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The liver performs a variety of essential functions; hence drug-induced liver injury (DILI) is a serious concern that can ultimately lead to the withdrawal of a drug from the market or discontinuation of drug development. However, the mechanisms of drug-induced liver injury are not always clear. We hypothesized that drugs may inhibit the liver recovery process, especially bile canalicular (BC) network reformation, leading to persistent liver injury and deterioration, and tested this hypothesis in the present work. The BC structure disappeared in mice following treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or thioacetamide (TAA) for 4 weeks, then reappeared after 4 weeks of receiving a normal diet. By contrast, reconstruction of the BC structure was suppressed in mice fed a diet containing 0.3% benzbromarone (BBR; which can induce fatal liver injury in clinical settings) after liver injury. Plasma ALT levels were increased significantly in mice treated with BBR after DDC or TAA treatment, compared with BBR alone. To confirm whether BBR has a direct inhibitory effect on hepatocytes, we also examined BC reformation in primary cultured mouse hepatocytes with a sandwich configuration. Under these culture conditions, the BC network rapidly reformed from days 2 and 3 after seeding. During the reformation period, BBR inhibited BC reformation significantly. These results suggest that BBR inhibits BC reconstruction and delays recovery from pre-existing liver injury., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Drug-induced liver injury is not readily detectable using conventional animal studies during pre-clinical drug development. To address this problem, other researchers have proposed the use of co-administration of lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, and a drug. Using this approach, liver injury that is otherwise not detected following drug administration alone can be successfully identified. Previous studies have demonstrated that such injury is suppressed by heparin; therefore, the mechanism may involve coagulation-dependent ischemia. However, it has not been established how LPS-induced ischemia might sensitize hepatocytes to a potentially hepatotoxic drug. In the present study, we aimed to determine the effect of LPS-induced ischemia on liver mitochondrial function and downstream toxicologic responses. Consistent with previous findings, plasma alanine transaminase (ALT) activity was higher in rats co-administered with LPS (1 mg/kg) and diclofenac (100 mg/kg), but reduced by heparin. Liver mRNA expression of Hmox1, encoding heme oxygenase-1, an oxidative stress indicator, was three times higher at 2 hr after LPS administration. Furthermore, respiratory activity via mitochondrial complex II, lipid peroxidation in mitochondria, and the susceptibility to mitochondrial permeability transition pore opening in response to diclofenac administration were significantly increased by LPS administration. The increase in plasma ALT activity and the sensitization to mitochondrial permeability transition pore opening were reduced by the co-administration of heparin. In conclusion, LPS-induced transient ischemia disrupts respiratory chain complex activities, enhances reactive oxygen species production, especially in mitochondria, and sensitizes mitochondria to permeability transition pore opening when testing a potentially hepatotoxic drug in vivo.

The risk of drug-induced liver injury (DILI) is of great concern to the pharmaceutical industry. It is well-known that metabolic activation of drugs to form toxic metabolites (TMs) is strongly associated with DILI onset. Drug-induced mitochondrial dysfunction is also strongly associated with increased risk of DILI. However, it is difficult to determine the target of TMs associated with exacerbation of DILI because of difficulties in identifying and purifying TMs. In this study, we propose a sequential in vitro assay system to assess TM formation and their ability to induce mitochondrial permeability transition (MPT) in a one-pot process. In this assay system, freshly-isolated rat liver mitochondria were incubated with reaction solutions of 44 test drugs preincubated with liver microsomes in the presence or absence of NADPH; then, NADPH-dependent MPT pore opening was assessed as mitochondrial swelling. In this assay system, several hepatotoxic drugs, including benzbromarone (BBR), significantly induced MPT in a NADPH-dependent manner. We investigated the rationality of using BBR as a model drug, since it showed the most prominent MPT in our assay system. Both the production of a candidate toxic metabolite of BBR (1',6-(OH)2 BBR) and NADPH-dependent MPT were inhibited by several cytochrome P450 (CYP) inhibitors (clotrimazole and SKF-525A, 100μM). In summary, this assay system can be used to evaluate comprehensive metabolite-dependent MPT without identification or purification of metabolites., (Copyright © 2015 Elsevier Inc. All rights reserved.)

