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1. Self-administration of complementary products concurrently with conventional medication is increasingly common. The potential for cytochrome P450 (CYP) inhibition requires investigation. The N-in-one assay with ten probe substrates for nine CYPs was used with human liver microsomes to investigate ten products. CYP inhibition was measured in a single liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis. Estimated IC50-values were determined for the extracts that produced significant inhibition (less than 100 μg ml-1). 2. Inhibition of CYP2C19 by dong quai (IC50 = 13.7-14.3 μg ml-1 for the methanolic extract) and CYP2D6 by goldenseal (IC50 = 6.7 and 6.3 μg ml-1 for the aqueous and methanolic extracts, respectively), are of particular concern as the potential for adverse interactions is high. The inhibition of CYP2C8 by horsetail (IC50 = 93 μg ml-1 for the aqueous extract) requires further investigation, as the potential for concurrent use with products that require CYP2C8 for metabolism is significant. CYP3A4 inhibition varied depending on the probe reaction being monitored. 3. The earlier reported findings of inhibition by black cohosh, goldenseal and gotu kola were confirmed. The present work has shown that the N-in-one cocktail is a rapid and reliable method that can be used as an initial screen to help prioritize products that require more detailed investigations and it can also be applied to monitor product variability. [ABSTRACT FROM AUTHOR]

Objective. Hepatic stellate cells only produce fibronectin isoforms in disease states. The isoform-defining domains can be detected in the blood circulation. This study examines whether circulating levels of fibronectin isoforms show a relationship with liver fibrosis on histology in patients with chronic hepatitis C. Material and methods. In a prospective study, 50 patients with chronic hepatitis C who underwent a liver biopsy were compared to 50 matched controls and 35 patients with other liver conditions. Results. Circulating levels of the fibronectin isoforms were significantly higher in patients with chronic hepatitis C compared to healthy controls [oncofetal fibronectin (oFN) 2.45 ± 0.17 versus 1.76 ± 0.16 mg/l, P < 0.005; extra domain-A (EDA) 1.05 ± 0.06 versus 0.86 ± 0.06 mg/l, P < 0.05; and extra domain-B (EDB) 14.55 ± 0.74 versus 9.31 ± 0.58 mg/l, P < 0.001], even though total fibronectin was lower (198.9 ± 3.5 versus 343.6 ± 14.5 mg/l, P < 0.001). A correlation with the fibrosis score was found for both oFN ( r = 0.46, P < 0.005) and EDA ( r = 0.51, P < 0.001). The combination of an elevation in both markers (oFN and EDA) in the upper quartile was associated with a specificity of > 99% for predicting significant fibrosis (stages 2–4) and 95% for predicting advanced fibrosis (stages 3–4). A combination of decreased values in the lowest tertile for both markers had a specificity of 94% for excluding significant fibrosis. Based on these findings, 30% of the patients scheduled for a liver biopsy could be correctly classified as having or not having significant fibrosis. The remainder would have to proceed with a biopsy. Conclusion. Circulating fibronectin isoforms produced by activated stellate cells represent a viable marker for the presence of significant fibrosis or a lack thereof. [ABSTRACT FROM AUTHOR]

The present study was carried out to investigate whether taurine plays any beneficial role in acetaminophen (APAP)-induced acute hepatotoxicity. APAP exposure increased the plasma levels of ALT, ALP, LDH, TNF-α and NO production. Moreover, APAP treatment reduced the glutathione level and antioxidant enzyme activities, increased lipid peroxidation and caused hepatic DNA fragmentation which ultimately leads to cellular necrosis. Also, incubation of hepatocytes with APAP reduced cell viability, enhanced ROS generation and increased CYP2E1 activity. APAP overdose caused injury in the hepatic tissue and hepatocytes via the upregulation of CYP2E1 and JNK. Taurine treatment was effective in counteracting APAP-induced hepatic damages, oxidative stress and cellular necrosis. Results indicate that APAP overdose caused hepatic injury due to its metabolism to hepatotoxic NAPQI (N-acetyl-p-benzoquinone imine), usually catalysed by CYP2E1, and via the direct activation of JNK-dependent cell death pathway. Taurine possesses prophylactic as well as therapeutic potentials against APAP-induced hepatic injury. [ABSTRACT FROM AUTHOR]

Primary mouse hepatocytes (between 10–250 cells per capsule) were immobilized within 1.0% w/v alginate microbeads. The textural properties of the alginate matrix were characterized and a full protocol based upon the measurement of the initial rate of Resazurin reduction was studied and standardized. Using this method, the decay rate constant ( Kd = 0.45 ± 0.01 days−1) and the time in which the cell viability decreases in half ( VI50 = 37 ± 0.7 h) have been measured. The method was compared with the analysis of cell vitality using Calcein A/M and Ethidium Homodimer I. Differences between the two methods were found in the viability profile due to the significant presence of double stained cells along the culture time. According to the author's knowledge, this is the first report of a systematic study and determination of the Kd value for immobilized hepatocytes, incorporating a wide range of cell concentrations within the alginate matrix. [ABSTRACT FROM AUTHOR]

1. 4-n-Nonylphenol and bisphenol A are endocrine disrupting chemicals that are mainly detoxified through glucuronidation. A factor that may modulate their glucuronidation rates is co-exposure to pharmaceuticals. 2. This study aimed to identify and characterize the potential metabolic interactions between 14 drugs and these two endocrine disruptors. 3. Nonylphenol and bisphenol A were co-incubated in freshly isolated rat hepatocytes with, drugs at a high concentration. Statistically significant metabolic inhibition of bisphenol A and nonylphenol biotransformation was observed with nine drugs (>50% inhibition by naproxen, salicylic acid, carbamazepine and mefenamic acid). Inhibition assays of UGT activity in rat liver microsomes revealed: 1) competitive inhibition by naproxen (Kiapp = 848.3 μM) and carbamazepine (Kiapp = 1023.1 μM), 2) no inhibition by salicylic acid suggesting another mechanism of inhibition. 4. Detoxification of nonylphenol and bisphenol A was shown to be impaired by excessive concentrations of many drugs and health risk assessment should therefore address this issue. [ABSTRACT FROM AUTHOR]

Galactose-specific lectins (Gal-lectins) were isolated from the mitochondrial fraction of prostate post-operational hyperplasic tissue of two diagnoses: benign prostate hyperplasic tissue with low-grade intraepithelial neoplasia (LGPIN) and benign prostate hyperplasic tissue with atypical adenomatous hyperplasia (AAH). They had similar molecular weight and other properties. Effects of these lectins were investigated in vitro model experiments on bovine liver cells mitochondrial properties. Time-dependent changes: (i) in the amount of H2O2; (ii) redox state of Cu in cytochrome oxidase and (iii) redox state of heme in cytochrome a+a3 (cyt a+a3) of cytochrome c oxidase complex were studied. Gal-lectins from both sources increase the amount of H2O2 and decrease the redox state of Cu in cytochrome oxidase and heme in cyt a+a3. However the Gallectin from tissue with more severed transformation (AAH) expresses significantly more strong and long-lasting influence. These effects are mediated by galactose binding domain of the lectins as are completely abolished by the inclusion of galactose in reaction medium. Accumulation of H2O2 and long-lasting decrease in the redox state of key enzymes of mitochondrial respiration chain could induce defective functioning of these organelles and whole cells. Obtained data point the possible way, which enhances further transformation of prostate tissue by release of Gallectins from damaged mitochondria. [ABSTRACT FROM AUTHOR]

