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Liver disease is a leading cause of morbidity and mortality. Liver transplantation remains the only proven treatment for end-stage liver failure but is limited by the availability of donor organs. Hepatocyte cell therapy, either with bioartificial liver devices or hepatocyte transplantation, may help address this by delaying or preventing liver transplantation. Early clinical studies have shown promising results, however in most cases, the benefit has been short lived and so further research into these therapies is required. Alternative sources of hepatocytes, including stem cell-derived hepatocytes, are being investigated as the isolation of primary human hepatocytes is limited by the same shortage of donor organs. This review summarises the current clinical experience of hepatocyte cell therapy together with an overview of possible alternative sources of hepatocytes. Current and future areas for research that might lead towards the realisation of the full potential of hepatocyte cell therapy are discussed. [ABSTRACT FROM PUBLISHER]

Mitochondrial oxidative damage is hypothesized to contribute to the pathogenesis of chronic cholestatic liver diseases. Melatonin, an indolamine synthesized in the pineal gland, shows a wide range of physiological functions, and is under clinical investigation for expanded applications. Melatonin has demonstrated efficient protective effects against various types of oxidative damage in the liver system. This study investigates the protective effects of melatonin pretreatment on glycochenodeoxycholic acid (GCDCA)-induced hepatotoxicity and elucidates the potential mechanism of melatonin-mediated protection. Melatonin markedly decreased mitochondrial ROS (mROS) production in L02 cells treated with 100 μM GCDCA, and inhibited GCDCA-stimulated cytotoxicity. Notably, melatonin exerted its hepatoprotective effects by upregulating sirtuin 3 (SIRT3) activity and its expression level, thus regulating superoxide dismutase 2 (SOD2) acetylation and inhibiting the production of mROS induced by GCDCA. Moreover, siRNA targeting SIRT3 blocked the melatonin-mediated elevation in mitochondrial function by inhibiting SIRT3/SOD2 signaling. Importantly, melatonin-activated SIRT3 activity was completely abolished by AMP-activated, alpha 1 catalytic subunit (AMPK) siRNA transfection. Similar results were obtained in rat with bile duct ligation or BDL. In summary, our findings indicate that melatonin is a novel hepatoprotective small molecule that functions by elevating SIRT3, stimulating SOD2 activity, and suppressing mitochondrial oxidative stress at least through AMPK, and that SIRT3 may be of therapeutic value in liver cell protection for GCDCA-induced hepatotoxicity. [ABSTRACT FROM AUTHOR]

Context: Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides (Sacc.) Nirenberg (Nectriaceae) mold that contaminates maize and other agricultural products. Although the effects of FB1 on sphingolipid metabolism are clear, little is known about early molecular changes associated with FB1 carcinogenicity. Objective: Alteration on DNA methylation, as an early event in non-genotoxic carcinogenesis, may play an important role in the mechanism of FB1 toxiciy. Materials and methods: Dose-related effects of FB1 (1-50 µM for 24 h) on global DNA methylation by using high-performance liquid chromatography with UV-diode array detection (HPLC-UV/DAD) and CpG promoter methylation by methylation-specific PCR (MSP) were performed in rat liver (Clone 9) and rat kidney (NRK-52E) epithelial cells. Results: Cell viability reduction is 39% and 34% by the XTT test and LDH release in the growth medium is 32% and 26% at 200 µM of FB1 treatment in Clone 9 and NRK-52E cells, respectively. No significant dose-related effects of FB1 on global DNA methylation which ranged from 4 to 5% were observed in both cells compared with controls. Promoter regions of c-myc gene were methylated (>33%) at 10 and 50 µM of FB1 treatment in Clone 9 cells while it was unmethylated in NRK-52E cells. Promoter regions of p15 gene were unmethylated while VHL gene were found to be methylated (>33%) at 10, 25, and 50 µM and 10 and 50 µM of FB1 treatment in Clone 9 and NRK-52E cells, respectively. Discussion and conclusion: Alteration in DNA methylation might play an important role in the toxicity of FB1 in risk assessment process. [ABSTRACT FROM AUTHOR]

Ethanol metabolism in hepatocytes causes the generation of reactive oxygen species, endoplasmic reticulum stress and alterations in mitochondrial energy and REDOX metabolism. In ethanol-exposed liver disease, autophagy not only acts as a cleanser to remove damaged organelles and cytosolic components, but also selectively clears specific targets such as lipid droplets and damaged mitochondria. Moreover, ethanol appears to play a role in protecting hepatocytes from apoptosis at certain concentrations. This article describes the evidence, function and potential mechanism of autophagy in ethanol-exposed liver disease and the controversy surrounding the effects of ethanol on autophagy. [ABSTRACT FROM AUTHOR]

Primary hepatocytes are highly differentiated cells and proliferatively quiescent. However, the stress produced during liver digestion seems to activate cell cycle entry by proliferative/dedifferentiation programs that still remain unclear. The aim of this work was to assess whether the oxidative stress associated with hepatocyte isolation affects cell cycle and particularly cytokinesis, the final step of mitosis. Hepatocytes were isolated from C57BL/6 mice by collagenase perfusion in the absence and presence of N-acetyl cysteine (NAC). Polyploidy, cell cycle, and reactive oxygen species (ROS) were studied by flow cytometry (DNA, phospho-histone 3, and CellROX® Deep Red) and Western blotting (cyclins B1 and D1, and proliferating cell nuclear antigen). mRNA expression of cyclins A1, B1, B2, D1, and F by reverse transcription (RT)-PCR was also assessed. Glutathione levels were measured by mass spectrometry. Here we show that hepatocyte isolation enhanced cell cycle entry, increased hepatocyte binucleation, and caused marked glutathione oxidation. Addition of 5 mM NAC to the hepatocyte isolation media prevented glutathione depletion, partially blocked ROS production and cell cycle entry of hepatocytes, and avoided the blockade of mitosis progression, abrogating defective cytokinesis and diminishing the formation of binucleated hepatocytes during isolation. Therefore, addition of NAC to the isolation media decreased the generation of polyploid hepatocytes confirming that oxidative stress occurs during hepatocyte isolation and it is responsible, at least in part, for cytokinesis failure and hepatocyte binucleation. [ABSTRACT FROM AUTHOR]

Oxidative stress and mitochondrial dysfunction are hypothesized to contribute to the pathogenesis of chronic cholestatic liver diseases. Silent information regulator 1 (SIRT1) attenuates oxidative stress and improves mitochondrial biogenesis in numerous mitochondrial-related diseases; however, a functional role for SIRT1 in chronic liver cholestasis, characterized by increased levels of toxic bile acids, remains unknown. We show decrease in SIRT1 levels and its activity and impairment of mitochondrial biogenesis in the liver of patients with extrahepatic cholestasis. Moreover, we found that glycochenodeoxycholic acid (GCDCA) stimulated cytotoxicity, disrupted the mitochondrial membrane potential, increased reactive oxygen species production, and decreased mitochondrial mass and mitochondrial DNA content in L02 cells. Consistent with this finding, GCDCA was found to decrease SIRT1 protein expression and activity, thus promoting the deacetylation of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α), a key enzyme involved in mitochondrial biogenesis and function. Conversely, GCDCA-induced mitochondrial injury was efficiently attenuated by SIRT1 overexpression. In summary, these findings indicate that the loss of SIRT1 may play a crucial role in the pathogenesis of liver damage observed in patients with extrahepatic cholestasis. The findings also indicate that genetic supplementation of SIRT1 can ameliorate GCDCA-induced hepatotoxicity through the activation of PGC-1α-dependent mitochondrial biogenesis. [ABSTRACT FROM AUTHOR]

