Increased Serum Alanine Aminotransferase Levels Research Articles

ROLE OF EMBLICA OFFICINALIS IN COPPER INDUCED LIVER TOXICITY IN ADULT ALBINO RATS

Background: Copper (Cu) is an important heavy metal used widely in all industries. However, its toxicity on various organs is established. Emblica officinalis, commonly known as Amla, is a potential antioxidant and can prevent Cu induced hepatotoxicity. 
 Objective: To evaluate the effects of Emblica officinalis extract on liver histology and Alanine Aminotransferase (ALT) levels after Cu induced toxicity on adult albino rats.
 Methods: This was an experimental study of 28 days duration. 36 adult male albino rats were divided into three groups of twelve animals each. Group A was control group in which rats were given 1.5 ml normal saline while group B rats were given CuSO4 200mg/kg b.w./day. Rats in Group C were given both CuSO4 (200mg/kg b.w/day) and Emblica officinalis fruit extract (300mg/kg bw/day). The agents were fed via orogastric tube for four weeks and rats were sacrificed 24 hours after administration of last dose.
 Results: Cu treated rats showed necrosis of hepatocytes and Increased serum ALT levels. Emblica officinalis co-treatment attenuated Cu-induced hepatic necrosis and variation in ALT levels. The difference among the groups was statistically significant with p-value <0.05.
 Conclusion: Emblica officinalis fruit extract improves liver function in Cu induced hepatotoxicity by limiting oxidative damage.

Loss of carnitine palmitoyltransferase 1a reduces docosahexaenoic acid-containing phospholipids and drives sexually dimorphic liver disease in mice

Background and aimsGenome and epigenome wide association studies identified variants in carnitine palmitoyltransferase 1a (CPT1a) that associate with lipid traits. The goal of this study was to determine the role of liver-specific CPT1a on hepatic lipid metabolism. Approach and resultsMale and female liver-specific knockout (LKO) and littermate controls were placed on a low-fat or high-fat diet (60% kcal fat) for 15 weeks. Mice were necropsied after a 16 h fast, and tissues were collected for lipidomics, matrix-assisted laser desorption ionization mass spectrometry imaging, kinome analysis, RNA-sequencing, and protein expression by immunoblotting. Female LKO mice had increased serum alanine aminotransferase levels which were associated with greater deposition of hepatic lipids, while male mice were not affected by CPT1a deletion relative to male control mice. Mice with CPT1a deletion had reductions in DHA-containing phospholipids at the expense of monounsaturated fatty acids (MUFA)-containing phospholipids in whole liver and at the level of the lipid droplet (LD). Male and female LKO mice increased RNA levels of genes involved in LD lipolysis (Plin2, Cidec, G0S2) and in polyunsaturated fatty acid metabolism (Elovl5, Fads1, Elovl2), while only female LKO mice increased genes involved in inflammation (Ly6d, Mmp12, Cxcl2). Kinase profiling showed decreased protein kinase A activity, which coincided with increased PLIN2, PLIN5, and G0S2 protein levels and decreased triglyceride hydrolysis in LKO mice. ConclusionsLiver-specific deletion of CPT1a promotes sexually dimorphic steatotic liver disease (SLD) in mice, and here we have identified new mechanisms by which females are protected from HFD-induced liver injury.

Selpercatinib Is a Promising Treatment Option for RET-Mutated Medullary and RET Fusion-Positive Thyroid Cancers

Selpercatinib Is a Promising Treatment Option for <i>RET</i>-Mutated Medullary and <i>RET</i> Fusion-Positive Thyroid Cancers

Postoperative Atrial Fibrillation and Flutter in Liver Transplantation: An Important Predictor of Early and Late Morbidity and Mortality.

