopy. Five centrilobular areas were counted per tissue sample.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

RNA was extracted from the 30 mg of tissue derived from the left lobe of liver samples using the Qiagen RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. RNA concentration was measured using ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and quality was verified using 2100 Bio-Analyzer (Agilent Technologies, Santa Clara, CA). Ten micrograms of total RNA was reverse transcribed in a final volume of 100 μl using the High Capacity Archive kit and random hexamers (Applied Biosystems, Foster City, CA), diluted 4-fold in water and stored at −80°C. For each sample, 2 μl of cDNA, corresponding to 25 ng of reverse transcribed RNA, was analyzed in duplicate, using the LightCycler®480 Instrument (Roche Applied Science, Indianapolis, IN). Following primers were obtained from Applied Biosystems: heme oxygenase (decycling) 1 (Hmox1, Mm00516005_m1) and DNA-damage inducible transcript 3 (Ddit3/Chop, Mm00492097_m1). The relative amount of target gene mRNAs was determined using the 2−ΔΔ CT method (Schmittgen and Livak, 2008) and expression was normalized to internal control TATA box binding protein (Tbp, Mm00446971_m1). Total serum RNA, including microRNA (miRNA), was isolated using QIAzol reagent (Qiagen) according to the manufacturer’s instructions with minor modifications as detailed in (Tryndyak et al., 2012). The efficiency of small RNA isolation was monitored by using spiked miRNA and TaqMan miRNA assays (Applied Biosystems). Total RNA (1.5 μl per reaction) was used for qRT-PCRs of miR-122, miR-34a, miR-200b, miR-192, miR-221, and miR-181a using TaqMan assays (Applied Biosystems), according to the manufacturer’s instructions. The relative amount of each miRNA was measured using the 2−ΔΔCt method and normalized to mmu-miR-16, a ubiquitous non-liver-specific miRNA.

Immuno-spin trapping detection of oxidative stress-induced protein and DNA adducts

A technique of immuno-spin trapping was used to evaluate hepatic oxidative stress levels (Chatterjee et al., 2011) following treatment with acetaminophen. Livers of the mice treated with DMPO before sacrifice (see procedure above) were fixed in 10% neutral buffered formalin, soaked in 30% sucrose for 24 hrs, embedded into OCT (optimal cutting temperature) compound and stored at −80°C. Frozen sections (10 μm) were cut using a frozen tissue processor (Leica Instruments, Bannockburn, IL, USA). Immunohistochemistry was performed as detailed in (Chatterjee et al., 2011) using antibody specific to DMPO nitrone adducts and Alexafluor 568 goat anti-rabbit secondary antibody (Molecular Probes/Invitrogen, Eugene, OR). Confocal images were taken on a Zeiss LSM510-UV Meta microscope (Carl Zeiss, Oberkochen, Germany). The 488-nm line from an argon laser was used for producing polarized light for a DIC image as well as fluorescence excitation of the Alexa 488 secondary antibody. Metamorph imaging software (v.7.7. Molecular Devices LLC, Sunnyvale, CA) was used to calculate the fluorescence intensity of the groups. Specifically, Metamorph was used to open each image and then apply an inclusive threshold between 30–255 gray levels. The total integrated intensity of each thresholded image was then calculated and exported for analysis.

Liver Extraction and NMR sample Preparation

The Folch method was used to extract the water-soluble metabolites (Folch et al., 1957). Aqueous portion of the liver extract were frozen and placed in the speedvac to lyophilize overnite. To each tube was added 30 μl of D2O containing 0.1% NaN3 and 1 mM trimethylsilyl propionic acid. Each tube was sonicated, then vortexed to effect dissolution. The tubes were place in a Fisher Scientific accuSpin Micro 17R centrifuge and spun at 5kG for 20 minutes to collect all the liquid at the bottom of each vial. A Neptune barrier pipette tip was cut off below the filter media and placed in the top of a 9 mm Microliter Analytical sample vial. Each sample was transferred to the top of a barrier pipette tip for filtration by centrifugation to force the sample through the filter media. The samples were spun for 10 minutes at 2200 rpm on a Beckman GS-6R centrifuge. The filter tips were removed and a cap was placed on each vial.

