Phosphatidylcholine (18:0/20:4), a potential biomarker to predict ethionamide-induced hepatic steatosis in rats.

Ethionamide (ETH), a second-line drug for multidrug-resistant tuberculosis, is known to cause hepatic steatosis in rats and humans. To investigate predictive biomarkers for ETH-induced steatosis, we performed lipidomics analysis using plasma and liver samples collected from rats treated orally with ETH at 30 and 100 mg/kg for 14 days. The ETH-treated rats developed hepatic steatosis with Oil Red O staining-positive vacuolation in the centrilobular hepatocytes accompanied by increased hepatic contents of triglycerides (TG) and decreased plasma TG and total cholesterol levels. A multivariate analysis for lipid profiles revealed differences in each of the 35 lipid species in the plasma and liver between the control and the ETH-treated rats. Of those lipids, phosphatidylcholine (PC) (18:0/20:4) decreased dose-dependently in both the plasma and liver. Moreover, serum TG-rich very low-density lipoprotein (VLDL) levels, especially the large particle fraction of VLDL composed of PC containing arachidonic acid (20:4) involved in hepatic secretion of TG, were decreased dose-dependently. In conclusion, the decreased PC (18:0/20:4) in the liver, possibly leading to suppression of hepatic TG secretion, was considered to be involved in the pathogenesis of the ETH-induced hepatic steatosis. Therefore, plasma PC (18:0/20:4) levels are proposed as mechanism-related biomarkers for ETH-induced hepatic steatosis.

Effects of Delgocitinib Ointment 0.5% on the Normal Mouse Skin and Epidermal Tight Junction Proteins in Comparison With Topical Corticosteroids.

Delgocitinib ointment 0.5% is the world's first topical Janus kinase inhibitor product and was approved for treatment of atopic dermatitis (AD) in Japan. Although topical corticosteroids (TCSs) have been the mainstay of pharmacotherapy in AD over the past decades, long-term use of TCSs causes skin atrophy and alteration of the epidermal tight junction (TJ) leading to epidermal barrier dysfunction. In this study, delgocitinib ointment 0.5% or representative TCSs of different potencies were applied dermally once daily to the ear pinna of normal ICR mice for 14 days, and ear pinna thickness, histopathology, and immunohistochemistry for epidermal TJ proteins claudin-1 and -4 were evaluated. All the TCSs caused decreases in ear pinna thickness with epidermal thinning, sebaceous gland atrophy, and atrophy/decreased number of the subcutaneous adipocytes and decreased immunohistochemical staining intensity for epidermal claudins. In contrast, delgocitinib ointment 0.5% did not cause any of those changes. In conclusion, once daily topical delgocitinib ointment 0.5% for 14 days did not cause skin atrophy or decreased immunohistochemical staining of epidermal claudins, which are common safety concerns associated with TCSs. These characteristics suggest that delgocitinib ointment 0.5% has an improved safety profile over currently available TCS therapies particular for the long-term AD treatment.

Specificity of transaminase activities in the prediction of drug-induced hepatotoxicity.

The activities of the transaminases (aminotransferases) alanine aminotransferase and aspartate aminotransferase in the blood (serum or plasma) are widely used as sensitive markers of possible tissue damage and, in particular for liver toxicity. On the other hand, an increase in transaminase activities is not always accompanied by findings suggestive of hepatotoxicity. Transaminases are some of the key enzymes in the gluconeogenesis and glycolysis pathways and exist in many organs and tissues which have high activities of the gluconeogenesis and glycolysis. The activities of transaminases are altered not only in the liver but also in other organs by modification of gluconeogenesis by nutritional or hormonal factors and this phenomenon leads to alteration of transaminase activity in the blood. Drugs, which are considered to directly or secondarily modify gluconeogenesis through lowering blood glucose levels or activating lipid metabolism, such as α-glucosidase inhibitors and fibrates, slightly increase transaminase activities in the blood but there is little evidence that the phenomenon is related to drug-induced liver injury (DILI). This type of elevations can be called pharmacology-related elevation. The pharmacology-related elevation of transaminase activities sometimes makes it difficult to assess precisely the potential hepatotoxicity of new investigational drugs. Considering the characteristic of transaminases, concomitant use of new biomarkers more specific to hepatic injury is needed in the assessment of DILI both in non-clinical and clinical studies. In this review, we will discuss the specificity of transaminases to DILI and future perspectives for transaminases in the estimation of risk of DILI.

