Transition between Acute and Chronic Hepatotoxicity in Mice Is Associated with Impaired Energy Metabolism and Induction of Mitochondrial Heme Oxygenase-1

<div><p>The formation of protein inclusions is frequently associated with chronic metabolic diseases. In mice, short-term intoxication with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to hepatocellular damage indicated by elevated serum liver enzyme activities, whereas only minor morphological changes are observed. Conversely, chronic administration of DDC for several weeks results in severe morphological damage, characterized by hepatocellular ballooning, disruption of the intermediate filament cytoskeleton, and formation of Mallory-Denk bodies consisting predominantly of misfolded keratins, Sqstm1/p62, and heat shock proteins. To evaluate the mechanistic underpinnings for this dichotomy we dissected the time-course of DDC intoxication for up to 10 weeks. We determined body weight change, serum liver enzyme activities, morphologic alterations, induction of antioxidant response (heme oxygenase-1, HO-1), oxidative damage and ATP content in livers as well as respiration, oxidative damage and the presence and activity of HO-1 in endoplasmic reticulum and mitochondria (mtHO-1). Elevated serum liver enzyme activity and oxidative liver damage were already present at early intoxication stages without further subsequent increase. After 2 weeks of intoxication, mice had transiently lost 9% of their body weight, liver ATP-content was reduced to 58% of controls, succinate-driven respiration was uncoupled from ATP-production and antioxidant response was associated with the appearance of catalytically active mtHO-1. Oxidative damage was associated with both acute and chronic DDC toxicity whereas the onset of chronic intoxication was specifically associated with mitochondrial dysfunction which was maximal after 2 weeks of intoxication. At this transition stage, adaptive responses involving mtHO-1 were induced, indirectly leading to improved respiration and preventing further drop of ATP levels. Our observations clearly demonstrate principally different mechanisms for acute and chronic toxic damage.</p></div

Oxidative damage to liver tissue during DDC-intoxication.

<p>MDA-protein adduct levels (pmol/mg protein) in (A) liver homogenate and (B) mitochondria. Concentrations of 8-OHdG (ng/µg DNA) in (C) liver homogenate, representing total DNA, and (D) mitochondria. Data are mean ± SD from 5 mice per treatment group. Significant differences were found compared to controls (*** p<0.001), but not between treatment groups.</p

Pathomorphological changes in mouse liver tissue during DDC-feeding.

<p>A: Hematoxylin-eosin staining. Pathomorphological changes in mouse liver tissue (white asterisks: portal tracts; black asterisks: central veins; black arrows: fibrosis and ductular reaction). During early stages (1, 2 and 5 weeks) of DDC-feeding, hepatocytes increase in size compared to control. Mild and focal inflammation was observed in the lobular parenchyma. Eight and 10 weeks of DDC-feeding shows additional occurrence of steatosis, hepatocellular ballooning and MDBs (enlarged inset left upper corner: ballooned hepatocyte containing an MDB; left lower corner: hepatocyte with steatosis). B: Double immunofluorescence (red: p62; green: keratin 8/18). In controls only the keratin cytoskeleton network (green) is visible in the hepatocytes, during DDC-intoxication (1–2 weeks) pericellular fibrosis is recognizable; at 5 weeks p62-containing aggregates appear which develop to MDBs, containing p62 and keratin 8/18 (green+red→yellow) at weeks 8 and 10. All scale bars are 20 µm.</p

Histopathological scoring of mouse liver after DDC-intoxication.

<p>Mouse liver sections were stained with H&E (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066094#pone-0066094-g002" target="_blank">Fig. 2A</a>) and scored for hepatocyte damage characteristics by a certified pathologist (C. L.). The average score of 5 animals (4 sections each) is shown together with the significance levels respective to controls (Error bars are SD; n.s.: not significant, * p<0.05; ** p<0.01; *** p<0.001).</p><p>Steatosis (0: <5%, 1: 5–33%, 2: >33–60%, 3: >60% of liver parenchyma).</p><p>Microvesicular steatosis (MVS; 0: none, 1: present).</p><p>Inflammation (0: none, 1: 1 focus per 200× field, 2: 2–4 foci, 3: >4 foci).</p><p>Hepatocyte ballooning (0: none, 1: few, 3: many).</p><p>Mallory-Denk bodies (MDBs; 0: none, 1: few, 2: many).</p

Change of body weight, hepatic ATP content and mitochondrial respiration and respiration control ratio (RCR) by DDC-intoxication.

<p>A: Percent body weight change, compared to week 0, monitored for each mouse over 10 weeks in the control and DDC intoxication groups. Data are mean ± SD of 5 mice per treatment group. B: Hepatic ATP content was quantified in control and DDC-treated mice. Each data point is the mean ± SD from three mice per treatment group. C: Respiration control ratio (RCR) with 5 mM succinate as substrate. D: RCR using 2.5 mM each glutamate/malate as substrate. E: Respiration rates with 5 mM succinate in absence (state4) and presence (state3) of 250 µM ADP. F: Respiration rates with 2.5 mM each glutamate/malate in absence (state4) and presence (state3) of 250 µM ADP. Data are mean ± SD from 6 mice per treatment group. Significances are shown versus controls: * p<0.05; ** p<0.01.</p

Expression of Nrf2 and heme oxygenase 1 in livers of DDC-treated mice.

<p>A: Nrf2 protein expression and (B) densitometry in hepatic nuclear fractions. The intensity of protein bands was quantified, and individual blot densities were normalized to loading control (Lamin A/C) C: Heme oxygenase-1 (HO-1) protein expression and (D) densitometry in total liver homogenate (HO-1) and mitochondrial fractions (mtHO-1). The intensity of protein bands was quantified, and individual blot densities were normalized to loading control (calnexin for total homogenate, Ponceau S for the mitochondrial fraction). (E) Activities of HO-1 in microsomes and mtHO-1 in mitochondria as pmol bilirubin/mg protein/h.</p

Serum parameters for tissue damage in DDC-treated mice.

<p>A: Aspartate transaminase (AST); B: alanine transaminase (ALT); C: alkaline phosphatase (ALP); D: lactate dehydrogenase (LDH). Data are mean ± SD from 6 mice per treatment group. Significances are shown versus controls: * p<0.05; *** p<0.001.</p