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Increasing evidence demonstrates a physiological role for the aryl hydrocarbon receptor (AhR) in regulating hepatocyte cell cycle progression. Previous studies have used a murine model of liver regeneration to show that exposure to the potent exogenous AhR ligand, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), suppresses hepatocyte proliferation in vivo. Based on recent reports that natural killer (NK) cells negatively regulate liver regeneration, coupled with the well-established immunomodulatory effects of TCDD, it was hypothesized that alterations in lymphocyte activation contribute to the suppression of liver regeneration in TCDD-treated mice. To test this, mice were treated with TCDD (20 μg/kg) 1 day prior to 70% partial hepatectomy (PH), in which two-thirds of the liver was surgically resected. Lymphocytes were collected from the remnant liver and analyzed by flow cytometry. Whereas exposure to TCDD did not alter the number of NK cells or CD3+ T-cells recovered from the regenerating liver, it reduced the percentage and number of intra-hepatic NKT cells 42 h after PH. With regard to lymphocyte activation, TCDD treatment transiently increased CD69 expression on NK and NKT cells 12 h after PH, but had no effect on intracellular levels of IFNγ in NK, NKT, or CD3+ T-cells. To determine the relevance of NK cells to the suppression of liver regeneration by TCDD, mice were treated with anti-Asialo GM-1 (ASGM-1) antibody to deplete NK cells prior to TCDD treatment and PH, and hepatocyte proliferation was measured using bromodeoxyuridine incorporation. Exposure to TCDD was found to inhibit hepatocyte proliferation in the regenerating liver of NK cell-depleted mice and control mice to the same extent. Hence, it is unlikely that enhanced numbers or increased activation of NK cells contribute to the suppression of liver regeneration in TCDD-treated mice. [ABSTRACT FROM AUTHOR]

In the present study we investigated the beneficial role of glycine in iron (FeSO4) induced oxidative damage in murine hepatocytes. Exposure of hepatocytes to 20 μM FeSO4 for 3 hours enhanced reactive oxygen species (ROS) generation and induced alteration in biochemical parameters related to hepatic oxidative stress. Investigating cell signalling pathway, we observed that iron (FeSO4) intoxication caused NF-κB activation as well as the phosphorylation of p38 and ERK MAPKs. Iron (FeSO4) administration also disrupted Bcl-2/Bad protein balance, reduced mitochondrial membrane potential, released cytochrome c and induced the activation of caspases and cleavage of PARP protein. Flow cytometric analysis also confirmed that iron (FeSO4) induced hepatocytes death is apoptotic in nature. Glycine (10 mM) supplementation, on the other hand, reduced all the iron (FeSO4) induced apoptotic indices. Combining, results suggest that glycine could be a beneficial agent against iron mediated toxicity in hepatocytes. [ABSTRACT FROM AUTHOR]

We describe the usability of human pooled hepatocytes for non-CYP metabolism evaluation and an in vivo–in vitro correlation analysis for aldehyde oxidase (AO) substrate compounds using pooled hepatocytes. By comparing intrinsic clearance values of 18 compounds primarily metabolized by AO, UDP-glucuronosyltransferase, carbonyl/aldo-keto reductase, flavin-containing monooxygenase, and monoamineoxidase in individual hepatocytes and pooled hepatocytes from the same individual donors, intrinsic clearance in the pooled hepatocytes was ±30% of the average clearance value in individuals for 15 of 18 compounds, suggesting that pooled hepatocytes maintained the average activity of the individual hepatocytes. Although the results of an in vivo–in vitro correlation analysis for AO substrate compounds showed a trend toward under-prediction, the underestimation ratios for all AO substrates were nevertheless comparable (7.2- to 14.9-fold), suggesting that hepatic clearance prediction for these compounds can be quantified using empirical scaling. These observations enabled us to obtain specific pooled hepatocytes that showed the expected non-CYP enzyme activities by pre-characterization and to quantify hepatic clearance prediction for AO compounds using an empirical scaling factor. [ABSTRACT FROM AUTHOR]

Systematic under-prediction of clearance is frequently associated with in vitro kinetic data when extrapolated using physiological scaling factors, appropriate binding parameters and the well-stirred model. The present study describes a method of removing this systematic bias through application of empirical correction factors derived from regression analyses applied to the in vitro and in vivo data for a defined set of reference compounds. Linear regression lines were established with in vivo intrinsic clearance (CLint), derived from in vivo clearance data and scaled in vitro intrinsic clearance from isolated hepatocyte incubations. The scaled CLint was empirically corrected to a predicted in vivo CLint using the slope and intercept from a uniform weighted linear regression applied to the in vitro to in vivo extrapolation. Cross validation of human data demonstrated that 66% of the reference compounds had a predicted in vivo CLint within two-fold of the observed value. The average absolute fold error (AAFE) for the in vivo CLint predictions was 1.90. For rat, 54% of the compounds had a predicted value within two-fold of the observed and the AAFE was 1.98. Three AstraZeneca projects are used to exemplify how a two-sided prediction interval, applied to the rat regression corrected reference data, can form the basis for assessing the likelihood that, for a given chemical series, the in vitro kinetic data is predictive of in vivo clearance and is therefore appropriate to guide optimisation of compound metabolic stability. [ABSTRACT FROM AUTHOR]

Drug metabolism studies in vitro were carried out inexpensively and readily to serve as an adequate mechanism to characterize drug metabolites, elucidate their pathways, and make suggestions for further testing in vivo. In this work, the comparative metabolism of mequindox (MEQ) was investigated in vitro by incubation with chicken liver microsomes, hepatocytes, and intestinal microflora, followed by analysis using ultra-performance liquid chromatography coupled with electrospray ionization hybrid quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) for structure identification. There were 12 metabolites detected when MEQ was incubated with liver microsomes, 6 metabolites with the hepatocytes and 4 metabolites with intestinal microflora, respectively. The major metabolites in liver microsomes were bideoxymequindox and 2-isoethanol-N1-deoxymequindox, and that in hepatocytes were 2-isoethanol mequindox and 2-isoethanol-N1-deoxymequindox, but in intestinal incubations, N1-deoxymequindox and bideoxymequindox were the major metabolites. The results indicated that the metabolism of MEQ was active in vitro; meanwhile, revealed the main metabolic pathways of MEQ were N→O group reduction, carbonyl reduction and hydroxylation reaction. The information regarding in vitro metabolism of MEQ provided a better understanding of the role of the liver and intestinal tract in the disposition of MEQ. [ABSTRACT FROM PUBLISHER]

Prediction of chemical-induced hepatotoxicity in humans from in vitro data continues to be a significant challenge for the pharmaceutical and chemical industries. Generally, conventional in vitro hepatic model systems (i.e. 2-D static monocultures of primary or immortalized hepatocytes) are limited by their inability to maintain histotypic and phenotypic characteristics over time in culture, including stable expression of clearance and bioactivation pathways, as well as complex adaptive responses to chemical exposure. These systems are less than ideal for longer-term toxicity evaluations and elucidation of key cellular and molecular events involved in primary and secondary adaptation to chemical exposure, or for identification of important mediators of inflammation, proliferation and apoptosis. Progress in implementing a more effective strategy for in vitro-in vivo extrapolation and human risk assessment depends on significant advances in tissue culture technology and increasing their level of biological complexity. This article describes the current and ongoing need for more relevant, organotypic in vitro surrogate systems of human liver and recent efforts to recreate the multicellular architecture and hemodynamic properties of the liver using novel culture platforms. As these systems become more widely used for chemical and drug toxicity testing, there will be a corresponding need to establish standardized testing conditions, endpoint analyses and acceptance criteria. In the future, a balanced approach between sample throughput and biological relevance should provide better in vitro tools that are complementary with animal testing and assist in conducting more predictive human risk assessment. [ABSTRACT FROM AUTHOR]