Postoperative atrial fibrillation/flutter (POAF) is the most common perioperative arrhythmia and may be particularly problematic after liver transplantation (LT). This study is a single-center retrospective analysis of POAF to determine its incidence following LT, to identify risk factors, to assess its impact on clinical outcomes, and to summarize management strategies. The records of all patients who underwent LT between 2010 and 2018 were reviewed. Extracted data included pre-LT demographics and cardiac evaluation, in-hospital post-LT cardiac events, early and late complications, and survival. Among 1011 patients, the incidence of post-LT POAF was 10%. Using binary logistic regression, pre-LT history of atrial fibrillation was the strongest predictor of POAF (odds ratio [OR], 6.72; 95% confidence interval [CI], 2.00-22.57; P<0.001), followed by history of coronary artery disease (CAD; OR, 2.52; 95% CI, 1.10-5.81; P=0.03). Cardiac stress testing abnormality and CAD on cardiac catheterization were also associated with higher risk. Median time to POAF onset after LT was 3days with 72% of cases resolving within 48hours. POAF patients had greater hospital length of stay, death during the LT admission, and 90-day and 1-year mortality. POAF was an independent risk factor for post-LT mortality (OR, 2.0; 95% CI, 1.3-3.0; P<0.01). Amiodarone was administered to 73% of POAF patients with no evidence of increased serum alanine aminotransferase levels. POAF occurred in 10% of post-LT patients with early onset and rapid resolution in most affected patients. POAF patients, however, had significant morbidity and mortality, suggesting that POAF is an important marker for worse early and late post-LT outcomes.

MicroRNA-10a Impairs Liver Metabolism in Hepatitis C Virus-Related Cirrhosis Through Deregulation of the Circadian Clock Gene Brain and Muscle Aryl Hydrocarbon Receptor Nuclear Translocator-Like 1.

The circadian rhythm of the liver plays an important role in maintaining its metabolic homeostasis. We performed comprehensive expression analysis of microRNAs (miRNAs) using TaqMan polymerase chain reaction of liver biopsy tissues to identify the miRNAs that are significantly up‐regulated in advanced chronic hepatitis C (CHC). We found miR‐10a regulated various liver metabolism genes and was markedly up‐regulated by hepatitis C virus infection and poor nutritional conditions. The expression of miR‐10a was rhythmic and down‐regulated the expression of the circadian rhythm gene brain and muscle aryl hydrocarbon receptor nuclear translocator‐like 1 (Bmal1) by directly suppressing the expression of RA receptor‐related orphan receptor alpha (RORA). Overexpression of miR‐10a in hepatocytes blunted circadian rhythm of Bmal1 and inhibited the expression of lipid synthesis genes (sterol regulatory element binding protein [SREBP]1, fatty acid synthase [FASN], and SREBP2), gluconeogenesis (peroxisome proliferator‐activated receptor gamma coactivator 1 alpha [PGC1α]), protein synthesis (mammalian target of rapamycin [mTOR] and ribosomal protein S6 kinase [S6K]) and bile acid synthesis (liver receptor homolog 1 [LRH1]). The expression of Bmal1 was significantly correlated with the expression of mitochondrial biogenesis‐related genes and reduced Bmal1 was associated with increased serum alanine aminotransferase levels and progression of liver fibrosis in CHC. Thus, impaired circadian rhythm expression of Bmal1 by miR‐10a disturbs metabolic adaptations, leading to liver damage, and is closely associated with the exacerbation of abnormal liver metabolism in patients with advanced CHC. In patients with hepatitis C‐related liver cirrhosis, liver tissue miR‐10a levels were significantly associated with hepatic reserve, fibrosis markers, esophageal varix complications, and hepatitis C‐related hepatocellular carcinoma recurrence. Conclusion: MiRNA‐10a is involved in abnormal liver metabolism in cirrhotic liver through down‐regulation of the expression of the circadian rhythm gene Bmal1. Therefore, miR‐10a is a possible useful biomarker for estimating the prognosis of liver cirrhosis.