Purpose: To characterize hepatocyte polyploidization induced by intermediate dose of γ-ray. Materials and methods: Male Wistar strain rats were whole-body irradiated (WBI) with 2 Gy of γ-ray at the age of 1 month, and 5-6 rats were sacrificed monthly at 0-25 months after irradiation. The nuclear DNA content of individual hepatocytes was measured by flow cytometry, then hepatocytes were classified into various ploidy classes. Results: Survival percentage, after exposure up to the end of the study, did not indicate any differences between the irradiated groups and controls. The degree of polyploidization in hepatocytes of irradiated rats, was significantly lower than that for the control after 1 month of exposure, and it continued to be lower after up to 8 months. Thereafter, the degree of polyploidization in the irradiated group slowly returned to the control level when the irradiated rats reached the age of 10 months. Conclusion: Intermediate dose of ionizing radiation, in contrast to high doses, decelerate hepatocyte polyploidization, which may coincides with the hypothesis of the beneficial effects of low doses of ionizing radiation. [ABSTRACT FROM AUTHOR]

Context: Indian mustard [ Brassica juncea (L.) Czern. & Coss. (Brassicaceae)] is reported to possess diverse pharmacological properties. However, limited information is available concerning its hepatoprotective activity and mechanism of action. Objective: To study the protective mechanism of mustard seed extract against acetaminophen (APAP) toxicity in a hepatocellular carcinoma (HepG2) cell line. Materials and methods: Hepatotoxicity models were established using APAP (2.5-22.5 mM) based on the cytotoxicity profile. An antioxidant-rich fraction from mustard seeds was extracted and evaluated for its hepatoprotective potential. The mechanism of action was elucidated using various in vitro antioxidant assays, the detection of intracellular generation of reactive oxygen species (ROS), and cell cycle analysis. The phytoconstituents isolated via HPLC-DAD were also evaluated for hepatoprotective activity. Results: Hydromethanolic seed extract exhibited hepatoprotective activity in post- and pre-treatment models of 20 mM APAP toxicity and restored the elevated levels of liver indices to normal values ( p < 0.05). Post-treatment suppressed the generation of ROS by 58.37% and pre-treatment effectively prevented the generation of ROS by 90.5%. The mechanism of ROS suppression was further supported by antioxidant activity (IC50) data from DPPH (103.37 ± 4.2 µg AAE/mg), FRAP (83.26 ± 1.1 µg AAE/mg), ORAC (1115 µM GAE/ml), ABTS (83.05 µg GAE/ml), and superoxide (345.22 ± 5.15 µg AAE/mg) scavenging assays and by the restoration of cell cycle alterations. HPLC-DAD analysis revealed the presence quercetin, vitamin E, and catechin, which exhibited hepatoprotective activity. Discussion and conclusions: A phytoextract of mustard seeds acts by suppressing the generation of ROS in response to APAP toxicity. [ABSTRACT FROM AUTHOR]

In this study, we used primary cultures of fish hepatic cells as a tool for evaluating the effects of environmental contamination. Primary hepatic cell cultures derived from the subtropical fishMetynnis rooseveltiwere exposed to different concentrations (0.275, 2.75 and 27.5 μg L−1) of the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA). Cellular respiratory activity was evaluated by polarography using three substrates: 0.5 M glucose, 0.5 M succinate and 0.5 M α-ketoglutarate. Significant changes were observed in cellular oxygen consumption with 0.5 M α-ketoglutarate. Even at low concentrations, 2,4-D and MCPA were potent uncouplers of oxidative phosphorylation. Primary cultures ofM. rooseveltiliver cells may provide a useful tool for the evaluation of environmental contaminant effects. A review of regulations regarding permitted concentrations of these herbicides is needed. [ABSTRACT FROM PUBLISHER]

Polymers have been utilized to deliver the drug to targeted site in controlled manner, achieving the high-therapeutic efficacy. Polymeric drug conjugates having variable ligands as attachments have been proved to be biodegradable, stimuli sensitive and targeted systems. Numerous polymeric drug conjugates having linkers degraded by acidity or intracellular enzymes or sensitive to over expressed groups of diseased organ/tissue have been synthesized during last decade to develop targeted delivery systems. Most of these organs have number of receptors attached with different cells such as Kupffer cells of liver have mannose-binding receptors while hepatocytes have asialoglycoprotein receptors on their surface which mainly bind with the galactose derivatives. Such ligands can be used for achieving high targeting and intracellular delivery of the drug. This review presents detailed aspects of receptors found in different cells of specific organ and ligands with binding efficiency to these specific receptors. This review highlights the need of further studies on organ-specific polymer-drug conjugates by providing detailed account of polymeric conjugates synthesized till date having organ-specific targeting. [ABSTRACT FROM AUTHOR]

Purpose of the study: The definitive treatment for liver failure is, currently, liver transplantation. Research into other possible treatments, focused on achieving regeneration of the liver parenchyma, have led to the development of methods to generate hepatocytes from stem cells. In our study, we transplant allogenic adipose-derived stem cells (ASCs), not previously differentiated to hepatocytes, to treat acute liver failure induced by intraperitoneal administration of carbon tetrachloride (CCl4) in a Sprague-Dawley rat model. Material and Methods: The ASCs were delivered via the tail vein, having previously been labeled with PKH26, a fluorescent membrane marker. Two control groups were established, Group 1( n = 15) consisting of olive oil (5 mL/kg) and Group 2( n = 15): 1 × 106 PKH26-labeled ASCs. Further, two study groups, Group 3( n = 30): CCl4 dissolved in olive oil and Group 4( n = 30): CCl4 dissolved in olive oil and 1 × 106 PKH26-labeled ASCs completed the experimental design. Results: Blood samples were analyzed, finding AST and ALT levels significantly higher in treatment over control groups at 24 and 48 hours. The mortality rates were statistically different between control groups and Group 3 (Group 1-3 p = .04, Group 2-3 p = .04) and between Groups 3 and 4 ( p = .02). Examining the liver parenchyma, a significantly higher number of ASCs were observed in Group 4 than in Group 2 at all time points ( p = .00). Conclusions: The intravenous injection of allogenic ASCs in this model of CCl4-induced liver failure reduced the mortality in treated animals. ASCs injected in the rat tail vein were found in the liver in animals with induced acute liver failure. [ABSTRACT FROM AUTHOR]

The aim of this study was to systematically study zebrafish model to predict drug-induced liver injury (DILI) with the drug acetaminophen (APAP) and find the sensitive early biomarkers. The concentration-toxicity relationship was determined at concentrations 1, 2, 4, 8, 16 mmol/L of APAP. APAP caused a concentration-dependent mortality of zebrafish. APAP caused a time-dependent mortality. Mild to severe vacuolar degeneration of liver cells were observed. APAP increased serum alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and xanthine oxidase (XOD) levels significantly, while it delevated argininosuccinic acid lyase (ASAL) and total bile acid (TBA) levels. TBA and ASAL were more sensitive than ALT in this process. Zebrafish may be a convenient, practical and valuable biological model to predict DILI. [ABSTRACT FROM AUTHOR]

Encapsulation techniques have the potential to protect hepatocytes from cryoinjury. In this study, we comparatively evaluated the viability and metabolic function of primary rat hepatocytes encapsulated in calcium alginate microbeads, in chitosan tripolyphosphate beads, and in three-layered alginate-chitosan-alginate (ACA) microcapsules, before and after cryopreservation at −80°C and in liquid nitrogen (LN2) for 1 and 3 months. Findings demonstrated that LN2 was atop of −80°C in regard to preservation of viability (> 90%) and hepatic functions. LN2-cryopreserved hepatocytes encapsulated in ACA microcapsules retained metabolic function post-thawing, with > 90% of the albumin, total protein and urea syntheses activities, and > 80% of oxidative function. [ABSTRACT FROM AUTHOR]