Enhancement of acetaminophen-induced chronic hepatotoxicity in spontaneously diabetic torii (SDT) rats.

Some patients encounter hepatotoxicity after repeated acetaminophen (APAP) dosing even at therapeutic doses. In the present study, we focused on the diabetic state as one of the suggested risk factors of drug-induced liver injury in humans and investigated the contribution of accelerated gluconeogenesis to the susceptibility to APAP-induced hepatotoxicity using an animal model of type 2 diabetes patients. Sprague-Dawley (SD) rats and spontaneously diabetic torii (SDT) rats were each given APAP at 0 mg/kg, 300 and 500 mg/kg for 35 days by oral gavage. Plasma and urinary glutathione-related metabolites, liver function parameters, and hepatic glutathione levels were compared between the non-APAP-treated SDT and SD rats and between the APAP-treated SDT and SD rats. Hepatic function parameters were not increased at either dose level in the APAP-treated SD rats, but were increased at both dose levels in the APAP-treated SDT rats. Increases in hepatic glutathione levels attributable to the treatment of APAP were noted only in the APAP-treated SD rats. There were differences in the profiles of plasma and urinary glutathione-related metabolites between the non-APAP-treated SD and SDT rats and the plasma/urinary endogenous metabolite profile after treatment with APAP in the SDT rats indicated that hepatic glutathione synthesis was decreased due to accelerated gluconeogenesis. In conclusion, SDT rats were more sensitive to APAP-induced chronic hepatotoxicity than SD rats and the high susceptibility of SDT rats was considered to be attributable to lowered hepatic glutathione levels induced by accelerated gluconeogenesis.

Estimation of potential risk of allyl alcohol induced liver injury in diabetic patients using type 2 diabetes spontaneously diabetic Torii-Leprfa (SDT fatty) rats.

In order to estimate the potential risk of chemicals including drug in patients with type 2 diabetes mellitus (T2DM), we investigated allyl alcohol induced liver injury using SD rats and Spontaneously Diabetic Torii-Leprfa (SDT fatty) rats as a model for human T2DM. The diabetic state is one of the risk factors for chemically induced liver injury because of lower levels of glutathione for detoxification by conjugation with chemicals and environmental pollutants and their reactive metabolites. Allyl alcohol is metabolized to a highly reactive unsaturated aldehyde, acrolein, which is detoxified by conjugation with glutathione. Therefore, we used allyl alcohol as a model compound. Our investigations showed that SDT fatty rats appropriately mimic the diabetic state in humans. The profiles of glucose metabolism, hepatic function tests and glutathione synthesis in the SDT fatty rats were similar to those in patients with T2DM. Five-week oral dosing with allyl alcohol to the SDT fatty rats revealed that the allyl alcohol induced liver injury was markedly enhanced in the SDT fatty rats when compared with the SD rats and the difference was considered to be due to lower hepatic detoxification of acrolein, the reactive metabolite of allyl alcohol, by depleted hepatic glutathione synthesis. Taking all the results of the present study into consideration, the potential for allyl alcohol to induce liver injury is considered to be higher in diabetic patients than in healthy humans.

Ether-phosphatidylcholine characterized by consolidated plasma and liver lipidomics is a predictive biomarker for valproic acid-induced hepatic steatosis.

Valproic acid (VPA) is known to induce hepatic steatosis due to mitochondrial toxicity in rodents and humans. In the present study, we administered VPA to SD rats for 3 or 14 days at 250 and 500 mg/kg and then performed lipidomics analysis to reveal VPA-induced alteration of the hepatic lipid profile and its association with the plasma lipid profile. VPA induced hepatic steatosis at the high dose level without any degenerative changes in the liver on day 4 (after 3 days dosing) and at the low dose level on day 15 (after 14 days dosing). We compared the plasma and hepatic lipid profiles obtained on day 4 between the VPA-treated and control rats using a multivariate analysis to determine differences between the two groups. In total, 36 species of plasma lipids and 24 species of hepatic lipids were identified as altered in the VPA-treated group. Of these lipid species, ether-phosphatidylcholines (ePCs), including PC(16:0e/22:4) and PC(16:0e/22:6), were decreased in both the plasma and liver from the low dose level on day 4, however, neither an increase in hepatic TG level nor histopathological hepatic steatosis was observed at either dose level on day 4. Hepatic mRNA levels of glycerone-phosphate O-acyltransferase (Gnpat), which is a key enzyme for biosynthesis of ePC, was also decreased by treatment with VPA along with the decrease in ePCs. In conclusion, the changes in ePCs, (PC[16:0e/22:4] and PC[16:0e/22:6]), have potential utility as predictive biomarkers for VPA-induced hepatic steatosis.

