Publications Search Results
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PublicationIn vitro biotransformation assays using fish liver cells: Comparing rainbow trout and carp hepatocytes( 2022-09-23)Biotransformation assays using primary hepatocytes from rainbow trout, Oncorhynchus mykiss, were validated as a reliable in vitro tool to predict in vivo bioconcentration factors (BCF) of chemicals in fish. Given the pronounced interspecies differences of chemical biotransformation, the present study aimed to compare biotransformation rate values and BCF predictions obtained with hepatocytes from the cold-water species, rainbow trout, to data obtained with hepatocytes of the warm-water species, common carp (Cyprinus carpio). In a first step, we adapted the protocol for the trout hepatocyte assay, including the cryopreservation method, to carp hepatocytes. The successful adaptation serves as proof of principle that the in vitro hepatocyte biotransformation assays can be technically transferred across fish species. In a second step, we compared the in vitro intrinsic clearance rates (CLin vitro, int) of two model xenobiotics, benzo[a]pyrene (BaP) and methoxychlor (MXC), in trout and carp hepatocytes. The in vitro data were used to predict in vivo biotransformation rate constants (kB) and BCFs, which were then compared to measured in vivo kB and BCF values. The CLin vitro, int values of BaP and MXC did not differ significantly between trout and carp hepatocytes, but the predicted BCF values were significantly higher in trout than in carp. In contrast, the measured in vivo BCF values did not differ significantly between the two species. A possible explanation of this discrepancy is that the existing in vitro-in vivo prediction models are parameterized only for trout but not for carp. Therefore, future research needs to develop species-specific extrapolation models.
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PublicationComparison of Alternative Methods for Bioaccumulation Assessment: Scope and Limitations of In Vitro Depletion Assays with Rainbow Trout and Bioconcentration Tests in the Freshwater Amphipod Hyalella azteca( 2020)Bioaccumulation assessment predominantly relies on the bioconcentration factor (BCF) as the sole decisive metric. The test guideline 305 by the Organisation for Economic Co‐operation and Development (OECD) provides the standard procedure for deriving this in vivo fish BCF, which is not only expensive and labor‐intensive, but also requires many animals. Accordingly, there is a great need for and interest in alternative methods that can help to reduce, replace, and refine vertebrate tests, as described in the 3R principles. Two alternative approaches have been developed: the bioconcentration test with the freshwater amphipod Hyalella azteca and the OECD test guideline 319 which provides a method to determine experimentally derived in vitro metabolism rates that can then be incorporated into in silico prediction models for rainbow trout BCF calculation. In the present study both alternative methods were applied to 5 substances of different physicochemical characteristics. The results were compared with literature values of fish in vivo BCFs and additional BCFs obtained with the alternative methods, if available. Potential differences between the results oft he test methods are discussed utilizing information such as in vivo metabolism rates. The currently available data set suggests that these 2 alternative methods pose promising alternatives to predict bioaccumulation in fish, although defined applicability domains have yet to be determined. Environ Toxicol Chem 2020;00:1-13.