Carnosic acid (CA), a phenolic diterpene isolated from rosemary, shows potential benefits in health promotion and disease prevention. In the present study, the cytotoxic and apoptotic-inducing effects of CA on human hepatocellular carcinoma HepG2 cells were investigated. The MTT assay results indicated that CA decreased cell viability in HepG2 cells in a dose-dependent manner. Treatment with CA caused a rapid Caspase-3 activation and subsequently proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), both of which were markers of cells undergoing apoptosis. CA also dissipated mitochondrial membrane potential and decreased the ratio of Bcl-2/Bax protein, which mediated cytosolic translocation of cytochrome c from the mitochondria. Furthermore, CA reduced the phosphorylation of Akt, which was partially inhibited by insulin, an activator of phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway. In conclusion, our data suggest that the mitochondrial dysfunction and deactivation of Akt may contribute to the apoptosis-inducing effects of CA. [ABSTRACT FROM AUTHOR]

1. 4-[2((1R,2R)-2-Hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine-2-carboxylic acid methylamide (BLZ945) is a small molecule inhibitor of CSF-1R kinase activity within osteoclasts designed to prevent skeletal related events in metastatic disease. Key metabolites were enzymatically and structurally characterized to understand the metabolic fate of BLZ945 and pharmacological implications. The relative intrinsic clearances for metabolites were derived from in vitro studies using human hepatocytes, microsomes and phenotyped with recombinant P450 enzymes. 2. Formation of a pharmacologically active metabolite (M9) was observed in human hepatocytes. The M9 metabolite is a structural isomer (diastereomer) of BLZ945 and is about 4-fold less potent. This isomer was enzymatically formed via P450 oxidation of the BLZ945 hydroxyl group, followed by aldo-keto reduction to the alcohol (M9). 3. Two reaction phenotyping approaches based on fractional clearances were applied to BLZ945 using hepatocytes and liver microsomes. The fraction metabolized (fm) or contribution ratio was determined for each metabolic reaction type (oxidation, glucuronidation or isomerization) as well as for each metabolite. The results quantitatively illustrate contribution ratios of the involved enzymes and pathways, e.g. the isomerization to metabolite M9 accounted for 24% intrinsic clearance in human hepatocytes. In summary, contribution ratios for the Phase I and Phase II pathways can be determined in hepatocytes. [ABSTRACT FROM AUTHOR]

Background and Aims:Using decellularized scaffold to reengineer liver tissue is a promising alternative therapy for end-stage liver diseases. Though the decellularized human liver matrix is the ideal scaffold for reconstruction of the liver theoretically, the shortage of liver donors is still an obstacle for potential clinical application. Therefore, an appropriate alternative scaffold is needed. In the present study, we used a tissue engineering approach to prepare a rat decellularized spleen matrix (DSM) and evaluate the effectiveness of this DSM for primary rat hepatocytes culture.Methods:Rat decellularized spleen matrix (DSM) was prepared by perfusion of a series of detergents through spleen vasculature. DSM was characterized by residual DNA and specific extracellular matrix distribution. Thereafter, primary rat hepatocytes were cultured in the DSM in a 3-dimensional dynamic culture system, and liver cell survival and biological functions were evaluated by comparison with 3-dimensional sandwich culture and also with cultured in decellularized liver matrix (DLM).Results:Our research found that DSM did not exhibit any cellular components, but preserved the main extracellular matrix and the intact vasculature evaluated by DNA detection, histology, immunohistochemical staining, vessel corrosion cast and upright metallurgical microscope. Moreover, the method of DSM preparation procedure was relatively simple with high success rate (100%). After seeding primary hepatocytes in DSM, the cultured hepatocytes survived inside DSM with albumin synthesis and urea secretion within 10 d. Additionally, hepatocytes in dynamic culture medium had better biological functions at day 10 than that in sandwich culture. Albumin synthesis was 85.67 ± 6.34 μg/107cell/24h in dynamic culture in DSM compared to 62.43 ± 4.59 μg/107cell/24h in sandwich culture (P< 0.01) and to 87.54 ± 5.25 μg/107cell/24h in DLM culture (P> 0.05); urea release was 32.14 ± 8.62 μg/107cell/24h in dynamic culture in DSM compared to 20.47 ± 4.98 μg/107cell/24h in sandwich culture (P< 0.05) and to 37.38 ± 7.29 μg/107cell/24h cultured in DLM (P> 0.05).Conclusion:The present study demonstrates that DSM can be prepared successfully using a tissue engineering approach. The DSM is an appropriate scaffold for primary hepatocytes culture. [ABSTRACT FROM PUBLISHER]

Bacillus anthracis, the causative agent of anthrax, has become an increasingly important scientific topic due to its potential role in bioterrorism. The lethal toxin (LT) of B. anthracis consists of lethal factor (LF) and a protective antigen (PA). This study investigated whether only lethal factor was efficient as a hepatotoxin in the absence of the PA. To achieve this aim, LF (100 µg/kg body weight, dissolved in sterile distilled water) or distilled water vehicle were intraperitoneally injected once into adult rats. At 24 h post-injection, the hosts were euthanized and their livers removed and tissue samples examined under light and electron microscopes. As a result of LF application, hepatic injury - including cytoplasmic and nuclear damage in hepatocytes, sinusoidal dilatation, and hepatocellular lysis - became apparent. Further, light microscopic analyses of liver sections from the LF-injected rats revealed ballooning degeneration and cytoplasmic loss within hepatocytes, as well as peri-sinusoidal inflammation. Additionally, an increase in the numbers of Kupffer cells was evident. Common vascular injuries were also found in the liver samples; these injuries caused hypoxia and pathological changes. In addition, some cytoplasmic and nuclear changes were detected within the liver ultrastructure. The results of these studies allow one to suggest that LF could be an effective toxicant alone and that PA might act in situ to modify the effect of this agent (or the reverse situation wherein LF modifies effects of PA) such that lethality results. [ABSTRACT FROM AUTHOR]

1. The quantitative prediction of the pharmacokinetic parameters of a drug from data obtained using human in vitro systems remains a significant challenge i.e. prediction of metabolic clearance in humans and estimation of the relative contribution of enzymes involved in the clearance. This has become particularly problematic for low turnover compounds. 2. Having human hepatocytes with stable cellular function over several days that adequately mimic the complexity of the physiological environment would be a major advance. Thus, we evaluated human hepatocytes, maintained in culture during 7 days in the microfluidic LiverChip™ system, in terms of morphological appearance, relative mRNA expression of phase I and II enzymes and transporters as a function of time, and metabolic capacity using probe substrates. 3. The results showed that mRNA levels of the major genes for enzymes involved in drug metabolism were well-maintained over a 7-day period of culture. Furthermore, after 4 days of culture, in the Liverchip™ device, human hepatocytes exhibited higher or similar CYPs activities compared to 1 day of culture in 2D-static conditions. 4. The functional data were supported by light/electron microscopies and immunohistochemistry showing viable tissue structure and well-differentiated human hepatocytes: presence of cell junctions, glycogen storage, and bile canaliculi. [ABSTRACT FROM AUTHOR]