An original ligand (Lac-10-Chol) designed to interact with asialoglycoprotein receptors to potentially target hepatocyte was synthesised by grafting a lactose head to a cholesteryl structure, which was then included in liposomes. Preliminary formulation tests led to the selection of conventional formulations based on soybean phosphatidylcholine/cholesterol/DOTAP (± DOPE) (± Lac-10-Chol) that present reproducible absolute entrapment value (1.45 ± 0.10%), with a size of 109 ± 7 nm and a slight positive charge (3.77 ± 1.59 mV). Cell viability (via the MTT test), expressed as the percentage of nontreated cells in HepG2 cells, was very close to the control. Internalization tests evidenced an intracellular penetration of fluorescent liposomes, but no specific ligand effect was demonstrated ( P > 0.05). Nevertheless, regarding the adenosine triphosphate (ATP) assay, a slight increase was obtained with liposome loaded with ATP incorporating Lac-10-chol after 24 hours ( P < 0.05). [ABSTRACT FROM AUTHOR]

Morinda citrifolia Linn. (Rubiaceae), common name noni, has been used as a herbal medicine for over 2000 years. The consumption of noni, and especially the fruit, stresses the importance, urgency, and possibility of the examination of drug interaction when concomitantly administered with a drug. The objectives of this study were to determine the effects of noni juice (NJ) on aminopyrine N-demethylase (APND), uridine diphosphoglucuronosyl-transferase (UGT), and cytosolic glutathione S-transferase (GST) drug metabolizing enzymes and the molecular mechanism elucidation of NJ on APND using different inhibitors and stimulators. The in vitro results for APND showed that different concentrations of NJ significantly increased the activity in isolated hepatocytes at 1.0 ng/mL, 10 ng/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL, and 100 μg/mL. The ex vivo results demonstrated that NJ (210 mg/kg) produced a statistically significant increase in APND activity following 1 day of NJ treatment. The results for UGT and GST showed a decrease in the activity of UGT at a dose of 21 mg/kg following 1 day of treatment, and at 2.1 and 21 mg/kg following 14 days of treatment. GST enzyme demonstrated an increase in activity by 100% for all doses following 1 day of treatment. Molecular mechanism elucidation of the ex vivo effect of NJ on phase I APND showed that KT5720 significantly reduced the activity as compared to control. A change in activity of APND, UGT, and GST following 1 day and 14 days of treatment suggests that all three metabolic pathways may play a role in herb–drug interaction by modulation of metabolic enzymes. [ABSTRACT FROM AUTHOR]

Glutathione S-transferase (GST) is a family of enzymes involved in the detoxification of electrophilic compounds. Different classes of GST are expressed in various organs, such as liver, lungs, stomach and others. Expression of GST can be modulated by diet components and plant-derived compounds. The importance of controlling GST expression is twofold: increasing levels of GST are beneficial to prevent deleterious effects of toxic and carcinogenic compounds, while inhibition of GST in tumor cells may help overcoming tumor resistance to chemotherapy. A screening of 16 plants used in the Brazilian pharmacopoeia tested their effects on GST expression in hepatocytes and Jurkat (leukemia) T-cells. The methanol extracts of five plants inhibited GST expression in hepatocytes. Three plants significantly inhibited and four others induced GST expression in Jurkat cells. Among these, the extracts of Bauhinia forficata Link. (Leguminosae) and Cecropia pachystachya Tréc. (Urticaceae) inhibited GST expression at relatively low concentrations. With the exception of B. forficata, all plants were cytotoxic when administered to Jurkat cells at high doses (1 mg/mL) and some extracts were considerably cytotoxic even at lower concentrations. [ABSTRACT FROM AUTHOR]

This study investigates the mechanism by which MnSOD exerts its protective effect in hypoxia-reoxygenation (H/R) injury in hepatocytes. Following induction of H/R, MnSOD expression and activity levels increased and remained high for over 24 h. Hepatocytes silenced for MnSOD (siMnSOD) demonstrated increased susceptibility to H/R-induced apoptotic cell death and a lower capacity to generate mitochondrial reactive oxygen species. Microarray and real time PCR analysis of gene expression from siMnSOD cells revealed a number of down-regulated protective genes, including hemeoxygenase-1, glutamate-cysteine ligase and Nrf2, a master regulator of cellular adaptation to stress. Decreased Nrf2 protein expression and nuclear translocation were also confirmed in siMnSOD cells. siMnSOD cells showed low glutathione (GSH) content with no oxidation to GSSG, lower lipid peroxidation levels than their controls and lower mitochondrial membrane potential, which all were even more salient after H/R. Therefore, MnSOD appears to act as a signalling mediator for the activation of survival genes following H/R injury in hepatocytes. [ABSTRACT FROM AUTHOR]

Augmenter of liver regeneration (ALR) enhances the proliferation of hepatocytes and accelerates recovery from acute liver failure in animal models. ALR is expressed in both the liver and kidney; however, the specific location of ALR expression and its biological effects in the kidney remain unknown. We aimed to investigate the efficacy of ALR in rats with gentamicin (GM)-induced acute renal failure (ARF). Rats were randomized into the normal group, GM+saline group, GM+vehicle group, and GM+rrALR group. Blood urea nitrogen, serum creatinine, and urine beta-N-acetyl-D-glucosaminidase were measured, and histological analyses of the kidneys were performed. The expression of ALR protein was determined by immunohistochemistry and Western blotting. In vitro incorporation of tritiated thymidine was used to measure the proliferation of renal tubular epithelial cells. In normal rats, the expression of ALR protein was faint in the medulla and absent in the cortex. However, in ARF rats, ALR expression increased significantly in both the renal cortex and medulla. Histological analyses revealed that treatment with recombinant rat ALR (rrALR) reduced the extent of injury of tubular cells in the renal cortex. Serum/urine biochemical parameters also showed that renal dysfunction was improved by the administration of rrALR. Intraperitoneal injection of rrALR enhanced the proliferation of tubular cells in vivo. We also confirmed that rrALR could promote the proliferation of renal tubular cells in vitro. These results indicate that rrALR effectively accelerates kidney recovery after ARF induced by gentamicin, and that the protective effect is associated with enhanced proliferation of renal tubular cells. [ABSTRACT FROM AUTHOR]

Trichloroethylene (TCE) is a widespread environmental contaminant that is carcinogenic when given in high, chronic doses to certain strains of mice and rats. The capacity of TCE to cause cancer in humans is less clear. The current maximum contaminant level (MCL) of 5 ppb (μg/L) is based on an US Environment Protection Agency (USEPA) policy decision rather than the underlying science. In view of major advances in understanding the etiology and mechanisms of chemically induced cancer, USEPA began in the late 1990s to revise its guidelines for cancer risk assessment. TCE was chosen as the pilot chemical. The USEPA (2005) final guidelines emphasized a “weight-of-evidence” approach with consideration of dose-response relationships, modes of action, and metabolic/toxicokinetic processes. Where adequate data are available to support reversible binding of the carcinogenic moiety to biological receptors as the initiating event (i.e., a threshold exists), a nonlinear approach is to be used. Otherwise, the default assumption of a linear (i.e., nonthreshold) dose-response is utilized. When validated physiologically based pharmacokinetic (PBPK) models are available, they are to be used to predict internal dosimetry as the basis for species and dose extrapolations. The present article reviews pertinent literature and discusses areas where research may resolve some outstanding issues and facilitate the reassessment process. Key research needs are proposed, including role of dichloroacetic acid (DCA) in TCE-induced liver tumorigenesis in humans; extension of current PBPK models to predict target organ deposition of trichloroacetic acid (TCA) and DCA in humans ingesting TCE in drinking water; use of human hepatocytes to ascertain metabolic rate constants for use in PBPK models that incorporate variability in metabolism of TCE by potentially sensitive subpopulations; measurement of the efficiency of first-pass elimination of trace levels of TCE in drinking water; and assessment of exogenous factors’ (e.g., alcohol, drugs) ability to alter metabolic activation and risks at such low-level exposure. [ABSTRACT FROM AUTHOR]

