Yoshiji Asaoka

Nrf2 Activators Attenuate the Progression of Nonalcoholic Steatohepatitis–Related Fibrosis in a Dietary Rat Model

Oxidative stress is considered to be a key mechanism of hepatocellular injury and disease progression in patients with nonalcoholic steatohepatitis (NASH). The transcription factor Nrf2 (nuclear factor-erythroid-2-related factor 2) plays a central role in stimulating expression of various antioxidant-associated genes in the cellular defense against oxidative stress. As the cytosolic repressor kelch-like ECH-associated protein 1 (Keap1) negatively regulates Nrf2, activation of Nrf2 facilitated by its release from Keap1 may represent a promising strategy in the treatment of NASH. To test this hypothesis, we used two chemically distinct types of Nrf2 activator. One is the thiol-reactive agent oltipraz (OPZ), a typical Nrf2 activator, and the other is a novel biaryl urea compound, termed NK-252 (1-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)-3-(pyridin-2-ylmethyl)urea). NK-252 exhibits a greater Nrf2-activating potential than OPZ. Furthermore, in vitro binding studies revealed that NK-252 interacts with the domain containing the Nrf2-binding site of Keap1, whereas OPZ does not. This finding indicates that NK-252 is more potent than OPZ due to its unique mechanism of action. For in vivo animal model studies, we used rats on a choline-deficient L-amino acid–defined (CDAA) diet, which demonstrate pathologic findings similar to those seen in human NASH. The administration of OPZ or NK-252 significantly attenuated the progression of histologic abnormalities in rats on a CDAA diet, especially hepatic fibrosis. In conclusion, by using Nrf2 activators with independent mechanisms of action, we show that, in a rat model of NASH, the activation of Nrf2 is responsible for the antifibrotic effects of these drugs. This strategy of Nrf2 activation presents new opportunities for treatment of NASH patients with hepatic fibrosis.

A possible mechanism for hepatotoxicity induced by BIRB‐796, an orally active p38 mitogen‐activated protein kinase inhibitor

BIRB‐796, a selective inhibitor of p38 mitogen‐activated protein kinase, has entered clinical trials for the treatment of autoimmune diseases. Levels of alanine transaminase, a biomarker of hepatic toxicity in clinical pathology, were found to be increased in Crohns disease patients treated with BIRB‐796. The purpose of the present study was to clarify the molecular mechanism(s) of this hepatotoxicity. A toxicogenomic analysis using a highly sensitive DNA chip, 3D‐Gene™ Mouse Oligo chip 24k, indicated that BIRB‐796 treatment activated the nuclear factor (erythroid‐derived 2)‐like 2 signaling pathway, which plays a key role in the response to oxidative stress. A reactive intermediate of BIRB‐796 was detected by the glutathione‐trapping method using mouse and human liver microsomes. The production of this reactive metabolite in the liver may be one of the causes of BIRB‐796s hepatotoxicity. Copyright

Immunohistochemistry of LAMP-2 and adipophilin for phospholipidosis in liver and kidney in ketoconazole-treated mice

Drug-induced phospholipidosis is an abnormal accumulation of phospholipids in the lysosomes following repeated administration of cationic amphiphilic drugs. Phospholipidosis is detected histopathologically as cytoplasmic vacuolation; however, it is difficult to distinguish from lipid accumulation since their morphological features are similar. In this study, we investigated the usefulness of immunohistochemistry for lysosome-associated membrane protein-2 (LAMP-2) and adipophilin, a membrane protein of cytosolic non-lysosomal lipid droplets, in the liver and kidneys of mice orally administered ketoconazole, an inducer of hepatic phospholipidosis. In 7-week-old mice administered ketoconazole (300 mg/kg/day) for 7 days, cytoplasmic vacuolation was histopathologically observed in centrilobular hepatocytes and proximal tubular epithelial cells under the fasted condition. The cytoplasmic vacuolation consisted of foamy vacuoles, which were revealed to be phospholipidosis-characteristic lamellar bodies by electron microscopy. Furthermore, lipid-like vacuoles were observed in the perilobular hepatocytes, and revealed to be lipid droplets by electron microscopy. In immunohistochemistry, the foamy vacuoles and lipid-like vacuoles were positive for LAMP-2 and adipophilin, respectively. These results indicate that immunohistochemistry for LAMP-2 and adipophilin could distinguish between phospholipidosis and lipid accumulation. Additionally, it could detect ketoconazole-induced phospholipidosis in the glycogen-rich livers of non-fasted mice. In conclusion, ketoconazole induced phospholipidosis in not only the liver but also the kidneys, and immunohistochemistry for LAMP-2 and adipophilin could be useful for the pathological evaluation of drug-induced phospholipidosis in mice.

Histopathological image analysis of chemical-induced hepatocellular hypertrophy in mice.