1. Raloxifene-6-glucuronide (R6G) is a substrate of rat multidrug resistance-associated protein 2 (Mrp2), a transporter responsible for biliary excretion of organic anions. 2. Pharmacokinetic modeling of R6G in Eisai hyperbilirubinemic rats (EHBRs), hereditary Mrp2-deficient rats, and wild-type Sprague-Dawley rats (SDRs) indicated that reduction in not only biliary excretion but also hepatic uptake of R6G influenced low clearance in EHBRs. 3. An integration plot study demonstrated that the hepatic uptake of R6G was 66% lower in EHBRs than that in SDRs. A reduction was observed for the other Mrp2 substrate Valsartan (95% lower) but not for estradiol-17β-glucuronide (E217βG). This variation may be associated with the difference in substrate specificity of transporters and/or inhibition of hepatic uptake of organic anions by endogenous substances such as bilirubin glucuronides. 4. In conclusion, incidental alteration of the hepatic uptake of organic anions should be considered as an explanation of their enhanced systemic exposure in EHBRs.

Recent studies have shown that medium-chain triacylglycerol (MCT) improved serum albumin concentration in elderly people with protein-energy malnutrition (PEM) and in malnourished rats. However, the mechanism for this effect has not been clarified. Dietary MCT promotes insulin secretion from the pancreas, and insulin activates mammalian target of rapamycin (mTOR) complex 1 (mTORC1) via the activation of phosphoinositide 3-kinase (PI3K) and its downstream effecter, Akt. mTORC1 promotes mRNA translation through S6K and 4E-BP1. Therefore, we hypothesized that dietary MCT elevates albumin synthesis through promotion of insulin-Akt-mTOR transduction in the liver. To test this hypothesis, we measured phosphorylated Akt, mTOR and albumin in the livers of malnourished rats. In the present study we examined rats fed low-protein diets containing either MCT or long-chain triacylglycerol (LCT) with energy restriction. The plasma and liver albumin levels were significantly higher in the MCT-fed group than in the LCT-fed group. In addition, plasma insulin concentration, liver phosphorylated Akt/Akt and phosphorylated mTOR/mTOR levels were significantly higher in the MCT-fed group than in the LCT-fed group. These results suggest that one of the mechanisms for the albumin improvement effect of dietary MCT is the promotion of albumin synthesis through the insulin-Akt-mTOR signaling pathway of the liver.

Multidrug resistance-associated protein 2 (MRP2) is a member of a family of efflux transporters that are involved in biliary excretion of organic anions from hepatocytes. Disrupted canalicular localization and decreased protein expression of MRP2 have been observed in patients with chronic cholestatic disorder and hepatic failure without a change in its mRNA expression. We have previously demonstrated that post-transcriptional regulation of the rapid retrieval of rat MRP2 from the canalicular membrane to the intracelluar compartment occurs under conditions of acute (~30min) oxidative stress. However, it is unclear whether MRP2 expression is decreased during its sustained internalization during chronic oxidative stress. The present study employed buthionine sulfoximine (BSO) to induce chronic oxidative stress in the livers of Sprague-Dawley rats and then examined the protein expression and localization of MRP2. Canalicular MRP2 localization was altered by BSO treatment for 2h without changing the hepatic protein expression of MRP2. While the 8h after exposure to BSO, hepatic MRP2 protein expression was decreased, and the canalicular localization of MRP2 was disrupted without changing the mRNA expression of MRP2. The BSO-induced reduction in MRP2 protein expression was suppressed by pretreatment with N-benzyloxycarbonyl (Cbz)-Leu-Leu-leucinal ( MG-132), a proteasomal inhibitor. Furthermore, the modification of MRP2 by small ubiquitin-relatedmodifier 1 (SUMO-1) was impaired in BSO-treated rat liver,while that by ubiquitin (Ub) and MRP2 was enhanced. Taken together, the results of this study suggest the sustained periods of low GSH content coupled with altered modification of MRP2 by Ub/SUMO-1 were accompanied by proteasomal degradation of MRP2., (© 2012 Elsevier B.V. All rights reserved.)