In order to study the liver targeting of the N-galactosylated chitosan (GC) polymer in liver, we first conjugated the lactobionic acid with chitosan (CS) to obtain the carrier of GC with different degree of substitution of lactosyl group. Western blot was performed to detect the expression levels of the asialoglycoprotein receptors (ASGPR) in the cell lines of HepG2, SMMC-7721, and HL-7702. The protein level of ASGPR was lower in HepG2 compared to HL-7702 and SMMC-7721. Although all treated by CS, viabilities of HL-7702 and HepG2 did not experience any significant drop, while viability of SMMC-7721 decreased 15% on average from control. It was the first data about the inhibitory effect of GC on the liver cells. Fluorescein isothiocyanate (FITC) labeled GC (GC-FITC) was injected intravenously into mice at a dose of 0.02 μmol/mouse. GC-FITC showed maximum liver localization at 5 min and even detectable at 48 h after injection. Further, the accumulation of GC in liver was about 5.4-fold higher than that of CS. In conclusion, GC demonstrated its higher efficacy in drug liver targeting and thus could be a more promising drug or gene carrier in future therapies. [ABSTRACT FROM AUTHOR]

The possible molecular mechanisms of Nano-selenium (nano-se) in attenuating hepatocellular carcinoma (HCC) was investigated in this study. To achieve this target, the apoptotic/necrotic rate in hepatic cells was investigated morphologically by double staining with acridine orange/ethidium bromide to address the type of cell death induced by nano-Se in HCC-bearing rats. To predict the oxidative stress and DNA damage, the generation of 8-hydroxy-2-deoxyguanosine (8-OHdG) and 2-deoxyguanosine (2-dG) was examined. Moreover, the expression of some HCC-related genes was investigated such as aldo-keto reductase 1B10 ( Akr1b10), ING3 and Foxp1 genes. As well as the histopathological study of liver tissue sections was performed. The results obtained from this study revealed that (HCC+Nano Se) group shows the highest number of damaged cancerous cells. Furthermore, the necrotic/apoptotic rate was significantly higher in (nano-Se+HCC), (HCC+Doxo) and (HCC+Doxo+nano-se) compared to that in the untreated HCC group. Treatment of HCC group with nano-se decreased the ratio of 8-OHdG/2-dG generation significantly with respect to the untreated HCC group. The opposite was observed regarding the gene expression of AKr1b10 and ING3. The treatment of HCC group with nano-se elicited significant increase in the expression of Akr1b10 and ING3 genes compared with untreated HCC group. On the other hand, the expression of Foxp1 gene was significantly decreased in HCC group treated with nano-se in comparison with the untreated HCC group. The histopathological study provided a supportive evidence for the molecular genetics study. Our data shed light on the molecular mechanisms of nano-se in attenuating HCC in the experimental model. [ABSTRACT FROM AUTHOR]

To understand the mechanism of hepatocyte differentiation during rat liver regeneration (LR) at the gene transcription level, Rat Genome 230 2.0 Array was used to determine the gene expression changes of hepatocytes. Then the genes associated with cell differentiation and their signaling pathways were searched by NCBI, QIAGEN and KEGG webs, and their signaling activities were calculated by spectral function Ep(t) based on the expression profiles. The key genes associated with the signaling pathways and cell differentiation were analyzed and verified by the same kind extraction method. The mechanism of hepatocyte differentiation during rat LR was analyzed by Ingenuity Pathway Analysis 9.0 (IPA). The results showed that 42 signaling pathways regulated cell differentiation and the Ep(t) values of IL-9 signaling pathway and its JAK/STAT and PI3K/AKT branches, PEDF signaling pathway and its NF-κB and Ras/ERK branches were significantly increased in the progression phase (6–72 h after partial hepatectomy [PH]) of rat LR, and the Ep(t) values of hepatocyte differentiation were also significantly increased. ERK/MAPK signaling pathway was significantly increased in the priming (0.5–6 h after PH) and progression phase, and hepatocyte differentiation were also significantly increased. In conclusion, among the 42 signaling pathways that regulated cell differentiation, the signaling activity of IL-9 signaling pathway and its JAK/STAT and PI3K/AKT branches, PEDF signaling activity and its NF-κB and Ras/ERK branches, and ERK/MAPK signaling activity were significantly increased and promoted hepatocyte differentiation in rat LR. Meanwhile, the expression of MYC involved in mentioned signaling pathways and expression of AGT, BCL2, IL10, CCND1, PLAT, and NF1 related to cell differentiation were increased significantly. [ABSTRACT FROM AUTHOR]

Background. Reduced glutathione (γ-glutamylcysteinylglycine), GSH, is essential when protecting cells from oxidative stress and also an indicator of disease risk. Reported concentrations of GSH and its oxidized form, glutathione disulfide (GSSG), varies considerably, primarily due to the instability of GSH and various analytical methods. Methods. We designed a sensitive method to analyze GSH and GSSG in porcine hepatocytes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after stabilization with N-ethylmaleimide (NEM). This method includes stable isotope labeled internal standards and simple synthesis of labeled GSSG which commercial sources rarely offer. GSH and GSSG were analyzed in porcine liver biopsies giving a reference interval based on a large number of samples (26 pigs; 3 parallels). Results. The LC-MS/MS results revealed excellent linearity for both GSH and GSSG, with interday coefficient of variation (%CV) for GSH-NEM and GSSG <10 %. Accuracy for recovery tests was between 95.6% and 106.7% ( n = 3) for GSH and between 92.3% and 107.7% ( n = 3) for GSSG. The limits of quantification were 0.1 μM for GSH-NEM and 0.08 μM for GSSG. The mean concentration of GSH was 3.5 (95% CI = 1.5-8.1) mmol/liter and of GSSG 0.0023 (95% CI = 0.0003-0.019) mmol/liter. Conclusion. For the first time GSH and GSSG are analyzed in porcine hepatocytes by LC-MS/MS yielding a reference level of GSH and GSSG. The method is reproducible in any laboratory with LC-MS/MS service and will probably be applicable in all soft tissues and cell suspensions, essentially with no modification. [ABSTRACT FROM AUTHOR]

1. To investigate the non-linear kinetics of in vitro hepatocyte uptake across species, the OATP substrate Pitavastatin was used as a probe. 2. Experiments were conducted at AstraZeneca (Alderley Park, Macclesfield) using freshly isolated rat, dog and human hepatocytes, utilising the 'oil spin' methodology described by Hassen et al. (1996). Very few mechanistic models have previously been used to characterise the uptake process. 3. Here two candidate pharmacokinetic non-linear compartmental models are proposed. Both models have been shown to be structurally identifiable and distinghishable previously, which establishes that all unknown parameters could be identified from the experimental observations available and that input/output relationships for both the candidate models were structurally different. 4. A kinetic modelling software package, FACSIMILE (MCPA Software, Faringdon, UK), was used to obtain numerical solutions for the system equations and for parameter estimation. Model fits gave good agreement with the in vitro data and suggest the current widely accepted assumption that the rate of diffusion across the hepatocyte cell membrane is the same at both 4 °C and 37 °C is not valid, at least for Pitavastatin. Although this finding has already been proposed, this is the first time it is comprehensively debunked using statistical testing. [ABSTRACT FROM AUTHOR]

Purpose: Exposure to radiation primes the liver for extensive replacement of the resident parenchymal cells by transplanted hepatocytes. The mechanisms underlying this repopulation remain to be clarified. In these studies, we examined the possible occurrence of cell senescence in vivo following radiation-associated preconditioning of the host liver. Materials and methods: Fischer 344 rats underwent external-beam, computed-tomography-based partial liver irradiation. A single dose of 25 Gy was delivered to the right liver lobes (40% of liver mass). An additional group of animals received a 1/3 partial hepatectomy (removal of the left anterior lobe) four days after irradiation. Non-irradiated groups served as controls. All rats were sacrificed four weeks after the initial treatment. Results: The irradiated livers displayed several markers of cell senescence, including expression of senescence-associated-β-galactosidase (SA-β-gal), increase in cell size, and up-regulation of cyclin-dependent kinase inhibitors (CDK-I) p16 and p21. Furthermore, quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis revealed activation of the senescence-associated secretory phenotype (SASP), including the cytokines interleukin 6 (IL6) and 1α (IL1α). The senescence-related changes were more prominent in rats undergoing partial hepatectomy (PH) following irradiation (IR). Conclusions: We conclude that priming with radiation for liver repopulation results in the induction of cell senescence and the up-regulation of a senescence-associated secretory phenotype. The latter can contribute to the extensive growth of transplanted cells in this system. [ABSTRACT FROM AUTHOR]

