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Homogeneous extremely low-frequency electromagnetic fields (ELF-EMFs) alter biological phenomena, including the cell phenotype and proliferation rate. Heterogenous vortex magnetic fields (VMFs), a new approach of exposure to magnetic fields, induce systematic movements on charged biomolecules from target cells; however, the effect of VMFs on living systems remains uncertain. Here, we designed, constructed, and characterized an ELF-VMF-modified Rodin's coil to expose SH-SY5Y cells. Samples were analyzed by performing 2D-differential-gel electrophoresis, identified by MALDI-TOF/TOF, validated by western blotting, and characterized by confocal microscopy. A total of 106 protein spots were differentially expressed; 40 spots were downregulated and 66 were upregulated in the exposed cell proteome, compared to the control cell proteome. The identified spots are associated with cytoskeleton and cell viability proteins, and according to the protein-protein interaction network, a significant interaction among them was found. Our data revealed a decrease in cell survival associated with apoptotic cells without effects on the cell cycle, as well as evident changes in the cytoskeleton. We demonstrated that ELF-VMFs, at a specific frequency and exposure time, alter the cell proteome and structurally affect the target cells. This is the first report showing that VMF application might be a versatile system for testing different hypotheses in living systems, using appropriate exposure parameters.© 2022 Bioelectromagnetics Society.

The online version of the original article can be found athttps://doi.org/10.1631/jzus.B2100201 Erratum to: J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2021 22(12):1045-1052 https://doi.org/10.1631/jzus.B2100201

The clinical phenotype of

Administration of carbon tetrachloride (CClAnimals were fed either a high fat, sucrose, and cholesterol diet (HFSCD) or a HFSCD plus intraperitoneal injections of low doses of CClOur experimental model reproduced several aspects of the human NASH pathophysiology. NLRP3 inflammasome activation was induced by the combined effect of HFSCD plus CClOur results indicate that NLRP3 inflammasome activation plays a key role and may be an important therapeutic target for NASH treatment.

Caffeine elicits protective effects against liver diseases, such as NASH; however, its mechanism of action involving the pyrin domain-containing-3 (NLRP3) inflammasome signaling pathway remains to be elucidated. This study aimed to evaluate the effect of caffeine on the NLRP3 inflammasome signaling pathway in a rat model of NASH. NASH was induced by feeding rats a high-fat, -sucrose, and -cholesterol diet (HFSCD) for 15 weeks along with a weekly low dose (400 mg/kg, i.p.) of CCl4. Caffeine was administered at 50 mg/kg p.o. The effects of HFSCD+CCl4 and caffeine on the liver were evaluated using biochemical, ultrastructural, histological, and molecular biological approaches. The HFSCD+CCl4-treated rats showed fat accumulation in the liver, elevated levels of inflammatory mediators, NLRP3 inflammasome activation, antioxidant dysregulation, and liver fibrosis. Caffeine reduced necrosis, cholestasis, oxidative stress, and fibrosis. Caffeine exhibited anti-inflammatory effects by attenuating NLRP3 inflammasome activation. Moreover, caffeine prevented increases in toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) protein levels and mitigated the phosphorylation of mitogen-activated protein kinase (MAPK). Importantly, caffeine prevented the activation of hepatic stellate cells. This study is the first to report that caffeine ameliorates NASH by inhibiting NLRP3 inflammasome activation through the suppression of the TLR4/MAPK/NF-κB signaling pathway.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide, often preceded by cirrhosis and usually diagnosed at advanced stages; therefore, identifying molecular changes at early stages is an attractive strategy for detection and timely treatment. Here, we investigated the progressive transcriptomic changes during experimental hepatocarcinogenesis to identify novel early tumor markers in an HCC model induced by chronic administration of sublethal doses of diethylnitrosamine. An analysis of differentially expressed genes showed that four processes associated with oxidation-reduction and detoxification were significantly over-represented during hepatocarcinogenesis progression, of which the Nuclear Factor, Erythroid 2 Like 2 pathway showed several dysregulated genes. Interestingly, we also identified 91 genes dysregulated at early HCC stages, but the expression of the indolethylamine N-methyltransferase gene (INMT), as well as the level of its encoding protein, were strongly downregulated. INMT was increased in perivenular hepatocytes of normal livers but decreased in livers of experimental HCC. Furthermore, a gene expression and survival analysis performed using data from the liver hepatocellular carcinoma project of The Cancer Genome Atlas Program revealed that INMT is also significantly downregulated in human HCC and is associated with poor overall survival. In conclusion, by performing a transcriptome analysis of the HCC progression, we identified that INMT is early downregulated in the rat hepatocarcinogenesis and is associated with poor prognosis in human HCC, suggesting that INMT downregulation may be a promising prognostic marker for HCC in high-risk populations.