Prescription Audit of Anti-fungal Treatment of Dermatophytoses in the Dermatology Out Patient Department of a Tertiary Care Hospital

Aims: To study the prescriptions of clinically diagnosed cases of dermatophytoses, to evaluate medical treatment of clinical failure cases, any rise in serum ALT (Alanine aminotransferase) levels associated with usage of oral anti-fungal drugs, availability of drugs in the hospital pharmacy and cost minimization of different oral treatment regimens with Griseofulvin, Fluconazole, Itraconazole and Terbinafine. Settings and design: Dermatology Out Patient Department (OPD) of a tertiary care hospital, single centre prospective cohort observational study. Materials and Methods: 370 patients from the Dermatology Out Patient Department (OPD) diagnosed of dermatophytoses were enrolled. Follow-up was done 3±1 weeks apart, twice for newly diagnosed patients and once for patients on ongoing therapy. Two separate blood samples were collected 3±1 weeks apart for estimation of serum ALT. The prime lesion in each patient was graded as mild, moderate and severe. Patients were categorized as clinically cured, clinically improved, or as clinical failure following treatment. Statistical analysis used: Descriptive statistics, Fischer’s exact test and Wilcoxon’s signed rank test. Results: Azoles were the most commonly prescribed antifungals. Clinical status of patients was independent of the oral antifungal drug received (p &gt; 0.05). A statistically significant difference in clinical status between compliant and non-compliant patients was seen (p &lt; 0.05). A statistically significant increase in serum ALT levels was seen (p &lt; 0.05) however, it was not clinically significant. 55.52% of drugs were unavailable in the hospital formulary. The average expenses per prescription was approximately INR 1123.55. Itraconazole was the most expensive treatment regimen followed by terbinafine and fluconazole. Conclusions: There exists a non-uniformity in the management of dermatophytoses due to lack of adequate guidance and nonavailability of drugs and clinical cure of patients depends upon compliance to therapy.

Palmitate-induced lipotoxicity is crucial for the pathogenesis of nonalcoholic fatty liver disease in cooperation with gut-derived endotoxin

Although previous studies have indicated important roles of palmitate, a saturated fatty acid, in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), it remains unclear how palmitate contributes to inflammation and fibrosis in the liver. Administration of palmitate in high fat diet (HFD)-fed but not basal diet (BD)-fed mice resulted in an increase in serum alanine aminotransferase (ALT) levels. Surprisingly, combined administration of very low dose lipopolysaccharide in palmitate-treated mice led to a marked increase in serum ALT levels despite BD-fed conditions. Administration of palmitate alone in BD-fed mice caused inflammatory cell infiltration and liver fibrosis mediated by the toll-like receptor 4 pathway without ALT elevation. In addition, a significant correlation between serum free fatty acid levels and liver fibrosis stage was observed in patients with NAFLD. These results indicate that palmitate may play crucial roles in the pathogenesis of NAFLD in the presence of gut-derived endotoxin.

Myeloid Notch1 deficiency activates the RhoA/ROCK pathway and aggravates hepatocellular damage in mouse ischemic livers.

Myeloid Notch1 deficiency activates the ras homolog gene family member A/ROCK pathway and exacerbates hepatocellular injury by inhibiting transcriptional repressor Hes1 and inducing scaffold protein JSAP1 in IR-triggered liver inflammation; our findings underscore the crucial role of the Notch-Hes1 axis as a novel regulator of innate immunity-mediated inflammation and imply the therapeutic potential for the management of organ IR injury in transplant recipients. (Hepatology 2018;67:1041-1055).