The proteins on lymphocyte surface play important roles in a wide range of immunological processes, but the profile and characterization of surface proteins remain to be further investigated, among which the method for fast screening of surface proteins needs to be established. In this study, a conventional cDNA clone library of hepatic lymphocytes from C57BL/6 mouse was constructed, and then the cDNA was inserted into a recombinant expression vector pSecTag-attR with a signal peptide and tag protein for fluorescence screening. The recombinant cDNA expression library was transfected into COS-1 cells, and the transfected cells with the expressed membrane proteins were labeled by fluorescence antibodies and isolated by fluorescence activated cell sorting. After two cycles of sorting, the purity of fluorescence positive cells with membrane proteins was up to 98%, and the representative membrane molecules on lymphocytes such as CD3, CD4, CD8, NK1.1 and NKG2D were detected in the library. These results demonstrated that the cDNA expression library containing transmembrane proteins provided an efficient and fast tool for the study of transmembrane proteins on hepatic lymphocytes. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B was characterized by fluctuant immune response to infected hepatocytes resulting in hepatic inflammation and virus persistence. Recently, Programmed Death-1 (PD-1) and its ligand PD-L1 have been demonstrated to play an essential role in balancing antiviral immunity and inflammation in the livers of acute hepatitis B patients, significantly influencing disease outcome. PD-1 up-regulation in peripheral T cells is associated with immune dysfunction in chronic hepatitis B patients. However, the effect of PD-1/PD-L1 on hepatic damage and chronic infective status is still unknown in patients with chronic HBV infection. Here, we report up-regulation of PD-1 and PD-L1 in liver biopsies from 32 chronic HBV patients compared to 4 healthy donors. PD-1/PD-L1 up-regulation was significantly associated with hepatic inflammation and ALT elevation. Moreover, appropriate up-regulation but not overexpression of PD-L1 in the active phase of chronic hepatitis B as well as lower expression of PD-L1 in the inactive phase in liver residential antigen presenting cells (including Kupffer cells and sinusoidal endothelial cells) may contribute to viral inhibition. Our data suggest that the intrahepatic interaction of PD-1 and PD-L1 might play an important role in balancing the immune response to HBV and immune-mediated liver damage in chronic HBV infection. [ABSTRACT FROM AUTHOR]

A class of imidazolium salts (IMSs) is routinely used in organic synthetic chemistry as precursors to generate N-heterocyclic carbenes (NHCs) with catalytic activity. However, their biological properties are largely unknown. The current study investigates the biological activity of a typical NHC precursor DBZIM and its trimer TDBZIM in hepatic stellate cells (HSCs), which is an in vitro model for studying liver fibrosis. The results show that HSCs treated with IMSs have an enhanced GSH/GSSG ratio and a reduced level of reactive oxygen species (ROS), which may consequently contribute to the attenuation in gene expression of fibrogenic molecules such as smooth muscle actin-α (SMAA), transforming growth factor-beta 1 (TGF-β1), procollagen αI(I) and fibronectin. Further, the in vivo experiments demonstrate that DBZIM is an anti-fibrotic agent in a mouse model of liver fibrosis. These findings suggest that the versatile IMSs could be a potential source for developing novel therapeutics to treat liver fibrosis and other fibrogenic disorders caused by oxidative stress and TGF-β1 mal-signalling. [ABSTRACT FROM AUTHOR]

1. 5-{2-[4-(3,4-Difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid (1) is a novel, potent, and selective agonist of the peroxisome proliferator-activated receptor alpha (PPAR-α). 2. In preclinical species, compound 1 demonstrated generally favourable pharmacokinetic properties. Systemic plasma clearance (CLp) after intravenous administration was low in Sprague-Dawley rats (3.2 ± 1. 4 ml min-1 kg-1) and cynomolgus monkeys (6.1 ± 1. 6 ml min-1 kg-1) resulting in plasma half-lives of 7.1 ± 0.7 h and 9.4 ± 0.8 h, respectively. Moderate bioavailability in rats (64%) and monkeys (55%) was observed after oral dosing. In rats, oral pharmacokinetics were dose-dependent over the dose range examined (10 and 50 mg kg-1). 3. In vitro metabolism studies on 1 in cryopreserved rat, monkey, and human hepatocytes revealed that 1 was metabolized via oxidation and phase II glucuronidation pathways. In rats, a percentage of the dose (approximately 19%) was eliminated via biliary excretion in the unchanged form. 4. Studies using recombinant human CYP isozymes established that the rate-limiting step in the oxidative metabolism of 1 to the major primary alcohol metabolite M1 was catalysed by CYP3A4. 5. Compound 1 was greater than 99% bound to plasma proteins in rat, monkey, mouse, and human. 6. No competitive inhibition of the five major cytochrome P450 enzymes, namely CYP1A2, P4502C9, P4502C19, P4502D6 and P4503A4 (IC50's > 30 μM) was discerned with 1. 7. Because of insignificant turnover of 1 in human liver microsomes and hepatocytes, human clearance was predicted using rat single-species allometric scaling from in vivo data. The steady- state volume was also scaled from rat volume after normalization for protein-binding differences. As such, these estimates were used to predict an efficacious human dose required for 30% lowering of triglycerides. 8. In order to aid human dose projections, pharmacokinetic/pharmacodynamic relationships for triglyceride lowering by 1 were first established in mice, which allowed an insight into the efficacious concentrations required for maximal triglyceride lowering. Assuming that the pharmacology translated in a quantitative fashion from mouse to human, dose projections were made for humans using mouse pharmacodynamic parameters and the predicted human pharmacokinetic estimates. 9. First-in-human clinical studies on 1 following oral administration suggested that the human pharmacokinetics/ dose predictions were in the range that yielded a favourable pharmacodynamic response. [ABSTRACT FROM AUTHOR]

01. Paeonol, the primary active component of a traditional Chinese medicine Moutan Cortex, has a wide range of pharmacological activities. In the present study, the metabolism of paeonol by cytochrome P450s (CYPs) was investigated in human liver microsomes. 2. One O-demethylated metabolite was detected in reaction catalysed by human liver microsomes, and was identified as resacetophenone by comparing the tandem mass spectra and the chromatographic retention time with that of the standard compound. 3. The study with a chemical selective inhibitor, cDNA-expressed human CYPs, a correlation assay, and a kinetics study demonstrated that CYP1A2 was the major isoform responsible for the paeonol O-demethylation in human liver microsomes. [ABSTRACT FROM AUTHOR]

1. We compared the intrinsic clearance (CLint) of a number of substrates in suspensions of fresh and cryopreserved human hepatocytes from seven donors. 2. CLint values for a cocktail incubation of phenacetin, diclofenac, diazepam, bufuralol, midazolam, and hydroxycoumarin were 4.9 ± 3.4, 18 ± 7.2, 5.1 ± 4.9, 6.3 ± 3.3, 9.8 ± 5.8 and 22 ± 14 μl min-1/106 cells, respectively, and they correlated well with corresponding CLint values using cryopreserved hepatocytes from 25 different donors. 3. CLint values of each cocktail substrate and 20 AstraZeneca new chemical entities were compared in fresh and cryopreserved hepatocytes from the same three donors. There was a statistically significant correlation between CLint in fresh and cryopreserved hepatocytes for each of the three livers (p < 0.002) and the geometric mean of the ratio of fresh to cryopreserved CLint values was 1.03. 4. In conclusion, the results add further support to the use of cryopreserved human hepatocytes as a screening model for the intrinsic clearance of new chemical entities. [ABSTRACT FROM AUTHOR]