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PublicationMitochondrial transfer by human mesenchymal stromal cells ameliorates hepatocyte lipid load in a mouse model of NASH( 2020)
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PublicationNADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies( 2019)BACKGROUND: Human hepatocyte in vitro cell culture systems are important models for drug development and toxicology studies in the context of liver xenobiotic metabolism. Often, such culture systems are used to elucidate the biotransformation of xenobiotics or drugs and further investigate drug and drug metabolite effects on biological systems in terms of potential therapeutic benefit or toxicity. Human hepatocytes currently used for such in vitro studies are mostly primary cells or cell lines derived from liver cancers. Both approaches have limitations such as low proliferation capacity and progressive dedifferentiation found in primary cells or lack of liver functions in cell lines, which makes it difficult to reliably predict biotransformation of xenobiotics in patients. In order to overcome these limitations, HepaFH3 cells and Upcyte® hepatocytes representing primary-like hepatocytes of the first and second generation are increasingly used. Based on primary human hepatocyte cells transduced for stable expression of Upcyte® proliferation genes, they are mitotically active and exhibit liver functions over an extended period, making them comparable to primary human hepatocytes. These hepatocyte models show active liver metabolism such as urea and glycogen formation as well as biotransformation of xenobiotics. The latter is based on the expression, activity and inducibility of cytochrome P450 enzymes (CYP) as essential phase I reaction components. However, for further characterisation in terms of performance and existing limitations, additional studies are needed to elucidate the mechanisms involved in phase I reactions. One prerequisite is sufficient activity of microsomal NADPH-cytochrome P450 reductase (POR) functionally connected as electron donor to those CYP enzymes. OBJECTIVE: For Upcyte® hepatocytes and HepaFH3 cells, it is so far unknown to what extent POR is expressed, active, and may exert CYP-modulating effects. Here we studied POR expression and corresponding enzyme activity in human hepatoblastoma cell line HepG2 and compared this with HepaFH3 and Upcyte® hepatocytes representing proliferating primary-like hepatocytes. METHODS: POR expression of those hepatocyte models was determined at mRNA and protein level using qRT-PCR, Western Blot and immunofluorescence staining. Kinetic studies on POR activity in isolated microsomes were performed by a colorimetric method. RESULTS: The investigated hepatocyte models showed remarkable differences at the level of POR expression. Compared to primary-like hepatocytes, POR expression of HepG2 cells was 4-fold higher at mRNA and 2-fold higher at protein level. However, this higher expression did not correlate with corresponding enzyme activity levels in isolated microsomes, which were comparable between all cell systems tested. A tendency of higher POR activity in HepG2 cells compared to HepaFH3 (p = 0.0829) might be present. Compared to primary human hepatocyte microsomes, POR activity was considerably lower in all hepatocyte models. CONCLUSION: In summary, our study revealed that POR expression and activity were clearly detectable in all in vitro hepatocyte models with the highest POR expression in cancer cell line HepG2. However, POR activity was lower in tested hepatocyte models when compared to human primary hepatocyte microsomes. Whether this was caused by e.g. polymorphisms or metabolic differences of investigated hepatocyte models will be target for future studies.
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PublicationToxicogenomics directory of rat hepatotoxicants in vivo and in cultivated hepatocytes( 2018)Transcriptomics is developing into an invaluable tool in toxicology. The aim of this study was, using a transcriptomics approach, to identify genes that respond similar to many different chemicals (including drugs and industrial compounds) in both rat liver in vivo and in cultivated hepatocytes. For this purpose, we analyzed Affymetrix microarray expression data from 162 compounds that were previously tested in a concentration-dependent manner in rat livers in vivo and in rat hepatocytes cultivated in sandwich culture. These data were obtained from the Japanese Toxicogenomics Project (TGP) and North Rhine-Westphalian (NRW) data sets, which represent 138 and 29 compounds, respectively, and have only 5 compounds in common between them. The in vitro gene expression data from the NRW data set were generated in the present study, while TGP is publicly available. For each of the data sets, the overlap between up- or down-regulated genes in vitro and in vivo was identified, and named in vitro-in vivo consensus genes. Interestingly, the in vivo-in vitro consensus genes overlapped to a remarkable extent between both data sets, and were 21-times (upregulated genes) or 12-times (down-regulated genes) enriched compared to random expectation. Finally, the genes in the TGP and NRW overlap were used to identify the upregulated genes with the highest compound coverage, resulting in a seven-gene set of Cyp1a1, Ugt2b1, Cdkn1a, Mdm2, Aldh1a1, Cyp4a3, and Ehhadh. This seven-gene set was then successfully tested with structural analogues of valproic acid that are not present in the TGP and NRW data sets. In conclusion, the seven-gene set identified in the present study responds similarly in vitro and in vivo to a wide range of different chemicals. Despite these promising results with the seven-gene set, transcriptomics with cultivated rat hepatocytes remains a challenge, because in general many genes are up- or downregulated by in vitro culture per se, respond differently to test compounds in vitro and in vivo, and/or show higher variability in the in vitro system compared to the corresponding in vivo data.