Introduction: Hepatocyte transplantation is proposed as a solution for liver failure. The allotransplantation of hepatocytes has been studied extensively, however, graft rejection remains a major problem. The aim of the present study was to evaluate the immunosuppressive activity of mycophenolate mofetil (MMF), sirolimus, and their combination in an experimental model of hepatocyte allotransplantation in rats with acute liver failure. Materials and methods: Five male Wistar rats were used as hepatocyte donors and 60 male Lewis rats as recipients. The recipients were divided into five groups of 12 animals each. Group 1: no treatment. Group 2: cyclosporine. Group 3: sirolimus. Group 4: MMF. Group 5: MMF and sirolimus. All surviving animals were preserved for 15 days. For the induction of acute liver failure the recipients were injected with N-dimethyl-nitrosamine 24 hr before transplantation. The isolated hepatocytes were transplanted intrasplenically. Results: Analysis of the results showed a statistically significant prolongation of animal survival for groups 3, 4, and 5. More animals in group 5 survived than those in groups 3 and 4, although the difference was not statistically significant. Transplant hepatocyte survival was significantly better in groups 3, 4, and 5. Hepatocytes transplanted in the spleen of animals of group 5 showed better survival compared with those of groups 3 and 4. Conclusion: Use of MMF and sirolimus, as monotherapy or in combination, is both effective and safe as immunosuppressive treatment in hepatocyte transplantation, as was proven in this experimental protocol. [ABSTRACT FROM AUTHOR]

In addition to their hypotensive properties, angiotensin receptor blockers (ARBs) have been shown to exert clinical antidyslipidemic effects. The mechanism underlying these ARB lipid metabolic effects remains unclear. Some ARBs, for example, telmisartan, activate peroxisome proliferator-activated activated receptor-gamma (PPAR-gamma). We hypothesized that PPAR-gamma-activating ARBs might exert antidyslipidemic effects via PPAR-alpha. In this study, we assessed the effect of telmisartan on the expression of PPAR-alpha and lipoprotein lipase (LPL). PPAR-alpha expression was detected by reverse-transcription polymerase chain reaction and Western blot in HepG2 hepatocytes as well as differentiated C2C12 myocytes treated with increasing concentrations of telmisartan (0.1-10 μmol/L) for 48 h. Results showed that 1 μmol/L and 10 μmol/L telmisartan significantly increased the expression of PPAR-alpha mRNA and protein in HepG2 cells ( p < 0.01). No effect was shown in differentiated C2C12 cells. Similarly, 1 µmol/L and 10 μmol/L telmisartan significantly increased the expression of LPL mRNA and protein in HepG2 cells ( p < 0.01), and this increase was significantly ( p < 0.01) inhibited by the PPAR-alpha-specific antagonist MK886. These results indicate that certain of the antidyslipidemic effects of telmisartan might be mediated via increased PPAR-alpha-dependent induction of LPL expression. [ABSTRACT FROM AUTHOR]

Short, middle, and long-term exercise, as well as the relative intensity of the physical effort, may influence a broad array of laboratory results, and it is thereby of pivotal importance to appropriately differentiate the 'physiologic' from the 'pathological' effects of exercise. Therefore, the values of some biomarkers in physically active subjects may be cautiously interpreted since the results may fall outside the conventional reference ranges. It has been demonstrated that middle and long-term endurance and/or strenuous exercise triggers transient elevations of muscular and cardiac biomarkers. However, no data have been published about the effect of short-term maximal exercise test on the most useful muscular, hepatic and cardiovascular biomarkers. The aim of the present study was to assess the baseline concentrations of muscular, hepatic, and cardiovascular makers between trained and untrained subjects, along with changes induced by maximal exercise test. We measured C reactive protein (CRP), procalcitonin (PCT), gamma glutamyltransferase (GGT), creatine kinase-MB isoenzyme (CK-MB), Hs-TnT, NT-proBNP, CK, LDH, AST, and ALT in serum samples of physically active (trained) and physically inactive (sedentary) male collected before, immediately after a maximal exercise test and after a 30-min recovery period. Trained subjects tend to have significantly raised base concentrations of CK, CK-MB, ALT, and LDH compared to sedentary individuals, and this can be clearly interpreted as a mild injury of skeletal muscle. A single maximal exercise was also effective to transiently increase the concentrations of NT-proBNP, but not those of Hs-TnT, thus suggesting that the cardiac involvement is mostly benign in nature. [ABSTRACT FROM AUTHOR]

Recent studies suggest that organ decellularization is a promising approach to facilitate the clinical application of regenerative therapy by providing a platform for organ engineering. This unique strategy uses native matrices to act as a reservoir for the functional cells which may show therapeutic potential when implanted into the body. appropriate cell sources for artificial livers have been debated for some time. The desired cell type in artificial livers is primary hepatocytes, but in addition, other supportive cells may facilitate this stem cell technology. In this context, the use of mesenchymal stem cells (MSC) is an option meeting the criteria for therapeutic organ engineering. Ideally, supportive cells are required to (1) reduce the hepatic cell mass needed in an engineered liver by enhancing hepatocyte function, (2) modulate hepatic regeneration in a paracrine fashion or by direct contact, and (3) enhance the preservability of parenchymal cells during storage. here, we describe enhanced hepatic function achieved using a strategy of sequential infusion of cells and illustrate the advantages of co-cultivating bone marrow-derived MSCs with primary hepatocytes in the engineered whole-liver scaffold. These co-recellularized liver scaffolds colonized by MSCs and hepatocytes were transplanted into live animals. after blood flow was established, we show that expression of adhesion molecules and proangiogenic factors was upregulated in the graft. [ABSTRACT FROM AUTHOR]

The liver is a target of in vitro tissue engineering despite its capability to regenerate in vivo. The construction of liver tissues in vitro remains challenging. in this review, conventional 3D cultures of hepatocytes are first discussed. Recent advances in the 3D culturing of liver cells are then summarized in the context of in vitro liver tissue reconstruction at the micro- and macroscales. The application of microfluidics technology to liver tissue engineering has been introduced as a bottom-up approach performed at the microscale, whereas whole-organ bioengineering technology was introduced as a top-down approach performed at the macroscale. Mesoscale approaches are also discussed in considering the integration of micro- and macroscale approaches. Multiple parallel multiscale liver tissue engineering studies are ongoing; however, no tissue-engineered liver that is appropriate for clinical use has yet been realized. The integration of multiscale tissue engineering studies is essential for further understanding of liver reconstruction strategies. [ABSTRACT FROM AUTHOR]

Columnar epithelia (e.g., kidney, intestine) and hepatocytes embody the two major organizational phenotypes of non-stratified epithelial cells. Columnar epithelia establish their apical and basal domains at opposing poles and organize in monolayered cysts and tubules, in which their apical surfaces form a single continuous lumen whereas hepatocytes establish their apical domains in the midst of their basolateral domains and organize a highly branched capillary luminal network, the bile canaliculi, in which a single hepatocyte can engage in lumen formation with multiple neighbors. To maintain their distinct tissue architectures, columnar epithelial cells bisect their luminal domains during symmetric cell divisions, while the cleavage furrow in dividing hepatocytes avoids bisecting the bile canalicular domains. We discuss recently discovered molecular mechanisms that underlie the different cell division phenotypes in columnar and hepatocytic model cell lines. The serine/threonine kinase Par1b determines both the epithelial lumen polarity and cell division phenotype via cell adhesion signaling that converges on the small GTPase rhoa. [ABSTRACT FROM AUTHOR]