The potential role of hepatocytes versus hepatic progenitor cells (HPC) on the onset and pathogenesis of hepatocellular carcinoma (HCC) has not been fully clarified. Because the administration of 2‐acetylaminofluorene (2AAF) followed by a partial hepatectomy, selectively induces the HPC proliferation, we investigated the effects of chronic 2AAF administration on the HCC development caused by the chronic administration of the carcinogen diethylnitrosamine (DEN) for 16 weeks in the rat. DEN + 2AAF protocol impeded weight gain of animals but promoted prominent hepatomegaly and exacerbated liver alterations compared to DEN protocol alone. The tumor areas detected by γ‐glutamyl transferase, prostaglandin reductase‐1, and glutathione S‐transferase Pi−1 liver cancer markers increased up to 80% as early as 12 weeks of treatment, meaning 6 weeks earlier than DEN alone. This protocol also increased the number of Ki67‐positive cells and those of CD90 and CK19, two well‐known progenitor cell markers. Interestingly, microarray analysis revealed that DEN + 2AAF protocol differentially modified the global gene expression signature and induced the differential expression of 30 genes identified as HPC markers as early as 6 weeks of treatment. In conclusion, 2AAF induces the early appearance of HPC markers and as a result, accelerates the hepatocarcinogenesis induced by DEN in the rat. Thus, since 2AAF simultaneously administrated with DEN enriches HPC during hepatocarcinogenesis, we propose that DEN + 2AAF protocol might be a useful tool to investigate the cellular origin of HCC with progenitor features.

Hepatocellular carcinoma (HCC) is a health problem worldwide due to its high mortality rate, and the tumor microenvironment (TME) plays a key role in the HCC progression. The current ineffective therapies to fight the disease still warrant the development of preventive strategies. Quercetin has been shown to have different antitumor activities; however, its effect on TME components in preneoplastic lesions has not been fully investigated yet. Here, we aimed to evaluate the effect of quercetin (10 mg/kg) on TME components during the early stages of HCC progression induced in the rat. Histopathological and immunohistochemical analyses showed that quercetin decreases the size of preneoplastic lesions, glycogen and collagen accumulation, the expression of cancer stem cells and myofibroblasts markers, and that of the transporter ATP binding cassette subfamily C member 3 (ABCC3), a marker of HCC progression and multi-drug resistance. Our results strongly suggest that quercetin has the capability to reduce key components of TME, as well as the expression of ABCC3. Thus, quercetin can be an alternative treatment for inhibiting the growth of early HCC tumors.

Hepatocellular carcinoma (HCC), which is the most frequent primary liver malignancy, is ranked as the sixth most common cancer and the third leading cause of cancer-related deaths worldwide, with its incidence expected to continue rising. One of the reasons is that most patients are diagnosed at an advanced stage when therapeutic options are ineffective. The development of HCC is attributed to a chronic exposition to either one or a combination of low amounts of different hepatotoxins, such as in hepatitis virus infection, alcohol consumption, aflatoxin from contaminated foods, metabolic factors, and exposure to chemical carcinogens from tobacco smoke (Forner et al., 2018). Integrative studies combining exome sequencing, transcriptome analysis, and the genomic characterization of HCC have shown that these etiological factors may raise the frequency of particular genetic alterations, resulting in intra-tumor heterogeneity that presents a huge challenge for treatment. For example, mutations in the catenin β-1 (CTNNB1) gene (a proto-oncogene in the WNT signaling pathway that encodes the β‍-catenin transcription factor) are strongly associated with alcohol-related HCC, whereas mutations in the telomerase reverse transcriptase (TERT) promoter and tumor protein p53 (TP53) genes are the most commonly observed in hepatitis B virus (HBV)‍-associated HCC (Calderaro et al., 2017; Cancer Genome Atlas Research Network, 2017). The above findings emphasize the molecular diversity of HCC and the associations of different etiologies with distinct mechanisms in HCC progression. Consequently, prevention strategies are still attractive for HCC management.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible, and highly fatal disease. It is characterized by the increased activation of both fibroblast and myofibroblast that results in excessive extracellular matrix (ECM) deposition. Extracellular vesicles (EVs) have been described as key mediators of intercellular communication in various pathologies. However, the role of EVs in the development of IPF remains poorly understood. This study aimed to characterize the differentially expressed proteins contained within EVs cargo derived from the fibroblast cell lines LL97A (IPF-1) and LL29 (IPF-2) isolated from lungs bearing IPF as compared to those derived from the fibroblast cell lines CCD8Lu (NL-1) and CCD19Lu (NL-2) isolated from healthy donors. Isolated EVs were subjected to label-free quantitative proteomic analysis by LC-MS/MS, and as a result, 331 proteins were identified. Differentially expressed proteins were obtained after the pairwise comparison, including all experimental groups. A total of 86 differentially expressed proteins were identified in either one or more comparison groups. Of note, proteins involved in fibrogenic processes, such as tenascin-c (TNC), insulin-like-growth-factor-binding protein 7 (IGFBP7), fibrillin-1 (FBN1), alpha-2 collagen chain (I) (COL1A2), alpha-1 collagen chain (I) (COL1A1), and lysyl oxidase homolog 1 (LOXL1), were identified in EVs cargo isolated from IPF cell lines. Additionally, KEGG pathway enrichment analysis revealed that differentially expressed proteins participate in focal adhesion, PI3K-Akt, and ECM–receptor interaction signaling pathways. In conclusion, our findings reveal that proteins contained within EVs cargo might play key roles during IPF pathogenesis.