Inhibition of Drp1 protects against senecionine-induced mitochondria-mediated apoptosis in primary hepatocytes and in mice

Pyrrolizidine alkaloids (PAs) are a group of compounds found in various plants and some of them are widely consumed in the world as herbal medicines and food supplements. PAs are potent hepatotoxins that cause irreversible liver injury in animals and humans. However, the mechanisms by which PAs induce liver injury are not clear. In the present study, we determined the hepatotoxicity and molecular mechanisms of senecionine, one of the most common toxic PAs, in primary cultured mouse and human hepatocytes as well as in mice. We found that senecionine administration increased serum alanine aminotransferase levels in mice. H&E and TUNEL staining of liver tissues revealed increased hemorrhage and hepatocyte apoptosis in liver zone 2 areas. Mechanistically, senecionine induced loss of mitochondrial membrane potential, release of mitochondrial cytochrome c as well as mitochondrial JNK translocation and activation prior to the increased DNA fragmentation and caspase-3 activation in primary cultured mouse and human hepatocytes. SP600125, a specific JNK inhibitor, and ZVAD-fmk, a general caspase inhibitor, alleviated senecionine-induced apoptosis in primary hepatocytes. Interestingly, senecionine also caused marked mitochondria fragmentation in hepatocytes. Pharmacological inhibition of dynamin-related protein1 (Drp1), a protein that is critical to regulate mitochondrial fission, blocked senecionine-induced mitochondrial fragmentation and mitochondrial release of cytochrome c and apoptosis. More importantly, hepatocyte-specific Drp1 knockout mice were resistant to senecionine-induced liver injury due to decreased mitochondrial damage and apoptosis. In conclusion, our results uncovered a novel mechanism of Drp1-mediated mitochondrial fragmentation in senecionine-induced liver injury. Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by PAs.

Autophagy confers resistance to lipopolysaccharide-induced mouse hepatocyte injury.

During sepsis, bacterial products, particularly LPS, trigger injury in organs such as the liver. This common condition remains largely untreatable, in part due to a lack of understanding of how high concentrations of LPS cause cellular injury. In the liver, the lysosomal degradative pathway of autophagy performs essential hepatoprotective functions and is induced by LPS. We, therefore, examined whether hepatocyte autophagy protects against liver injury from septic levels of LPS. Mice with an inducible hepatocyte-specific knockout of the critical autophagy gene Atg7 were examined for their sensitivity to high-dose LPS. Increased liver injury occurred in knockout mice, as determined by significantly increased serum alanine aminotransferase levels, histological evidence of liver injury, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, and effector caspase-3 and -7 activation. Hepatic inflammation and proinflammatory cytokine induction were unaffected by the decrease in hepatocyte autophagy. Although knockout mice had normal NF-κB signaling, hepatic levels of Akt1 and Akt2 phosphorylation in response to LPS were decreased. Cultured hepatocytes from knockout mice displayed a generalized defect in Akt signaling in response to multiple stimuli, including LPS, TNF, and IL-1β. Akt activation mediates hepatocyte resistance to TNF cytotoxicity, and anti-TNF antibodies significantly decreased LPS-induced liver injury in knockout mice, indicating that the loss of autophagy sensitized to TNF-dependent liver damage. Hepatocyte autophagy, therefore, protects against LPS-induced liver injury. Conditions such as aging and steatosis that impair hepatic autophagy may predispose to poor outcomes from sepsis through this mechanism.

Effect of dietary cottonseed meal on growth performance, physiological response, and gossypol accumulation in pre-adult grass carp, Ctenopharyngodon idellus

Cottonseed meal (CM) was used at up to 36.95% content in the diet (replacing 60% of dietary fish meal protein) without any negative eff ects on growth performance of pre-adult grass carp (initial body weight, 761 g) under outdoor conditions. A culture trial was conducted in net cages installed in a large concrete pond. Seven isonitrogenous and isoenergetic diets containing a gradient of CM concentrations (0, 12.2%, 24.4%, 36.6%, 48.8%, 54.8%, and 61.0%) as replacement for dietary fish meal protein (0, 20%, 40%, 60%, 80%, 90%, and 100%) were formulated. Dietary non-resistant starch (from maize) was inverse to dietary CM. Growth performance and feed utilization of fish fed the diets containing CM replacing 0–40% fishmeal protein were not aff ected after the 6-week feeding trial. Accumulation of hepatopancreatic total gossypol in the hepatopancreas was significantly correlated with free gossypol content in the diets (HTG=88.6+1.5×DFG, R2=0.89, P<0.05). Intestinal α-amylase and γ-glutamyl transpeptidase activities rose along with increasing dietary CM level. The structure of the mid-intestinal tissues and the ultrastructure of the enterocyte microvilli were normal when dietary CM was <36.6% (60% protein replacement). Increasing dietary CM content increased serum alanine aminotransferase levels but decreased serum alkaline phosphatase, cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and albumin (P<0.05).