A pharmacologic increase in activity of plasma transaminase derived from small intestine in animals receiving an acyl CoA: diacylglycerol transferase (DGAT) 1 inhibitor.

Acyl CoA: diacylglycerol acyltransferase (DGAT) 1 is an enzyme that catalyzes the re-synthesis of triglycerides (TG) from free fatty acids and diacylglycerol. JTT-553 is a DGAT1 inhibitor and exhibits its pharmacological action (inhibition of re-synthesis of TG) in the enterocytes of the small intestine leading to suppression of a postprandial elevation of plasma lipids. After repeated oral dosing JTT-553 in rats and monkeys, plasma transaminase levels were increased but there were neither changes in other hepatic function parameters nor histopathological findings suggestive of hepatotoxicity. Based on the results of exploratory studies for investigation of the mechanism of the increase in transaminase levels, plasma transaminase levels were increased after dosing JTT-553 only when animals were fed after dosing and a main factor in the diet contributing to the increase in plasma transaminase levels was lipids. After dosing JTT-553, transaminase levels were increased in the small intestine but not in the liver, indicating that the origin of transaminase increased in the plasma was not the liver but the small intestine where JTT-553 exhibits its pharmacological action. The increase in small intestinal transaminase levels was due to increased enzyme protein synthesis and was suppressed by inhibiting fatty acid-transport to the enterocytes. In conclusion, the JTT-553-related increase in plasma transaminase levels is considered not to be due to release of the enzymes from injured cells into the circulation but to be phenomena resulting from enhancement of enzyme protein synthesis in the small intestine due to the pharmacological action of JTT-553 in this organ.

High fructose diet feeding accelerates diabetic nephropathy in Spontaneously Diabetic Torii (SDT) rats.

Diabetic nephropathy (DN) is one of the complications of diabetes and is now the most common cause of end-stage renal disease. Fructose is a simple carbohydrate that is present in fruits and honey and is used as a sweetener because of its sweet taste. Fructose has been reported to have the potential to progress diabetes and DN in humans even though fructose itself does not increase postprandial plasma glucose levels. In this study, we investigated the effects of high fructose intake on the kidney of the Spontaneously Diabetic Torii (SDT) rats which have renal lesions similar to those in DN patients and compared these with the effects in normal SD rats. This study revealed that a 4-week feeding of the high fructose diet increased urinary excretion of kidney injury makers for tubular injury and accelerated mainly renal tubular and interstitial lesions in the SDT rats but not in normal rats. The progression of the nephropathy in the SDT rats was considered to be related to increased internal uric acid and blood glucose levels due to the high fructose intake. In conclusion, high fructose intake exaggerated the renal lesions in the SDT rats probably due to effects on the tubules and interstitium through metabolic implications for uric acid and glucose.

Evaluation of the Potential Risk of Drugs to Induce Hepatotoxicity in Human-Relationships between Hepatic Steatosis Observed in Non-Clinical Toxicity Study and Hepatotoxicity in Humans.

In the development of drugs, we sometimes encounter fatty change of the hepatocytes (steatosis) which is not accompanied by degenerative change in the liver in non-clinical toxicity studies. In this study, we investigated the relationships between fatty change of the hepatocytes noted in non-clinical toxicity studies of compound X, a candidate compound in drug development, and mitochondrial dysfunction in order to estimate the potential risk of the compound to induce drug-induced liver injury (DILI) in humans. We conducted in vivo and in vitro exploratory studies for this purpose. In vivo lipidomics analysis was conducted to investigate the relationships between alteration of the hepatic lipids and mitochondrial dysfunction. In the liver of rats treated with compound X, triglycerides containing long-chain fatty acids, which are the main energy source of the mitochondria, accumulated. Accumulation of these triglycerides was considered to be related to the inhibition of mitochondrial respiration based on the results of in vitro mitochondria toxicity studies. In conclusion, fatty change of the hepatocytes (steatosis) in non-clinical toxicity studies of drug candidates can be regarded as a critical finding for the estimation of their potential risk to induce DILI in humans when the fatty change is induced by mitochondrial dysfunction.