Oxidative stress is a feature of cholestatic syndrome and induces multidrug resistance-associated protein 2 (Mrp2) internalization from the canalicular membrane surface. We have previously shown that the activation of a novel protein kinase C (nPKC) by oxidative stress regulates Mrp2 internalization. The internalized Mrp2 was recycled to the canalicular surface in a protein kinase A (PKA)-dependent manner after intracellular glutathione (GSH) levels were replenished. However, the putative phosphorylation targets of these protein kinases involved in reversible Mrp2 trafficking remain unclear. In this study, we investigated the effect of changing the intrahepatic redox status on the C-terminal phosphorylation status of radixin (p-radixin), which links Mrp2 to F-actin, and the interaction of p-radixin with Mrp2 in rat hepatocytes. We detected a significant decrease in the amount of p-radixin that co-immunoprecipitated with Mrp2 after tertiary-butylhydroperoxide (t-BHP) treatment. After treatment with GSH-ethylester (GSH-EE), the phosphorylation level became the same as that of the control. A PKC and protein phosphatase (PP)-1/2A inhibitor, but not a PP-2A selective inhibitor, prevented the t-BHP-induced decrease of p-radixin and subsequent canalicular Mrp2 localization. In contrast, a PKA inhibitor affected the recovery process facilitated by GSH-EE treatment. In conclusion, the interaction of p-radixin with Mrp2 was decreased by the activation of PKC and PP-1 under oxidative stress conditions which subsequently led to Mrp2 internalization, whereas the interaction of p-radixin and Mrp2 was increased by the activation of PKA during recovery from oxidative stress., (2011 Elsevier B.V. All rights reserved.)

Cholestasis develops during inflammation and is characterized as occurring under oxidative stress. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid or lipopolysaccharide (LPS)-induced acute oxidative stress in rat liver. However, it remains unclear whether canalicular Mrp2 internalization is observed in human liver under conditions of acute oxidative stress. In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and found in traditional Chinese medicine, on endotoxin-induced Mrp2 internalization in rat and human liver slices. At 1.5h following LPS treatment (100microg/mL), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of filamentous actin in the rat and human liver slices. Pretreatment with DMA (10microM) counteracted LPS-induced subcellular distribution of Mrp2. Our data clearly indicated that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress in rat and human liver slices., (Copyright 2010 Elsevier Inc. All rights reserved.)

A number of genes and their protein products are expressed within the liver lobules in a region-specific manner and confer heterogeneous metabolic properties to hepatocytes; this phenomenon is known as 'metabolic zonation'. To elucidate the roles of Dicer, an endoribonuclease III type enzyme required for microRNA biogenesis, in the establishment of liver zonation, we examined the distribution of proteins exhibiting pericentral or periportal localization in hepatocyte-specific Dicer1 knockout mouse livers. Immunohistochemistry showed that the localization of pericentral proteins was mostly preserved in Dicer1-deficient livers. However, glutamine synthetase, whose expression is normally confined to a few layers of hepatocytes surrounding the central veins, was expressed in broader pericentral areas. Even more striking was the observation that all the periportal proteins that were examined, including phosphoenolpyruvate carboxykinase, E-cadherin, arginase 1, and carbamoyl phosphate synthetase-I, lost their localized expression patterns and were diffusely expressed throughout the entire lobule. Thus, with regard to periportal protein expression, the consequences of Dicer loss were similar to those caused by the disruption of beta-catenin. An analysis of livers deficient in beta-catenin did not identify the down-regulation of Dicer1 or any microRNAs, indicating that they are not directly activated by beta-catenin. Thus, the present study illustrates that Dicer plays a pivotal role in the establishment of liver zonation. Dicer is essential for the suppression of periportal proteins by Wnt/beta-catenin/TCF signalling, albeit it likely acts in an indirect manner.

