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The present study identified genes showing promoter region hypermethylation by CpG island microarrays in the liver of rats treated with hepatocarcinogen thioacetamide (TAA) for 28 days. Among 47 hypermethylated genes, Hist1h2aa , Tmem70 , Ube2e2 , and Slk were confirmed to show hypermethylation by methylation-specific quantitative polymerase chain reaction (PCR) and pyrosequencing analyses as well as downregulation of transcript levels by real-time reverse transcription-PCR analysis in the livers of rats treated with TAA. All gene products of the 4 selected genes showed decreased immunoreactivity forming negative liver cell foci in a subpopulation of glutathione S -transferase placental form (GST-P) + foci in TAA-promoted rat livers in a two-stage hepatocarcinogenesis model. Among them, TMEM70 and UBE2E2 showed increased incidences of negative foci in GST-P + foci by promotion of all examined TAA, β-naphthoflavone, piperonyl butoxide, fenbendazole and phenobarbital, while HIST1H2AA and SLK did not respond to all promotive treatments. In the late stage of tumor promotion by TAA, the incidence of GST-P + proliferative lesions with downregulation of TMEM70 or UBE2E2 was higher in adenomas and carcinomas than liver cell foci. TMEM70 plays a role in mitochondrial oxidative phosphorylation, and UBE2E2 participates in the stabilization of cell cycle regulatory proteins. Therefore, our results indicate that aberrant epigenetic gene downregulation suggestive of a metabolic shift of cellular respiration from oxidative phosphorylation to glycolysis and aberrant cell cycle regulation facilitating cell proliferation from as early as 28 days after hepatocarcinogen treatment contribute to tumor development. [ABSTRACT FROM AUTHOR]

Highlights: [•] ACME could restore fatty acid storage in non-induced HSCs. [•] ACME inhibited several biomarker of the activated HSC. [•] Antrodia cinnamomea could use as an functional food for the prevention of liver fibrosis. [Copyright &y& Elsevier]

Abstract: Background: Mammalian thioredoxin reductases (TrxR) are selenoproteins with important roles in antioxidant defense and redox regulation, principally linked to functions of their main substrates thioredoxins (Trx). All major forms of TrxR are intracellular while levels in serum are typically very low. Methods: Serum TrxR levels were determined with immunoblotting using antibodies against mouse TrxR1 and total enzyme activity measurements were performed, with serum and tissue samples from mouse models of liver injury, as triggered by either thioacetamide (TAA) or carbon tetrachloride (CCl4). Results: TrxR levels in serum increased upon treatment and correlated closely with those of alanine aminotransferase (ALT), an often used serum biomarker for liver damage. In contrast, Trx1, glutathione reductase, superoxide dismutase or selenium-containing glutathione peroxidase levels in serum displayed much lower increases than TrxR or ALT. Conclusions: Serum TrxR levels are robustly elevated in mouse models of chemically induced liver injury. General significance: The exaggerated TrxR release to serum upon liver injury may reflect more complex events than a mere passive release of hepatic enzymes to the extracellular milieu. It can also not be disregarded that enzymatically active TrxR in serum could have yet unidentified physiological functions. [Copyright &y& Elsevier]

Highlights: [•] Curcumin, silybin-phytosome and alpha-R lipoic acid protect against the hepatotoxic effects of thioacetamide. [•] These dietary supplements prevented the development of chemically induced liver cirrhosis. [•] The beneficial effects of these supplements are mediated via their antioxidant and antifibrotic potentials. [ABSTRACT FROM AUTHOR]

Abstract: The current study was conducted to investigate the anti-fibrotic effect and its possible underlying mechanisms of thymoquinone (TQ) against hepatic fibrosis in vivo. TQ is the major active compound derived from the medicinal Nigella sativa. Liver fibrosis was induced in male Kunming mice by intraperitoneal injections of thioacetamide (TAA, 200mg/kg). Mice were treated concurrently with TAA alone or TAA plus TQ (20mg/kg or 40mg/kg) given daily by oral gavage. Our data demonstrated that TQ treatment obviously reversed liver tissue damage compared with TAA alone group, characterized by less inflammatory infiltration and accumulation of extracellular matrix (ECM) proteins. TQ significantly attenuated TAA-induced liver fibrosis, accompanied by reduced protein and mRNA expression of α-smooth muscle actin (α-SMA), collagen-І and tissue inhibitor of metalloproteinase-1 (TIMP-1). TQ downregulated the expression of toll-like receptor 4 (TLR4) and remarkably decreased proinflammatory cytokine levels as well. TQ also significantly inhibited phosphatidylinositol 3-kinase (PI3K) phosphorylation. Furthermore, TQ enhanced the phosphorylation adenosine monophosphate-activated protein kinase (AMPK) and liver kinase B (LKB)-1. In conclusion, TQ may reduce ECM accumulation, and it may be at least regulated by phosphorylation of AMPK signaling pathways, suggesting that TQ may be a potential candidate for the therapy of hepatic fibrosis. [Copyright &y& Elsevier]