1H and 13C Nuclear Magnetic Resonance (NMR) Spectroscopy

All 1H NMR spectroscopy was performed on a Varian INOVA spectrometer (Agilent, formerly Varian Inc., Palo Alto, CA) operating at 599.64 MHz (1H frequency) and 25°C. The micro-coil probe is a Protasis/MRM 10 ll capillary NMR probe (Magnetic Resonance Micro- sensors, Savoy, IN). Samples were introduced into the micro-coil using a Protasis High-throughput Sample Automation System (Protasis, Marlboro, MA). The pw90 was measured to be 11.1 μsec at tpwr=45. An inversion recovery experiment was acquired to measure the T1 values, which was approximately 2.1 sec. This information was used to calculate the Ernst angle. A 70° flip angle was used of 8 μs with 2048 transients, using a one pulse sequence with a 2 sec presaturation pulse and 3 sec repetition time. For 13C NMR spectroscopy, the same samples run for 1H NMR spectroscopy were dried and resuspended in 25 μl of D2O. All 13C NMR spectroscopy was performed on a Varian INOVA spectrometer (Agilent) operating at 150 MHz (13C frequency) and 25°C. A Varian broadband cryoprobe was used (Magnetic Resonance Micro-sensors, Savoy, IN). A 30° flip angle was used of 2 μs with 13000 transients, using a one pulse sequence with a 3 sec repetition time with WALTZ 1H decoupling during acquisition.

Statistical analysis

Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test. Results were considered significant at p < 0.05.

Results

Liver toxicity was assessed in fed wild type C57BL/6J and HCV-Tg mice at 24 hrs after i.g. administration of a single large dose (300 mg/kg) of acetaminophen. In both mouse groups, liver injury was observed and was histopathologically characterized as centrilobular necrosis with inflammatory cell infiltration (Figure 1A). Serum markers of liver injury, activity of ALT (Figure 1B) and AST (data not shown), were markedly elevated in both WT and HCV-Tg groups receiving acetaminophen. No significant effect was observed on the serum levels of ALP and albumin (data not shown). The extent of liver necrosis, assessed both as a qualitative pathology score, and as a quantitative measure of percent necrotic liver (Figure 1C) was also similar in both wild type and HCV-Tg mice. While Figure 1 presents data from all animals in each group, it should be noted that the characteristic histological changes were observed in 5/10 mice in the wild type group and in 5/8 mice in HCV-Tg group, an observation similar to the known animal-to-animal variability in the extent of acetaminophen-induced liver necrosis in the C57BL/6J mice (Beyer et al., 2007). The incidence of liver necrotic lesions was significantly increased in both APAP-treated groups compared to corresponding control groups (p<0.05).

Figure 1
Liver toxicity phenotypes 24 hrs after a single dose of acetaminophen (300 mg/kg, APAP) in fed wild type (WT) and HCV-Tg mice

Several recent reports have demonstrated that circulating miRNAs may be a sensitive and independent indicator of liver injury (Cermelli et al., 2011; Tryndyak et al., 2012). Hence, we evaluated the level of miRNAs in serum of control and acetaminophen-treated mice. Serum levels of miR122 and miR192 (Figure 1D), as well as miR34a, miR200b, miR221, and miR181a (data not shown) were markedly increased in both acetaminophen-treated groups compared to corresponding control groups. Interestingly, the increase in serum levels of liver-specific miRNAs miR122 and miR192 (Gatti et al., 2011) was significantly greater in acetaminophen-treated HCV-Tg mice than in acetaminophen-treated wild type animals.