Metabolic diseases reach epidemic proportions. A better knowledge of the associated alterations in the metabolic pathways in the liver is necessary. These studies need in vitro human cell models. Several human hepatoma models are used, but the response of many metabolic pathways to physiological stimuli is often lost. Here, we characterize two human hepatocyte cell lines, IHH and HepaRG, by analysing the expression and regulation of genes involved in glucose and lipid metabolism. Our results show that the glycolysis pathway is activated by glucose and insulin in both lines. Gluconeogenesis gene expression is induced by forskolin in IHH cells and inhibited by insulin in both cell lines. The lipogenic pathway is regulated by insulin in IHH cells. Finally, both cell lines secrete apolipoprotein B-containing lipoproteins, an effect promoted by increasing glucose concentrations. These two human cell lines are thus interesting models to study the regulation of glucose and lipid metabolism. [ABSTRACT FROM AUTHOR]

Objective. The remarkable ability of liver to regenerate after insults has been harnessed by surgeons when designing techniques for liver resection or transplantation. However, the underlying mechanisms of liver regeneration are not fully clarified. On the other hand, aquaporins (AQPs) are small transmembrane proteins with unexpected physiological roles in addition to water transport. For example, they play pivotal roles in cell migration, angiogenesis, and cell proliferation, events that are also occurred during liver regeneration. We thus examined the possible involvement of AQPs in this regenerative process. Material and methods. A two-thirds partial hepatectomy (PH) rat model was employed. The temporal expression of various AQPs in the liver following PH was determined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. The localization of AQPs was evaluated by immunohistochemistry. Results. As anticipated, AQP0, 8, 9, and 11 were detected mainly in hepatocytes; unexpectedly, Kupffer cells were observed to express AQP8 during a specific period of time in the regenerative process. AQP9 protein was shown to be expressed in a progressively enhanced pattern at early time points after PH. A transient expression of AQP11 in the nucleus of hepatocytes was observed. Conclusion. These findings suggest the possibility that AQP might be involved in the PH-induced liver regeneration. [ABSTRACT FROM AUTHOR]

The ways, how to increase effectiveness of doxorubicin (DOX) in cancer cells and decrease its toxicity in normal cells, have been intensively studied. In breast cancer cells MCF-7, isoquinoline derivative oracin (ORC) inhibited DOX reduction and increased DOX antiproliferative effect. The aim of this study was to test the influence of ORC on the reduction of DOX and its toxicity in hepatocytes and non-tumourous breast cells. The kinetics of DOX reduction was measured in cytosols from rat liver, human liver and human mammary epithelial cells MCF-10A. Activity and expression of carbonyl reductase 1 (CBR1) were assayed using menadione as a substrate and western blot analysis. End-point tests of viability served for study of cytotoxicity of DOX, ORC and DOX+ORC combinations in rat hepatocytes and MCF-10A cells. The inhibitory effect of ORC on DOX reductases was almost none in MCF-10A cells and mild in liver. CBR1 expression and activity was lower in non-tumourous MCF-10A cells than in cancer MCF-7 cells. Cytotoxicity tests showed that DOX+ORC combinations had significantly lower toxicity than DOX alone in MCF-10A cells as well as in hepatocytes. ORC significantly decreases DOX toxicity in MCF-10A and in hepatocytes. Therefore, concomitant use of ORC and DOX may protect normal cells against DOX toxicity. [ABSTRACT FROM AUTHOR]

Context: Ursolic acid is a pentacyclic triterpenoid which has hepatoprotective and antihepatotoxic activities. Objective: This study investigated whether ursolic acid is able to stimulate liver regeneration in partially hepatectomized mice. Materials and methods: Ursolic acid or the vehicle solution was orally administered to the experimental, sham-operated and vehicle-treated group mice for 7 days, positive control animal (mice) was treated with recombinant human hepatocyte growth factor (rhHGF), and then the 70% liver partial hepatectomy was performed. The liver mass recovery rate was estimated by measuring the ratios of mice liver weight to body weight. The liver cells undergoing DNA synthesis were identified by immunohistochemistry analysis using monoclonal anti-BrdU antibodies. The expression levels of cyclin D1, cyclin E and C/EBP proteins (C/EBPα and C/EBPβ) were detected by the Western blotting technique. Results: Our results showed administration of ursolic acid significantly increased the ratio of the liver to body weight and BrdU labeling index at 36 and 48 h after partial hepatectomy, and the potency of UA is similar to rhHGF treated positive control mice. In addition, ursolic acid treatment significantly increased cyclin D1, cyclin E and C/EBPβ protein expression levels at 36 h after liver PHx compared with the vehicle-treated control mice. Discussion and conclusion: All these results suggest that ursolic acid stimulates liver proliferation after partial hepatectomy, and this effect may be associated with the stimulation of C/EBPβ expression. [ABSTRACT FROM AUTHOR]

Liver parenchymal cells are an important target for the treatment of several metabolic and viral disorders. Corrective gene delivery for this purpose is an avenue that is receiving increasing attention. In the present study, we report a novel neo glycolipid that may be formulated into cationic liposomes with or without poly(ethylene glycol) decoration. Lipoplexes formed with plasmid DNA are nuclease resistant and are targeted to the human hepatoblastoma cell line HepG2 by selective asialoglycoprotein receptor mediation. Transfection levels achieved by lipoplexes containing the targeting ligand cholesteryl-3β-N-(4-aminophenyl-β-D-galactopyranosyl) carbamate were sixfold greater than those obtained with similar but untargeted lipoplexes. [ABSTRACT FROM PUBLISHER]

Although some drugs cause drug-induced liver injury (DILI) through direct damage to hepatocytes or intereference with bile secretion, others cause delayed, often idiosyncratic, DILI with clinical features, such as mild lymphocytic infiltrate, that are reminiscent of allergic reactions involving activation of the adaptive immune system. Even in cases of direct drug-induced hepatotoxicity, infiltration of inflammatory cells into the liver is often observed, suggesting a role for the innate immune system (e.g., neutrophils, macrophages, and so on). Therefore, a variety of hypotheses for the pathogenesis of DILI center around a pathogenic role of drug- (or drug-metabolite-) specific adaptive immune cells, as well as hepatic-injury-induced innate immune responses in the development, progression, and/or resolution of DILI. [ABSTRACT FROM AUTHOR]