To date, there is no effective treatment for alcoholic liver disease, despite its prevalence world-wide. Because alcohol consumption is associated with oxidative stress-induced liver injury and pro-inflammatory responses, naturally occurring antioxidants and/or anti-inflammatories may be potential therapeutics. Spermidine is an abundant, ubiquitous polyamine that has been found to display strong antioxidant and anti-inflammatory properties. To further investigate whether spermidine is an effective intervention for alcohol-induced liver disease, we examined its hepatoprotective properties using a two-hit, chronic ethanol and acute lipopolysaccharide (LPS)-induced mouse model of liver injury. We determined that spermidine administration prevented ethanol and LPS-induced increases in liver injury using plasma ALT as a readout. Furthermore, histological analysis of tissue from control and treated animals revealed that the pathology associated with ethanol and LPS treatment was prevented in mice additionally treated with spermidine. As predicted, spermidine also prevented ethanol and LPS-induced oxidative stress by decreasing the levels of both reactive oxygen species (ROS) and lipid peroxidation. We further determined that spermidine treatment prevented the nuclear translocation of nuclear factor κB (NFκB) by blocking the phosphorylation of the inhibitory protein, IκB, thereby preventing expression of pro-inflammatory cytokines. Finally, by measuring expression of known markers of hepatic stellate cell activation and monitoring collagen deposition, we observed that spermidine also prevented alcohol and LPS-induced hepatic fibrosis. Together, our results indicate that spermidine is an antioxidant thereby conferring anti-inflammatory and anti-fibrotic effects associated with alcoholic liver injury.

The activation of Nuclear Factor, Erythroid 2 Like 2 - Kelch Like ECH Associated Protein 1 (NRF2-KEAP1) signaling pathway plays a critical dual role by either protecting or promoting the carcinogenesis process. However, its activation or nuclear translocation during hepatocellular carcinoma (HCC) progression has not been addressed yet. This study characterizes the subcellular localization of both NRF2 and KEAP1 during diethylnitrosamine-induced hepatocarcinogenesis in the rat. NRF2-KEAP1 pathway was continuously activated along with the increased expression of its target genes, namely Nqo1, Hmox1, Gclc, and Ptgr1. Similarly, the nuclear translocation of NRF2, MAF, and KEAP1 increased in HCC cells from weeks 12 to 22 during HCC progression. Likewise, colocalization of NRF2 with KEAP1 was higher in the cell nuclei of HCC neoplastic nodules than in surrounding cells. Moreover, immunofluorescence analyses revealed that the interaction of KEAP1 with filamentous Actin was disrupted in HCC cells. This disruption may be contributing to the release and nuclear translocation of NRF2 since the cortical actin cytoskeleton serves as anchoring of KEAP1. In conclusion, this evidence indicates that NRF2 is progressively activated and promotes the progression of experimental HCC.

Alcoholic liver disease (ALD) may be attributed to multiple hits driving several alterations. The aim of this work was to determine whether nucleoredoxin (NXN) interacts with flightless-I (FLII)/actin complex and how this ternary complex is altered during ALD progression induced by different ALD models. ALD was recapitulated in C57BL/6J female mice by the well-known ALD Lieber-DeCarli model, and by an in vitro human co-culture system overexpressing NXN. The effects of ethanol and low doses of lipopolysaccharides (LPS) and diethylnitrosamine (DEN) were also evaluated in vivo as a first approach of an ALD multi-hit protocol. We demonstrated that NXN interacts with FLII/actin complex. This complex was differentially altered in ALD in vivo and in vitro, and NXN overexpression partially reverted this alteration. We also showed that ethanol, LPS and DEN synergistically induced liver structural disarrangement, steatosis and inflammatory infiltration accompanied by increased levels of proliferation (Ki67), ethanol metabolism (CYP2E1), hepatocarcinogenesis (GSTP1) and LPS-inducible (MYD88 and TLR4) markers. In summary, we provide evidence showing that NXN/FLII/actin complex is involved in ALD progression and that NXN might be involved in the regulation of FLII/actin-dependent cellular functions. Moreover, we present a promising first approach of a multi-hit protocol to better recapitulate ALD pathogenesis.

Hepatocellular carcinoma (HCC), the most common primary liver cancer, arises after a long period of exposure to etiological factors. Nonalcoholic steatohepatitis (NASH) is ranked as the main risk factor for developing HCC; hence, experimental models of NASH leading to HCC have become key tools both to investigate the molecular mechanisms underlying the pathophysiology and to evaluate new putative drugs for treating chronic liver diseases in humans. Animal models of NASH induced by a high-fat diet (HFD) plus chemical inducers, such as the NASH-HCC (STAM), high-fat diet/diethylnitrosamine (HFD/DEN), choline-deficient high-fat diet/DEN (CDHFD/DEN), and Western diet/carbon tetrachloride (WD/CCl4) models, are promising because they exacerbate liver damage and significantly shorten the experimental time. In this review, we critically summarize and discuss the ability of these models to recapitulate the liver alterations that precede and lead to HCC progression, as well as the impact of the diet in promoting liver injury progression. We also emphasize the strengths and weaknesses of the models' ability to closely mimic the stages of liver injury development that occur in humans. Based on the molecular mechanisms induced by the currently available NASH models leading to HCC, we argue that although several NASH models have importantly contributed to describing the disease chronology, the progress in emulating the progression from NASH to HCC has been partial. Thus, the development of novel NASH/HCC models remains an unmet need.