Highlights: [•] We investigated the role of ghrelin in treatment of chronic liver injury, and whether ghrelin's action is related to modulation of nitric oxide. [•] Ghrelin acts as anti-inflammatory and anti-oxidant agent by improving liver functions and decreasing serum TNF-α levels. [•] Ghrelin has an anti-fibrotic effect evidenced by the reversal of increased collagen content, and histological studies. [•] Ghrelin possesses an anti-apoptotic effect shown by reversal of increased Bax and decreased Bcl-2. [•] Ghrelin's hepatoprotective effect was partially due to increased nitric oxide release. [Copyright &y& Elsevier]

Abstract: Given the involvement of oxidative stress in liver-disease- or hepato-toxicant-induced hepatic damage and fibrosis, antioxidants are an effective preventive and therapeutic tool. The beneficial results of vitamin C, one of the physiological antioxidants, have been observed both in experimental animals and in humans. However, most of these studies have been concerned with supplementary vitamin C; the effects of under vitamin C insufficiency, which humans sometimes confront, have not been substantially investigated. In the present study, we established a vitamin C-insufficient animal model (half-to-normal serum vitamin C concentration) with gulo -/- mice that cannot synthesize vitamin C, and induced hepatotoxicity by means of thioacetamide (TAA) injections twice a week for 18 weeks. Additionally, we explored the direct effects of vitamin C both on immortalized human hepatic stellate LX-2 cells and on rat primary hepatic stellate cells. Vitamin C insufficiency resulted in a decreased survival rate and increased serum markers for hepatocyte damage, such as alanine aminotransferase and aspartate aminotransferase. Concomitantly, the levels of reactive oxygen species (ROS) and lipid peroxides in the liver were increased. Histological examinations of the vitamin C-insufficient liver revealed increases in collagen fiber deposition and activated-hepatic-stellate-cell number. Vitamin C, when directly applied to the LX-2 cells as well as the rat primary hepatic stellate cells, suppressed not only proliferation but hydrogen peroxide-induced collagen expression as well. In conclusion, vitamin C insufficiency exacerbated TAA-induced hepatotoxicity. These effects seem to be mainly from insufficient scavenging of ROS in the liver, and possibly in part, by directly affecting hepatic stellate cells. [Copyright &y& Elsevier]

Background & Aims: In vertebrates, canonical Hedgehog (Hh) pathway activation requires Smoothened (SMO) translocation to the primary cilium (Pc), followed by a GLI-mediated transcriptional response. In addition, a similar gene regulation occurs in response to growth factors/cytokines, although independently of SMO signalling. The Hh pathway plays a critical role in liver fibrosis/regeneration, however, the mechanism of activation in chronic liver injury is poorly understood. This study aimed to characterise Hh pathway activation upon thioacetamide (TAA)-induced chronic liver injury in vivo by defining Hh-responsive cells, namely cells harbouring Pc and Pc-localised SMO. Methods: C57BL/6 mice (wild-type or Ptc1 +/−) were TAA-treated. Liver injury and Hh ligand/pathway mRNA and protein expression were assessed in vivo. SMO/GLI manipulation and SMO-dependent/independent activation of GLI-mediated transcriptional response in Pc-positive (Pc+) cells were studied in vitro. Results: In vivo, Hh activation was progressively induced following TAA. At the epithelial-mesenchymal interface, injured hepatocytes produced Hh ligands. Progenitors, myofibroblasts, leukocytes and hepatocytes were GLI2+. Pc+ cells increased following TAA, but only EpCAM+/GLI2+ progenitors were Pc+/SMO+. In vitro, SMO knockdown/hGli3-R overexpression reduced proliferation/viability in Pc+ progenitors, whilst increased proliferation occurred with hGli1 overexpression. HGF induced GLI transcriptional activity independently of Pc/SMO. Ptc1 +/− mice exhibited increased progenitor, myofibroblast and fibrosis responses. Conclusions: In chronic liver injury, Pc+ progenitors receive Hh ligand signals and process it through Pc/SMO-dependent activation of GLI-mediated transcriptional response. Pc/SMO-independent GLI activation likely occurs in Pc−/GLI2+ cells. Increased fibrosis in Hh gain-of-function mice likely occurs by primary progenitor expansion/proliferation and secondary fibrotic myofibroblast expansion, in close contact with progenitors. [Copyright &y& Elsevier]