Our aim was to examine the relation of serum osteocalcin (OCN) levels with the clinical, biochemical, and histological characteristics of patients with biopsy-proven nonalcoholic fatty liver disease (NAFLD). We carried out a case-control study including 99 patients with biopsy-proven NAFLD and 75 age- and sex-matched controls. Concentrations of OCN were measured in aprotinin-treated serum samples using a solid-phase enzyme amplified sensitivity immunoassay. Serum OCN levels were significantly lower in patients with NAFLD than in healthy controls. In patients with NAFLD, serum OCN levels were inversely associated with ALT ( r ==−0.36, p < 0.001), AST ( r ==−0.39, p < 0.001), HOMA-IR ( r ==−0.30, p < 0.01) and the degree of hepatocyte ballooning ( r ==−0.20, p < 0.05). Serum OCN was the only independent predictor of the degree of hepatocyte ballooning in NAFLD patients (β ==−0.24; t == −2.146, p < 0.05). Compared with controls, NAFLD patients have a decrease in serum OCN concentrations, which is significantly associated with serum transaminases and the extent of hepatocyte ballooning. [ABSTRACT FROM AUTHOR]

Aflatoxins are one of the most widespread and worrisome sources of feed contamination worldwide, and have a considerable impact on fish farm production, leading to high mortality and a gradual decline in fish stock quality in aquaculture. In this study, we investigated, for the first time, the effects induced in vitro by aflatoxin B1 (AFB1) on Sparus aurata hepatocyte culture and we compared our results with the Microtox® system using Vibrio fischeri. At AFB1 doses ranging from 1 to 10 μg·mL−1, the results showed signs of primary necrotic cell death in hepatocytes and a very toxic evaluation with Microtox®; between 0.005 and 1 μg·mL−1, the cytotoxic effects and apoptotic delayed death in eukaryotic cells corresponded with an evaluation of no toxicity or biostimulatory effect using V. fischeri. Overall, our results highlighted equivalent toxic responses and overlapped with values of EC50/IC50. Hence, these two in vitro systems could be considered as a useful starting point in the design of new batteries to evaluate the toxicity of potentially dangerous feed-borne substances. [ABSTRACT FROM PUBLISHER]

LY377604 has a potential to form 4-hydroxycarbazole, which was reported in the literature as a mutagen. This safety concern led to our investigation of the metabolism and carcinogenicity of LY377604. In in vitro studies with LY377604, 4-hydroxycarbazole was detected in the presence of liver microsomes prepared from different species. When incubated with liver slices, only the conjugate of 4-hydroxycarbazole was detected. Subsequent in vivo radio-labelled studies were conducted to characterise the formation of 4-hydroxycarbazole from LY377604. Free 4-hydroxycarbazole was not detected in vivo, but the O-glucuronide conjugate was identified as a minor metabolite in urine samples, representing 0.2% and 0.9% of the radioactive dose in rats and monkeys. The low level of circulating 4-hydroxycarbazole glucuronide conjugate was also detected in plasma. LY377604 was negative in all genetic toxicology assays and was not associated with tumour induction in a 6-month carcinogenicity study using RasH2+/− mouse model. The exposure to free 4-hydroxycarbazole was not measurable after one dose and was about 0.1%−0.2% of the parent exposure at the end of the 6-month study. These data suggested that 4-hydroxycarbazole was formed as a minor metabolite in vivo, but it was primarily conjugated and excreted in urine as the glucuronide conjugate. The absence of tumours in the carcinogenicity study combined with the exposure data suggested that the low level of free 4-hydroxycarbazole did not represent a carcinogenic risk. [ABSTRACT FROM AUTHOR]

Objective. In the industrialized world, obesity is an increasing socioeconomic health problem. Obese subjects have a higher risk of developing several types of cancer. NK cells are an integral component of the innate immune system, able to destruct tumor cells. The adipokine leptin plays a crucial role in the development of obesity and its related diseases. Peripheral leptin signaling is modulated by the liver. Methods. The aim of this study was to evaluate the number of hepatic NK cells (CD56++) and the number of leptin-receptor positive (Ob-R++) cells in the livers of five normal-weight and five obese humans. Livers were removed during autopsy and accurately defined sections were stained immunohistochemically and CD56++, Ob-R++, and double-positive cells were quantified. Results. Results revealed a dramatic reduction of NK cells and Ob-R-expressing NK cells in the livers of obese individuals. Conclusions. The present study demonstrates, for the first time, body-weight-dependent numbers of hepatic NK cells. This supports the hypothesis of obesity-associated alterations of immune cell numbers in different human organs. [ABSTRACT FROM AUTHOR]

This article describes hepatocyte metabolism mathematical model (HEMETβ), which is an improved version of HEMET, an effective and versatile virtual cell model based on hepatic cell metabolism. HEMET is based on a set of non-linear differential equations, implemented in Simulink®, which describes the biochemical reactions and energetic cell state, and completely mimics the principal metabolic pathways in hepatic cells. The cell energy function and modular structure are the core of this model. HEMETβ as HEMET model describes hepatic cellular metabolism in standard conditions (cell culture in a plastic multi-well placed in an incubator at 37°C with 5% of CO2) and with excess substrates concentration. The main improvements in HEMETβ are the introductions of Michaelis–Menten models for reversible reactions and enzymatic inhibition. In addition, we eliminated hard non-linearities and modelled cell proliferation and every single aminoacid degradation pathway. All these innovations, combined with a user-friendly aspect, allow researchers to create new cell types and validate new experimental protocols just varying ‘peripheral’ pathways or model inputs. [ABSTRACT FROM PUBLISHER]

Bisphenol F (BPF) is present in the environment and as a contaminant of food. Humans may, therefore, be exposed to BPF, and an assessment of this risk is required. BPF has been shown to have genotoxic and endocrine-disruptor properties in a human hepatoma cell line (HepG2), which is a model system for studies of xenobiotic toxicity. In this study, we investigated the ability of HepG2 cells to biotransform BPF, because metabolism may affect the observed effects of BPF, and we compared this metabolic capacity with that of human hepatocytes. Cells were incubated for 24 hours with [3H]-BPF. The culture medium was then concentrated and its metabolites were isolated by high-performance liquid chromatography and identified by mass spectrometry. BPF was largely metabolized into the corresponding sulfate by the HepG2 cell line. BPF was metabolized into both sulfate and glucuronide by human hepatocytes, but with differences between individuals. The metabolism of BPF in both HepG2 cells and human hepatocytes suggests the existence of a detoxification pathway. Thus, these two cell models differ in metabolic capacity. It is, therefore, very important, when assessing the toxic effects of substances in vitro, to determine, in parallel, the biotransformation capacities of the model used to extrapolate in vivo. [ABSTRACT FROM AUTHOR]

In this study, NOH (NOH == N-octadecyl-4-[(D-galactopyranosyl)oxy]-2,3,5,6-tetrahydroxy hexanamide) was enzymatically synthesized as a targeting molecule and incorporated into liposomes to prepare a liposome surface modified with galactose. Glycyrrhetinic-acid--loaded liposome (GA-LP) and glycyrrhetinic-acid--loaded liposome surface modified with galactose (NOH-GA-LP) were prepared by the ethanol-injection method. NOH-GA-LP was characterized by morphology, particle size, zeta potential, encapsulation efficiency, release in vitro, and stability. The size of spherical particles was in the range of 179--211 nm. Spherical particles exhibit a positive electrical charge (38.7 mV) and possess high encapsulation efficiency (91.3%) and show sustained release (72% over 48 hours) in vitro. This novel approach for the liposome surface modified with galactose by enzymatic synthesis is expected to provide potential application as a drug carrier for active targeted delivery to hepatocytes. [ABSTRACT FROM AUTHOR]