We have hypothesized a suppressive mechanism against dietary docosahexaenoic acid (22:6n-3; DHA)-induced tissue lipid peroxidation, in which the degradation products, including their conjugates, are excreted into the urine by xenobiotic or organic anion transporters. In this study, we employed parent-strain Sprague-Dawley rats (SDRs), together with their mutant strain, Eisai hyperbilirubinuria rats (EHBRs). EHBRs are deficient in multidrug resistance-associated protein (MRP) 2, and show defective urinary excretion of numerous xenobiotics and organic anions. Both strains of rats were fed a diet containing DHA at 8.4% of total energy for 31 d. In the livers of the DHA-fed rats, the level of free malondialdehyde (MDA) + 4-hydroxy-2-alkenals (HAE) fell, and conversely glutathione S-transferase (GST) activity increased in MRP2-deficient EHBRs as compared to the SDRs, suggesting that the glutathione (GSH)-conjugation reaction for the aldehydes generated on DHA intake was accelerated in the MRP2-deficient EHBRs. Since the gene expression of liver MRP3 in the MRP2-deficient EHBRs was amplified to compensate for DHA intake, it is thought that the transport of MRP3 substrates into the bloodstream, rather than MRP2-mediated excretion of its substrates into the bile, was promoted. Indeed, excretion of mercapturic acid (acetylcysteine conjugates derived metabolically from the conjugate of each aldehyde with GSH) into the urine increased significantly in MRP2-deficient EHBRs fed DHA.

The ATP-binding cassette transporter family C 2 (Abcc2) is a member of efflux transporters involved in the biliary excretion of organic anions from hepatocytes. Posttranslational regulation of Abcc2 has been implicated, although the molecular mechanism is not fully understood. In the present study, we performed yeast two-hybrid screening to identify novel protein(s) that particularly interacts with the linker region of Abcc2 located between the NH(2)-terminal nucleotide binding domain and the last membrane-spanning domain. The screening resulted in the identification of a series of small ubiquitin-like modifier (SUMO)-related enzymes and their substrates. In yeast experiments, all of these interactions were abolished by substituting the putative SUMO consensus site in the linker region (IKKE) in Abcc2 to IRKE. In vitro SUMOylation experiments confirmed that the Abcc2 linker was a substrate of Ubc9-mediated SUMOylation. It was also found that the IKKE sequence is the target of SUMOylation, since a mutant with IKKE is substituted by IRKE was not SUMOylated. Furthermore, we demonstrated for the first time that Abcc2, endogenously expressed in rat hepatoma-derived McARH7777 cells, is SUMOylated. Suppression of endogenous Ubc9 by small interfering RNA resulted in a selective 30% reduction in Abcc2 protein expression in the postnuclear supernatant, whereas subcellular localization of Abcc2 confirmed by semiquantitative immunofluorescence analysis was minimally affected. This is the first demonstration showing the regulation of ABC transporter expression by SUMOylation.

The optimal dietary n-6/n-3 ratio has not been fully elucidated. To investigate the influence of the dietary n-6/n-3 ratio on this ratio in the body and on liver beta-oxidation peroxisomal activity, rats were fed diets containing fat at an n-6/n-3 ratio of 1 to 16 for 4 weeks. To investigate whether elevation of the liver peroxisomal beta-oxidation activity increases the n-6/n-3 ratio in the body, rats were fed a diet containing a peroxisome-activating agent, bezafibrate, for 2 weeks, and its influence on the liver n-6/n-3 ratio was examined. The slope of the regression line between the dietary and liver total lipid n-6/n-3 ratios was significantly smaller when the dietary n-6/n-3 ratio was 4 or greater than when it was smaller than 4. Peroxisomal beta-oxidation and acyl CoA oxidase activities were significantly lower in rats fed a diet with an n-6/n-3 ratio of 16 than in those fed a diet with a ratio of 1. The peroxisomal beta-oxidation activity in the bezafibrate-supplemented group was significantly higher than that in the control group. The serum and liver total lipid n-6/n-3 ratios were significantly higher in the 0.015% bezafibrate-supplemented group than in the control group. These findings suggest that the liver n-6/n-3 ratio might be controlled via peroxisomal beta-oxidation in rats.