Arachidonic acid-containing phosphatidylcholine characterized by consolidated plasma and liver lipidomics as an early onset marker for tamoxifen-induced hepatic phospholipidosis.

Lipid profiling has emerged as an effective approach to not only screen disease and drug toxicity biomarkers but also understand their underlying mechanisms of action. Tamoxifen, a widely used antiestrogenic agent for adjuvant therapy against estrogen-positive breast cancer, possesses side effects such as hepatic steatosis and phospholipidosis (PLD). In the present study, we administered tamoxifen to Sprague-Dawley rats and used lipidomics to reveal tamoxifen-induced alteration of the hepatic lipid profile and its association with the plasma lipid profile. Treatment with tamoxifen for 28 days caused hepatic PLD in rats. We compared the plasma and liver lipid profiles in treated vs. untreated rats using a multivariate analysis to determine differences between the two groups. In total, 25 plasma and 45 liver lipids were identified and altered in the tamoxifen-treated group. Of these lipids, arachidonic acid (AA)-containing phosphatidylcholines (PCs), such as PC (17:0/20:4) and PC (18:1/20:4), were commonly reduced in both plasma and liver. Conversely, tamoxifen increased other phosphoglycerolipids in the liver, such as phosphatidylethanolamine (18:1/18:1) and phosphatidylinositol (18:0/18:2). We also examined alteration of AA-containing PCs and some phosphoglycerolipids in the pre-PLD stage and found that these lipid alterations were initiated before pathological alteration in the liver. In addition, changes in plasma and liver levels of AA-containing PCs were linearly associated. Moreover, levels of free AA and mRNA levels of AA-synthesizing enzymes, such as fatty acid desaturase 1 and 2, were decreased by tamoxifen treatment. Therefore, our study demonstrated that AA-containing PCs might have potential utility as novel and predictive biomarkers for tamoxifen-induced PLD. Copyright © 2017 John Wiley & Sons, Ltd.

Usefulness of in vitro combination assays of mitochondrial dysfunction and apoptosis for the estimation of potential risk of idiosyncratic drug induced liver injury.

Drug-induced liver injury (DILI) is one of the serious and frequent drug-related adverse events. This adverse event is a main reason for regulatory action pertaining to drugs, including restrictions in clinical indications and withdrawal from clinical trials or the marketplace. Idiosyncratic DILI especially has become a major clinical concern because of its unpredictable nature, frequent hospitalization, need for liver transplantation and high mortality. The estimation of the potential for compounds to induce idiosyncratic DILI is very difficult in non-clinical studies because the precise mechanism of idiosyncratic DILI is still unknown. Recently, many in vitro assays which indicate a possibility of the prediction of the idiosyncratic DILI have been reported. Among these, some in vitro assays focus on the effects of compounds on mitochondrial function and the apoptotic effects of compounds on human hepatocytes. In this study, we measured oxygen consumption rate (OCR) and caspase-3/7 activity as an endpoint of mitochondrial dysfunction and apoptosis, respectively, with human hepatocytes after treatment with compounds causing idiosyncratic DILI (troglitazone, leflunomide, ranitidine and diclofenac). Troglitazone and leflunomide decreased the OCR but did not affect caspase-3/7 activity. Ranitidine increased caspase-3/7 activity but did not affect the OCR. Diclofenac decreased the OCR and increased caspase-3/7 activity. Acetaminophen and ethanol, which are also hepatotoxicants but do not induce idiosyncratic DILI, did not affect the OCR or caspase-3/7 activity. These results indicate that a combination assay of mitochondrial dysfunction and apoptosis is useful for the estimation of potential risk of compounds to induce idiosyncratic DILI.