The effects of demethylbellidifolin (DMB), a xanthone compound extracted from Swertia davidi Franch, on activation of hepatic stellate cells (HSC) were investigated. Rat HSC line HSC-T6 cultured in vitro showed an activated status, which expressed αα-smooth muscle actin (αα-SMA) and synthesized collagen I. Treated with different concentrations (10, 20, or 40 μμM) of DMB for 12 --48 hr could markedly inhibit cell proliferation reflected by MTT and [[3H]]thymidine incorporation assays, and downregulate the expressions of both αα-SMA and collagen I in HSC-T6 cells. Also, such treatment concentration-dependently downregulated both mRNA and protein expressions of transforming growth factor-ββ1 (TGF-ββ1) and connective tissue growth factor (CTGF). The expression of peroxisome proliferator-activated receptor-γγ (PPAR-γγ) was very low in activated HSC-T6, which could be significantly upregulated by treatment with DMB. Furthermore, PPAR-γγ antagonist PD68235 (5--20 μμM) markedly blocked these effects of DMB mentioned above. In summary, DMB inhibits HSC proliferation and activation, which may be related to activating PPAR-γγ-mediated pathway. [ABSTRACT FROM AUTHOR]

The pathophysiology of hepatic ischemia/reperfusion injury (IRI) includes a number of complex and diverse mechanisms involving interactions between hepatocytes, Kupffer cells, neutrophils, macrophages, sinusoidal endothelial cells, and platelets. Tumor Necrosis Factor-αα (TNF-αα), a cytokine produced by numerous cell types in response to inflammatory stimuli, is a well-known mediator during ischemia/reperfusion (IR) that plays a central role in injury to hepatocytes. TNF-αα has a multifactorial effect in hepatic IRI because of the many interactions between TNF-αα and reactive oxygen species, nitric oxide, adhesion molecules, and various cytokines and chemokines. The intracellular cascades that TNF-αα triggers and their downstream effects are clearly presented throughout this manuscript. With these mechanisms in mind, ischemic preconditioning and pharmacological interventions with potential clinical application to prevent or attenuate IRI will be emphasized. [ABSTRACT FROM AUTHOR]

Chemokines are a family of small heparin-binding proteins that orchestrate the infiltration of immune cells into the liver, but also directly influence the biology of resident liver cells. A nonredundant role of different chemokines and their receptors has been identified within the last years in animal models of acute and chronic liver diseases. Chemokine receptors that have been shown to play an import pathophysiological role in the liver are CCR1, CCR2, CCR5, D6, CXCR2 and CXCR3. Interestingly, most of these receptors have already been targeted by specific antagonists in early human trials and a CCR5 antagonist is already licensed for use in HIV infection. Most of these trials have been performed in autoimmune and infectious human diseases, but no controlled clinical trials have yet been performed in patients with liver diseases. Nevertheless, in light of growing evidence that chemokines are important mediators of liver damage, these trials seem to be on the clinical horizon. [ABSTRACT FROM AUTHOR]

We investigated diet supplementation with shiitake mushroom fruiting bodies on biochemical and histological changes in hypercholesterolemic rats. Six-wk old female Sprague-Dawley albino rats were divided into three groups of 10 rats each. A diet containing 5% Lentinus edodes fruiting bodies given to hypercholesterolemic rats reduced plasma total cholesterol, triglyceride, low-density lipoprotein (LDL), total lipid, phospholipids, and the LDL/high-density lipoprotein ratio by 34.33, 53.21, 75.00, 34.66, 25.73, and 71.43%, respectively. Feeding mushroom also significantly reduced body weight in hypercholesterolemic rats. However, it had no detrimental effects on plasma albumin, total bilirubin, direct bilirubin, creatinine, blood urea nitrogen, uric acid, glucose, total protein, calcium, sodium, potassium, chloride, inorganic phosphate, magnesium, or enzyme profiles. Feeding mushroom increased total lipid and cholesterol excretion in feces. The plasma lipoprotein fraction, separated by agarose gel electrophoresis, indicated that L. edodes significantly reduced plasma β and pre-β-lipoprotein but increased α-lipoprotein. A histological study of hepatic cells by conventional hematoxylin-eosin and oil red-O staining showed normal findings for mushroom-fed hypercholesterolemic rats. These results suggest that shiitake mushrooms could be recommended as a natural cholesterol lowering substance in the diet. [ABSTRACT FROM AUTHOR]

The advantages of MRI in the investigation of liver disease are well documented. Recent developments, including fast scanning technique and new MRI contrast agents, enable improved detection and characterization of focal liver lesions. Therefore, a definitive diagnosis can be made avoiding invasive procedures, such as liver biopsy. In this article, a special emphasis is placed on the clinical use of combined perfusional and hepatocyte-selective MRI contrast agents, which allow us to obtain morphologic and vascular information, owing to the dynamic study, as well as functional information, owing to the hepatocyte-selective phase of enhancement. Different clinical scenarios are considered in order to highlight the proper use of the hepatocyte phase to noninvasively characterize and detect different focal liver lesions. [ABSTRACT FROM AUTHOR]

The purpose of this study was to assess the impact of interferon-α2b (IFN-α2b) on the expression of various drug-metabolizing enzymes and transporters in freshly prepared co-cultures (parenchymal and non-parenchymal cells) of human primary hepatocytes. At therapeutically relevant concentrations (from 1000 to 3000 IU/mL), IFN-α2b up-regulated STAT1 (signal transducer and activator of transcription factor 1) mRNA expression. Conversely, three cytochrome P450s (CYP1A2, CYP2B6, CYP2E1), a UDP-glucuronosyltransferase (UGT2B7), a sulphotransferase (SULT1A1) and organic anion transporter (OAT2) were significantly down-regulated (~50%; P < 0.05). Western blot analysis of CYP1A2, UGT2B7 and OAT2 protein supported the mRNA data. Two peroxisome proliferator activator receptor alpha (PPARα)-controlled genes (pyruvate dehydrogenase kinase 4 and adipose differentiation-related protein), CYP3A4 and multidrug resistance-associated protein 2 were significantly up-regulated (up to 223%; P < 0.05). On the other hand, SULT2A1, carboxylesterase 2, organic anion transporting peptide (OATP1B1, OATP1B3, OATP2B1), organic cation transporter 1, P-glycoprotein and breast cancer resistance protein mRNA expression was not significantly affected. Western blot analysis of CYP3A4 supported the mRNA data also. The present results demonstrated complex interactions between IFN-α2b and hepatocytes and the observed down-regulation of CYP1A2, OAT2 and UGT2B7 is consistent with reports of drug interactions between IFN-α2b and drugs such as theophylline, clozapine and gemfibrozil. [ABSTRACT FROM AUTHOR]

The aim of this research was to study the application and effectiveness of Enzyme Linked Immuno Receptor Assay (ELIRA) method for understanding the bioactivity of human Growth Hormone (hGH) in micro-titer plates. For this purpose, rabbit hepatocyte microsomes which contained hGH receptors were used for coating of ELISA micro-titer plates. Then hGH was interacted with coated receptors. Fractions of bounded complexes were identified by antibodies in an Enzyme-based substrate detection system. Different assay conditions such as: buffers, blocking agents, temperatures and times of incubation were analyzed. Our result indicated that, the carbonate coating buffer was not effective in receptor coating in ELIRA. Overnight incubation of hGH and hGH receptors in HEPES assay buffer and BSA blocking resulted in the lower linearity and correlations (R2 == 0.46 to 0.85). However, 3 h incubation in Tris-HCl assay buffer at 30°°C resulted in higher linearity and correlations (R2 == 0.95 to 0.97). Finally, the coating of microwells by 250 μμg//ml of microsome membranes in Tris buffer at 30°°C for 3 hr and blocking by skim milk resulted to the best linearity and higher correlation, (R2 == 0.985) and lower detection limit about 2 ng//ml of bioactive hGH. [ABSTRACT FROM AUTHOR]