The intrinsic heterogeneity of hepatocellular carcinoma (HCC) represents a great challenge for its molecular classification and for detecting predictive biomarkers. Aldo-keto reductase (Akr) family members have shown differential expression in human HCC, while AKR1B10 overexpression is considered a biomarker; AKR7A3 expression is frequently reduced in HCC. To investigate the time-course expression of Akr members in the experimental hepatocarcinogenesis. Using DNA-microarray data, we analyzed the time-course gene expression profile from nodules to tumors (4–17 months) of 17 Akr members induced by the resistant hepatocyte carcinogenesis model in the rat. The expression of six members (Akr1c19, Akr1b10, Akr7a3, Akr1b1, Akr1cl1, and Akr1b8) was increased, comparable to that of Ggt and Gstp1, two well-known liver cancer markers. In particular, Akr7a3 and Akr1b10 expression also showed a time-dependent increment at mRNA and protein levels in a second hepatocarcinogenesis model induced with diethylnitrosamine. We confirmed that aldo-keto reductases 7A3 and 1B10 were co-expressed in nine biopsies of human HCC, independently from the presence of glypican-3 and cytokeratin-19, two well-known HCC biomarkers. Because it has been suggested that expression of Akr members is regulated through NRF2 activity at the antioxidant response element (ARE) sequences, we searched and identified at least two ARE sites in Akr1b1, Akr1b10, and Akr7a3 from rat and human gene sequences. Moreover, we observed higher NRF2 nuclear translocation in tumors as compared with non-tumor tissues. Our results demonstrate that Akr7a3 mRNA and protein levels are consistently co-expressed along with Akr1b10, in both experimental liver carcinogenesis and some human HCC samples. These results highlight the presence of AKR7A3 and AKR1B10 from early stages of the experimental HCC and introduce them as a potential application for early diagnosis, staging, and prognosis in human cancer.

Chronic liver injury promotes the molecular alterations that precede the establishment of cancer. Usually, several decades of chronic insults are needed to develop the most common primary liver tumor known as hepatocellular carcinoma. As other cancer types, liver cancer cells are governed by a common set of rules collectively called the hallmarks of cancer. Although those rules have provided a conceptual framework for understanding the complex pathophysiology of established tumors, therapeutic options are still ineffective in advanced stages. Thus, the molecular alterations that precede the establishment of cancer remain an attractive target for therapeutic interventions. Here, we first summarize the chemopreventive interventions targeting the early liver carcinogenesis stages. After an integrative analysis on the plethora of molecular alterations regulated by anticancer agents, we then underline and discuss that two critical processes namely oxidative stress and genetic alterations, play the role of 'dirty work laborer' in the initial cell damage and drive the transformation of preneoplastic into neoplastic cells, respectively; besides, the activation of cellular senescence works as a key mechanism in attempting to prevent the onset and establishment of liver cancer. Whereas the detrimental effects of the binomial made up of oxidative stress and genetic alterations are either eliminated or reduced, senescence activation is promoted by anticancer agents. We argue that collectively, oxidative stress, genetic alterations, and senescence are key events that influence the fate of initiated cells and the establishment of the hallmarks of cancer.

Pulmonary fibrosis is an emerging disease with a poor prognosis and high mortality rate that is even surpassing some types of cancer. This disease has been linked to the concomitant appearance of liver cirrhosis. Bleomycin-induced pulmonary fibrosis is a widely used mouse model that mimics the histopathological and biochemical features of human systemic sclerosis, an autoimmune disease that is associated with inflammation and expressed in several corporal systems as fibrosis or other alterations. To determine the effects on proliferation, redox and inflammation protein expression markers were analyzed by immunohistochemistry. Analyses showed a significant increase in protein oxidation levels by lipoperoxidation bio-products and in proliferation and inflammation processes. These phenomena were associated with the induction of the redox status in mice subjected to 100 U/kg bleomycin. These findings clearly show that the bleomycin model induces histopathological alterations in the liver and partially reproduces the complexity of systemic sclerosis. Our results using the bleomycin-induced pulmonary fibrosis model provide a protocol to investigate the mechanism underlying the molecular alteration found in the liver linked to systemic sclerosis.

Alcoholic liver disease (ALD) is closely linked to oxidative stress induction. Antioxidant enzymes balance oxidative stress and function as intermediary signaling regulators. Nucleoredoxin (NXN), an antioxidant enzyme, regulates physiological processes through redox-sensitive interactions. NXN interacts with myeloid differentiation primary response gene-88 (MYD88) and flightless-I (FLII) to regulate toll-like receptor 4 (TLR4)/MYD88 pathway activation, but FLII also regulates key cell processes and is secreted into the bloodstream. However, the effects of chronic ethanol consumption recapitulated by either ethanol alone or in combination with lipopolysaccharides (LPS), as a two-hit ALD model, on FLII/NXN/MYD88 complex and FLII secretion have not been explored yet. In this study, we have demonstrated that ethanol feeding increased FLII protein levels, its nuclear translocation and plasma secretion, and modified its tissue distribution in both in vivo and in vitro ALD models. Ethanol increased MYD88/FLII interaction ratio, and decreased NXN/MYD88 interaction ratio but this was partially reverted by two-hit model. While ethanol and two-hit model increased MYD88/TLR4 interaction ratio, two-hit model significantly decreased FLII nuclear translocation and its plasma secretion. Ethanol and LPS provoked similar effects in vitro; however, NXN overexpression partially reverted these alterations, and ethanol alone increased FLII secretion into culture medium. In summary, by analyzing the response of FLII/NXN/MYD88 complex during ALD early progression both in vivo and in vitro, we have discovered that the effects of chronic ethanol consumption disrupt this complex and identified FLII as a candidate non-invasive plasma biomarker for the early detection of ALD.