It has been suggested that ageing HCV-Tg mice may be prone to developing oxidative stress (Korenaga et al., 2005), we used animals that were 15 and 30 weeks of age in these experiments. No overt liver histopathological changes were found in vehicle-treated HCV-Tg mice of either age group, similar to observations with other HCV-Tg models (Koike et al., 2010). There was no difference in acetaminophen-induced liver injury between different age groups in either wild type or HCV-Tg mice, therefore data on mice of all ages tested are combined in Figures 1 and and22.

Figure 2
Liver markers of inflammation, oxidative and ER stress 24 hrs after a single dose of APAP in wild type and HCV-Tg mice

Activation of the hepatic macrophages and recruitment of inflammatory cells from systemic circulation are known hallmarks of acetaminophen-induced liver injury (Adams et al., 2010). Since it has been suggested that HCV-Tg mice may be susceptible to disease-causing factors through liver inflammation (Korenaga et al., 2005; Nishina et al., 2008), we evaluated the number of mature Kupffer cells in the liver of treated mice (Figure 2A–B). Immunohistochemistry for F4/80, a specific marker of quiescent Kupffer cells, showed a significant decrease in the number of positively labeled cells in the livers of mice treated with acetaminophen. This observation is similar to that reported by (Dambach et al., 2002). While statistical significance was observed in the comparison between acetaminophen-treated and corresponding control groups, there was no significant difference in the comparison between HCV-Tg and wild type mice.

Oxidative stress has been also suggested as a mechanism for increased susceptibility of HCV-Tg mice (Korenaga et al., 2005; Nishina et al., 2008) and acetaminophen is well-known to cause liver injury via reactive oxygen species (Powell et al., 2006). To assess the degree of oxidative stress, several approaches were used. Lipid peroxidation was evaluated using 4-HNE-protein adducts as a marker (Figure 2C). Hepatocytes around the central vein exhibited weakly positive staining of 4-HNE in both vehicle-treated groups. Acetaminophen treatment resulted in an intense staining of hepatocytes, primarily in the centrilobular region with some animals exhibiting positive staining even in mid-lobular and periportal regions. There was no difference in 4-HNE staining intensity or zonality between HCV-Tg and wild type groups. In addition, we determined the mRNA level of heme oxygenase-1 (Hmox1), one of several coordinately regulated proteins involved in protecting against liver injury which is inducible by a variety of hepatotoxicants (Choi et al., 2003). While acetaminophen induced Hmox1 (Figure 2D), the magnitude of the effect was similar between wild type and HCV-Tg mice. Next, we used a method of immuno-spin trapping for detecting and localizing protein free radicals in tissues (Chatterjee et al., 2011). The sites of generation of free radical species in liver tissues post acetaminophen administration were localized with anti-DMPO antibody and confocal microscopy (Figure 3A). Results indicated that the accumulation of the radical adducts was most pronounced in the cells bordering the central vein (Figure 3B) and that the amount of the radical adducts formed was significantly elevated in HCV-Tg mice treated with acetaminophen as compared to vehicle-treated HCV-Tg mice (Figure 3C).

Figure 3
Protein radical formation in APAP-treated mouse liver

Unfolded protein response (endoplasmic reticulum [ER] stress) has been shown to be induced in the liver by a number of toxicants, including acetaminophen (Nagy et al., 2007). In turn, HCV has also been shown to cause ER stress both in vitro (Chan and Egan, 2005) and in vivo (Nishina et al., 2010), yet the latter findings have not been observed in all studies with HCV-Tg mice (Lerat et al., 2009). Thus, we investigated whether HCV-Tg mice exhibit signs of liver ER stress, and whether it may be potentiated by acetaminophen. While acetaminophen caused an increase (Figure 2E) in mRNA for CCAAT/enhancer-binding protein-homologous protein (Chop), as expected (Nagy et al., 2007), there was no difference in expression level of Chop or spliced X-box DNA binding protein-1 (xXbp1, data not shown) between wild type and HCV-Tg mice treated with vehicle or acetaminophen.

