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. 2013 Jan 15;266(2):224-32.
doi: 10.1016/j.taap.2012.11.019. Epub 2012 Nov 29.

Acetaminophen-induced Acute Liver Injury in HCV Transgenic Mice

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Free PMC article

Acetaminophen-induced Acute Liver Injury in HCV Transgenic Mice

Takeki Uehara et al. Toxicol Appl Pharmacol. .
Free PMC article

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 24h 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.

Conflict of interest statement

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

Figures

Figure 1
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
(A) Representative photomicrographs of the liver sections (H&E, 100×). Asterisks indicate centrilobular necrosis with inflammatory cellular infiltration. CV, central vein. (B) Serum ALT levels. (C) Liver necrosis score. (D) Serum expression of miR122 and miR192 relative to levels in wild type control animals. Treatment groups are denoted as indicated in the graphical legend. Data shown are mean±SE (n=5–10/group). Asterisks denote statistical significance from other groups (as indicated by the lines and brackets) at p<0.05.
Figure 2
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
(A–B) Liver immunohistochemistry for F4/80. (A) Representative photomicrographs of the liver section (100×); CV, central vein. (B) F4/80 labeling index. The number of F4/80-positive cells per 600× centrilobular field was determined under light microscopy (mean±SE, n = 5–10/group). Five centrilobular areas were counted per tissue sample. (C) Representative photomicrographs (100×) of immunohistochemical detection of 4-HNE in mouse liver. (D–E) Liver expression of Hmox1 and Chop, relative to levels in wild type control animals. Treatment groups are denoted as indicated in the graphical legend. Data shown are mean±SE (n=4–5/group). Asterisks indicate significant differences between APAP-treated and corresponding control groups (p<0.05).
Figure 3
Figure 3. Protein radical formation in APAP-treated mouse liver
(A) Liver sections of DMPO-exposed mice, treated with APAP or vehicle, immunolocalized by anti-DMPO antibody (red) by confocal laser scanning microscopy (40× magnification). Bottom panel shows the overlay of DAPI-stained (nucleic acids stained in blue) and anti-DMPO-stained serial liver sections. (B) Representative magnified image (40× zoom) of the liver section from HCV-Tg mice treated with APAP showing protein radical formation (anti-DMPO; red) in the centrolobular region. (C) Quantification of anti-DMPO fluorescence intensities in WT and HCV-Tg with or without APAP treatment. Treatment groups are denoted as indicated in the graphical legend. Data shown are mean±SE (n=3/group) expressed as percent change over wild type control groups. Asterisks indicate significant differences between APAP-treated and corresponding control groups (p<0.05).
Figure 4
Figure 4. Liver toxicity phenotypes 24 hrs after a single dose of acetaminophen in fasted and fed wild type and HCV-Tg mice
(A) Serum ALT levels and liver necrosis scores in fasted mice treated with 200 mg/kg acetaminophen. (B) Serum ALT levels and liver necrosis scores in fed mice treated with 500 mg/kg acetaminophen. Treatment groups are denoted as indicated in the graphical legend. Data shown are mean±SE (n=3–5/group). Asterisks denote statistical significance from other groups (as indicated by the lines and brackets) at p<0.05.
Figure 5
Figure 5. Liver toxicity phenotypes 4 hrs after a single dose of APAP in wild type and HCV-Tg mice
(A) Representative photomicrographs of the liver section (H&E, 100×), CV, central vein. (B) Serum ALT level (mean±SE, n=6/group). (C) Total and oxidized (GSSG) glutathione levels, and the GSH/GSSG ratio in isolated liver mitochondria (mean±SE, n=3/group). Treatment groups are denoted as indicated in the graphical legend. Asterisks indicate significant differences between groups (p<0.05).
Figure 6
Figure 6. Fluxomic analysis of liver energy metabolism
(A) Labeling of metabolites from [U-13C]glucose via pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC). [4,5-13C]Glutamate is exclusively formed via PDH by the first turn of the Krebs cycle where the isotopomer [2,3-13C]glutamate is exclusively formed via PC. Repeat turns of the Krebs cycle result in related 13C isotopomers of glutamine, but these are at low fractional enrichment and can be neglected (see Supplementary Information for details). (B) A representative 13C NMR spectrum of liver from wild-type control with enlargements of the NMR peaks representing the C2 and C4 positions of glutamine and glutamate (inset). This illustrates the 13C-13C couplings of the peaks representing the 13C isotopomers, 2,3-13C-glutamine, 2,3-13C-glutamate, and 4,5-13C-glutamine. The assignment of isotopomers is based on comparison with coupling constants from the literature (Carvalho et al., 1999).

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