1. Human-chimeric mice with humanized liver have been constructed by transplantation of human hepatocytes into several types of mice having genetic modifications that injure endogenous liver cells. Here, we focus on liver urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice, which are the most widely used human-chimeric mice. Studies so far indicate that drug metabolism, drug transport, pharmacological effects and toxicological action in these mice are broadly similar to those in humans. 2. Expression of various drug-metabolizing enzymes is known to be different between humans and rodents. However, the expression pattern of cytochrome P450, aldehyde oxidase and phase II enzymes in the liver of human-chimeric mice resembles that in humans, not that in the host mice. 3. Metabolism of various drugs, including S-warfarin, zaleplon, ibuprofen, naproxen, coumarin, troglitazone and midazolam, in human-chimeric mice is mediated by human drug-metabolizing enzymes, not by host mouse enzymes, and thus resembles that in humans. 4. Pharmacological and toxicological effects of various drugs in human-chimeric mice are also similar to those in humans. 5. The current consensus is that chimeric mice with humanized liver are useful to predict drug metabolism catalyzed by cytochrome P450, aldehyde oxidase and phase II enzymes in humans in vivo and in vitro. Some remaining issues are discussed in this review. [ABSTRACT FROM AUTHOR]

Placental mesenchymal dysplasia (PMD) is characterized by placentomegaly and grapelike vesicles resembling a partial molar pregnancy and in most cases, a phenotypically normal fetus. Hepatic mesenchymal hamartoma (HMH) is a benign hamartomatous proliferation of mesenchymal liver tissue. PMD has been associated with HMH. Although rare, in combination, it is known to carry a poorer prognosis than in fetuses without structural abnormalities. There are only a few reported cases of PMD and associated HMH with varying management strategies and outcomes, precluding ascertainment of the most appropriate treatment plan. We present a case of PMD with associated cystic HMH resulting in fetal death. We also reviewed the published literature on this issue and explored possible management strategies to prevent adverse fetal and neonatal outcomes. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the protective effect of gossypetin (GTIN) against gamma (γ)-radiation-mediated DNA damage. Materials and methods: Increasing concentrations (10-150 μM) of GTIN were incubated with supercoiled DNA 1 h prior exposure to γ-radiation in the range of 5-Gy absorbed dose from Co60 γ source. To establish the effective protective concentration of GTIN, supercoiled DNA was pre-incubated with 50 μM of GTIN for 1 h followed by exposure of 5, 10 and 20 Gy doses of γ-radiation. Moreover, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical, hydroxyl radical, nitric oxide (NO) scavenging, metal chelating activity and ferric reducing antioxidant power (FRAP) of GTIN were measured and compared with standards. The flowcytometric analysis and radiation-induced genomic DNA damage by comet assay were employed to estimate the level of intracellular reactive oxygen species (ROS) using isolated murine hepatocytes. Results: GTIN was able to effectively scavenge different free radicals in in vitro situations. It could significantly prevent radiation induced supercoiled and genomic DNA damage with reduced comet parameters. It also acted as a potent scavenger of the radiation induced ROS. Conclusions: GTIN ameliorated radiation-induced oxidative stress and DNA damage by its free-radical scavenging activity. [ABSTRACT FROM AUTHOR]

Acute liver failure is a rare and often devastating condition consequent on massive liver cell necrosis that frequently affects young, previously healthy individuals resulting in altered cognitive function, coagulopathy and peripheral vasodilation. These patients frequently develop concurrent acute kidney injury (AKI). This abrupt and sustained decline in renal function, through a number of pathogenic mechanisms such as renal hypoperfusion, direct drug-induced nephrotoxicity or sepsis/systemic inflammatory response contributes to increased morbidity and is strongly associated with a worse prognosis. Improved understanding of the pathophysiology AKI in the context of acute liver failure may be beneficial in a number of areas; the development of new and sensitive biomarkers of renal dysfunction, refining prognosis and organ allocation, and ultimately leading to the development of novel treatment strategies, these issues are discussed in more detail in this expert review. [ABSTRACT FROM AUTHOR]

p38 MAPKs are important mediators of signal transduction that respond to a wide range of extracellular stressors such as UV radiation, osmotic shock, hypoxia, pro-inflammatory cytokines, and oxidative stress. The most abundant family member is p38α, which helps to couple cell proliferation and growth in response to certain damaging stimuli. In fact, increased proliferation and impaired differentiation are hallmarks of p38α-deficient cells. It has been reported that reactive oxygen species (ROS) play a critical role in cytokine-induced p38α activation. Under physiological conditions, p38α can function as a mediator of ROS signaling and either activate or suppress cell cycle progression depending on the activation stimulus. The interplay between cell proliferation, p38 MAPK activation, and ROS production plays an important role in hepatocytes. In fact, low levels of ROS seem to be needed to activate several signaling pathways in response to hepatectomy and to orchestrate liver regeneration. p38 MAPK works as a sensor of oxidative stress and cells that have developed mechanisms to uncouple p38 MAPK activation from oxidative stress are more likely to become tumorigenic. So far, p38α influences the redox balance, determining cell survival, terminal differentiation, proliferation, and senescence. Further studies would be necessary in order to clarify the precise role of p38 MAPK signaling as a redox therapeutical target. [ABSTRACT FROM AUTHOR]

1. Human chimeric mice (h-PXB mice) having humanized liver, constructed by transplantation of human hepatocytes, were evaluated as an experimental model for predicting human drug metabolism. Metabolism of zaleplon in h-PXB mice was compared with that in rat chimeric mice (r-PXB mice) constructed by transplantation of rat hepatocytes. 2. Zaleplon is metabolized to 5-oxo-zaleplon by aldehyde oxidase and to desethyl-zaleplon by cytochrome P450 (CYP3A4) in rat and human liver preparations. 3. Liver S9 fraction of h-PXB mice metabolized zaleplon to 5-oxo-zaleplon and desethyl-zaleplon in similar amounts. However, liver S9 fractions of r-PXB and control (urokinase-type plasminogen activator-transgenic severe combined immunodeficient) mice predominantly metabolized zaleplon to desethyl-zaleplon. 5-Oxo-zaleplon was detected as a minor metabolite. 4. Oxidase activity of h-PXB mouse liver cytosol toward zaleplon was about 10-fold higher than that of r-PXB or control mice. In contrast, activities for desethyl-zaleplon formation were similar in liver microsomes from these mice, as well as rat and human liver microsomes. 5. In vivo, the level of 5-oxo-zaleplon in plasma of h-PXB mice was about 7-fold higher than that in r-PXB or control mice, in agreement with the in vitro data. Thus, aldehyde oxidase in h-PXB mice functions as human aldehyde oxidase, both in vivo and in vitro. 6. In contrast, the plasma level of desethyl-zaleplon in r-PXB and control mice was higher than that in h-PXB mice. 7. These results suggest h-PXB mice with humanized liver could be a useful experimental model to predict aldehyde oxidase- and CYP3A4-mediated drug metabolism in humans. [ABSTRACT FROM AUTHOR]