Chemical-induced hepatocellular hypertrophy is frequently observed in rodents, and is mostly caused by the induction of phase I and phase II drug metabolic enzymes and peroxisomal lipid metabolic enzymes. Liver weight is a sensitive and commonly used marker for detecting hepatocellular hypertrophy, but is also increased by a number of other factors. Histopathological observations subjectively detect changes such as hepatocellular hypertrophy based on the size of a hepatocyte. Therefore, quantitative microscopic observations are required to evaluate histopathological alterations objectively. In the present study, we developed a novel quantitative method for an image analysis of hepatocellular hypertrophy using liver sections stained with hematoxylin and eosin, and demonstrated its usefulness for evaluating hepatocellular hypertrophy induced by phenobarbital (a phase I and phase II enzyme inducer) and clofibrate (a peroxisomal enzyme inducer) in mice. The algorithm of this imaging analysis was designed to recognize an individual hepatocyte through a combination of pixel-based and object-based analyses. Hepatocellular nuclei and the surrounding non-hepatocellular cells were recognized by the pixel-based analysis, while the areas of the recognized hepatocellular nuclei were then expanded until they ran against their expanding neighboring hepatocytes and surrounding non-hepatocellular cells by the object-based analysis. The expanded area of each hepatocellular nucleus was regarded as the size of an individual hepatocyte. The results of this imaging analysis showed that changes in the sizes of hepatocytes corresponded with histopathological observations in phenobarbital and clofibrate-treated mice, and revealed a correlation between hepatocyte size and liver weight. In conclusion, our novel image analysis method is very useful for quantitative evaluations of chemical-induced hepatocellular hypertrophy.

Analysis of gene expression for microminipig liver transcriptomes using parallel long-read technology and short-read sequencing

The microminipig is one of the smallest minipigs that has emerged as a possible experimental animal model, because it shares many anatomical and/or physiological similarities with humans, including the coronary artery distribution in the heart, the digestive physiology, the kidney size and its structure, and so on. However, information on gene expression profiles, including those on drug‐metabolizing phase I and II enzymes, in the microminipig is limited. Therefore, the aim of the present study was to identify transcripts in microminipig livers and to determine gene expression profiles. De novo assembly and expression analyses of microminipig transcripts were conducted with liver samples from three male and three female microminipigs using parallel long‐read and short‐read sequencing technologies. After unique sequences had been automatically aligned by assembling software, the mean contig length of 50843 transcripts was 707 bp. The expression profiles of cytochrome P450 (P450) 1A2, 2C, 2E1 and 3A genes in livers in microminipigs were similar to those in humans. Liver carboxylesterase (CES) precursor, liver CES‐like, UDP‐glucuronosyltransferase (UGT) 2C1‐like, amine sulfotransferase (SULT)‐like, N‐acetyltransferases (NAT8) and glutathione S‐transferase (GST) A2 genes, which are relatively unknown genes in pigs and/or humans, were expressed strongly. Furthermore, no significant gender differences were observed in the gene expression profiles of phase I enzymes, whereas UGT2B17, SULT1E1, SULT2A1, amine SULT‐like, NAT8 and GSTT4 genes were different between males and females among phase II enzyme genes under the present sample conditions. These results provide a foundation for mechanistic studies and the use of microminipigs as model animals for drug development in the future. Copyright

Aminoglutethimide-induced lysosomal changes in adrenal gland in mice

Aminoglutethimide is a steroidogenesis inhibitor and inhibits a cholesterol side-chain cleavage enzyme (CYP11A1) that converts cholesterol to pregnenolone in mitochondria. We investigated histopathological changes induced by 5-day administration of AG in mice. Cytoplasmic vacuoles of various sizes and single cell necrosis were found in zona fasciculata cells in AG-treated mice. Some vacuoles were positive for adipophilin, whereas others were positive for lysosome-associated membrane protein-2 on immunohistochemical staining, indicating they were enlarged lipid droplets and lysosomes, respectively. Electron microscopy revealed enlarged lysosomes containing damaged mitochondria and lamellar bodies in zona fasciculata cells, and they were considered to reflect the intracellular protein degradation processes, mitophagy and lipophagy. From these results, we showed that AG induces excessive lipid accumulation and mitochondrial damage in zona fasciculata cells, which leads to an accelerated lysosomal degradation in mice.

Spontaneous Accumulation of Globotriaosylceramide (Gb3) in Proximal Renal Tubules in an ICR Mouse.

This report describes spontaneous cytoplasmic vacuolation in the proximal renal tubules of a 7-week-old male ICR [Crlj:CD1(ICR)] mouse. The contents of vacuoles were positively stained with periodic acid-Schiff (PAS) and Sudan black, and the membranes were positive on immunohistochemical staining for lysosomal-associated membrane protein-2 (LAMP-2), a marker of lysosomal membrane. Electron microscopy revealed electron-dense lamellar bodies in the proximal tubular epithelial cells. These histopathological features are similar to those in α-galactosidase A-deficient mice, in which globotriaosylceramide (Gb3), a glycosphingolipid, accumulates in lysosomes. When we performed immunohistochemical staining for Gb3, the contents of vacuoles were positively stained. From these results, spontaneous cytoplasmic vacuolation in the proximal renal tubules in the mouse was identified as lysosomal accumulation of Gb3.