Abstract: Thioacetamide (TAA) induces oxidative stress and hepatocarcinogenicity in rats. We previously reported that TAA promotion caused various disruptions in cell cycle protein expression in rats, including downregulation of p16Ink4a, which is associated with intraexonic hypermethylation in hepatocellular proliferative lesions. This study further investigated the contribution of cell cycle aberrations associated with early hepatocarcinogenic processes induced by TAA using antioxidants, enzymatically modified isoquercitrin (EMIQ) and α-lipoic acid (ALA), in a two-stage rat hepatocarcinogenesis model. TAA-promotion after initiation with N-diethylnitrosamine increased the number and area of hepatocellular foci immunoreactive for glutathione S-transferase placental form (GST-P) and the numbers of proliferating and apoptotic cells. Co-treatment with EMIQ and ALA suppressed these increases. TAA-induced formation of p16Ink4a− foci in concordance with GST-P+ foci was not suppressed by co-treatment with EMIQ or ALA. TAA-promotion increased cellular distributions of cell proliferation marker Ki-67, G2/M and spindle checkpoint proteins (phosphorylated checkpoint kinase 1 and Mad2), the DNA damage-related protein phosphorylated histone H2AX, and G2-M phase-related proteins (topoisomerase IIα, phosphorylated histone H3 and Cdc2) within GST-P+ foci, and co-treatment with EMIQ or ALA suppressed these increases. These results suggest that downregulation of p16Ink4a may allow selective proliferation of preneoplastic cells by TAA promotion. However, antioxidants did not counteract this gene control. Moreover, effective suppression of TAA-induced cellular population changes within preneoplastic lesions by antioxidants may reflect facilitation of cell cycling and accumulation of DNA damage causing the activation of cell cycle checkpoints, leading to G2 and M phase arrest at the early stages of hepatocarcinogenesis promoted by TAA. [Copyright &y& Elsevier]

Highlights: [•] We examined the modifying effect of a-LA on tumor promotion by thioacetamide. [•] A-LA suppressed the number of ED2+ hepatic macrophages increased by thioacetamide. [•] A-LA suppressed tumor-promoting activity through anti-inflammatory action. [•] A-LA suppressed apoptosis and subsequent regeneration of non-transformed liver cells. [ABSTRACT FROM AUTHOR]

Abstract: Aim of present study was to investigate the effect of NAC on experimental chronic hepatotoxicity models induced by carbon tetrachloride (CCl4) and thioacetamide (TAA). CCl4 toxicity was induced by administering 200μl CCl4 (diluted 2:3 in coconut oil)/100g body weight, p.o., twice weekly for 8 weeks. TAA toxicity was induced by administering 150mg/kg b. wt. of TAA i.p., twice weekly for 8 weeks. NAC treatment was started along with toxicants (CCl4 and TAA) for 8 weeks and continued for further 4 weeks. Self reversal group was kept without any treatment for 4 weeks after completion of toxicant treatments. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Bilirubin were measured in serum. Hydroxyproline (HP), lipid peroxidation (LPO), catalase (CAT), Glutathione peroxidase (GPx) and Glutathione (GSH) were determined in liver samples by colorimetric methods. Cytochrome P450 2E1 (CYP 450 2E1), activity was determined as hydroxylation of aniline in liver microsomes. General examination and histological analysis were also performed. Serum markers of liver damage (AST, ALT, ALP and Bilirubin) were increased by CCl4 and TAA intoxication (p <0.001), whereas co-treatment with NAC reversed such changes (p <0.001). HP was enhanced in toxicant groups (p <0.001 in CCl4 and TAA), but inhibited by NAC (p <0.001). LPO was increased while as GSH, CAT and GPx decreased by the administration of CCl4 and TAA (p <0.001); co-administration of NAC restored these liver markers to normal levels (p <0.001). Biochemical determinations were corroborated by general and histological findings. Keeping in view the biochemical and histopathological studies, it was concluded that CCl4 and TAA are strong hepatotoxic agents that produce liver fibrosis with close proximity to human etiology (micronodular cirrhosis) and NAC has a significant protective activity against CCl4 and TAA. NAC has also been validated as a model against oxidative burden in chronic liver pathology. [Copyright &y& Elsevier]