ATF3-Mediated NRF2/HO-1 Signaling Regulates TLR4 Innate Immune Responses in Mouse Liver Ischemia/Reperfusion Injury

Activating transcription factor 3 (ATF3) is a stress-induced transcription factor that has been shown to repress inflammatory gene expression in multiple cell types and diseases. However, little is known about the roles and mechanisms of ATF3 in liver ischemia/reperfusion injury (IRI). In warm and cold liver IRI models, we showed that ATF3 deficiency significantly increased ischemia/reperfusion (IR)-stressed liver injury, as evidenced by increased serum alanine aminotransferase levels, histological liver damage, and hepatocellular apoptosis. These may correlate with inhibition of the intrahepatic nuclear factor erythroid-derived 2-related factor 2/heme oxygenase-1 (NRF2/HO-1) signaling pathway leading to enhancing Toll-like receptor 4/nuclear factor kappa beta (TLR4/NF-κB) activation, pro-inflammatory programs and macrophage/neutrophil trafficking, while simultaneously repressing anti-apoptotic molecules in ischemic liver. Interestingly, activation of NRF2/HO-1 signaling using an NRF2 activator, oltipraz (M2), during hepatic IRI-rescued ATF3 anti-inflammatory functions in ATF3-deficient mice. For in vitro studies, ATF3 ablation in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMMs) depressed levels of NRF2/HO-1 and PI3K/AKT, resulting in enhanced TLR4/NF-κB activation. Pretreatment of LPS-stimulated BMMs with M2 increased NRF2/HO-1 expression, promoted PI3K/AKT, which in turn suppressed TLR4/NF-κB-mediated proinflammatory mediators. Thus, our results first demonstrate ATF3-mediated NRF2/HO-1 signaling in the regulation of TLR4-driven inflammatory responses in IR-stressed livers. Our findings provide a rationale for a novel therapeutic strategy for managing IR-induced liver injury.

Changes in the incidence of severe thrombocytopenia and its predisposing conditions in HIV-infected patients since the introduction of highly active antiretroviral therapy.

Severe thrombocytopenia (TCP, platelets <50 × 10⁹/L) is relatively frequent during HIV infection and is associated with bleeding risk and disease progression. We investigated the changes in the incidence of severe TCP and its predisposing conditions in a cohort of HIV-positive subjects. The incidence and predictors of platelet counts <50 × 10⁹/L were investigated in all patients enrolled at our institution between 1985 and 2012. Three different periods were considered on the basis of the available antiretroviral regimens: P1 (1985-1989), P2 (1990-1996), and P3 (1997-2012). Incidence rates were assessed using Poisson regression models and the predictors by means of Cox regression. The study involved 5137 patients with platelet counts >50 × 10⁹/L at enrollment. Severe TCP occurred in 597 subjects, and its incidence decreased over time. The incidence decreased in patients with opportunistic diseases and malignancies but increased in patients with chronic liver disease; TCP unrelated to any cause other than HIV infection remained stable. Multivariate analysis showed that injected drug use, a diagnosis of AIDS, low CD4⁺ cell counts, increased serum alanine aminotransferase levels, and an earlier year of enrollment were predictors of an increased risk of severe TCP, whereas the use of highly active antiretroviral therapy (HAART) was associated with a reduced risk. A considerable reduction in the incidence of severe TCP after the introduction of HAART was found, probably because of its ability to limit bone marrow damage induced by uncontrolled HIV replication and opportunistic infections. On the contrary, HAART may have a reduced impact on TCP related to chronic liver disease.