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PublicationImpaired cell viability and functionality of hepatocytes after incubation with septic plasma( 2018)Liver dysfunction (LD) and liver failure are associated with poor outcome in critically ill patients. In patients with severe sepsis or septic shock, LD occurred in nearly 19% of patients. An early diagnosis of LD at time of initial damage of the liver can lead to a better prognosis of these patients because an early start of therapy is possible. We performed a second prospective study with septic patients to test a new cell-based cytotoxicity device (biosensor) to evaluate clinical relevance for early diagnosis of LD and prognostic capacity. In the clinical study, 99 intensive care unit patients were included in two groups. From the patients of the septic group (n = 51, SG), and the control (non-septic) group [n = 49, control group (CG)] were drawn 20 ml blood at inclusion, after 3, and 7 days for testing with the biosensor. Patients' data were recorded for hospital survival, organ function, and demographic data, illness severity [acute physiology and chronic health evaluation (APACHE) II-, sepsis-related organ failure assessment (SOFA) scores], cytokines, circulating-free deoxyribonucleic acid/neutrophil-derived extracellular traps (cf-DNA/NETs), microbiological results, and pre-morbidity. For the developed cytotoxicity test, the human liver cell line HepG2/C3A was used. Patients' plasma was incubated in a microtiter plate assay with the test cells and after 6 days incubation the viability (trypan blue staining, XTT-test) and functionality (synthesis of albumin, cytochrome 1A2 activity) was analyzed. An impairment of viability and functionality of test cells was only seen in the SG compared with the CG. The plasma of non-survivors in the SG led to a more pronounced impairment of test cells than the plasma of survivors at inclusion. In addition, the levels of cf-DNA/NETs were significantly higher in the SG at inclusion, after 3, and after 7 days compared with the CG. The SG showed an in-hospital mortality of 24% and the values of bilirubin, APACHE II-, and SOFA scores were markedly higher at inclusion than in the CG. Hepatotoxicity of septic plasma was already detected with the liver cell-based biosensor at inclusion and also in the course of disease. The biosensor may be a tool for early diagnosis of LD in septic patients and may have prognostic relevance.
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PublicationExtracellular-signal regulated kinase (Erk1/2), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and tristetraprolin (TTP) comprehensively regulate injury-induced immediate early gene (IEG) response in in vitro liver organ culture( 2016)Differentiated hepatocytes are long-lived and normally do not undergo cell division, however they have the unique capacity to autonomously decide their replication fate after liver injury. In this context, the key players of liver regeneration immediately after injury have not been adequately studied. Using an in vitro liver culture system, we show that after liver injury, p38 mitogen-activated protein kinase (p38MAPK), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and extracellular-signal regulated kinase (Erk)1/2 were activated within 15 min and continued to be phosphorylated for more than 2 h. Both p38MAPK and Erk1/2 were activated at the edge of the cut as well as on the liver surface where the mesothelial cell sheet expresses several cytokines. Notably, in human liver Erk1/2 was also activated under the mesothelial cell sheet shortly after liver resections. Furthermore, in in vitro liver slice culture immediate early genes (IEGs) were upregulated within 1-2 h and the S phase marker proliferation-cell-nuclear-antigen (PCNA) appeared 24 h after injury. Although Erk1/2 was activated after injury, in MK2 depleted liver a set of IEGs, such as Dusp1, Cox2, or c-Myc and proliferation marker gene Ki67 were not induced. In addition, in immortalized hepatocyte cells, THLE-2, the same subset of genes was upregulated upon stimulation with lipopolysaccharide (LPS), but not in the presence of MK2 inhibitor. The protein level of tristetraprolin (TTP), a substrate for MU that plays a role in mRNA degradation, was increased in the presence of MK2 inhibitor. In this context, the depletion of TIP gene rescued Dusp1, Cox2, or c-Myc upregulation in the presence of MK2 inhibitor. These data imply that MK2 pathway is positively involved in Erk1/2 induced IEG response after liver injury. These data also suggest that in vitro liver culture may be a useful tool for measuring the proliferation potential of hepatocytes in individual liver.