Abstract: To investigate the protective effect of enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and thioacetamide (TAA)-promoted rats. We examined the modifying effect of co-administration with EMIQ on the liver tissue environment including hepatic macrophages and lymphocytes and on the induction mechanism of preneoplastic cell apoptosis during early stages of hepatocellular tumor promotion. TAA increased the number and area of glutathione S-transferase placental form (GST-P)+ liver cell foci and the numbers of proliferating and apoptotic cells in randomly selected areas in liver sections. Co-administration with EMIQ suppressed these effects. TAA also increased the numbers of ED2+, cyclooxygenase-2+, and heme oxygenase-1+ liver cells, as well as the number of CD3+ lymphocytes. These effects were also suppressed by EMIQ. EMIQ increased liver levels of thiobarbituric acid-reactive substance and 8-hydroxydeoxyguanosine, and TUNEL+ apoptotic cells, death receptor 5 (DR5)+ cells and 4-hydroxy-2-nonenal+ cells within GST-P+ foci. Outside the GST-P+ foci, EMIQ decreased the numbers of apoptotic cells and DR5+ cells. These results suggest that TAA-induced tumor promotion involves activation of hepatic macrophages producing proinflammatory factors. EMIQ may suppress the TAA-induced tumor-promoting activity by an anti-inflammatory mechanism mediated by suppressing the activation of these macrophages. Furthermore, EMIQ may suppress tumor-promoting activity differentially between the inside and outside of GST-P+ foci. Within GST-P+ foci, EMIQ facilitates the apoptosis of preneoplastic cells through the upregulation of DR5. Outside the GST-P+ foci, EMIQ suppresses apoptosis and the subsequent regeneration of non-transformed liver cells. [Copyright &y& Elsevier]

Abstract: Cell cycle aberration was immunohistochemically examined in relation to preneoplastic liver cell foci expressing glutathione S-transferase placental form (GST-P) at early stages of tumor-promotion in rats with thioacetamide (TAA), a hepatocarcinogen facilitating liver cell regeneration. Immunoexpression of p16Ink4a following exposure to other hepatocarcinogens/promoters and its DNA methylation status were also analyzed during early and late tumor-promotion stages. GST-P+ liver cell foci increased cell proliferation and decreased apoptosis when compared with surrounding liver cells. In concordance with GST-P+ foci, checkpoint proteins at G1/S (p21Cip1, p27Kip1 and p16Ink4a) and G2/M (phospho-checkpoint kinase 1, Cdc25c and phospho-Wee1) were either up- or downregulated. Cellular distribution within GST-P+ foci was either increased or decreased with proteins related to G2-M phase or DNA damage (topoisomerase IIα, phospho-histone H2AX, phospho-histone H3 and Cdc2). In particular, p16Ink4a typically downregulated in GST-P+ foci and regenerative nodules at early tumor-promotion stage with hepatocarcinogens facilitating liver cell regeneration and in neoplastic lesions at late tumor-promotion stage with hepatocarcinogens/promoters irrespective of regenerating potential. Hypermethylation at exon 2 of Cdkn2a was detected at both early- and late-stages. Thus, diverse disruptive expression of G1/S and G2/M proteins, which allows for clonal selection of GST-P+ foci, results in the acquisition of multiple aberrant phenotypes to disrupt checkpoint function. Moreover, increased DNA-damage responses within GST-P+ foci may be the signature of genetic alterations. Intraexonic hypermethylation may be responsible for p16Ink4a-downregulation, which facilitates cell cycle progression in early preneoplastic lesions produced by repeated cell regeneration and late-stage neoplastic lesions irrespective of the carcinogenic mechanism. [Copyright &y& Elsevier]

This study examines the expression and cellular distribution pattern of nitric oxide synthase (NOS) isoforms, nitrotyrosine-derived complexes, and the nitric oxide (NO) production in the cerebellum of rats with cirrhosis induced by thioacetamide (TAA). The results showed local changes in the tissue distribution pattern of the NOS isoforms and nitrated proteins in the cerebellum of these animals. Particularly, eNOS immunoreactivity in perivascular glial cells of the white matter was detected only in TAA-treated animals. In addition, although neither neuronal NOS (nNOS) nor inducible NOS (iNOS) cerebellar protein levels appeared to be affected, the endothelial NOS (eNOS) isoform significantly increased its expression, and NO production slightly augmented in TAA-treated rats. These NOS/NO changes may contribute differently to the evolution of the hepatic disease either by maintaining the guanosine monophosphate–NO signal transduction pathways and the physiological cerebellar functions or by inducing oxidative stress and cell damage. This model gives rise to the hypothesis that the upregulation of the eNOS maintains the physiological production of NO, while the iNOS is silenced and the nNOS remains unchanged. The differential NOS-distribution and expression pattern may be one of the mechanisms involved to balance cerebellar NO production in order to minimize TAA toxic injury. These data help elucidate the role of the NOS/NO system in the development and progress of hepatic encephalopathy associated with TAA cirrhosis. [Copyright &y& Elsevier]