It is still unclear how Interferon-alfa (IFN-α) acts on preventing the appearance of hepatocarcinogenesis. We have demonstrated that IFN-α2b induces hepatocytic transforming growth factor-beta1 (TGF-β1) production and secretion by inducing reactive oxygen species (ROS) formation through the activation of NADPH oxidase. This TGF-β1, alters antioxidant defences and induces programmed cell death. Since it was demonstrated that IFN-α induces apoptosis through the activation of p38 mitogen-activated protein kinase (p38 MAPK), this study was aimed to assess the role of this kinase in the IFN-α2b-induced apoptosis in rat liver preneoplasia; and to further evaluate the participation of NADPH oxidase. p38 MAPK pathway was activated during the IFN-α2b-induced apoptosis in rat liver preneoplasia. This activation was accompanied with phosphorylation of different transcription factors, depending on the time of IFN-α2b stimulus. Our data suggest that NADPH oxidase is activated by IFN-α2b through p38 MAPK. p38 MAPK-induced activation of NADPH oxidase is accomplished by a two-step pathway: first, ROS-independent and second ROS- and TGF-β1-dependent. [ABSTRACT FROM AUTHOR]

Purpose: To identify transcriptional gene-networks involved in the early in vivo response of liver cells to radiation exposure and improve our understanding of the molecular processes responsible for tissue radiosensitivity. Materials and methods: Transcriptome variations of liver RNA samples were measured 3 hours post-irradiation using microarray technology. The results were confirmed and extended using real-time polymerase-chain-reaction (RT-PCR). Results: We identified quantitative changes in the expression of 126 genes, most of which were observed for the first time. We show that some modifications, such as the upregulation of the cyclin-dependent kinase inhibitor 1A (Cdkn1A) gene, persisted for at least two months after the initial exposure. Other genes regulated by the transformation-related protein 53 (Trp53/p53) such as Bcl2-associated X protein (Bax) or etoposide-induced-2.4 (Ei24/PIG8) were not upregulated. Grouping differentially expressed genes into functional categories revealed that the primary response of liver cells to radiation exposure was the enhancement of oxidoreductase activity and inhibition of cell proliferation, involving cell cycle progression and apoptosis-related genes. Conclusions: The data provides evidence of gene expression modifications associated with the hepatic response to radiation exposure. One of the main differences observed with radiation-sensitive tissues such as the spleen was cell proliferation. The comparison of our data with transcriptome modifications in different biological models enabled the identification of networks of genes that might be co-regulated. Overall, our expression data revealed genes and cellular pathways that might help to improve our understanding of the molecular basis underlying tissue radiosensitivity and to identify possible targets for novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

An attempt was made to develop quantitative structure-toxicity relationships (QSTRs) for acute rat hepatocyte cytotoxicity of a series of non-steroidal anti-inflammatory drugs (NSAIDs) with extended topochemical atom (ETA) indices. The ETA models were compared with those developed with a pool of other topological indices. Finally, attempt was made to develop models from the combined pool of topological (ETA and non-ETA) descriptors. The chemometric tools used for model development were multiple linear regression with factor analysis as the variable selection tool, stepwise regression, principal component regression analysis, partial least squares (PLS), genetic function approximation (GFA) and genetic PLS (G/PLS). Use of ETA descriptors along with non-ETA ones increased the statistical quality of the non-ETA models in different techniques except stepwise regression and GFA. The best three models came from GFA, G/PLS and PLS techniques (leave-one-out Q2 values of 0.871, 0.854 and 0.834, respectively, using combined set of descriptors except for GFA). Use of the ETA parameters suggests that the toxicity increases with bulk and degree of branching. Moreover, heteroatom count and degree of unsaturation are also important for the toxicity. [ABSTRACT FROM AUTHOR]

It has previously been reported that a peptide sequence of T7 phage protein p17 mediates uptake of its cargo by liver parenchymal cells. The aim of this study was to identify the phage-binding receptor. The involvement of LRP was confirmed by the observations that phage binding to Hepa 1c1c7 cells was inhibited by the LRP-binding receptor-associated protein, LRP-deficient mouse embryonic fibroblasts bound phage with lower efficiency than their wild-type counterparts, and using mouse models with ablated LRP liver expression. The identification of LRP as a cognate receptor for this sequence offers a new ligand-receptor combination for hepatocyte delivery of therapeutic agents. [ABSTRACT FROM AUTHOR]

Hepatocyte transplantation is an alternative to transplantation of the whole liver. Compared with xenogeneic hepatocytes, primary hepatocytes have some advantages, such as a more powerful function and a smaller frequency of rejection caused by the host. Cell microencapsulation prevents direct access of host cells to the graft but cannot impede transfer of transplant-derived peptides, which can cross the physical barrier. Sertoli cells are central to the immune privilege demonstrated in the testis, and their actions have been utilized to protect cell transplants. Co-microencapsulating Sertoli cells with HepG2 cells has proved to be a valuable strategy in hepatocyte transplantation. Thus mixed microcapsules of primary rat hepatocytes and primary Sertoli cells may improve metabolic function in a d-galactosamine and lipopolysaccharide-induced rat model of acute liver failure. [ABSTRACT FROM AUTHOR]

It was found previously that membranes based on co-polymers of acrylonitrile (AN) and 2-acrylamido-2-methyl-propansulfonic acid (AMPS) greatly stimulated the functionality and survival of primary hepatocytes. In those studies, however, the pure AN–AMPS co-polymer had poor membrane-forming properties, resulting in quite dense rubber-like membranes. Hence, membranes with required permeability and optimal biocompatibility were obtained by blending the AN–AMPS co-polymer with poly(acrylonitrile) homopolymer (PAN). The amount of PAN (P) and AN–AMPS (A) in the blend was varied from pure PAN (P/A-100/0) over P/A-75/25 and P/A-50/50 to pure AN–AMPS co-polymer (P/A-0/100). A gradual decrease of molecular cut-off of membranes with increase of AMPS concentration was found, which allows tailoring membrane permeability as necessary. C3A hepatoblastoma cells were applied as a widely accepted cellular model for assessment of hepatocyte behaviour by attachment, viability, growth and metabolic activity. It was found that the blend P/A-50/50, which possessed an optimal permeability for biohybrid liver systems, supported also the attachment, growth and function of C3A cells in terms of fibronectin synthesis and P-450 isoenzyme activity. Hence, blend membranes based on a one to one mixture of PAN and AN–AMPS combine sufficient permeability with the desired cellular compatibility for application in bioreactors for liver replacement. [ABSTRACT FROM AUTHOR]

In order to define clearly the conditions leading to the outcome of acute duck hepatitis B virus (DHBV) infection, 1-day-old Pekin ducklings were infected with DHBV by different routes and given different doses of inoculum. Groups of 24 ducklings were inoculated either intravenously via the vena cruralis, or intraperitoneally with pooled serum containing either 1.6 x 107 or 1.6 x 104 DHBV genomes. One control duck from each group was inoculated with an equal volume of normal duck serum. A sensitive and reproducible real-time polymerase chain reaction assay based on TaqMan technology was developed for the detection and quantitation of DHBV DNA in the serum and liver. DHBAg was observed in the hepatocytes by immunohistochemistry. Histological changes in the liver tissue were also observed. The results demonstrate that ducklings at each time point and in all groups developed detectable viraemia. In each group, DHBV DNA in the liver was at a lower level than in serum and the peak DNA titre was found in serum earlier than in the liver. In the low-dose groups it was always at a lower level than in the high-dose groups. The DHBV replication levels appeared to be directly related to the number of DHBAg-positive hepatocytes. The variation trends of DHBAg-positive hepatocytes were similar in the high-dose groups. Histological changes were associated with liver viral DNA levels. We suggest that this dose and route of inoculation can be used as a model to study acute DHBV infections. [ABSTRACT FROM AUTHOR]