Hepatic lipogenesis-induced de novo by glucocorticoids (GCs) is associated with the development of obesity and diabetes mellitus. The interaction of GCs and insulin in the regulation of hepatic lipogenesis remains unclear. The effect of exogenous GC administration on hepatic lipogenesis and fat deposition was studied in broiler chickens ( Gallus gallus domesticus), and the role of insulin in the effect of GCs on hepatic lipogenesis was evaluated. Dexamethasone (DEX, 2  mg/kg body mass (BM)) administration for 3-d resulted in BM loss and increased liver and cervical adipose tissue mass compared to control and pair-fed counterparts. DEX treatment significantly ( P  <  0.05) increased plasma level of insulin in either the fed or fasting state, whereas plasma glucose level was only increased in the fed state. In fasted chickens, DEX treatment significantly ( P  <  0.01) upregulated the hepatic mRNA levels of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). In the fed state, the mRNA levels of ACC and FAS were not significantly influenced by DEX treatment, nor was FAS activity. In cultured primary hepatocytes, combined DEX and insulin significantly upregulated the transcription of the genes for FAS (1.34-fold) and malic enzyme (1.72-fold). By contrast, the expression of sterol response element-binding protein-1 (SREBP-1) was significantly upregulated by insulin (1.67-fold) regardless of DEX. In abdominal adipose tissue, DEX treatment had no significant ( P>0.05) effect on the activities and transcription of FAS. The expressions of lipoprotein lipase and peroxisome proliferator-activated receptor-γγ were not significantly ( P>0.05) affected by DEX treatment in either the fasting or fed state. The results indicate that DEX increased hepatic de novo lipogenesis via the increased activity and expression of lipogenic enzymes. Insulin-activated gene expression for SREBP-1 is suggested to be involved in stress-augmented hepatic lipogenesis. [ABSTRACT FROM AUTHOR]

Background. Both non-alcoholic steatohepatitis (NASH) and polycystic ovary syndrome (PCOS) are associated with metabolic syndrome (MS) and insulin resistance (IR). Except for a few case reports, there are no systematic assessments of NASH in PCOS patients. Aim. To determine the prevalence of NASH and independent factors associated with NASH in a cohort of patients with documented PCOS. Methods. Patients with established diagnosis of PCOS and matched controls (matched for gender, age, and body mass index (BMI)) were included in the study. Causes of other liver diseases were systematically excluded by clinical and laboratory tests. Excessive alcohol use was defined as alcohol consumption of greater than 10 g/day. All liver biopsies were read by a single pathologist blinded to the clinical data. Histologic NASH was defined as steatosis with lobular inflammation and ballooning degeneration of hepatocytes with or without Mallory--Denk bodies or pericellular fibrosis. Univariate and multivariate analyses with logistic regression were performed to compare PCOS to matched controls. Results. Sixty-six patients were included in the study (34 PCOS and 32 matched controls). Of PCOS patients, 73% had a liver biopsy while 78% of the matched controls had a liver biopsy. In comparing PCOS patients to the matched controls, clinical (BMI, waist circumference, type 2 diabetes, MS, or its components, any alcohol consumption in the prior year, ethnic background, age, gender, etc.) and laboratory data (aminotransferases, ferritin, glucose, etc.) were not significantly different ( p > 0.05). However, PCOS patients tended to have more histologic NASH on their liver biopsies (44.0% vs. 20.8%, p == 0.08). Independent predictors of histologic NASH in PCOS patients were elevated aspartate aminotransferase (AST), high triglycerides and small amounts of alcohol consumption ( p == 0.019, 10-fold cross-validated AUC == 0.80, 95% CI == 0.56--0.94). Although about half of PCOS patients did not report any alcohol consumption, 50% did report rare alcohol use. In fact, PCOS patients with histologic NASH tended to report higher alcohol consumption per week than PCOS without NASH (3.80 ±± 6.16 vs. 1.11 ±± 1.87 g/week, p == 0.1). Nevertheless, these amounts of alcohol consumption were quite minimal. Conclusions. Despite similar clinical and laboratory profiles to the matched controls, PCOS patients seem to have more histologic NASH. Although alcohol consumption was rare for both PCOS and controls, even rare alcohol consumption in PCOS patients was independently associated with histologic NASH. [ABSTRACT FROM AUTHOR]

Cell cycle progression is dependent on the intracellular iron level and chelators can lead to iron depletion and decrease cell proliferation. This antiproliferative effect can be inhibited by exogenous iron. In this work, we present the synthesis of some new synthetic calix[4]arene podands bearing diamino-tetraesters, diamino-tetraalcohols, diamino-tetraacid and tetraaryloxypentoxy groups at the lower rim, designed as potential iron chelators. We report their effect on cell proliferation, in comparison with the new oral chelator ICL670A (4-[3,5-bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid). The antiproliferative effect of these new compounds was studied in the human hepatocarcinoma HepaRG cell cultures using cell nuclei counting after staining with the DNA intercalating fluorescence dye, Hoechst 33342. Their cytotoxicity was evaluated by the extracellular LDH activity. Preliminary results indicated that their antiproliferative effect was mainly due to their cytotoxicity. The efficiency of these compounds, being comparable to that of ICL670, was independent of iron depletion. This effect remains to be further explored. Moreover, it also shows that the new substituted calix[4]arenes could open the way to valuable new approaches for medicinal chemistry scaffolding. [ABSTRACT FROM AUTHOR]

Objective: Punarnavashtak kwath (PNK) is a classical Ayurvedic formulation, mentioned in Ayurvedic literature Bhaishajya Ratnavali, for hepatic disorders and asthma. This study investigated the hepatoprotective activity of PNK to validate the traditional use of this formulation. Materials and methods: PNK was prepared in the laboratory according to the method given in Ayurvedic literature. Phytochemical screening was performed to determine the presence of phytoconstituents. Hepatoprotective activity was evaluated against CCl4-induced hepatotoxicity in rats and by its effect on the HepG2 cell line. Results: Preliminary phytochemical screening revealed the presence of alkaloids, tannins, flavonoids, saponins, and a bitter principle in PNK. Administration of PNK produced significant hepatoprotective effect as demonstrated by decreased levels of serum liver marker enzymes such as aspartate transaminase, serum alanine transaminase, serum alkaline phosphatase, and serum bilirubin and an increase in protein level. Thiopentone-induced sleeping time was also decreased in the PNK-treated animals compared with the CCl4-treated group. It also showed antioxidant activity by increase in activity of glutathione, superoxide dismutase, and catalase and by a decrease in thiobarbituric acid reactive substance level compared with the CCl4-treated group. Results of a histopathological study also support the hepatoprotective activity of PNK. Investigation carried out on the HepG2 cell line depicted significant increase in viability of cells exposed to PNK as compared with CCl4-treated cells. Discussion and Conclusion: It can be concluded that PNK protects hepatocytes from CCl4-induced liver damages due to its antioxidant effect on hepatocytes. An in vitro study on HepG2 cell lines also supports its protective effect. [ABSTRACT FROM AUTHOR]

