Abstract
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.
Highlights
The formation of hepatocellular protein inclusions consisting of misfolded proteins is frequently observed in chronic metabolic diseases
hematoxylin and eosin (H&E)-stained sections of FFPE mouse liver sections (Fig. 1A) revealed that, compared to controls, during the first week of DDCfeeding centrolobular and midzonal hepatocytes increased in size, and mild and focal inflammation was observed in the lobular parenchyma
In mitochondria (Fig. 3B) we found a 2.5-fold increase during the first 2 weeks, subsequently increasing to 4-fold concentration compared to controls. 8-OHdG in homogenate, representing a mixture of damage to nuclear and mitochondrial DNA, increased 2.5-fold after 5 weeks and 4-fold thereafter (Fig. 3C). 8-OHdG in mtDNA alone increased almost 2fold already after one week, subsequently remaining at this level (Fig. 3D)
Summary
The formation of hepatocellular protein inclusions consisting of misfolded proteins is frequently observed in chronic metabolic diseases In human steatohepatitis such inclusions are called Mallory-Denk bodies (MDBs) and consist of misfolded cytoskeletal keratins, sequestosome 1 (Sqstm1/p62), heat shock proteins, and others [1,2,3]. MDBs are regarded as a characteristic feature of human steatohepatitis, together with other morphological changes, such as disruption of the keratin intermediate filament cytoskeleton, hepatocellular ballooning, inflammation and steatosis. DDC induces several of the other characteristics of human steatohepatitis, ballooning, inflammation and intermediate filament disruption It complements other frequently used models, such as methionine-choline-deficient or high-fat diet, both of which do not induce ballooning and formation of MDBs, and in the case of high-fat diet only lead to mild steatosis
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