Rabbits were fed a diet containing 1% cholesterol for 8 weeks and the levels of iron and oxidized lipids in liver analysed using atomic absorption spectroscopy and gas chromatography-mass spectrometry. A non-significant trend to an increase in iron level, but significant increases in the lipid peroxidation products, F2-isoprostanes and the cholesterol oxidation products 7 beta hydroxycholesterol, 7 ketocholesterol and cholesterol 5,6-alpha epoxide were detected in the liver of the cholesterol-fed rabbits. Histological analysis showed greater accumulation of lipids by Sudan red labelling in hepatocytes of zone I than zones II and III of the liver acinus. The increase in lipids coincided with an increase in iron staining in macrophages around liver microvessels and increased immunostaining to melanotransferrin and the lipid peroxidation product, 4-hydroxynonenal (4-HNE), in zone 1. The results are suggestive of microvascular damage associated with iron accumulation and oxidative stress in the liver during hypercholesterolemia. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is a chronic disease which affects millions of people worldwide. The prevalence of this disease is increasing annually and the number of diabetics is projected to rise above 300 million before 2025. The growing number of diabetics, coupled with the harsh side effects of some synthetic drugs has led to the increasing search for more natural products of plant origin. Vernonia amygdalina Del. (Asteraceae) is one of the plants commonly used for the treatment of diabetes in Africa. This study evaluated the effect of leaf extracts of this plant on glucose utilization in 3T3-L1, C2C12 muscle, and Chang-liver cells. Treatment of the cells with the acetone, methanol, water, and n-hexane/isopropanol extracts of V. amygdalina leaves significantly increased glucose utilization in the C2C12 muscle and Chang-liver cells but showed no effect on the 3T3-L1 cells. The water and n-hexane/isopropanol extracts were the most active in the C2C12 cells with a response of 78.3 and 95.6% above the control, respectively, while in the Chang-liver cells, water and acetone extracts had a response of 65.8 and 59.6% above the control, respectively. The results, especially of the water extract, strongly corroborate the ethnomedical uses of V. amygdalina as an antidiabetic plant. [ABSTRACT FROM AUTHOR]

The present study investigated the effect of fenugreek seed polyphenolic extract (FPEt) on ethanol-induced protein expression in Chang liver cells. Cells were incubated with either 30 mM EtOH alone or together in the presence of FPEt for 24 h. Cells were harvested and assessed for expression of alcohol metabolizing enzymes-alcohol dehydrogenase (ADH2 isoform), aldehyde dehydrogenase (ALDH2 isoform), cytochrome P450 (CYP2E1), the electron transport component (cytochrome-c), and the heat shock proteins. The expression of ADH2, ALDH2, and CYP2E1 were upregulated, whereas the expression of cytochrome-c was downregulated in the ethanol-treated cells. The expression of cellular heat shock proteins-HSP70, HSC70, HSC92, and mitochondrial protein mtHSP70 were induced in ethanol-treated Chang liver cells. FPEt modulated the protein expression changes induced by ethanol and had no effect when incubated with normal Chang liver cells. FPEt might exert cytoprotective action on ethanol-induced liver cell damage, possibly by enhancing cellular redox status. [ABSTRACT FROM AUTHOR]

Penitrem A is a potent neurotoxin produced by several species in the genus Penicillium, which primarily affects the central nervous system. The toxin has several effects on neurotransmitter release, both at the central and peripheral level, as well as on ion channels. We have evaluated the hepatic metabolism of penitrem A by rat hepatocytes and rat-liver microsomes in vitro. In addition, we have conducted an in vivo study in mice and determined metabolites in several organs. According to our results, penitrem A is extensively metabolized in the liver to at least five metabolites more hydrophilic than the parent compound. [ABSTRACT FROM AUTHOR]

Background Although different strategies have been established for hepatic differentiation of mesenchymal stromal cells (MSC), further studies are requiredto define an efficient strategy to produce hepatocytes from stem cells and uncover the mechanisms of hepatic differentiation. Methods Bone marrow mesenchymal stromal cells (BMMSC), isolated from ICR mice, were induced by fetal liver-conditioned medium from different developmental stages, embryonic days (ED) 9.5, 11.5 and 13.5 and newborn (1 day). Differentiated cells were characterized by morphologic changes, liver-specific gene expression at mRNA and/or protein levels and in vitro functional features. Results BMMSC morphologically became epithelioid and binucleated after 7 days' exposure to fetal liver-conditioned medium from ED13.5, expressed liver-specific genes (AFP, HNF-3β, TTR, CK18, ALB and CK19) at mRNA and/or protein levels and acquired in vitro functions characteristic of liver cells, including glycogen storage, urea production and albumin secretion. Conditioned medium derived from fetal liver at ED13.5 was most efficient on hepatic differentiation of BMMSC compared from the other three developmental stages. Discussion The present study not only provides a high-performance strategy for hepatic differentiation from BMMSC, but also implies liver at different developmental stages might secrete different types of cytokines that have diverse effects on hepatic differentiation, which could support further investigation to provide insight into fundamental processes that govern development and regeneration of the liver. [ABSTRACT FROM AUTHOR]

A purified saponin mixture (PSM) isolated from Astragalus corniculatus Bieb. (Fabaceae) was investigated for its protective effect in two models of toxicity, carbon tetrachloride (CCl4) and tert-butyl hydroperoxide (t-BuOOH), using isolated rat hepatocytes. CCl4 undergoes dehalogenation in the liver endoplasmic reticulum. This process leads to trichlormethyl radical (CCl3) formation, initiation of lipid peroxidation, and measurable toxic effects on the hepatocytes. Oxidative damage is widely recognized as being involved in the development of many pathological conditions. In our experiment, t-BuOOH was used as a model of oxidative stress. The hepatocytes were incubated with the PSM alone (0.01-100 μ M) and along with CCl4 (86 μ M) and t-BuOOH (75 μ M). As a sign of cytotoxicity, cell viability was used. CCl4 and t-BuOOH significantly decreased hepatocyte viability. Our data indicate that PSM showed lower toxic effects compared to CCl4 and t-BuOOH and in combination exerted statistically significant protection of cell viability against the toxic agents. [ABSTRACT FROM AUTHOR]

Objective. To assess the production of the endogenous IL-1 modulators IL-1 receptor antagonist (IL-1Ra), type I and II soluble IL-1 receptors (IL-1sRI and II) in patients with chronic liver disease (CLD). Material and methods. Plasma levels of IL-1beta (IL-1β) and IL-1 modulators were assessed in 126 CLD patients and 39 healthy controls. IL-1sRII was also measured in the supernatants of primary hepatocyte cultures. Results. Plasma IL-1sRI and IL-1Ra levels were significantly higher in cirrhotic CLD patients than in non-cirrhotic CLD patients and in controls. Levels did not depend on the etiology of CLD. Likewise, plasma IL-1β levels were elevated in CLD patients compared with those in controls. In contrast, IL-1sRII levels did not differ between CLD patients and controls. Cultures of human primary hepatocytes showed that IL-1sRII is induced by IL-1β, but not IL-6. Conclusions. In cirrhotic CLD patients elevated plasma IL-1β is not counteracted by endogenous levels of IL-1sRII, whereas high IL-1sRI is expected to neutralize the naturally occurring antagonist IL-1Ra, resulting in a dysregulation of the IL-1 system that might enhance pro-inflammatory activity of IL-1. [ABSTRACT FROM AUTHOR]

1. In vivo clearance predictions from in vitro assays require scaling factors to relate the concentrations of hepatocytes or microsomal protein to the intact liver. 2. The aims were to measure the variability in scaling factors for Wistar rat and beagle dog for which the literature is particularly scarce and determine any sex differences. 3. Scaling factors were determined by comparing the cytochrome P450 (P450) content in hepatocytes or microsomes against the P450 content of fresh liver homogenate. The use of fresh homogenate is recommended as freezing can increase contamination and affect the P450 assay. 4. Meangeo hepatic microsomal concentrations in Wistar rats were 61 mg g-1 liver (95% confidence interval (CI); 47-75 mg g-1 liver) and in beagle dogs 55 mg g-1 liver (95% CI = 48-62 mg g-1 liver). Meangeo hepatocellularity was 163 × 106 cells g-1 liver for Wistar rats (95% CI = 127-199 × 106 cells g-1 liver) and 169 × 106 cells g-1 liver (95% CI = 131-207 × 106 cells g-1 liver) for beagle dogs. The data generated in this study indicate a consistency in scaling factors between rat and dog. No sex differences were observed. [ABSTRACT FROM AUTHOR]