In the present study we analyzed VEGF-A expression in adult male and female C3H/S mice along a circadian time span. The animals were sacrificed at 00:00, 04:00, 08:00, 12:00, 16:00 and 20:00 h. Samples of the liver from each animal were processed for immunohistochemistry. The VEGF expression index was calculated as positives immunostaining hepatocytes x 100 and analyzed by ANOVA, and the Student-Newman-Keuls Multiple Comparison Test. The results show that in both male and female mice there were significant differences between the values corresponding to all the time points studied. In male mice the values of VEGF expression of 20:00 h are significantly higher than all the others analyzed while in females the value of the 20:00 h was significantly different from the value of the 04:00 h. In conclusion, the hepatocytes of the perivenular areas in the liver of intact adult male and female mice expressed VEGF with a defined circadian rhythm. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) is an important complication of the metabolic syndrome. The increasing prevalence of the metabolic syndrome is paralleled by an increasing prevalence of NAFLD, which has become one of the most common chronic liver diseases. NAFLD comprises a morphological spectrum ranging from nonalcoholic fatty liver (NAFL), characterized by accumulation of fat in hepatocytes, to nonalcoholic steatohepatitis (NASH). The key histological features of NASH accepted by most pathologists include steatosis, hepatocellular ballooning and lobular inflammation, whereas, like in other chronic liver diseases, the presence of fibrosis is not considered a requirement for the diagnosis. The diagnosis of NASH and the distinction from NAFL carries important prognostic and therapeutic implications because NASH, in contrast to NAFL, is associated with an increased risk of progression to cirrhosis and hepatocellular carcinoma. Hepatocellular ballooning is a key feature required for the diagnosis of NASH and a component of currently used histological grading and staging systems of NAFLD. However, it represents an ill-deined form of liver cell injury associated with cell swelling and rounding of the cytoplasm, the detection of which is prone to intra- as well as inter-observer variation. Some of the factors that may contribute to ballooning are the rearrangement of the intermediate filament cytoskeleton, accumulation of small-droplet fat in the cytoplasm and dilation of the endoplasmic reticulum. The rearrangement of the intermediate filament cytoskeleton can be demonstrated by the loss of keratin 8/18 immunostaining of the cytoplasm, and may thus be evaluated in the future as a marker for the more objective detection of hepatocellular ballooning in NASH. [ABSTRACT FROM AUTHOR]

This study examined the effects of (−−)schisandrin B [(−−)Sch B] on MAPK and Nrf2 activation and the subsequent induction of glutathione antioxidant response and cytoprotection against apoptosis in AML12 hepatocytes. Pharmacological tools, such as cytochrome P-450 (CYP) inhibitor, antioxidant, MAPK inhibitors and Nrf2 RNAi, were used to delineate the signalling pathway. (−−)Sch B caused a time-dependent activation of MAPK in AML12 cells, particularly the ERK1/2. The MAPK activation was followed by an enhancement in Nrf2 nuclear translocation and the eliciting of a glutathione antioxidant response. Reactive oxygen species arising from a CYP-catalysed reaction with (−−)Sch B seemed to be causally related to the activation of MAPK and Nrf2. ERK inhibition by U0126 or Nrf2 suppression by Nrf2 RNAi transfection almost completely abrogated the cytoprotection against menadione-induced apoptosis in (−−)Sch B-pre-treated cells. (−−)Sch B pre-treatment potentiated the menadione-induced ERK activation, whereas both p38 and JNK activations were suppressed. Under the condition of ERK inhibition, Sch B treatment did not protect against carbon tetrachloride-hepatotoxicity in an in vivo mouse model. In conclusion, (−−)Sch B triggers a redox-sensitive ERK/Nrf2 signalling, which then elicits a cellular glutathione antioxidant response and protects against oxidant-induced apoptosis in AML12 cells. [ABSTRACT FROM AUTHOR]

Here we report the use of natural, chemically-unmodified, microfibrillated cellulose (MFC) as a matrix for hepatocyte culture. We developed an original cell-culture design composed of a thin 3D-microstructured fibrous substrate consisting of a MFC sheet coating a highly O2-permeable polydimethylsiloxane (PDMS) membrane. The MFC-coated PDMS membranes were obtained according to a simple process where cellulose fibres were deposited from an aqueous suspension on the PDMS surfaces and the films were dried under mild conditions. To enable oxygen diffusion through the membranes, they were assembled on bottomless frames ('O2+' condition). Rat hepatocytes primary-cultured on such MFC-PDMS membranes quickly organized themselves into large hemispherical 3D aggregates which were tightly anchored to the MFC sheets. In contrast, hepatocytes cultured on smooth PDMS membranes in the O2+ system (O2+, PDMS) organized into unstable 2D monolayers which easily detached from the surfaces. Hepatocyte 3D cultures obtained on MFC-PDMS membranes exhibited higher liver-specific functions over a 2-week culture period, as assessed by both the higher albumin secretion and urea synthesis rate. The MFC-PDMS membranes appear suitable for obtaining stably-attached and functional hepatocyte 3D cultures and appear interesting for drug/chemical screenings in a microplate format, but also for microfluidic applications. [ABSTRACT FROM AUTHOR]

&agr;1-antitrypsin deficiency is an autosomal recessive disorder that results from point mutations in the SERPINA1 gene. The Z mutation (Glu342Lys) accounts for the majority of cases of severe &agr;1-antitrypsin deficiency. It causes the protein to misfold into ordered polymers that accumulate within the endoplasmic reticulum of hepatocytes. It is these polymers that form the periodic acid Schiff positive inclusions that are characteristic of this condition. These inclusions are associated with neonatal hepatitis, cirrhosis and hepatocellular carcinoma. The lack of circulating &agr;1-antitrypsin exposes the lungs to uncontrolled proteolytic attack and so can predispose the Z &agr;1-antitrypsin homozygote to early-onset emphysema. &agr;1-antitrypsin polymers can also form in extracellular tissues where they activate and sustain inflammatory cascades. This may provide an explanation for both progressive emphysema in individuals who receive adequate replacement therapy and the selective advantage associated with &agr;1-antitrypsin deficiency. Therapeutic strategies are now being developed to block the aberrant conformational transitions of mutant &agr;1-antitrypsin and so treat the associated disease. INSET: Key issues. [ABSTRACT FROM AUTHOR]

Acetaminophen (APAP) hepatotoxicity is the main cause of acute liver failure in humans. Although mitochondrial oxidant stress and induction of the mitochondrial permeability transition (MPT) have been implicated in APAP-induced hepatotoxicity, the link between these events is unclear. To investigate this, this study evaluated APAP hepatotoxicity in mice deficient of cyclophilin D, a protein component of the MPT. Treatment of wild type mice with APAP resulted in focal centrilobular necrosis, nuclear DNA fragmentation and formation of reactive oxygen (elevated glutathione disulphide levels) and peroxynitrite (nitrotyrosine immunostaining) in the liver. CypD-deficient (Ppif−/−) mice were completely protected against APAP-induced liver injury and DNA fragmentation. Oxidant stress and peroxynitrite formation were blunted but not eliminated in CypD-deficient mice. Thus, mitochondrial oxidative stress and induction of the MPT are critical events in APAP hepatotoxicity in vivo and at least part of the APAP-induced oxidant stress and peroxynitrite formation occurs downstream of the MPT. [ABSTRACT FROM AUTHOR]