[Investigation of Predisposition Biomarkers to Identify Risk Factors for Drug-induced Liver Injury in Humans: Analyses of Endogenous Metabolites in an Animal Model Mimicking Human Responders to APAP-induced Hepatotoxicity].

Drug-induced liver injury is a main reason of regulatory action pertaining to drugs, including restrictions to clinical indications and withdrawal from the marketplace. Acetaminophen (APAP) is a commonly used and effective analgesic/antipyretic agent and relatively safe drug even in long-term treatment. However, it is known that APAP at therapeutic doses may cause hepatotoxicity in some individuals. Hence great efforts have been made to identify risk factors for APAP-induced chronic hepatotoxicity. We investigated the contribution of undernourishment to susceptibility to APAP-induced chronic hepatotoxicity using an animal model. We employed daytime restricted fed (RF) rats as a modified-nutritional state model for human APAP-induced hepatotoxicity. RF and ad libitum fed (ALF) rats were given APAP at 0, 300, and 500 mg/kg for 3 months. Plasma and urinary glutathione-related metabolomes and liver function parameters were measured during the dosing period. Endogenous metabolites forming at different levels between the RF and ALF rats could be potential predisposition biomarkers for APAP-induced hepatotoxicity. In addition, RF rats were considered a useful model to estimate the contribution of nutritional state of patients to APAP-induced chronic hepatotoxicity. In this article we report our current research focusing on nutritional state as risk factor for APAP-induced chronic hepatotoxicity and our findings of hepatotoxicity biomarkers.

Enhancement of acetaminophen-induced chronic hepatotoxicity in restricted fed rats: a nonclinical approach to acetaminophen-induced chronic hepatotoxicity in susceptible patients.

Acetaminophen (APAP) is a commonly used and effective analgesic and antipyretic agent. However, some patients encounter hepatotoxicity after repeated APAP dosing at therapeutic doses. In the present study, we focused on the nutritional state as one of the risk factors of APAP-induced chronic hepatotoxicity in humans and investigated the contribution of undernourishment to susceptibility to APAP-induced chronic hepatotoxicity using an animal model mimicking undernourished patients. Rats were divided into 2 groups: the ad libitum fed (ALF) and the restricted fed (RF) rats and were assigned to 3 groups (n = 8/group) for each feeding condition. The animals were given APAP at 0, 300 and 500mg/kg for 99 days under each feeding condition. Plasma and urinary glutathione-related metabolites and liver function parameters were measured during the dosing period and hepatic glutathione levels were measured at the end of the dosing period. In the APAP-treated ALF rats hepatic glutathione levels were increased and hepatic function parameters were not changed, but in the APAP-treated RF rats hepatic glutathione levels were decreased at 500mg/kg and hepatic function parameters were increased at 300 and 500mg/kg. Moreover the urinary endogenous metabolite profile after long-term treatment with APAP in the ALF and RF rats was similar to that in human non-responders and responders to APAP-induced chronic hepatotoxicity, respectively. In conclusion, the RF rats were more sensitive to APAP-induced chronic hepatotoxicity than the ALF rats and were considered to be a useful model to estimate the contribution of the nutritional state of patients to APAP-induced chronic hepatotoxicity.

Effects of spaced feeding on gene expression of hepatic transaminase and gluconeogenic enzymes in rats.

Blood alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities are widely used as sensitive markers of liver toxicity. However, these activities are also recognized to be altered by hormonal and nutritional modifications. We investigated the relationships between the activity and gene expression of the hepatic transaminases and the state of hepatic amino acid/glucose/fatty acid metabolism in the ad libitum fed (ALF) and spaced-fed (SF) rats. Acceleration of hepatic gluconeogenesis and fatty acid oxidation was noted in the SF rats. Expression of hepatic clock gene was also altered in the SF rats. Hepatic transaminase activities in the SF rats were higher than those in the ALF rats. These alterations were due to increases in the synthesis of hepatic ALT and AST proteins. In conclusion, the increased transaminase protein synthesis in the liver of the SF rats was considered to be related to the acceleration of hepatic gluconeogenesis under the conditions of spaced feeding.

Relationships between plasma and tissue transaminase activities in rats maintained under different feeding conditions.