Portal vein arterialization promotes liver regeneration after extended partial hepatectomy in a rat model.

In the current study, we sought to establish a novel rat model of portal vein arterialization (PVA) and evaluate its impact on liver regeneration after extended partial hepatectomy (PH). A total of 105 Sprague-Dawley rats were randomly assigned to three groups: 68% hepatectomy (the PH group), portal arterialization after 68% hepatectomy (the PVA group), and right nephrectomy only (the control group). Liver regeneration rate (LRR), 5-bromo-2-deoxyuridine (BrdU) labeling index, and liver functions were assessed on postoperative day 2, 7, 14 and 28. The 28-day survival rates were compared among the three groups. The 28-day survival rates were similar in all groups (P = 0.331), and the anastomotic patency was 100%. The LRR in the PVA group was significantly higher than that of the PH group within postoperative 14 days (P < 0.05). The PVA and PH group had increased serum alanine aminotransferase levels (232 ± 61 U/L and 212 ± 53 U/L, respectively) compared with the control group (101 ± 13 U/L) on postoperative day 2, whereas from postoperative day 7 to day 28 there were no differences among the three groups. Serum albumin values were higher after the PVA procedure within postoperative day 14, which gradually became comparable on postoperative day 28 among the three groups. The peaks of BrdU labeling index appeared on postoperative day 2 in all rats, and the PVA procedure was associated with increased BrdU labeling index from postoperative day 7 to 28. The 28-day survival of the PVA rats was comparable. Our findings demonstrate that the PVA procedure utilizing portal vein trunk-renal artery microvascular reconstruction promotes remnant liver regeneration and confers beneficial effects on maintaining and even optimizing liver function after extended partial hepatectomy in rats.

Toxicity of new emerging pollutant tris-(2,3-dibromopropyl) isocyanurate on BALB/c mice.

The emerging heterocyclic brominated flame retardant tris-(2,3-dibromopropyl) isocyanurate (TBC), widely used in reinforced plastics, has demonstrated toxicity to fish. However, little is known about its toxicity in rodents. This study aims to determine the effect of TBC on growth, biochemical parameters in serum, organs and related gene expression of both male and female BALB/c mice after gastro-gavage administration of 0, 2, 10 and 50 mg kg⁻¹ TBC for 28 days. Results indicated that exposure to TBC had no effects on basic growth and food intake of mice, but significantly increased serum alanine aminotransferase levels in male mice. Histopathological analyses showed that focal necrosis (2, 10 and 50 mg kg⁻¹ TBC-exposed groups) and ballooning degeneration (10 and 50 mg kg⁻¹ TBC-exposed groups) were found in mouse liver, whereas transmission electron microscopy revealed dose-dependent hepatocyte apoptosis, mitochondrial degeneration and endoplasmic reticulum dilation. Histopathological and ultrastructural assessments in the lung showed dose-dependent hyperplasia of pulmonary alveolar epithelium, bronchial congestion, infiltration of inflammatory cells and mitochondrial swelling following TBC exposure. Our results also indicated that mitochondria are one of the major target cytoplasmic organelles for TBC, suggesting that damage in mitochondria is one of the pathways that led to toxic effects in the liver and lung of TBC-treated groups. Moreover, TBC effectively activated the gene expression of p53 in mice liver. Our findings provide strong evidence that TBC induces significant toxicity in mice organs, especially in liver and lung, which play vital roles in detoxification and gas exchange, respectively. This research will contribute to characterize the toxic effects of TBC, which was introduced as one of the candidates for brominated flame retardant replacement.