Objective. The novel adipokines omentin, chemerin, and adipsin are associated with insulin resistance and the components of the metabolic syndrome. We assayed circulating levels of these molecules and examined their association with clinical, biochemical, and histological phenotypes in patients with nonalcoholic fatty liver disease (NAFLD). Material and methods. Serum levels of omentin, chemerin, and adipsin were assayed by enzyme-linked immunosorbent assay in 99 patients with biopsy-proven NAFLD and 75 control subjects. We analyzed associations between adipokines and the characteristics of patients with NAFLD using multivariable linear regression models. Results. Adipsin levels did not differ between patients and controls, whereas both omentin and chemerin levels were significantly higher in patients with biopsy-proven NAFLD than in controls (both p values <0.001). Serum omentin levels were significantly associated with C-reactive protein (r= 0.29, p < 0.01) and the degree of hepatocyte ballooning (r= 0.27, p < 0.01), whereas chemerin showed a modest association with liver fibrosis (r = 0.22, p = 0.04). After stepwise linear regression analysis adjusting for potential confounders, serum omentin levels retained their independent significance as a predictor of hepatocyte ballooning in patients with NAFLD (β = 1.42; t- 2.79, p < 0.01). Conclusions. Our results suggest that serum omentin levels are raised in patients with NAFLD regardless of potential confounders and represent an independent predictor of hepatocyte ballooning. [ABSTRACT FROM AUTHOR]

Massive application of pesticides had generated a considerable concern in the public. Potentials of chlorpyrifos [ O,O-diethyl- O-(3,5,6-trichloro-2-pyridinyl) phosphorothionate] and cypermethrin [( RS)- α-cyano-3-phenoxybenzyl (1 RS)- cis- trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate] to induce the excision-repairable DNA damage, DNA strand breakage, and DNA hypomethylation in ICR mouse hepatocytes were investigated. It was showed that chlorpyrifos and cypermethrin didn't increase the incorporation of 3H-TdR into DNA of ICR mouse hepatocytes but increased the frequency of comet cells and decreased the 5MeC percentage of ICR mouse hepatocytes. In conclusion, chlorpyrifos and cypermethrin induced no excision-repairable DNA damage but led to DNA strand breakage and DNA hypomethylation in ICR mouse hepatocytes. [ABSTRACT FROM AUTHOR]

Excessive flux of free fatty acids (FFA) into the liver contributes to liver impairment in non-alcoholic fatty liver disease (NAFLD). It remains unclear how FFA contribute to impairment of hepatocytes. This study treated hepatocytes with linoleic acid and palmitate to investigate the early event triggering FFA-mediated impairment. It determined cell viability, content of nitrite/nitrate and triacylglycerides (TG), inducible nitric oxide synthase (iNOS) protein, oxidation of cardiolipin (CL) as well as formation of F2-isoprostanes in the presence of insulin and glucose. Linoleic acid caused significant decrease in cell viability. It is shown that palmitate caused induction of iNOS resulting in increased nitrite/nitrate concentration and slight increase in TG content. Linoleic acid led to a decrease in nitrite/nitrate concentration parallelled by massive TG accumulation in combination with increased oxidation of CL and increased F2-isoprostane levels. It is concluded that nitric oxide (NO) concentration regulates FFA-dependent TG accumulation and oxidative stress in rat hepatocytes. [ABSTRACT FROM AUTHOR]

Inferior vena cava (IVC) obstruction is uncommon in children. We report a patient with liver within a IVC extending to the right atrium who underwent successful surgical resection. A 12-year-old boy with an Arnold Chiari malformation was admitted for seizures. Premature ventricular contractions prompted an echocardiogram. This revealed a pedunculated mass in the right atrium and an IVC producing turbulent flow. He underwent a mass excision that was continuous with the liver. Histology demonstrated normal liver parenchyma. Based on the embryologic intimacy between the caudate lobe and the IVC, we postulate that the ectopic hepatic nodule was due to aberrant migration of hepatocytes into the IVC during embryogenesis. [ABSTRACT FROM AUTHOR]

Recently the authors have reported the potent beneficial effect of caffeic acid phenethyl ester (CAPE) on cholestatic oxidative liver injury induced by acute bile ligation in Swiss albino rats. Herein, they report the ultrastructural hepatocellular alterations induced by acute bile duct ligation and the effect of CAPE administration on these alterations. Bile duct ligation resulted in many degenerative changes, such as vacuolization, mitochondrial degeneration, endoplasmic reticulum dilatation, and lysosome accumulation within the cytoplasm of hepatocytes. Mitochondrial degeneration was also observed within the cytoplasm of the cells of biliary ductular epithelium. CAPE potentially protected the hepatocytes from the cholestasis-induced cellular injury. [ABSTRACT FROM AUTHOR]

1. We compare three different approaches to scale clearance (CL) from human hepatocyte and microsome CLint (intrinsic CL) for 52 drug compounds. 2. By using the well-stirred model with protein binding included only 11% and 30% of the compounds were predicted within 2-fold and the average absolute fold errors (AAFE) for the predictions were 5.9 and 4.1 for hepatocytes and microsomes, respectively. 3. When predictions were performed without protein binding, 59% of the compounds were predicted within 2-fold using either hepatocytes or microsomes and the AAFE was 2.2 and 2.3, respectively. 4. For hepatocytes and microsomes there were significant correlations (P = 8.7 × 10-13, R² = 0.72; P = 2.8 × 10-9, R² = 0.61) between predicted CLint in vivo (obtained from in vitro CLint) and measured CLint in vivo (obtained using the well-stirred model). When CL was calculated from the regression, 76% and 70% of the compounds were predicted within 2-fold and the AAFE was 1.6 and 1.8 for hepatocytes and microsomes, respectively. We demonstrate that microsomes and hepatocytes are in many cases comparable when scaling of CL is performed from regression. 5. By using the hepatocyte regression, CL for 82% of the compounds in an independent test set (n = 11) were predicted within 2-fold (AAFE 1.4). We suggest that a regression line that adjusts for systematic under-predictions should be the first-hand choice for scaling of CL. [ABSTRACT FROM AUTHOR]

ABCC2/Abcc2 (MRP2/Mrp2) is expressed at major physiological barriers, such as the canalicular membrane of liver cells, kidney proximal tubule epithelial cells, enterocytes of the small and large intestine, and syncytiotrophoblast of the placenta. ABCC2/Abcc2 always localizes in the apical membranes. Although ABCC2/Abcc2 transports a variety of amphiphilic anions that belong to different classes of molecules, such as endogenous compounds (e.g., bilirubin-glucuronides), drugs, toxic chemicals, nutraceuticals, and their conjugates, it displays a preference for phase II conjugates. Phenotypically, the most obvious consequence of mutations in ABCC2 that lead to Dubin-Johnson syndrome is conjugate hyperbilirubinemia. ABCC2/Abcc2 harbors multiple binding sites and displays complex transport kinetics. [ABSTRACT FROM AUTHOR]