Nucleoredoxin (NXN) is a redox-regulating protein potentially targeted by reactive oxygen species (ROS). It regulates molecular pathways that participate in several key cellular processes. However, the role of NXN in the alcohol liver disease (ALD) redox regulation has not been fully understood. Here, we investigated the effects of ethanol and ethanol plus lipopolysaccharide, a two-hit liver injury model (Ethanol/LPS), on NXN/dishevelled (DVL) interaction and on DVL-dependent phosphoinositides production both in mouse liver and in a co-culture system consisting of human hepatic stellate cells (HSC) and ethanol metabolizing-VL17A human hepatocyte cells. Ethanol and two-hit model increased Nxn protein and mRNA expression, and 4-hydroxynonenal adducts. Two-hit model promoted Nxn nuclear translocation and Dvl/Phosphatidylinositol 4-kinase type-IIα (Pi4k2a) interaction ratio but surprisingly decreased Dvl protein and mRNA levels and reverted ethanol-induced Nxn/Dvl and Dvl/frizzled (Fzd) interaction ratios. Ethanol resulted in a significant increase of Dvl protein and mRNA expression, and decreased Nxn/Dvl interaction ratio but promoted the interaction of Dvl with Fzd and Pi4k2a; formation of this complex induced phosphatidylinositol 4-phosphate [PI(4)P] production. Ethanol and LPS treatments provoked similar alterations on NXN/DVL interaction and its downstream effect in HSC/VL17A co-culture system. Interestingly, ROS and glutathione levels as well as most of ethanol-induced alterations were modified by NXN overexpression in the co-culture system. In conclusion, two-hit model of ethanol exposure disrupts NXN/DVL homeostatic status to allow DVL/FZD/PI4K2A complex formation and stimulates PI(4)P production. These results provide a new mechanism showing that NXN also participates in the regulation of phosphoinositides production that is altered by ethanol during alcoholic liver disease progression.

Liver cancer is a highly heterogeneous disease produced by a variety of risk factors including viral infection and hepatotoxins. The hepatocarcinogenesis process is associated to oxidative stress and takes several steps: initiation, promotion, and progression that have been well characterized in vivo models, such as in protocols with rodents. Also cell culture of human-derived cell lines of liver has been considered of great value as in vitro models for cancer research. Several pharmacological studies with potential antineoplastic and antioxidant compounds have been successfully performed in cultured cell lines because they provide advantages for experimentation with highly reproducible results. The limited treatment options in cancer patients are due to drug resistance and elevated heterogeneity of liver neoplasm; this encourages the investigation of synergistic antineoplastic effect of pharmacological combination with antioxidants in vitro cell models; this may provide additional options for treatment of cancer patients.

Cellular senescence (CS) is a phenomenon characterized by the permanent cell growth arrest in normal and altered physiological processes. A myriad of factors stimulates the appearance of this phenomenon, which has been placed in different scenarios, such as aging, tissue repair, tumor suppressor, and tumor promoter. The fact that CS is involved in multiple processes confers an intriguing complexity worthy to be summarized in terms of liver diseases. Since experimental and clinical studies have revealed a strong association of the CS with chronic liver diseases, in this chapter we discuss the current status of the field with a focus on the senescence molecular pathways altered in chronic liver diseases and propose a hypothetical model about senescence and reactive oxidative stress participation as determinant factors on the appearance and progression of liver cancer, the last step in chronic liver diseases.

Alcohol-induced liver fibrosis and eventually cirrhosis is a leading cause of death. Acetaldehyde, the first metabolite of ethanol, up-regulates expression of the human α2(I) collagen gene ( COL1A2 ). Early acetaldehyde-mediated effects involve phosphorylation and nuclear translocation of SMAD3/4–containing complexes that bind to COL1A2 promoter to induce fibrogenesis. We used human and mouse hepatic stellate cells to elucidate the mechanisms whereby acetaldehyde up-regulates COL1A2 by modulating the role of Ski and the expression of SMADs 3, 4, and 7. Acetaldehyde induced up-regulation of COL1A2 by 3.5-fold, with concomitant increases in the mRNA (threefold) and protein (4.2- and 3.5-fold) levels of SMAD3 and SMAD4, respectively. It also caused a 60% decrease in SMAD7 expression. Ski, a member of the Ski/Sno oncogene family, is colocalized in the nucleus with SMAD4. Acetaldehyde induces translocation of Ski and SMAD4 to the cytoplasm, where Ski undergoes proteasomal degradation, as confirmed by the ability of the proteasomal inhibitor lactacystin to blunt up-regulation of acetaldehyde-dependent COL1A2 , but not of the nonspecific fibronectin gene ( FN1 ). We conclude that acetaldehyde up-regulates COL1A2 by enhancing expression of the transactivators SMAD3 and SMAD4 while inhibiting the repressor SMAD7, along with promoting Ski translocation from the nucleus to cytoplasm. We speculate that drugs that prevent proteasomal degradation of repressors targeting COL1A2 may have antifibrogenic properties.