Background. It has been reported that hemodialysis (HD) stimulates hepatocyte growth factor (HGF) release, but it is not clear if this stimulation is due to HD itself or to heparin used during HD. To clarify this issue, we undertook the present study. Methods. We studied 18 HD patients using high-flux dialyzers, during a single 4-hr hemodialysis session (session A). The dialyzers were pre-rinse with normal saline without heparin, and HD was started with zero ultrafiltration and without anticoagulation. Anticoagulation was administered as IV injection (80 IU/kg of LMWH enoxaparin sodium) 10 min after the beginning of HD. HD was continued for 10 more minutes and then as prescribed. HGF serum levels were measured before the beginning of the HD session (sample t0) as well as 10 and 20 minutes after the beginning of the session (samples t10 and t20). In six more patients (controls), the same study was repeated but without the administration of LMWH during the first 20 min of HD initiation (session B). Results. In comparison with t0, t10 HGF serum levels changed significantly in neither session A nor in session B. However, at t20, HGF levels increased significantly in session A compared with t0 (increment 666.3 ± 211.0%, p < 0.0001) and t10 (increment 894.2 ± 506.0%, p < 0.0001), but not in session B. No differences were found between sessions A and B at samples t0 and t10 (p = NS). HGF serum levels at t20 in session A were found to be higher compared with corresponding levels in session B (p < 0.0001). Conclusion. Elevated HGF serum levels at the beginning of high-flux HD session are due to LMWH administration. [ABSTRACT FROM AUTHOR]

This study is aimed to evaluate the role of intracellular GSH in pyrrolizidine alkaloid clivorine-induced toxicity on human normal liver L-02 cells. Clivorine induced profound intracellular GSH depletion in a concentration-dependent manner. The GSH synthesis inhibitor L-Buthionine (S, R)-sulfoximine (BSO) significantly potentiated toxicity of clivorine, while GSH and GSH synthesis precursors N-Acetyl-L-cysteine (NAC) and S-adenosyl-L-methionine (SAM) protected cells against toxicity of clivorine. Overall, the results suggest that intracellular GSH plays an important role in the toxicity of clivorine on human hepatocytes. [ABSTRACT FROM AUTHOR]

A total of 110 drugs, selected to cover a range of physicochemical and pharmacokinetic properties, were used to explore standard approaches to the prediction of in vivo metabolic clearance using drug-depletion profiles from human liver microsomes (HLMs) and cyropreserved hepatocytes. A total of 41 drugs (37% of the compounds tested) showed measurable depletion rates using HLMs (depletion by 20% or more over the time course). The most reliable correlations in terms of bias (average fold error (AFE) = 2.32) and precision (root mean square error (RMSE) = 3501) were observed by comparing in vivo intrinsic clearance (CLint), calculated using the parallel-tube model and incorporating the fraction unbound in blood, with in vitro CLint adjusted for microsomal binding. For these reference drugs, 29% of predictions were within two-fold of the observed values and 66% were within five-fold. Compared with HLMs, clearance predictions with cryopreserved hepatocytes (57 drugs) were of similar precision (RMSE = 3608) but showed more bias (AFE = 5.21) with 18% of predictions within two-fold of the observed values and 46% within five-fold. However, with a broad complement of drug-metabolizing enzymes, hepatocytes catalysed measurable CLint values for a greater proportion (52%) of the reference compounds and were particularly proficient at defining metabolic rates for drugs with predominantly phase 2 metabolic routes. [ABSTRACT FROM AUTHOR]

The purpose of this study was to examine the lethal and sublethal toxicity of cadmium telluride (CdTe) quantum dots (Q-dots) in primary cultures of rainbow trout hepatocytes. Hepatocytes were exposed to concentrations of positively coated and aged (2 months and 2 years) Q-dots for 48 h at 15°C. Post treatment, cellular analysis was done of cell viability, lipid peroxidation (LPO), DNA damage, metallothioneins (MT), labile zinc/cadmium and heat shock proteins (chaperones). Q-dots were found to be toxic to fish hepatocytes at a threshold concentration of 0.1 mg/l. These nanoparticles increased the levels of MT, labile zinc, DNA strand breaks and heat shock proteins of the 70 kDa family. The strongest response was observed with the molecular chaperone of the 70 kDa family, reaching a 7-fold induction in exposed cells. Overall, the assessment of multiple biomarkers in trout hepatocytes exposed to differently 'aged' Q-dots suggests that the cytotoxic responses to two-year-old positively coated CdTe Q-dots were largely due to the liberation of Cd2+. [ABSTRACT FROM AUTHOR]

Objective. A rat model was designed to determine if fetal dexamethasone exposure is associated with decreased insulin sensitivity in adulthood, manifested as decreased binding of p85 to phosphorylated insulin receptor substrate-1 (IRS-1) within the phosphatidylinositol 3-kinase (PI 3-kinase) pathway of insulin signaling. Additionally, the study investigated whether a differential effect exists in male and female rodent offspring, such that females demonstrate decreased binding of p85 to IRS-1 after exposure to dexamethasone in utero. Methods. Timed-pregnant Sprague-Dawley rats received either tap water or dexamethasone in drinking water from day 13 of gestation until delivery. Fasting male and female offspring from each group were sacrificed on day 50 of life, and half of these rats were given insulin subcutaneously prior to sacrifice. Adipocyte, skeletal muscle, and liver cell lysates were analyzed by immunoprecipitation and Western blotting of IRS-1 and IRS-1-associated p85. Results. In all tissues examined, a significant inverse relationship was found between dexamethasone treatment in utero and association of p85 with IRS-1 in adulthood. p85 association with IRS-1 was similar in tissues from fasting and insulin-stimulated states. Furthermore, tissues of male and female rats demonstrated similar binding of p85 to IRS-1. Conclusions. In a rat model, fetal exposure to dexamethasone was associated with decreased insulin signaling at the level of p85 binding to IRS-1, a proximal step in insulin signaling within the PI 3-kinase pathway. This effect did not appear to be enhanced by administration of insulin prior to sacrifice, nor was a sex-dependent differential effect noted. [ABSTRACT FROM AUTHOR]

A porous chitosan-gelatin scaffold with specific geometry and complex internal microarchitectures was fabricated via a novel indirect rapid prototyping technique. A stereolithography (SL) technique was employed to build the resin mold, based on which a high-resolution poly-dimethylsilicone (PDMS) mold was microreplicated. The chitosan-gelatin hybrid solution was cast on the PDMS mold for pre-freezing and the monolayer scaffold with predefined features and highly porous structures was produced upon lyophilization. These monolayer structures along with the special interface layers can be stacked together to form a three-dimensional (3D) scaffold. The morphologies of the predefined features maintain well during the assembly process. Hepatocyte culture experiment indicates that the novel microstructured chitosan-gelatin porous scaffold could facilitate cells to migrate and proliferate into the deep layers of the scaffold in comparison with the traditional porous only scaffold. This approach provides a new way to fabricate a complex 3D scaffold using various natural biomaterials for vital organ engineering. [ABSTRACT FROM AUTHOR]