Hepatocytes express various transporters in the sinusoidal and canalicular membrane, which mediate hepatic uptake and canalicular efflux, forming directional transport from the sinusoid to the bile. Drug-drug interactions and genetic polymorphisms of the transporters are known to cause variations in transporter function. This review focuses on pharmacokinetic modeling of hepatobiliary transport of drugs to explain the alteration of the disposition of drugs caused by such variations, based on the clearance concept. For modeling and simulation, pravastatin and dibromosulfophthalein have been used as model compounds which are known to undergo transpoter-mediated hepatic uptake followed by biliary excretion. Pharmacokinetic modeling of hepatobiliary transport illustrates the concept of the rate-determining process in overall hepatobiliary transport. [ABSTRACT FROM AUTHOR]

Small hepatocytes (SHs) are “committed progenitor cells” that can further differentiate into mature hepatocytes (MHs). SHs can proliferate to form colonies, and the maturation of SHs occurs with the alteration of the cell shape from small and flat to large and rising/piled-up. The hepatic organoids consisting of rising/piled-up cells possess highly differentiated functions like those of MHs and anastomosing networks of bile canaliculi (BC) are developed. The cells can make bile, secrete it into BC, and the bile can be carried without leaking. Thus, the organoids consist of polarized hepatocytes and possess biochemical and physiological functions as hepatic tissue. [ABSTRACT FROM AUTHOR]

The asialoglycoprotein receptor, which is abundantly and near exclusively expressed on hepatocytes, has received much attention in the design of non-viral hepatotropic DNA delivery systems. Thus, asialoglycoproteins and hexopyranosyl ligands have been coupled to DNA-binding cationic polymers and liposomes in the assembly of complexes intended for uptake by liver parenchymal cells. The aim of the study was to construct a hepatocyte-targeted multimodular liposome-based transfecting complex, in which the biotin-streptavidin interaction provides the cohesive force between the ligand asialorosomucoid and the liposome bilayer, and to evaluate its transfection capabilities in the hepatocyte-derived human transformed cell line HepG2. Dibiotinylated asialoorosomucoid was attached to cationic liposomes constructed from 3 β[N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T):dioleoylphosphatidylethanolamine:biotinylcholesterylformylhydrazide (MSB1) (48:50:2 mole ratio) through streptavidin interposition. Liposome-pGL3 DNA interactions were studied by gel band shift and ethidium displacement assays. The cytotoxicity of assemblies was evaluated in the HepG2 cell line and transfection capabilities determined by measuring the activity of the transgene luciferase. Binding assays showed that all DNA was liposome associated at a DNA (negative):liposome (positive) charge ratio of 1:1. Accommodation of a streptavidin dibiotinylated asialoorosomucoid assembly was achieved at a DNA:liposome:streptavidin dibiotinylated asialoorosomucoid ratio of 1:4:9 (weight basis). Complexes showed optimal transfection activity at this ratio, which was reduced 10-fold by the presence of the competing ligand asialofetuin. The streptavidin-biotin interaction has been applied for the first time to the assembly of hepatocyte-targeted lipoplexes that display asialoorosomucoid and that are well tolerated by a human hepatoma cell line in which transfection is demonstrably achieved by receptor mediation. Favorable size and charge ratio characteristics suggest that this system may be suitable for in vivo application. [ABSTRACT FROM AUTHOR]

Hepatitis C (HCV) is the disease that has affected around 200 million people globally. HCV is a life threatening human pathogen, not only because of its high prevalence and worldwide burden but also because of the potentially serious complications of persistent HCV infection. Chronicity of the disease leads to cirrhosis, hepatocellular carcinoma and end-stage liver disease. HCV positive hepatocytes vary between less than 5% and up to 100%, indicating the high rate of replication of viral RNA. HCV has a very high mutational rate that enables it to escape the immune system. Viral diversity has two levels; the genotypes and Quasiaspecies. Major HCV genotypes constitute genotype 1, 2, 3, 4, 5 and 6 while more than 50 subtypes are known. All HCV genotypes have their particular patterns of geographical distribution and a slight drift in viral population has been observed in some parts of the globe. [ABSTRACT FROM AUTHOR]

Buagafuran (BF), derived from α-agarofuran, is a promising anti-anxiety drug in phase I clinical trials. The present study was undertaken to examine the regulation of BF on liver cytochrome P450 (CYP) isoforms in rats. After being administered (4, 16, and 64 mg/kg) by gavage for 7 continuous days, the activities of CYP isoforms were measured by the qualification of six metabolites from CYP probe substrates using LC-MS/MS analysis. The mRNA and protein levels of CYPs were detected by reverse transcription polymerase chain reaction and Western blotting assay, respectively. Using phenacetin and chlorzoxazone as probe drugs, the activities of CYP1A2 and CYP2E1 were monitored in vivo. The result indicated that BF significantly increased the activity and protein levels of CYP1A2 and CYP2E1, while the mRNA levels were elevated to a certain extent. CYP2C6 and CYP2C11 were also slightly induced by BF, but no effect on liver CYP3A was detected in rats. Treatment of BF orally resulted in the decreasing of AUC, MRT and increasing of CL/F of phenacetin as well as production of acetaminophen in rats. The similar pharmacokinetic changes were also observed when using chlorzoxazone as a probe drug. Collectively, BF has inducing potential of liver CYP1A2 and CYP2E1 and may influence the corresponding pharmacokinetics of other drugs. [ABSTRACT FROM AUTHOR]

Cell cycle progression is dependent on the intracellular iron level, and chelators lead to iron depletion and decrease cell proliferation. This antiproliferative effect can be inhibited by exogenous iron. In this work, we present the synthesis of new synthetic calix[4]arene podands bearing alkyl acid and alkyl ester groups at the lower rim, designed as potential iron chelators. We report their effect on cell proliferation, in comparison with the new oral chelator ICL670 (4-[3,5-bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid). The antiproliferative effect of these new compounds was studied in human hepatocarcinoma HepaRG cell cultures using the MTT assay. Their cytotoxicity was evaluated by extracellular LDH activity. Preliminary results indicate that their antiproliferative effect is due to their cytotoxicity. The efficiency of these compounds, comparable to that of ICL670, was independent of iron depletion. This effect remains to be further explored. Moreover, it also shows that novel substituted calix[4]arenes could open the way to new valuable medicinal chemistry scaffolding. [ABSTRACT FROM AUTHOR]

1. Non-linear dose-exposure (supra-proportionality) occurs when plasma drug concentrations increase in a non-linear fashion with increasing dose. To predict the likelihood of this, an understanding is required of the KM, which reflects a drug ability to saturate a specific enzyme involved in its metabolism. 2. This study assessed the accuracy of KM and Vmax determinations for compounds using a substrate- depletion approach with those determined using the product-formation approach, using both recombinant human cytochrome P450 (CYP) enzymes and human liver microsomes. 3. For the vast majority of the compounds studied, the KM's using recombinant CYPs and human liver microsomes in the two approaches predicted within two-fold. Further comparisons between the KM and Vmax-values were made between those measured using the product-formation approach and those estimated following simultaneous fitting of the Michaelis-Menten equation to all substrate depletion plots. In each case values were comparable. 4. In conclusion, the current study showed the substrate-depletion approach can be used to estimate KM and Vmax using both human liver microsomes and recombinant P450s. Estimation of these parameters during early discovery will aid in the understanding of dosages at which non-linearity may occur, but potentially aid predictions of likely clinical drug-drug interactions. [ABSTRACT FROM AUTHOR]