This study examines how treatment with a single dose of thioacetamide, a known experimental hepatotoxin, alters the content of dolichol, dolichol isoprene units and retinol in isolated rat parenchymal and non-parenchymal liver cells at different times and when the animals are supplemented with Vitamin A.Thioacetamide (300 mg/kg i.p.) was administered in a single injection to rats, sacrificed at intervals of 0.5, 1, 2, 3, 4, 15 and 30 days. Rats supplemented, following thioacetamide, with Vitamin A, 3 days before sacrifice showed increased mortality and cellular necrosis on the third and fourth days. Parameters indicating tissue necrosis returned to normal values in surviving animals. Dolichol and retinol content showed a variable, reversible decrease, with normal levels being restored in 15–30 days. After Vitamin A, dolichol content only in hepatic stellate cells (HSC) was lower then the controls 3 and 4 days after thioacetamide treatment, in parallel with the decrease of retinol storage.The percentage of dolichol-18 is not modified by thioacetamide alone. When supplemented with Vitamin A the percentage of dolichol-18 always decreased after thioacetamide, showing that damage was still present. Mechanisms that might be operative in liver cells are briefly discussed. This approach would provide an indication to investigate how the length of the dolichol chain is determined. [Copyright &y& Elsevier]

Summary: The phenotype and function of liver dendritic cells (LDCs) are poorly understood. This Snapshot summarizes our current knowledge on LDCs in the healthy and injured liver, and their role in fibrosis progression and reversal. It also draws attention to various pitfalls in the current experimental design and conclusions based on available data. [Copyright &y& Elsevier]

Summary: Natural killer T cells (NKT) are innate-like cells which are abundant in liver sinusoids and express the cell surface receptors of NK cells (e.g., NK1.1 (mouse) or CD161+/CD56+(human)) as well as an antigen receptor (TCR) characteristic of conventional T cells. NKT cells recognize lipid antigens in the context of CD1d, a non-polymorphic MHC class I-like molecule. Activation of NKT cells has a profound influence on the immune response against tumors and infectious organisms and in autoimmune diseases. NKT cells can be categorized into at least two distinct subsets: iNKT or type I use a semi-invariant TCR, whereas type II NKT TCRs are more diverse. Recent evidence suggests that NKT-cell subsets can play opposing roles early in non-microbial liver inflammation in that type I NKT are proinflammatory whereas type II NKT cells inhibit type I NKT-mediated liver injury. [ABSTRACT FROM AUTHOR]

Background & Aims: Hepatocellular carcinoma develops in patients with chronic hepatitis or cirrhosis via a stepwise accumulation of various genetic alterations. To explore the genetic basis of development of hepatocellular carcinoma in hepatitis C virus (HCV)-associated chronic liver disease, we evaluated genetic variants that accumulate in nontumor cirrhotic liver. Methods: We determined the whole exome sequences of 7 tumors and background cirrhotic liver tissues from 4 patients with HCV infection. We then performed additional sequencing of selected exomes of mutated genes, identified by whole exome sequencing, and of representative tumor-related genes on samples from 22 cirrhotic livers with HCV infection. We performed in vitro and in vivo functional studies for one of the mutated genes. Results: Whole exome sequencing showed that somatic mutations accumulated in various genes in HCV-infected cirrhotic liver tissues. Among the identified genes, the leptin receptor gene (LEPR) was one of the most frequently mutated in tumor and nontumor cirrhotic liver tissue. Selected exome sequencing analyses detected LEPR mutations in 12 of 22 (54.5%) nontumorous cirrhotic livers. In vitro, 4 of 7 (57.1%) LEPR mutations found in cirrhotic livers reduced phosphorylation of STAT3 to inactivate LEPR-mediated signaling. Moreover, 40% of Lepr-deficient (C57BL/KsJ-db/db) mice developed liver tumors after administration of thioacetamide compared with none of the control mice. Conclusions: Based on analysis of liver tissue samples from patients, somatic mutations accumulate in LEPR in cirrhotic liver with chronic HCV infection. These mutations could disrupt LEPR signaling and increase susceptibility to hepatocarcinogenesis. [Copyright &y& Elsevier]