Hepatocellular carcinoma (HCC) arises after a long period of exposition to etiological factors that might be either independent or collectively contributing. Several rodent models resemble human HCC; however, the major limitation of these models is the lack of chronic injury that reproducibly mimics the molecular alterations as it occurs in humans. Thus, we hypothesized that chronic administration of different DEN treatments identifies the best-fit dose to induce the HCC and/or to determine whether small DEN doses act synergistically with other known hepatotoxins to induce HCC in mice. C57BL/6 J male mice were intraperitoneally injected twice a week for 6 weeks with different DEN doses ranging from 2.5 to 40 mg/kg body weight; then, selected doses (2.5, 5 and 20 mg/kg) for 6, 10, 14, and 18 weeks. We demonstrated that DEN at 20 mg/kg promoted reactive oxygen species and 4-hydroxynonenal production, cell proliferation inflammatory infiltrate, and fibrosis, which in turn induced liver cancer by week 18. These parameters were established by evaluating histopathological changes, HCC markers such as glutathione S-transferase placental-1 (Gstp1), Cytokeratin-19 (Ck19) and prostaglandin reductase-1 (Ptgr1); that of Cyp2e1, a DEN metabolizing enzyme; and the expression of the proliferation marker Ki67. While DEN at 2.5 and 5 mg/kg increased Gstp1 and Ck19, DEN at 20 mg/kg decreased them and Cyp2e1 expression and activity. In summary, our results demonstrate that DEN chronically administrated at 20 mg/kg induces the HCC, while DEN at 2.5 and 5 mg/kg could be useful in elucidating its synergistic effect with other hepatotoxic agents in mice.

We investigated whether the fibrogenic actions of acetaldehyde, the immediate oxidation product of ethanol, are mediated via Wingless (WNT) and/or β-catenin pathways in human hepatic stellate cells (HSC). First, we show that both β-catenin small inhibitory RNA and a dominant negative-MYC expression vector markedly down-regulated the expressions of fibrogenic genes in freshly isolated HSC. We further show that acetaldehyde up-regulated platelet-derived growth factor receptor beta mRNA and protein expressions ranging from 4.0- to 7.2-fold (P

Conversion of protein -SH groups to disulfides is an early event during protein oxidation, which has prompted great interest in the study of thiol proteins. Chemical carcinogenesis is strongly associated with the formation of reactive oxygen species (ROS). The goal of this study was to detect thiol proteins that are sensitive to ROS generated during diethylnitrosamine (DEN) metabolism in the rat liver. DEN has been widely used to induce experimental hepatocellular carcinoma. We used modified redox-differential gel electrophoresis (redox-DIGE method) and mass spectrometry MALDI-TOF/TOF to identify differential oxidation protein profiles associated with carcinogen exposure. Our analysis revealed a time-dependent increase in the number of oxidized thiol proteins after carcinogen treatment; some of these proteins have antioxidant activity, including thioredoxin, peroxirredoxin 2, peroxiredoxin 6 and glutathione S-transferase alpha-3. According to functional classifications, the identified proteins in our study included chaperones, oxidoreductases, activity isomerases, hydrolases and other protein-binding partners. This study demonstrates that oxidative stress generated by DEN tends to increase gradually through DEN metabolism, causes time-dependent necrosis in the liver and has an oxidative effect on thiol proteins, thereby increasing the number of oxidized thiol proteins. Furthermore, these events occurred during the hepatocarcinogenesis initiation period.

Thymosin β4 (Tβ4) plays a role in fibrosis, inflammation, and in the reparative process of injured cells and tissues. Here, we discuss our preliminary work on the protective effect of Tβ4 on carbon tetrachloride (CCl(4) )-induced acute hepatotoxicity. Our studies thus far indicate that Tβ4 can prevent necrosis, inflammatory infiltration, and upregulation of α1(and 2) collagen, α-SMA, PDGF-β receptor, and fibronectin mRNA expression; in addition, Tβ4 can prevent downregulation of PPARγ and upregulation of MECP2 mRNA levels in acute liver injury. Our initial work therefore indicates that Tβ4 can prevent the alteration of markers of hepatic stellate cell transdifferentiation, which suggests that Tβ4 could maintain the quiescent phenotypic state of hepatic stellate cells in the rat livers by restoring PPARγ and downregulating MeCP2 expression levels. More specifically, these preliminary studies suggest that Tβ4 might be an effective anti-inflammatory and antifibrotic drug for the treatment of liver fibrogenesis.

There is a correlation between oxidative stress generated by diethylnitrosamine (DEN) metabolism and liver cancer development. Quercetin is a flavonoid with anti-carcinogenic and antioxidant properties. This study demonstrates the mechanism of action for the chemopreventive effect of quercetin. A 10 mg/kg dose of quercetin produced drastic effect, when it is administrated 2 h before DEN; at 24 days post-DEN, a 70.3% and 66.2% decrease in total area and number of preneoplastic lesions were observed, respectively. At 12 h post-DEN, quercetin inhibited levels of lipid peroxidation by 40%. Quercetin increased the levels of both GSH and of total glutathione, it increased the GSH/GSSG index and it caused a rapid and simultaneous elevation in the activities of superoxide dismutase, glutathione peroxidase and catalase. In conclusion, the quercetin mechanism of action is due to promote the enzymatic and non-enzymatic antioxidant defense system during the initiation of hepatocarcinogenesis.

S-adenosylmethionine (SAM), N-acetylcysteine (NAC) and quercetin exhibit a chemoprotective effect. Likely this effect is mediated by counteracting, oxidative stress and NF-kB activation. To test this hypothesis F344 rats were subjected to hepatocarcinogenesis with or without antioxidants. NAC decreased foci in number and area, SAM and quercetin decreased area. Lipid-peroxidation was decreased by antioxidants, but only SAM increased glutathione. SAM, in its regulation from IKK downwards, abolished the NF-kB activation. NAC decreased IKK and IkB-a phosphorylation, and Rel-A/p65 and NF-kB binding, though the last two were affected with less intensity compared to the NF-kB inhibitor. Quercetin decreased Rel-A/p65, without modifying upstream signalling. Although all antioxidants inhibited oxidative stress as shown by reduction of lipid peroxidation, not all exerted the same effect on NF-kB signalling pathway and only SAM increased GSH. The mechanisms exerted by SAM in the reduction of foci makes this compound a potential liver cancer therapeutic agent.