In separate experiments, we evaluated the effect of dose and fasting on potential differences in acetaminophen-induced liver injury among wild type and HCV-Tg mice (Figure 4). In animals that were fasted overnight and then treated for 24 hrs with 200 mg/kg of acetaminophen by gavage, we observed significant elevations in the degree of liver necrosis in both groups, and this effect in HCV-Tg animals was significantly greater than that in wild type mice (Figure 4A). In fed animals treated with 500 mg/kg of acetaminophen for 24 hrs (Figure 4B), significant liver necrosis was observed in both wild type and HCV-Tg mice, albeit the differences between wild type and HCV-Tg animals were not significant.

Figure 4
Liver toxicity phenotypes 24 hrs after a single dose of acetaminophen in fasted and fed wild type and HCV-Tg mice

While the histopathological evidence of acetaminophen-induced centrilobular liver injury is most pronounced 24 hrs after administration of 300 mg/kg dose, the effects on liver and mitochondrial glutathione pool occur within hours of dosing (Mitchell et al., 1973). Thus, we also compared acetaminophen’s liver effects at 4 hrs after dosing with 300 mg/kg (in fed animals). As expected, no histopathological signs of overt liver injury were observed in both wild type and HCV-Tg mice (Figure 5A). Serum enzyme markers levels were higher in acetaminophen-treated groups, albeit the changes were not significant (Figure 5B). In liver mitochondria from vehicle-treated HCV-Tg mice, the amount of total glutathione was significantly higher than that in the wild type mice (Figure 5C). Treatment with acetaminophen led to a significant decrease in total mitochondrial GSH in HCV-Tg, but not wild type mice (Figure 5C).

Figure 5
Liver toxicity phenotypes 4 hrs after a single dose of APAP in wild type and HCV-Tg mice

Mitochondria are known to be the target for HCV proteins (Wang and Weinman, 2006) and recent in vitro evidence shows that HCV has a profound effect on cellular metabolism and energy homeostasis (Diamond et al., 2010). Thus, we investigated what is the outcome of HCV-Tg and acetaminophen treatment (300 mg/kg in fed mice) on the mitochondrial energy metabolism using [U-13C]glucose and nuclear magnetic resonance detection of Krebs cycle intermediates (Saito et al., 2010). The relative flux of carbon from [U-13C]glucose through pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) pathways was evaluated (Figure 6A). Specifically, we performed isotopomer analysis of glutamine moieties (Figure 6B) as detailed in (Carvalho et al., 1999). Our results show that in livers of vehicle-treated HCV-Tg mice, as compared to wild type mice, there is a significant elevation in relative flux through PDH as compared to PC (Figure 6C). As shown previously (Saito et al., 2010), acetaminophen treatment does not result in a diminished flux through PDH 4 hrs after treatment with acetaminophen as no change in the relative flux through PC and PDH was observed in either HCV-Tg or wild type mice.

Figure 6
Fluxomic analysis of liver energy metabolism

Discussion

HCV infection leads to progressive liver disease and a variety of underlying mechanisms including oxidative stress, steatosis, impaired immune and apoptotic responses, and iron accumulation have been implicated in the development of steatohepatitis, cirrhosis and hepatocellular carcinomas (Tsai and Chung, 2010). While the majority of HCV-infected individuals do not exhibit signs of liver disease (e.g., elevations of serum enzymes or signs of liver inflammation or fibrosis) (Alberti et al., 2002), it is becoming increasingly clear that even in the absence of clinical signs of liver injury, HCV-infected subjects may be more susceptible to other environmental or biological factors associated with liver disease (Nguyen et al., 2008; Puoti et al., 2010). Acute infection with viruses that target the liver also has been shown to increase the risk of liver injury from acetaminophen (Yaghi et al., 2006; Maddox et al., 2010). Thus, the goal of this study was to use an animal transgenic model of HCV to examine potential susceptibility to acetaminophen-induced liver injury, even though HCV-Tg mice do not exhibit signs of liver inflammation.