Unlabelled: Recent studies have suggested that beta-catenin is involved in the regulation of hepatocyte proliferation in multiple contexts, including organ development and tumorigenesis. We explored the role of beta-catenin during liver regeneration using T cell factor/lymphoid enhancer factor (TCF/LEF)-reporter mice (TOPGal mice) and liver-specific beta-catenin knockout mice. Liver-specific beta-catenin knockout mice showed a delayed onset of DNA synthesis after hepatectomy, whereas recovery of liver mass was not affected. Among putative beta-catenin target genes examined, the induction of Ccnd1 expression was reduced, whereas the expression of Myc and Egfr was unaffected. Furthermore, cyclin D1 protein levels were not induced, and the expression of cyclins A, E, and proliferating cell nuclear antigen was delayed. Intriguingly, the analysis of TOPGal mice showed that hepatocytes with active TCF/LEF transcription are confined to the pericentral zone and are not increased in number during regeneration, indicating an uncoupling between beta-catenin/TCF signaling activity and hepatocyte proliferation., Conclusion: Our results indicate that beta-catenin is critical for the proper regulation of hepatocyte proliferation during liver regeneration; however, the activity of beta-catenin/TCF signaling does not correlate with hepatocyte proliferation, suggesting that this regulation might be indirect/secondary.

To clarify the alternative mechanisms to vitamin E (VE) regulating lipid peroxide accumulation in the liver after docosahexaenoic acid (DHA) ingestion, we examined the relationship between the DHA-induced lipid peroxide formation and induction of the xenobiotic transporters, Ral-binding GTPase-activating protein (RalBP1) and multidrug resistance-associated proteins 1, 2 and 3 (MRP1-3), in the liver of rats fed with DHA. The test diets contained DHA and linoleic acid (LA) (8.7% and 2.1% of total energy, respectively) with different levels of dietary VE (normal and low: 68 and 7.7 mg of alpha-tocopherol equivalent per kg diet, respectively), and the control diet contained LA alone (11.5% of total energy). The rats were fed with these experimental diets for 14 d. The proportions of DHA in the liver, kidney and heart were higher in the DHA-fed groups than in the LA-fed group. The tissue thiobarbituric acid values as an index of lipid peroxidation were also significantly higher in the DHA-fed groups, but the value did not differ between the DHA-fed groups with different VE levels. In the liver, there were no significant differences in the glutathione S-transferase (GST) and aldehyde dehydrogenase (ALDH) activities or in the expression of GST M2, RalBP1, MRP1 and MRP2 mRNA. However, the obvious induction of expression of liver MRP3 mRNA and tendency to produce the protein were recognized after DHA ingestion. This study is the first to report the gene expression of MRP3 by DHA ingestion. There might exist, therefore, some relationship between the DHA intake and MRP3 induction in regulating lipid peroxide accumulation in the liver.

Oxidative stress in the liver is sometimes accompanied by cholestasis. We have described the internalization of multidrug resistance-associated protein 2/ATP-binding cassette transporter family 2 (Mrp2/Abcc2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress in rat liver. However, the signaling pathway and regulatory molecules have not been investigated. In the present study, we investigated the mechanism of EA-induced Mrp2 internalization using isolated rat hepatocyte couplets (IRCHs). The Mrp2 index, defined as the ratio of Mrp2-positive canalicular membrane staining in IRCHs per number of cell nuclei, was significantly reduced by treatment with EA. This reduction was abolished by a nonspecific protein kinase C (PKC) inhibitor Gö6850, a Ca(2+) chelator, EGTA, but not by a protein kinase A (PKA)-selective inhibitor, a Ca(2+)-dependent conventional PKC (cPKC) inhibitor Gö6976, or a protein kinase G (PKG) inhibitor (1 microM). Moreover, an increase in the intracellular Ca(2+) level and NO release into medium were observed shortly after the EA treatment. Both of these increases, as well as Mrp2 internalization, were completely blocked by EGTA. In conclusion, EA produced a reduction in GSH, Ca(2+) elevation, NO production, and nPKC activation in a sequential manner, finally leading to Mrp2 internalization.