In order to verify the nutritional aspect of alterations of the plasma and tissue transaminase activities, rats were fed 4 hr per day for 35 days (the spaced-fed (SF) rats) and the time course of the alterations in plasma and tissue transaminase activity was compared with those in the ad libitum fed (ALF) rats. Plasma transaminase activities were stable throughout the experiment period in the ALF rats. In the SF rats there were alterations in the plasma alanine aminotransferase (ALT) activities, the direction of which was different between the early phase and late phase of the experiment period; plasma ALT activities decreased in the early phase and gradually increased in the late phase. Plasma aspartate aminotransferase (AST) activities were stable in the SF rats throughout the experiment period as well as the ALF rats. The decreases in plasma ALT activities in the early phase were considered to be related to decreases in ALT activities in the small intestinal mucosa (SI mucosa). On the other hand, the increases in plasma ALT activities in the late phase were considered to be related to increases in ALT activities in the liver. Multiple regression analyses (MRAs) revealed that plasma ALT activities in the SF rats could be estimated by the ALT activities in the SI mucosa and liver. From these results, the alterations of the plasma ALT activities in the SF rats could be explained by those in the SI mucosa and liver under the conditions in our study.

Effects of fenofibrate on plasma and hepatic transaminase activities and hepatic transaminase gene expression in rats.

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels are widely used as sensitive markers of possible tissue damage, particularly liver toxicity. Lipid-lowering drugs, such as fibrates, slightly increase serum transaminase levels in humans, but there is little evidence that the phenomenon is related to drug-induced liver injury (DILI). Some in vitro studies have indicated that the elevations of serum transaminase activities after treatment of humans with fenofibrate, one of the fibrates, are related to increased transaminase synthesis in the hepatocytes rather than to transaminase leakage from the hepatocytes associated with cell lysis. In this study, male F344/DuCrlCrlj (Fischer) rats were treated once with fenofibrate at a dose level of 400 mg/kg and the relationships between the pharmacological effects, blood and hepatic transaminase activities and the gene expression of the transaminases in the liver were investigated. Fenofibrate treatment slightly increased plasma transaminase activities in rats with the findings directly related to the pharmacological action of the drug. The increases were in parallel with increases in hepatic transaminase activities associated with increases in the transaminase genes in the liver and were not considered to be a consequence of hepatotoxicity from the drug. The modification in transaminase gene expression is likely to be secondary to the pharmacological action of fenofibrate. The evidence obtained in our study underlines the importance of gene regulation as a possible alternative mechanism for increased blood transaminase activities.

Urinary excretion of oxidative metabolites of bilirubin in fenofibrate-treated rats.

Bilirubin oxidative metabolites (BOM) were shown to be excreted into the urine in rats in which exaggerated oxidative stress was induced. We measured bilirubin (BR) and biopyrrins in the urine of rats treated with fenofibrate, a peroxisome proliferator, which is known to cause oxidative stress. Male Crj:CD(SD)IGS rats aged 6 weeks were treated orally with fenofibrate at 10, 400 and 800 mg/kg for 2 weeks. Urinary excretion of BR and BOM, and the plasma BOM levels were determined after the first dose and after 1-week and 2-week treatment. Urinary excretion of BOM was significantly and dose-dependently increased by fenofibrate treatment at 400 and 800 mg/kg. This became more prominent as the dosing period progressed and reached an 8-fold increase in the 400 mg/kg group and 11-fold increase in the 800 mg/kg group compared with the data before dosing on Day 14. Plasma BOM levels were increased 1.8-fold and 2.7-fold, respectively, at 400 and 800 mg/kg in fenofibrate-treated rats. At 800 mg/kg, there was also increased urinary excretion of BR (2-fold) on Day 14. These changes of BOM in the urine and plasma indicated that BR was oxidized by reactive oxygen species (ROSs), which were produced by treatment with fenofibrate. In conclusion, urinary excretion of BOM, which is a marker for oxidative stress, urinary excretion of BR and the plasma BOM levels were increased in rats treated with fenofibrate. Increased urinary excretions of BR and BOM, and increased plasma BOM levels are likely to be the consequence of physiological protection against the oxidative stress produced by fenofibrate. These findings suggest a possibility that analysis of BOM in the urine and plasma could be helpful in evaluating the degree of oxidative stress in vivo.