Few experimental models may faithfully replicate the pathophysiological state of the liver in HCV carriers who do not exhibit symptoms of liver disease, yet are in need of careful surveillance. On one hand, important discoveries have been made in our understanding of the role that inflammation plays in acute liver injury through co-exposures to a drug of interest and lipopolysaccharide or other inflammogens (Ganey et al., 2004; Jaeschke, 2008; Han et al., 2010). Inflammation is one of the most common pathways suggested as being responsible for interactions that could enhance liver injury (Roth and Ganey, 2010). Chronic inflammation may lead to sensitization to secondary xenobiotic-induced stress because many molecular events involved in the progression of liver injury are shared (Gunawan and Kaplowitz, 2007). However, it is not clear whether an animal model where acute liver injury is introduced as a co-factor is relevant to address the mechanisms of potential susceptibility in patients with chronic HCV infection who show persistently normal ALT levels. On the other hand, transgenic mice expressing various HCV proteins do not show signs of liver injury, inflammation or ER stress, at least in the first 12 months of age (Koike et al., 2010). The HCV-Tg mice are, however, known to be susceptible to exposures promoting liver disease and carcinogenesis (Furutani et al., 2006; Machida et al., 2009; Jeannot et al., 2012). Thus, we posit that while the HCV-Tg mouse model that was used in this study is not one to investigate the inflammation-related potentiation of liver injury, it may be highly relevant for recapitulating the state of the liver in human “asymptomatic” carriers of HCV.

Several experimental designs were used in our study to evaluate putative susceptibility of HCV-Tg mice to acetaminophen. In fed mice, we did not observe a significant difference in the severity of liver histology or serum markers of liver injury between wild type and HCV-Tg mice that were dosed with either 300 or 500 mg/kg of acetaminophen. Importantly, under overnight fasting conditions, a significant difference in liver injury among wildtype and HCV-Tg mice was observed when 200 mg/kg of acetaminophen was administered for 24 hrs.

The mitochondria are an early target for and a sensitive bellwether of acetaminophen-induced liver injury (Heinloth et al., 2004). Acetaminophen-induced mitochondrial dysfunction has been reported in vivo in mice (Jaeschke and Bajt, 2006), in vitro in human metabolically-active HepaRG cells (McGill et al., 2011), and in human subjects (McGill et al., 2012). In addition, mitochondria are a well-known target for HCV proteins (Sheikh et al., 2008). Mitochondrial energy metabolism is affected (Korenaga et al., 2005) and permeability transition induced (Machida et al., 2006) by HCV. Fasting has a profound impact on liver metabolism (Kopelovich and Sabine, 1970) and glutathione levels (Maruyama et al., 1968). Thus, even though greater liver injury was observed in fasted HCV-Tg mice, we reasoned that mechanistic studies of the role of glutathione should be conducted under physiological fed conditions because of the potential central role of mitochondrial glutathione in the mechanism of susceptibility of HCV-Tg mice to acetaminophen-induced liver injury.

Increased oxidative stress was confirmed by intense positive staining of 4-HNE in the livers of both wild type and HCV-Tg mice treated with acetaminophen. Decreased number of F4/80 positive Kupffer cells is explained by the evidence that activation of Kupffer cells due to inflammation changes the pattern of expression of its membrane protein (Dambach et al., 2002). Furthermore, induction of Hmox1, protein involved in the mechanisms of NF-E2-related factor 2 (Nrf2)-related oxidative stress and Chop, a marker for ER stress, was observed in acetaminophen-treated groups. Interestingly, the results from immuno-spin trapping technique showed an increase in peri-central reactive oxygen species production in HCV-Tg mice treated with acetaminophen, but not in wild type animals. The apparent inconsistency between the results from different assays used here is not unexpected. First, we confirm a widely-known observation that oxidative stress is induced in the liver upon treatment with acetaminophen. Second, the immuno-spin trapping technique was used to successfully confirm the precise site in the liver lobule where reactive oxygen species have been postulated to be produced. This technique is assessing oxidant production in a very narrow window of time (2 hrs before sacrifice), while 4-HNE and gene expression assays are reflective of the effects occurring over the entire 24 hr treatment period. Still, a detailed time-course investigation may be necessary to fully characterize the impact and kinetics of HCV-Tg effect on formation of liver reactive oxygen species, a key mechanism of acetaminophen-induced liver injury.