Cellular senescence is characterized by irreversible cell arrest and is associated with the development of chronic diseases, including cancer. Here, we investigated the induction of cellular senescence during liver carcinogenesis. Liver cancer was induced in Fischer 344 rats with a weekly intraperitoneal injection of diethylnitrosamine (50mg/kg body weight) for 16 weeks. Double-detection of β-galactosidase with Ki67 for cell proliferation; a-SMA and Pdgfrb for cell specificity; p53, p21, p16, and cyclin D1, CDK2, and CDK4 for senescence-associated molecular pathways and γ-glutamyltranspeptidase (GGT) for hepatocarcinogenesis was assessed to determine the association of these markers with cellular senescence. DNA damage was measured through senescence-associated heterochromatin foci (SAHF) detection. Progressive cellular senescence was observed in both fibrotic septa and hepatocytes from week 10 to 18. The maximum peak of positive senescent and fibrotic cells was observed at week 16 and decreased at week 18, but cell proliferation remained high. Whereas the increased p16 expression and SAHF were concomitant with that of β-galactosidase, those of p53 and p21 were barely detected. Furthermore, β-galactosidase positive myofibroblast-like cells were mainly surrounding GGT-positive tumors. Our findings showed that in hepatocarcinogenesis by diethylnitrosamine, cellular senescence is associated with p16 pathway activation and is mainly localized in myofibroblast-like cells.

Alcoholic steatosis, instead of being innocuous, plays a critical role in liver inflammation and fibrogenesis. The severity of fatty liver is governed by the concerted balance between lipid transport, synthesis, and degradation. Whereas scavenger receptor class B, type I (SR-B1) is critical for reverse cholesterol uptake by the liver, peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator-1α and -β (PGC1α and PGC1β) are critical for lipid degradation and synthesis, respectively. Because betaine is a lipotropic agent, we have evaluated its effects on alcoholic steatosis. Betaine effectively prevented chronic alcohol-mediated (i) impaired SR-B1 glycosylation, plasma membrane localization, and consequent impaired cholesterol transport; and (ii) up regulation of PGC-1β, sterol regulatory element-binding protein 1c and downstream lipogenic genes with concomitant increased liver cholesterol, triglycerides and hepatic lipid score. Similarly, because of its anti-inflammatory and anti-fibrotic effects in other organs, we evaluated the protective effects of thymosin β4 (Tβ4) against carbon tetrachloride (CCl4)-induced hepatotoxicity in rat. Tβ4 prevented CCl4-induced (i) necrosis, inflammatory infiltration and up-regulation of α1(2)collagen, alpha-smooth muscle actin (α-SMA), platelet derived growth factor beta (PDGF-β) receptor and fibronectin mRNA expression; (ii) down-regulation of adipogenic gene, PPARγ and the up-regulation of epigenetic repressor gene, methyl CpG binding protein 2 (MeCP2) mRNA levels, suggesting that the anti-fibrogenic actions of Tβ4 involve the prevention of trans-differentiation of quiescent hepatic stellate cells into myo-fibroblasts largely by up-regulating PPARγ and by down-regulating MeCP2 genes. We therefore conclude that betaine and Tβ4 can effectively protect against alcoholic hepatosteatosis and hepatic fibrogenesis, respectively.

Alcohol-induced liver fibrosis and eventually cirrhosis is a leading cause of death. Acetaldehyde, the first metabolite of ethanol, up-regulates expression of the human α2(I) collagen gene (COL1A2). Early acetaldehyde-mediated effects involve phosphorylation and nuclear translocation of SMAD3/4–containing complexes that bind to COL1A2 promoter to induce fibrogenesis. We used human and mouse hepatic stellate cells to elucidate the mechanisms whereby acetaldehyde up-regulates COL1A2 by modulating the role of Ski and the expression of SMADs 3, 4, and 7. Acetaldehyde induced up-regulation of COL1A2 by 3.5-fold, with concomitant increases in the mRNA (threefold) and protein (4.2- and 3.5-fold) levels of SMAD3 and SMAD4, respectively. It also caused a 60% decrease in SMAD7 expression. Ski, a member of the Ski/Sno oncogene family, is colocalized in the nucleus with SMAD4. Acetaldehyde induces translocation of Ski and SMAD4 to the cytoplasm, where Ski undergoes proteasomal degradation, as confirmed by the ability of the proteasomal inhibitor lactacystin to blunt up-regulation of acetaldehyde-dependent COL1A2, but not of the nonspecific fibronectin gene (FN1). We conclude that acetaldehyde up-regulates COL1A2 by enhancing expression of the transactivators SMAD3 and SMAD4 while inhibiting the repressor SMAD7, along with promoting Ski translocation from the nucleus to cytoplasm. We speculate that drugs that prevent proteasomal degradation of repressors targeting COL1A2 may have antifibrogenic properties.