Lack of interleukin-1α in Kupffer cells attenuates liver inflammation and expression of inflammatory cytokines in hypercholesterolaemic mice

The role of Kupffer cell interleukin (IL)-1 in non-alcoholic steatohepatitis development remains unclear. To evaluate the role of Kupffer cell IL-1α, IL-1β or IL-1 receptor type-1 (IL-1R1) in steatohepatitis. C57BL/6 mice were irradiated and transplanted with bone marrow-derived cells from WT, IL-1α-/-, IL-1β-/- or IL-1R1-/- mice combined with Kupffer cell ablation with Gadolinium Chloride, and fed atherogenic diet. Plasma and liver triglycerides and cholesterol, serum alanine aminotransferase (ALT), liver histology and expression levels of inflammatory genes were assessed. The ablation and replacement of Kupffer cells with bone marrow-derived cells was confirmed. The atherogenic diet elevated plasma and liver cholesterol, reduced plasma and liver triglycerides and increased serum ALT levels in all groups. Steatosis and steatohepatitis were induced, but without liver fibrosis. A reduction in the severity of portal inflammation was observed only in mice with Kupffer cell deficiency of IL-1α. Accordingly, liver mRNA levels of inflammatory genes encoding for IL-1α, IL-1β, TNFα, SAA1 and IL-6 were significantly lower in mice with Kupffer cell deficiency of IL-1α compared to WT mice. Selective deficiency of IL-1α in Kupffer cells reduces liver inflammation and expression of inflammatory cytokines, which may implicate Kupffer cell-derived IL-1α in steatohepatitis development.

Comparison of Hepatic Transcription Profiles of Locked Ribonucleic Acid Antisense Oligonucleotides: Evidence of Distinct Pathways Contributing to Non-target Mediated Toxicity in Mice

Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice administered non-toxic and toxic LNA gapmers. After repeated administration, a toxic LNA gapmer (TS-2), but not a non-toxic LNA gapmer (NTS-1), caused hepatocyte necrosis and increased serum alanine aminotransferase levels. Microarray data revealed that, in addition to gene expression patterns consistent with hepatotoxicity, 17 genes in the clathrin-mediated endocytosis (CME) pathway were altered in the TS-2 group. TS-2 significantly down-regulated myosin 1E (Myo1E), which is involved in release of clathrin-coated pits from plasma membranes. To map the earliest transcription changes associated with LNA gapmer-induced hepatotoxicity, a second microarray analysis was performed using NTS-1, TS-2, and a severely toxic LNA gapmer (HTS-3) at 8, 16, and 72 h following a single administration in mice. The only histopathological change observed was minor hepatic hypertrophy in all LNA groups across time points. NTS-1, but not 2 toxic LNA gapmers, increased immune response genes at 8 and 16 h but not at 72 h. TS-2 significantly perturbed the CME pathway only at 72 h, while Myo1E levels were decreased at all time points. In contrast, HTS-3 modulated DNA damage pathway genes at 8 and 16 h and also modulated the CME pathway genes (but not Myo1E) at 16 h. Our results may suggest that different LNAs modulate distinct transcriptional genes and pathways contributing to non-target mediated hepatotoxicity in mice.

Induction of autophagy, a promising approach for treating liver injury

Recent investigations have demonstrated a complex interrelationship between autophagy and cell death. A common mechanism of cell death in liver injury is tumor necrosis factor (TNF) cytotoxicity. To better delineate the in vivo function of autophagy in cell death, we examined the role of autophagy in TNF-induced hepatic injury. Atg7Δhep mice with a hepatocyte-specific knockout of the autophagy gene atg7 were generated and cotreated with D-galactosamine (GalN) and lipopolysaccharide (LPS). GalN/LPS-treated Atg7Δhep mice had increased serum alanine aminotransferase levels, histological injury, numbers of TUNEL (terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling)-positive cells and mortality as compared with littermate controls. Loss of hepatocyte autophagy similarly sensitized to GalN/TNF liver injury. GalN/LPS injury in knockout animals did not result from altered production of TNF or other cytokines. Atg7Δhep mice had accelerated activation of the mitochondrial death pathway and caspase-3 and −7 cleavage. Increased cell death did not occur from direct mitochondrial toxicity or a lack of mitophagy, but rather from increased activation of initiator caspase-8 causing Bid cleavage. GalN blocked LPS induction of hepatic autophagy, and increased autophagy from beclin 1 overexpression prevented GalN/LPS injury. Autophagy, therefore, mediates cellular resistance to TNF toxicity in vivo by blocking activation of caspase-8 and the mitochondrial death pathway, suggesting that autophagy is a therapeutic target in TNF-dependent tissue injury.