In developing therapeutic alternatives to liver transplantation, we have used the strategy of applying a small intestinal segment as a scafold for hepatocyte transplantation and also as a portocaval shunt (pcs) system to address both liver dysfunction and portal hypertension. The aim of this study was to investigate the feasibility of such an intestinal segment in animal models. hepatocytes isolated from luciferase-transgenic Lewis rats were transplanted into jejunal segments of wild-type Lewis rats with mucosa removal without pcs application. Luciferase-derived luminescence from transplanted hepatocytes was stably detected for 30 d. Then, we performed autologous hepatocyte transplantation into the submucosal layer of an isolated and vascularized small intestinal segment in pigs. Transplanted hepatocytes were isolated from the resected left-lateral lobe of the liver. On day 7, hepatocyte clusters and bile duct-like structures were observed histologically. To create an intestinal pcs system in pigs, an auto-graft of the segmental ileum and interposing vessel graft were anastomosed to the portal vein trunk and inferior vena cava. however, thrombi were observed in vessels of the intestinal pcss. We measured the correlation between infusion pressure and low volume in whole intestines ex vivo in both species and found that the high pressure corresponding to portal hypertension was still insuicient to maintain the patency of the intestinal grafts. In conclusion, we demonstrated the feasibility of the small intestine as a scafold for hepatocyte transplantation in rat and pig models, but pcs using an intestinal graft failed to maintain patency in a pig model. [ABSTRACT FROM AUTHOR]

In a previous study, we noticed that some ascitic cells isolated from melanoma patients survive, proliferate, and differentiate into giant phagocytes after the other cells died. Similar phenomena were observed in the primary cultures of mouse lung and liver cells. In the present study, the effects of dying cells on abdominal exudate cells, and the biological characteristics of the differentiated cells were studied. The results indicate that necrotic cells induce CD14−/CD68 + fraction of abdominal exudate cells to proliferate and differentiate into giant phagocytes; however, apoptotic cells had no such effect. Morphologic studies revealed that the large phagocytes possess characteristics of macrophages. Moreover, necrotic cells enhance the expression of CD14, CD68, CD80, and CD86, and the differentiated cells expressed high levels of CD68 and CD86. Our results indicate that necrotic cells induce CD14−/CD68+ fraction of abdominal exudate cells to proliferate and differentiate, and the differentiated cells possess characteristics similar to macrophages. [ABSTRACT FROM AUTHOR]

1. Organic anion transporting polypeptide 1B1 plays a pivotal role in the disposition of many anionic drugs. Significant overlap in substrate specificity between individual OATP isoforms has hampered the identification of the relative importance of individual isoforms for hepatic uptake of xenobiotics. 2. The present study focused on the use of siRNA technology to decrease OATP1B1 selectively in human hepatocytes. Following delivery of siRNA by the novel lipid, AtuFECT01, mRNA expression of OATP1B1 was reduced by 94%-98% with no significant toxicity. Off-target effects were also shown to be minimal as evidenced by the expression of common drug metabolizing enzymes, transporters, nuclear receptors and associated co-regulators. Uptake of estrone-3-sulfate (5 nM) by OATP1B1 was reduced by 82%-95%. This methodology was subsequently used to assess the relative contribution of OATP1B1 uptake in human hepatocytes for olmesartan (42%-62%), valsartan (28%-81%), rosuvastatin (64%-72%), pitavastatin (84%-98%) and lopinavir (64%-89%). These data are consistent with previous values obtained using a relative activity factor approach. 3. The siRNA approach provides a robust and reproducible method for assessing the relative contribution of OATP1B1 to hepatic uptake of new chemical entities. The technique also has potential utility in facilitating detailed characterization of drug-drug interactions involving hepatic drug transporters. [ABSTRACT FROM AUTHOR]

Cyclosporin A is an immunosuppressant drug used to prevent graft rejection in organ transplantation. Side effects include toxicity to the kidney and liver. Recently, many experimental and clinical data have demonstrated that CsA-induced toxicity is associated with increased production of reactive oxygen species (ROS) and lipid peroxidation in the kidney and liver. The aim of the study was ultrastructural examination of renal and liver epithelial cells in the course of chronic experimental CsA treatment. Rats were treated with CsA in a dose of 25 mg/kg/day. Animals developed failure of the kidney and the liver functions manifested by an increase in serum levels of creatinine, urea, uric acid, bilirubin, AST, and ALT and a decrease in total proteins. Ultrastructural examination of tubular epithelial cells and hepatocytes revealed dilatation of endoplasmic reticulum and injury to mitochondria, formation of autolysosomes, and presence of single apoptotic cells. On the basis of a review of the literature the authors suggest that mitochondrial and cell membrane system changes are, at least in part, a result of oxidative damage to the liver and renal tubular epithelial cells. [ABSTRACT FROM AUTHOR]

The concentration of hexabromocyclododecanes (HBCDs) in the environment media and organism samples are gradually rising with the increase of HBCDs usage. This study is designed to investigate the cytological effects of HBCDs on human hepatocyte L02 and explore the potential molecular mechanism. The results of CCK-8 assay showed that high concentration of HBCDs (>20 μM) significantly suppressed cell survival, while comparatively lower dose of HBCDs (10−13–10−7M) slightly stimulated cell proliferation (P< 0.05). In the mean time, high concentration HBCDs markedly induced cell apoptosis and DNA damage, accompanying with increase of intracellular Ca2+level and decrease of mitochondrial membrane potential (P< 0.05). ROS level induced by low concentration of HBCDs was comparatively lower than that by high concentration of HBCDs. In addition, low concentration HBCDs exposure (10−13–10−7M) resulted in up-regulation of PCNA protein expression level in a time-dependent manner. However, high concentration HBCDs exposure led to increase of Apaf-1 expression level. In conclusion, loss of mitochondrial membrane potential and activation of Apaf-1 mediated pathway involve the L02 cell apoptosis induced by high concentration HBCDs exposure. However, low concentration HBCDs exposure could stimulate cell proliferation of L02 cells, which might be associated with enhancement of PCNA expression. [ABSTRACT FROM AUTHOR]

Context: Rubus aleaefolius Poir. (Rosaceae) is used as a folk medicine to treat various types of hepatitis with significant effects in Fujian Province of China. The ethyl acetate fraction of R. aleaefolius root ethanol extract proved effective after our testing in vivo animal experiments. Objective: The protective effects of a major constituent, 1β-hydroxyeuscaphic acid isolated from R. aleaefolius was first investigated against carbon tetrachloride (CCl4)-induced injury in liver cells from hepatocytes cell line (BRL-3A). Materials and methods: Treatment of BRL-3A with CCl4 led to generation of free radicals detected after a 2 h incubation and produced cell injury demonstrated by increased leakage of alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) in the media. Exposure to CCl4 caused apoptosis to cells but did not induce lipid peroxidation. Following treatment with 1β-hydroxyeuscaphic acid at doses ranging from 1 to 100 µg/mL for 24 h, cellular morphology, cell growth function (MTT assay), ALT, AST, malondialdehyde (MDA) and superoxide dismutase (SOD) were assessed and evaluated under control and exposed conditions. Results: The IC50 of 1β-hydroxyeuscaphic acid was 15 μg/mL. Exposure of injured BRL-3A at 20 μg/mL changed abnormal size, cellular shrinkage, and enhanced regulation. ALT, AST, MDA enzyme levels were reduced and SOD activity was increased. Discussion and conclusion: Treatment with 1β-hydroxyeuscaphic acid has significant hepatoprotective activity by lowering the leakage of intracellular enzymes, reducing the oxidation of proteins and decreasing the incidence of apoptosis. These results provide a basis for confirming the traditional uses of R. aleaefolius in treating hepatic diseases. [ABSTRACT FROM AUTHOR]