We observed that HCV-Tg mice had higher basal total mitochondrial glutathione content than wild type mice, consistent with the observation that HCV-Tg mice may be prone to mitochondrial oxidative stress (Korenaga et al., 2005). Importantly, mitochondrial glutathione was affected by acetaminophen treatment in HCV-Tg, but not in wild type, mice 4 hrs after dosing. While these results are supportive of the HCV-induced sensitization of the mitochondria to exogenous stressors, we also determined whether mitochondrial energy metabolism may be affected in HCV-Tg mice. Similar to the observations of (Saito et al., 2010), acetaminophen did not result in a diminished flux through pyruvate dehydrogenase in wild type mice. Interestingly, the ratio of flux through pyruvate carboxylase versus pyruvate dehydrogenase was reduced significantly in HCV-Tg mice, as compared to wild type mice, which is indicative of an increase in pyruvate entry into the Krebs cycle via pyruvate dehydrogenase. In turn, this shows that more glucose-derived pyruvate is used for ATP generation rather than amino acid synthesis in HCV-Tg mice, even though the total mitochondrial glutathione pool is greater than that in wild type mice. The increased demand for ATP in HCV-Tg mice is consistent with the negative impact that HCV core protein has on the mitochondrial electron transport (Korenaga et al., 2005).

In summary, this study used HCV-Tg mouse model to investigate the molecular mechanisms that may underlie the clinical observation of an increased risk of acute liver injury following acetaminophen overdose among subjects with coexistent HCV. This study is clinically relevant given the rising prevalence of HCV in the general population and that we used the animal model that is aimed to be reflective of the majority of HCV-infected subjects (Alberti et al., 2002) who do not exhibit signs of liver disease but may be susceptible to co-exposures. While we observed that the degree of acetaminophen-induced liver injury in HCV-Tg mice is not markedly exaggerated as compared to the wild-type mice, except under conditions of fasting, we show that liver mitochondria in HCV-Tg mice exhibited signs of functional strain both prior to, and following a toxic insult. In addition, albeit not addressed by the current study design, a possibility exists that the reported sensitization to acetaminophen in HCV-infected patients may be due to events associated with viral replication, chronic inflammation, or viral proteins other than those tested using the HCV-Tg model that was used. While encouraging, these findings, together with the clinical observation (Nguyen et al., 2008), require further confirmation from prospective clinical and mechanistic studies before they may be considered in clinical decision-making.

Supplementary Material

01

Acknowledgments

Supported, in part, by grants from the National Institutes of Health P42 ES005948 and R01 ES015241. Saurabh Chatterjee was a recipient of NIH K99/R00 award ES019875. The authors wish to acknowledge technical assistance from Drs. John Grimes, Marc ter Horst, and Thomas O’Connell. We are also grateful to Dr. Steven Weinman for providing the HCV-Tg animals and for fruitful discussions.

Abbreviations

HCV
Hepatitis C virus
NAPQI
N-acetyl-p-benzoquinone imine
ALT
alanine aminotransferase
GSH
glutathione
NMR
nuclear magnetic resonance

Footnotes

Conflict of Interest: The authors declare that there are no conflicts of interest.

Note: The views expressed in this manuscript do not necessarily represent those of the U.S. Food and Drug Administration.

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