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PublicationHepatocytes as in vitro test system to investigate metabolite patterns of pesticides in farmed rainbow trout and common carp: Comparison between in vivo and in vitro and across species( 2016)In vitro tools using isolated primary fish hepatocytes have been proposed as a useful model to study the hepatic metabolism of xenobiotics in fish. In order to evaluate the potential of in vitro fish hepatocyte assays to provide information on in vivo metabolite patterns of pesticides in farmed fish, the present study addressed the following questions: Are in vitro and in vivo metabolite patterns comparable? Are species specific differences of metabolite patterns in vivo reflected in vitro? Are metabolite patterns obtained from cryopreserved hepatocytes comparable to those from freshly isolated cells? Rainbow trout and common carp were dosed orally with feed containing the pesticide methoxychlor (MXC) for 14 days. In parallel, in vitro incubations using suspensions of freshly isolated or cryopreserved primary hepatocytes obtained from both species were performed. In vivo and in vitro samples were analyzed by thin-layer chromatography with authentic standards supported by HPLC-MS. Comparable metabolite patterns from a qualitative perspective were observed in liver in vivo and in hepatocyte suspensions in vitro. Species specific differences of MXC metabolite patterns observed between rainbow trout and common carp in vivo were well reflected by experiments with hepatocytes in vitro. Finally, cryopreserved hepatocytes produced comparable metabolite patterns to freshly isolated cells. The results of this study indicate that the in vitro hepatocyte assay could be used to identify metabolite patterns of pesticides in farmed fish and could thus serve as a valuable tool to support in vivo studies as required for pesticides approval according to the EU regulation 1107.
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PublicationToxicogenomics applied to cultures of human hepatocytes enabled an identification of novel petasites hybridus extracts for the treatment of migraine with improved hepatobiliary safety( 2009)Butterbur extracts (petasites hybridus) are recommended for the prevention of migraine, but pharmacovigilance reports may be suggestive for rare hepatobiliary toxicity. In order to evaluate its hepatotoxic potential a series of in vivo and in vitro studies were carried out. Essentially, there were no signs of hepatocellular toxicity at estimated therapeutic Cmax levels of 60 ng/mL. Nonetheless, in a 28-day toxicity study at approximately 200-fold of therapeutic doses induced liver transaminases and bilirubin elevations were observed. In a subsequent 6-months chronic toxicity study the initial hepatobiliary effects were reproduced, but at the end of the study liver function recovered and returned to normal as evidenced by clinical chemistry measurements. To identify possible mechanisms of hepatotoxicity, we investigated liver function in vitro at >170-fold of therapeutic Cmax levels, including cytotoxicity (LDH, MTT, ATP), transaminase activities (ALT, AST), albumin synthesis, urea and testosterone metabolism to assay for CYP monooxygenase activity. Only with extracts rich in petasin (37% petasin) and at high well above therapeutic doses liver toxicity was observed. A toxicogenomic approach applied to hepatocyte cultures enabled hypothesis generation and was highly suggestive for extracts high in petasin content to impair bile acid transport, lipid and protein metabolism. Importantly, neither chronic rat in vivo nor rat in vitro studies predicted reliably hepatotoxicity, therefore re-emphasising the utility of human-based in vitro investigations for the development of safe medicinal products. Finally, toxicogenomics enabled the characterization of a novel butterbur extract with no signals for hepatotoxicity.
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