Effects on the liver C3A cell line treated with a panel of engineered nanomaterials (NMs) consisting of two zinc oxide particles (ZnO; coated 100 nm and uncoated 130 nm), two multi- walled carbon nanotubes (MWCNTs), one silver (Ag < 20 nm), one 7 nm anatase, two rutile TiO2 nanoparticles (10 and 94 nm) and two derivatives with positive and negative covalent functionalisation of the 10 nm rutile were evaluated. The silver particles elicited the greatest level of cytotoxicity (24 h LC5O - 2 μg/cm2). The silver was followed by the uncoated ZnO (24 h 1C50 - 7.5 μg/cm2) and coated ZnO (24 h LC5O - 15 ~ig/cm2) particles with respect to cytotoxicity. The ZnO NMs were found to be about 50-60% soluble which could account for their toxicity. By contrast, the Ag was <1% soluble. The 1C50 was not attained in the presence of any of the other engineered NMs (up to 80 μg/cm2). All NMs significantly increased 11-8 production. Meanwhile, no significant change in TNF-a, IL-6 or CRP was detected. Urea and albumin production were measured as indicators of hepatic function. These markers were only altered by the coated and uncoated ZnO, which significantly decreased albumin production. [ABSTRACT FROM AUTHOR]

1. A fast, straightforward and cost-effective assay was validated for the assessment of CYP induction in cryopreserved human hepatocytes cultured in 48-well plates. The cocktail strategy ( in situ incubation) was used to assess the induction of CYP1A2, CYP2B6, CYP2C9 and CYP3A4 by using the recommended probe substrate, i.e. phenacetin, bupropion, diclofenac and midazolam, respectively. 2. Cryopreserved human hepatocytes were treated for 72 h with prototypical reference inducers, β-naphthoflavone (25 µM), phenobarbital (500 µM) and rifampicin (10 µM) as positive controls for CYP induction. The use of a cocktail strategy has been validated and compared to the classical approach (single incubation). The need of using phase II inhibitor (salicylamide) in CYP induction assay was also investigated. 3. By using three different batches of cryopreserved human hepatocytes and our conditions of incubations, we showed that there was no relevant drug-drug interaction using the cocktail strategy. The same conclusions were observed when a broad range of enzyme activity has to be assessed (wide range of reference inducers, i.e. EC50-Emax experiment). In addition, the interassay reproducibility assessment showed that the day-to-day variability was minimal. 4. In summary, the study showed that the conditions used (probe substrates, concentration of probe substrate and time of incubation) for the cocktail approach were appropriate for investigations of CYP induction potential of new chemical entities. In addition, it was also clear that the use of salicylamide in the incubation media was not mandatory and could generate drug-drug interactions. For this reason, we recommend to not use salicylamide in CYP induction assay. [ABSTRACT FROM AUTHOR]

Alpha-1 antitrypsin Deficiency (AATD) is a common, but under recognized metabolic genetic disease. Although many mutations in the alpha-1 antitrypsin (AAT) gene are described, the Z variant is the allele overwhelmingly associated with liver disease. PI*ZZ homozygotes occur in approximately 1 in 2,000-5,000 births in North American and European populations. The AAT protein is synthesized in large quantities by the liver, and then secreted into serum. Its physiologic function is to inhibit neutrophil proteases in order to protect host tissues from non-specific injury during periods of inflammation. The mutant Z gene of AAT directs the synthesis of a mutant protein which folds abnormally during biogenesis in the endoplasmic reticulum of hepatocytes and is retained intracellularly, rather than efficiently secreted. Intracellular proteolysis pathways, including the proteasome and autophagy, are activated as a response to the intracellular burden of misfolded protein. The lack of circulating anti-protease activity leaves the lung vulnerable to injury and the development of emphysema. The intracellular accumulation of AAT mutant Z protein within hepatocytes can cause liver injury, cirrhosis and hepatocellular carcinoma by triggering a cascade of chronic hepatocellular apoptosis, regeneration, and end organ injury. There is no specific treatment for PI*ZZ associated liver disease, other than standard liver supportive care and liver transplantation. There is a high degree of variability in the clinical manifestations among PI*ZZ homozygous patients, suggesting a strong influence of as yet poorly characterized, genetic and environmental disease modifiers. Studies of the processes of intracellular injury have led to a new era of rational therapeutic development. [ABSTRACT FROM AUTHOR]

1. In this manuscript we describe a non-radioactive, high-throughput method to evaluate hepatic uptake using cryopreserved hepatocytes. We have validated the uptake of pravastatin with different amounts of hepatocytes and the impact of the oil layer used in separation. The time- and concentration-dependent uptake profiles of several anionic and cationic charged drugs were evaluated. The results with our method compare favourably with the literature for pravastatin, atorvastatin and estrone 3-sulfate. 2. Two approaches for kinetic determination (temperature difference and fitting the linear and non-saturable passive diffusion rate in the equation, i.e. V = ( Vmax × S)/(Km + S) + Pdif × S) have been evaluated. Kinetic studies indicate that the different approaches for determining passive diffusion can affect Km and Vmax, but not the clearance of active uptake ( Vmax/Km). 3. Using pravastatin as a probe substrate, species differences were observed in the organic anion-transporting polypeptide (OATP) 1B1 and 1B3 activities. Plasma protein significantly reduced the uptake of atorvastatin, but not pravastatin. 4. Our data suggests that evaluation of the role of active uptake in hepatic clearance in humans should consider the relative ratio of active uptake to passive diffusion, species differences and plasma protein binding when applying in vitro uptake data. [ABSTRACT FROM AUTHOR]

In the present study, cytotoxic effects of the polycyclic aromatic hydrocarbons benzo[a] pyrene (B[a]P) were investigated in Sparus aurata hepatocytes primary cultures after acute and chronic exposure. Cells were treated with a wide range of B[a]P doses (1 pg/mL to 100 μg/mL) for 24, 48 and 72 h. B[a]P toxicity was quantified in sea bream hepatocytes by MTT assay and immunofluorescence analysis of apoptosis after the various exposure periods, in order to evaluate the hepatic damage and toxicity range. Results showed three cytotoxic responses: B[a]P cell death for primary necrosis after exposure to high concentrations for short times, apoptosis induction with the use of sublethal doses and cell proliferation allied with neoplastic foci formation after exposure to low concentrations for long times. This responses provided an interesting correlation between the damage caused on hepatocytes and the metabolism of this toxic compound, to date mainly studied in vivo. Additionally, the statistical analysis revealed that the effects of time and dose were significant for both parameters and especially the time was extremely significant (P<0.0001), in fact B[a]P induced damage that increased over time. Our findings demonstrated and confirmed that S. aurata is a very sensitive species to B[a]P exposure since adverse effects were found at all tested doses. Furthermore, the new in vitro animal model can be considered a useful tool for studying the cellular effects induced by any contaminant harmful for farmed fish. [ABSTRACT FROM AUTHOR]

Pseudoxanthoma elasticum (PXE) is a serious genetic disorder with ectopic mineralization affecting the skin, the eye and the cardiovascular system. The disease is predominantly caused by mutations in the transmembrane ABC protein ABCC6, a putative small substrate transporter. Interestingly, ABCC6 seems virtually absent in the affected organs, whereas a high expression is seen in hepatocytes. This and further published experimental evidence indicate that PXE is a systemic, metabolic liver disease where circulatory changes affect the peripheral mineralization process. Owing to the well-characterized transport of organic substrates by related ABC proteins, it has been proposed that PXE is caused by impaired export of an antimineralization compound to the blood. The authors here present an alternative hypothesis that explains ectopic mineralization in PXE as a consequence of hepatic accumulation of ABCC6 substrate(s) that via gene-regulating effects leads to altered hepatic secretion and activation of antimineralization/anticalcification proteins such as fetuin-A and Gla proteins. [ABSTRACT FROM AUTHOR]