There is accumulating evidence that Wnt/beta-catenin signaling is involved in the regulation of liver development and physiology. The presence of genetic alterations resulting in constitutive beta-catenin stabilization in human and murine liver tumors also implicates this pathway in hepatocyte proliferation. In the present study, we generated hepatocyte-specific beta-catenin knockout mice to explore the role of beta-catenin in liver function. Conditional knockout mice were born at the expected Mendelian ratio and developed normally to adulthood, indicating beta-catenin is dispensable for essential liver function under normal breeding conditions. However, the liver mass of knockout mice was 20% less than those of mice in the control groups. Expression analysis revealed loss of genes required for glutamine synthesis in knockout mice. Loss of the liver glutamine synthesis pathway did not affect the blood ammonia level in mice fed a standard diet, yet, knockout mice showed significantly elevated blood ammonia levels with high-protein dietary feeding. Furthermore, the expression of two cytochrome P450 enzymes, CYP1A2 and CYP2E1, was almost completely abolished in livers from hepatocyte-specific beta-catenin knockout mice. Consequently, these mice were resistant to acetaminophen challenge, confirming the requirement of these cytochrome P450 enzymes for metabolism of xenobiotic substances. In conclusion, in addition to regulating hepatocyte proliferation, beta-catenin may also control multiple aspects of normal liver function.

Oxidative stress in the liver is sometimes accompanied by cholestasis. We investigated the localization and role of multidrug-resistance-associated protein (Mrp) 2, a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress. Normal Sprague-Dawley rat (SDR) and Mrp2-deficient Eisai hyperbilirubinemic rat (EHBR) livers were perfused with 500 microM EA. The release of glutamic pyruvic transaminase (GPT) and thiobarbituric-acid-reactive substances (TBARS) from EHBR liver was markedly delayed compared with that from SDR liver. This is mainly due to the higher basal level of glutathione (GSH) in EHBR liver (59.1 +/- 0.3 nmol/mg protein) compared with SDR liver (39.7 +/- 1.5 nmol/mg protein). EA similarly induced a rapid reduction in GSH followed by mitochondrial permeability transition in the isolated mitochondria from both SDR and EHBR. Internalization of Mrp2 was detected before nonspecific disruption of the canalicular membrane and GPT release in SDR liver perfused with 100 microM EA. SDR liver preperfused with hyperosmolar buffer (405 mosmol/L) for 30 min induced internalization of Mrp2 without changing the basal GSH level, while elimination of hepatic GSH by 300 microM EA perfusion was significantly delayed thereafter. Concomitantly, hepatotoxicity assessed by the release of GPT and TBARS was also significantly attenuated under hyperosmolar conditions. In conclusion, preserved cytosolic and intramitochondrial GSH is the key factor involved in the acute hepatotoxicity induced by EA and its susceptibility could be altered by the presence of Mrp2.

In previous studies, we showed that docosahexaenoic acid (DHA) ingestion enhanced the susceptibility of rat liver and kidney to lipid peroxidation, but did not increase lipid peroxide formation to the level expected from the relative peroxidizability index (P-index) of the total tissue lipids. The results suggested the existence of some suppressive mechanisms against DHA-induced tissue lipid peroxide formation, as increased tissue ascorbic acid (AsA) and glutathione levels were observed. Therefore, we focused initially on the role of AsA for the suppressive mechanisms. For this purpose, we examined the influence of different levels of dietary AsA (low, moderate, high and excessive levels were 100, 300 (control), 600 and 3000 mg/kg diet respectively) on the tissue lipid peroxide and antioxidant levels in AsA-requiring Osteogenic Disorder Shionogi/Shi-od/od (ODS) rats fed DHA (6.4 % total energy) for 32 or 33 d. Diets were pair-fed to the DHA- and 100 mg AsA/kg diet-fed group. We found that the lipid peroxide concentrations of liver and kidney in the DHA-fed group receiving 100 mg AsA/kg diet were significantly higher or tended to be higher than those of the DHA-fed groups with AsA at more than the usual control level of 300 mg/kg diet. Contrary to this, the liver alpha-tocopherol concentration was significantly lower or tended to be lower in the DHA and 100 mg AsA/kg diet-fed group than those of the other DHA-fed groups. However, tissue lipid peroxide formation and alpha-tocopherol consumption were not suppressed further, even after animals received higher doses of AsA. The present results suggest that higher than normal concentrations of tissue AsA are not necessarily associated with the suppressive mechanisms against dietary DHA-induced tissue lipid peroxide formation.