The inhibitory effects of cationic drugs (β-adrenoreceptor antagonists, calcium (Ca)-channel blocker, If channel inhibitor, antiarrhythmic drugs, and antibacterial drugs) that inhibit 1-methyl-4-phenylpyridinium (MPP) and/or metformin uptake into hOCT1-3/rOct1-3-expressing cells and human/rat hepatocytes were investigated in this study. The drug-drug interaction (DDI) potential of these drugs for the hOCT/rOct-mediated hepatic/renal uptake process was also assessed. The IC50 values of cardiovascular drugs, including an If channel inhibitor with a new mechanism of action, were greater for hOCT2/rOct2 than those for hOCT1/rOct1 or hOCT3/rOct3. No species differences in these values were observed between hOCTs and rOcts. As for hOCT2-mediated uptake, the IC50 values of quinidine and the If channel inhibitor for metformin uptake were lower than those for MPP uptake. However, previous clinical studies found that the IC50 values of these drugs for hOCT1/rOct1 and hOCT2/rOct2 were much greater than their unbound plasma concentrations, which suggests that the DDIs of these cationic compounds may not be related to hOCT/rOct-mediated hepatic/renal uptake pathways. In addition, investigation of the luminal transporters of cationic compounds in the kidney, as well as the in vitro DDI potential of their inhibitors, is important for the clarification of cationic compound DDIs in humans. [ABSTRACT FROM AUTHOR]

The linkage point between kinetics and dynamics of drugs and other foreign chemicals is at the active site of the target. The response is determined within certain limits by the concentration of the substance at the target and its mechanism of action. However, the concentration of the substance at the active site under in vivo conditions cannot be easily measured, and surrogates such as plasma concentration are used instead. The emergence of in vitro approaches to study some primary pharmacokinetic (PK) and pharmacodynamic (PD) processes in simpler experimental setups could provide ways to estimate the concentration at the active site, which then can be used in a more reliable extrapolation and prediction of in vivo behavior and action of the substance. Imaging and other novel analytical techniques are providing direct or indirect measurements of in vivo concentrations in tissues. In this review, processes such as transport, metabolism, etc. affecting the concentration of the substance at the active site are discussed, analytical possibilities to measure directly or estimate indirectly the concentration at the active site are illustrated, the overall design and performance of physiologically based PK-PD models are described, and some examples concerning blood-brain barrier, liver, and lungs are presented. In conclusion, several important recommendations are made: (1) In vitro systems should be characterized in a much more detailed way than has been done so far, especially with respect to unbound concentrations at the sites of disposition and action; (2) kinetics of the compound in the in vitro system have to be more thoroughly characterized; (3) local models encompassing the in vitro system should be developed and used; and (4) PBPK/PD models should incorporate these local models. [ABSTRACT FROM AUTHOR]

Hepatic hollow fiber bioreactors are a promising class of bioartificial liver assist device (BLAD). The development of this type of device is currently hindered by limited oxygen transport to cultured hepatocytes, due to low solubility of oxygen in aqueous media. In order to increase the oxygen spectrum to cultured hepatocytes housed within a hollow fiber bioreactor, several different engineering strategies were explored in this study. These included: supplementing the circulating media stream of the hollow fiber bioreactor with a hemoglobin-based oxygen carrier (bovine red blood cells) with defined oxygen binding and release kinetics and operating the bioreactor with media flow through the hollow fiber membrane into the extracapillary space (ECS). We hypothesize that these two strategies can be used to improve hepatocyte oxygenation and possibly attain an in vivo-like pO2 spectrum, similar to that observed in vivo in the liver sinusoid. This work is significant, since provision of an in vivo-like pO2 spectrum should create a fully functional BLAD that could potentially bridge thousands of liver failure patients towards native liver regeneration of damaged tissue or, if necessary, orthotopic liver transplantation. [ABSTRACT FROM AUTHOR]

Fumonisin B1 (FB1), the most prevalent member of toxins produced by several species of Fusarium molds, which occur mainly in maize, causes several fatal hepatopathies and nephropathies of animals. The current study was scrutinized to ascertain different cytotoxic and morphological transformations in rat hepatocytes induced by the treatments of diverse concentrations (300, 500, or 1000 μM) of fumonisin B1 in vitro, using both monolayer and spheroid cultures. In each hepatocyte culture, the cytotoxicity of FB1 was augmented in dose- and time-response manners. Morphological transformations among FB1-treated groups integrated accumulation of lipid droplets, cytoplasmic vacuolation in hepatocyte monolayers, and bleb formation in the hepatocyte spheroids. Additionally, electron microscopy revealed the loss of microvilli, mitochondrial swelling, and formation of lamellar membranous whorl in the vacuoles and bile canaliculi-like structures. Appearance of electron dense bodies in the monolayers, and loss of cell-to-cell contact in spheroids were depicted in 1000 μM FB1-treated hepatocytes. These outcomes insinuate different vital events in explaining morphological transformations in the cell membrane and organelles, induced by fumonisins in rat hepatocytes. [ABSTRACT FROM AUTHOR]

Objective. In infection with hepatitis C virus (HCV), spontaneous clearance of the virus occurs in 30-40% of cases. By contrast, in chronic infection, this is rare. The basis for viral clearance in acute disease is unknown. Whereas cellular immune responses have been studied in detail, few data exist on the role of viral structural proteins, such as the core protein. The purpose of this study was to investigate the effects of core produced de novo within adult mouse hepatocytes by using a new transgenic mouse line in which expression of HCV core is regulated by tetracycline (tet-off). Material and methods. In this work, transgenic mice with conditional HCV core were created, to study the acute expression of HCV core protein in the context of the mature liver. The subcellular distribution of the core, hepatocellular oxidative stress and apoptosis were monitored. Results. Core protein is readily detectable and strongly associated with cytoplasmic lipid vesicles, endoplasmic reticulum and mitochondria. Mitochondrial oxidative stress was evidenced by a reduction in thioredoxin-2 (trx2). Concurrently, caspase-3 activity and TUNEL increased and, over time, the level of core protein in the liver declined. Conclusions. Mice that are conditionally transgenic for HCV core protein, which is readily detected and morphologically associated with steatosis in individual hepatocytes, were developed. Acute expression of core protein causes mitochondrial stress, as demonstrated by a reduction in trx2 and in the apoptosis of core-positive hepatocytes. We speculate that these events could be involved in the clearance of virus during acute hepatitis C, by both reducing the burden of virus in the liver and effectively priming the immune response. [ABSTRACT FROM AUTHOR]

Chimeric mice with a humanized liver have been previously established by the transplantation of human hepatocytes to urokinase-type plasminogen activator/severe combined immunodeficiency mice. A non-invasive method to detect the induction of cytochrome P450 (CYP) 3A4 was evaluated in chimeric mice with a humanized liver. Dexamethasone (DEX) was used as a probe drug to detect induction; and rifampicin was used as a model drug to induce CYP3A4. Before and after rifampicin treatment (50 mg kg-1, intraperitoneal injection once a day for 4 days) in the chimeric mice, DEX was subcutaneously injected and the urinary excretion of 6β-hydroxydexamethason (6βOHD) and DEX was determined. The metabolic ratio (6βOHD/DEX) significantly increased after rifampicin treatment. Livers from the control and rifampicin-treated chimeric mice were stained immunohistolochemically with antibodies against CYP3A4 and CYP3A5. CYP3A4 and CYP3A5 were detected in the area of humanized liver, but staining was intense for CYP3A4 and very weak for CYP3A5. Only the staining of CYP3A4 was increased after rifampicin treatment. Formation of 6βOHD by human liver microsomes was higher than that formed by mouse liver microsomes. Metabolite formation was catalysed by both CYP3A4 and CYP3A5 and the intrinsic clearance (Vmax/Km) by CYP3A4 was found to be 50-fold higher than that of CYP3A5. The results of the present study indicate that estimation of the changes of the urinary metabolic ratio (6βOHD/DEX) in the chimeric mice with a humanized liver is a very useful tool for detecting the induction of CYP3A4 by a non-invasive method. [ABSTRACT FROM AUTHOR]