Background Transforming growth factor beta 1 (TGF-β1) is a pleiotropic cytokine that activates hepatic stellate cell (HSC) proliferation, but inhibits parenchymal cell proliferation. Therefore, we hypothesize that TGF-β1 regulates HSC proliferation and elucidated its molecular action. Methods In order to elucidate the molecular mechanism whereby TGF-β1 up-regulates platelet derived growth factor beta (PDGF-β) receptor mRNA and induces a delayed proliferation of HSC, we used proliferation and apoptosis assays as well as RT-PCR, Western blot analysis, immunostaining, and flow cytometry in mouse and rat HSC. Results We show that TGF-β1 markedly induces the proliferation of mouse HSC in culture with concomitant 2.1-fold (p

Thymosin β4 (Tβ4) plays a role in fibrosis, inflammation, and in the reparative process of injured cells and tissues. Here, we discuss our preliminary work on the protective effect of Tβ4 on carbon tetrachloride (CCl(4) )-induced acute hepatotoxicity. Our studies thus far indicate that Tβ4 can prevent necrosis, inflammatory infiltration, and upregulation of α1(and 2) collagen, α-SMA, PDGF-β receptor, and fibronectin mRNA expression; in addition, Tβ4 can prevent downregulation of PPARγ and upregulation of MECP2 mRNA levels in acute liver injury. Our initial work therefore indicates that Tβ4 can prevent the alteration of markers of hepatic stellate cell transdifferentiation, which suggests that Tβ4 could maintain the quiescent phenotypic state of hepatic stellate cells in the rat livers by restoring PPARγ and downregulating MeCP2 expression levels. More specifically, these preliminary studies suggest that Tβ4 might be an effective anti-inflammatory and antifibrotic drug for the treatment of liver fibrogenesis.

We have previously evaluated the chemopreventive effect of celecoxib on preneoplastic lesions in rat liver. However, though the effects of celecoxib have been tested in a variety of carcinomas, there has not been a study on the modulation of gene expression in response to this drug. Here, we evaluated the effect of celecoxib on the gene expression profile associated with hepatocarcinogenesis. Male Sprague-Dawley rats underwent the modified resistant hepatocyte model and were fed a diet containing 1500 ppm of celecoxib. Gene expression profiles were evaluated using DNA microarrays and further validations were performed using quantitative PCR, western blotting and immunohistochemical staining. Celecoxib modulated the expression of 46 genes, and those regulated by growth hormone were selected for further analysis. Celecoxib significantly upregulated the expression of the Cyp2b1/2, Cyp3a1, and alpha2-urinary globulin (alpha2uG) genes and restored the expression of Cyp2b3 to normal. The protein expression of Cyp2b1/2 was increased, but the expressions of Cyp3a1 and alpha2uG were only restored to normal levels. The increased Cyp2b1/2 expression in response to celecoxib was mainly confined to preneoplastic lesions. A search for the upstream mediator of these genetic alterations found that carcinogenesis inactivated by 87% the signal transducer and activator of transcription 5 (Stat5), a transcription factor that is activated by growth hormone signaling, but celecoxib treatment restored its activation. In conclusion, these results suggest that celecoxib exerts anticancer effects on altered hepatic cells by restoring mRNA and the protein expression levels of specific genes, in part through the reactivation of Stat5.

Caffeic acid phenethyl ester (CAPE), a natural component of propolis, shows anticarcinogenic properties in the modified resistant hepatocyte model when administered before initiation or promotion of hepatocarcinogenesis process; however, information about the mechanism underlying this chemoprotection is limited. The aim of this work was to characterize the effect of CAPE on cytochrome P450 (CYP), which is involved in diethylnitrosamine (DEN) metabolism during the initiation stage of chemical hepatocarcinogenesis. Male Fischer-344 rats were treated as in the modified resistant hepatocyte model. Liver samples were obtained at four different times: at 12 h after pretreatment with CAPE and at 12 and 24 h and 25 days after DEN administration. Liver damage was determined by histology with hematoxylin and eosin, measurement of total CYP levels and enzyme activity, and gamma-glutamyl transpeptidase-positive (GGT+) staining of hepatocyte foci. CAPE administration prevented DEN-induced necrosis at 24 h. It also decreased O-dealkylation of 7-ethoxy-resorufin (EROD), O-dealkylation of 7-methoxyresorufin (MROD), and 7-pentoxy-resorufin activities at 12 h after its administration and EROD and MROD activities at 12 h after administration of DEN. CAPE treatment decreased GGT+ foci by 59% on day 25. Our results suggest that CAPE modifies the enzymatic activity of CYP isoforms involved in the activation of DEN, such as CYP1A1/2 and CYP2B1/2. These findings describe an alternative mechanism for understanding the ability of CAPE to protect against chemical hepatocarcinogenesis.

Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, may reduce the risk and mortality of certain types of human cancer. The chemopreventive effect of celecoxib on preneoplastic lesions induced by chemical hepatocarcinogenesis was investigated.Male Sprague Dawley rats were fed a celecoxib-supplemented diet between days 18 and 26 post-initiation (1500 ppm) and sacrificed on day 26. The effects of celecoxib on proliferation, apoptosis, COX-2 activity and liver function were evaluated by immunohistochemistry, TUNEL assay, enzyme-immunoassay and spectrophotometry, respectively.Celecoxib decreased, in area and number, gamma-glutamyltranspeptidase and glutathione S-transferase placental-positive lesions, below levels found after 18 days, by 55.2% and 62.2%, and by 50.5% and 71.1%, respectively, (p0.05). Celecoxib neither induced apoptosis nor altered the levels of prostaglandin E2, bilirubin or alanine aminotransferase in the plasma; however, proliferating cell nuclear antigen and cyclin D1 decreased by 77.7% and 94.9%, respectively, (p0.05).Celecoxib regresses existing preneoplastic liver lesions through antiproliferative processes, without altering liver function.