A high‐fat diet and multiple administration of carbon tetrachloride induces liver injury and pathological features associated with non‐alcoholic steatohepatitis in mice

The aim of the present study was to establish a progressive steatohepatitis mouse model because few reported animal models of non-alcoholic steatohepatitis (NASH) show the progression from fatty liver to steatohepatitis. C57BL/6N mice were fed a high-fat diet (HFD) to develop obesity and were either administered carbon tetrachloride (CCl4 ) eight times (0.05mL/kg, s.c., once, followed by 0.1mL/kg, s.c., seven times) or not. Serum parameters and hepatic histopathology were examined. In a separate experiment, CCl4 was administered subcutaneously from 0 to eight times to HFD-fed obese mice to investigate progressive changes. Markers of oxidative stress, inflammation and apoptosis, as well as histopathological changes in the liver, were analysed. The HFD-fed obese mice showed fatty liver but not steatohepatitis. In contrast, HFD-fed mice administered CCl4 eight times showed histopathological features of steatohepatitis (fatty liver, inflammation, hepatocellular ballooning and fibrosis) and increased serum alanine aminotransferase levels. However, the multiple administration of CCl4 to obese mice reduced the ratio of reduced glutathione to oxidized glutathione, superoxide dismutase activity and mitochondrial DNA copy number, leading to the development of chronic oxidative stress, increased numbers of apoptotic cells and increased levels of both tumour necrosis factor-α and transforming growth factor-β mRNA. The resulting inflammation led to increased hydroxyproline content in the liver and fibrosis. The present study demonstrates that multiple administration of CCl4 to HFD-fed obese mice induces chronic oxidative stress that triggers inflammation and apoptosis and leads to the development of fibrosis in the liver, resulting in progression from fatty liver to steatohepatitis. This murine model will be useful in the research of hepatic disorders.

Inhibition of hepatocyte autophagy increases tumor necrosis factor-dependent liver injury by promoting caspase-8 activation.

Recent investigations have demonstrated a complex interrelationship between autophagy and cell death. A common mechanism of cell death in liver injury is tumor necrosis factor (TNF) cytotoxicity. To better delineate the in vivo function of autophagy in cell death, we examined the role of autophagy in TNF-induced hepatic injury. Atg7Δhep mice with a hepatocyte-specific knockout of the autophagy gene atg7 were generated and cotreated with D-galactosamine (GalN) and lipopolysaccharide (LPS). GalN/LPS-treated Atg7Δhep mice had increased serum alanine aminotransferase levels, histological injury, numbers of TUNEL (terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling)-positive cells and mortality as compared with littermate controls. Loss of hepatocyte autophagy similarly sensitized to GalN/TNF liver injury. GalN/LPS injury in knockout animals did not result from altered production of TNF or other cytokines. Atg7Δhep mice had accelerated activation of the mitochondrial death pathway and caspase-3 and -7 cleavage. Increased cell death did not occur from direct mitochondrial toxicity or a lack of mitophagy, but rather from increased activation of initiator caspase-8 causing Bid cleavage. GalN blocked LPS induction of hepatic autophagy, and increased autophagy from beclin 1 overexpression prevented GalN/LPS injury. Autophagy, therefore, mediates cellular resistance to TNF toxicity in vivo by blocking activation of caspase-8 and the mitochondrial death pathway, suggesting that autophagy is a therapeutic target in TNF-dependent tissue injury.