Usnic acid is a component of nutritional supplements promoted for weight loss that have been associated with liver-related adverse events including mild hepatic toxicity, chemical hepatitis, and liver failure requiring transplant. To determine if metabolism factors might have had a role in defining individual susceptibility to hepatotoxicity, in vitro metabolism studies were undertaken using human plasma, hepatocytes, and liver subcellular fractions. Usnic acid was metabolized to form three monohydroxylated metabolites and two regio-isomeric glucuronide conjugates of the parent drug. Oxidative metabolism was mainly by cytochrome P450 (CYP) 1A2 and glucuronidation was carried out by uridine diphosphate-glucuronosyltransferase (UGT) 1A1 and UGT1A3. In human hepatocytes, usnic acid at 20 µM was not an inducer of CYP1A2, CYP2B6, or CYP3A4 relative to positive controls omeprazole, phenobarbital, and rifampicin, respectively. Usnic acid was a relatively weak inhibitor of CYP2D6 and a potent inhibitor of CYP2C19 (the concentration eliciting 50% inhibition (IC50) = 9 nM) and CYP2C9 (IC50 = 94 nM), with less potent inhibition of CYP2C8 (IC50 = 1.9 µM) and CYP2C18 (IC50 = 6.3 µM). Pre-incubation of microsomes with usnic acid did not afford any evidence of time-dependent inhibition of CYP2C19, although evidence of slight time-dependent inhibition of CYP2C9 (KI = 2.79 µM and Kinact = 0.022 min-1) was obtained. In vitro data were used with SimCYPRto model potential drug interactions. Based on usnic acid doses in case reports of 450 mg to >1 g day-1, these in vitro data indicate that usnic acid has significant potential to interact with other medications. Individual characteristics such as CYP1A induction status, co-administration of CYP1A2 inhibitors, UGT1A1 polymorphisms, and related hyperbilirubinaemias, or co-administration of low therapeutic index CYP2C substrates could work alone or in consort with other idiosyncrasy risk factors to increase the risk of adverse events and/or hepatotoxicity. Thus, usnic acid in nutritional supplements might be involved as both victim and/or perpetrator in clinically significant drug-drug interactions. [ABSTRACT FROM AUTHOR]

Cultured rat hepatocytes and human hepatoma HepG2 cells were used to evaluate the hepatoprotective properties of polyphenolic extracts from the edible part of artichoke (AE). The hepatocytes were exposed to H2O2generated in situ by glucose oxidase and were treated with either AE, or pure chlorogenic acid (ChA) or with the well known antioxidant, N, N'-diphenyl-p-phenilenediamine (DPPD). Addition of glucose oxidase to the culture medium caused depletion of intracellular glutathione (GSH) content, accumulation of malondialdehyde (MDA) in the cultures, as a lipid peroxidation indicator, and cell death. These results demonstrated that AE protected cells from the oxidative stress caused by glucose oxidase, comparable to DPPD. Furthermore, AE, as well as ChA, prevented the loss of total GSH and the accumulation of MDA. Treatment of HepG2 cells for 24 h with AE reduced cell viability in a dose-dependent manner, however, ChA had no prominent effects on the cell death rate. Similarly, AE rather than ChA induced apoptosis, measured by flow cytometric analysis of annexin and by activation of caspase-3, in HepG2 cells. Our findings indicate that AE had a marked antioxidative potential that protects hepatocytes from an oxidative stress. Furthermore, AE reduced cell viability and had an apoptotic activity on a human liver cancer cell line. [ABSTRACT FROM AUTHOR]

Hepatic clearance prediction, using scaled data obtained from hepatocytes and microsomes, often under-predicts the eventual observed clearance. This occurs commonly where the compound is highly bound and/or a sinusoidal transporter substrate. The authors' own laboratory observations and those reported in the literature indicate that consideration of transporter effects in vitro is not sufficient to provide a direct, quantitative estimate of hepatic clearance in vivo. The physiology of contributing processes has been reviewed and the processes were compiled into a kinetic model of compound disposition for a hepatocyte compartment. The model has variables describing the kinetic effects of plasma protein binding, sinusoidal uptake, passive permeability, and cellular disposition. Parameters were determined experimentally requiring, in some instances, assays less familiar or routine to Drug metabolism and pharmacokinetics (DMPK) laboratories. The model accurately fitted data for the hepatic disposition of a UCB-proprietary compound that did not undergo metabolism but was a substrate for protein binding and sinusoidal uptake. On addition of bovine serum albumin to the assay, the uptake kinetics approximated neither those for the free fraction nor the total concentration. However, the model accurately predicted the intermediate observed kinetics and illustrated the kinetic effects of plasma protein binding and the complex interplay between various competing and collaborating processes. Through the use of simulations the model illustrated the influence of different combinations and influences of hepatic kinetic processes. Possible causes behind the extraction of highly bound compounds were identified as (1) low compound permeability facilitating active uptake and (2) high permeability facilitating the role of intracellular metabolism. In almost all circumstances koff is not low enough (<1 s-1) to limit the extraction of plasma protein bound compounds; however, kon still exerts an effect through competition with the uptake process. Crucially the model indicated the complex nature of the combined processes, whose broad range of effects on hepatic disposition could only be accurately determined with a kinetic model. [ABSTRACT FROM AUTHOR]

The in vitro metabolism of M445526 (ZD6126 phenol) was investigated by incubating [14C]-M445526 at a concentration of 10 µg ml-1 with human hepatic microsomes (4 mg ml-1) or human hepatocytes (2 × 106 cells ml-1) for up to 180 min. Following incubation with microsomes and hepatocytes, up to 78% and 40% of [14C]-M445526 was metabolized after 180 and 120 min, respectively. High-performance liquid chromatography (HPLC) with radiochemical detection confirmed extensive metabolism of [14C]-M445526 by microsomes and hepatocytes. Mass spectrometry and 1H-NMR spectroscopy enabled structural identification of up to eight metabolites. Human liver microsomes formed one major (O-desmethyl) and three minor (a further O-desmethyl and two different hydroxylated) phase I metabolites. Human hepatocytes produced one major metabolite, a sulphate conjugate of the major O-desmethyl metabolite formed by microsomes. Four minor metabolites were also formed, primarily by O-demethylation with subsequent glucuronidation. Taken collectively, [14C]-M445526 underwent extensive in vitro metabolism by human liver fractions. These data were confirmed by subsequent human in vivo studies. [ABSTRACT FROM AUTHOR]

Polychlorinated biphenyls (PCBs) are a group of persistent and widely distributed environmental pollutants that have various deleterious effects, e.g., neurotoxic, endocrine disruption and reproductive abnormalities, including cancers. Chronic exposure to environmentally hazardous chemicals like PCBs is of great concern to human health. It has been reported earlier that apoptotic proteins change in rats under chronic PCB treatment. It is of importance to determine if chronically exposed human cells develop a different protein expression. In the present study, the authors chronically exposed metabolically competent human liver (HepG2) cells at 50 to 100 μM to examine the role of the well-known environmentally hazardous pollutant non-coplanar 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) to study cell death. After 12 weeks of exposure these cells showed significant changes in apoptotic death in subsequent trypan blue growth assay, fluorescence microscopy, DNA fragmentation, and immunoblotting studies. Interestingly, chronically exposed cells showed marked differences in apoptotic and/or death-related proteins (e.g., Bcl2, Bak, and the pro and active forms of caspase-9, which were up-regulated), in contrast to acutely exposed (i.e., 48-h PCB-153 exposed) cells, which maintained linear growth despite repeated exposures. Similarly, tumor suppressor protein p53, proto-oncogene c-myc, and cell cycle regulator protein p21 were also up-regulated compared to nonchronically exposed HepG2 Cells. The results indicated that PCB-153-induced chronic exposure significantly altered different apoptotic (e.g., Bcl2, Bak, caspase-3) and tumor suppressor (e.g., p21, p53, and c-myc) proteins in the cellular model. These results suggest that chronic exposure to PCB-153 can induce cell survival by altering several apoptotic and tumor suppressor proteins. [ABSTRACT FROM AUTHOR]