BACKGROUND: Individual hepatocyte cell tradition systems are important models for drug development and toxicology studies in the context of liver xenobiotic metabolism. prolonged period, making them comparable to primary human being hepatocytes. These hepatocyte models display active liver rate of metabolism such as urea and glycogen formation as well as biotransformation of xenobiotics. Clofilium tosylate 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 Clofilium tosylate 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 Clofilium tosylate levels in isolated microsomes, which were comparable between all cell systems tested. A tendency of higher POR activity in HepG2 cells compared to HepaFH3 (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. hepatocyte metabolism models for preclinical screening of drug conversion, clearance and potential hepatotoxicity. A clear understanding of the enzymatic interplay to enable complete liver phase I and phase II reactions is crucial for the prediction of drug pharmacokinetics. This can be affected by dynamic variability within and between individuals, age-related modifications as well as by genetic polymorphisms of relevant enzymes [2C4]. In phase I metabolism, cytochrome P450 monooxygenases (CYPs) represent the most prominent enzyme family for oxidative biotransformation of drugs and other lipophilic xenobiotics [5, 6]. From the 57 known human CYPs only about a handful enzymes, mostly PRKM1 belonging to CYP-families 1, 2 and 3, are responsible for the metabolisation of more than three quarters of FDA-approved drugs [7, 8]. Preclinical evaluation of novel drug candidates and scientific investigation of already used drugs rely on physiologically relevant models of human hepatocytes for metabolism, biotransformation and toxicology studies. Currently, primary human hepatocytes (pHHs) are the gold standard for studies on hepatic metabolism, clearance, hepatotoxicity and drug-drug interaction . However, this research is still restricted by pHH scarcity, donor variability and their rapid dedifferentiation [10C14]. An inflammatory response by endotoxin contamination [15, 16] originating from bacterial collagenase preparations, loss of normal cell polarity when dissolving them from liver tissue or down-regulation of liver-specific transcription factors influencing phase I/II protein expression were discussed as you can causes [17C19]. To conquer these limitations, many liver organ cancer-derived cell lines such as for example HepG2 and HepaRG had been formulated to serve as surrogate for pHHs. Advantages are their unlimited availability, easy proliferative and handling capacity [20C24]. A definite disadvantage can be their hereditary instability because of the cancer origin, making them nearly unusable for medical applications such as for example disease-related liver organ repopulation. Used HepG2 cells Widely, a human being hepatoblastoma cell range, exhibit just low expressions and actions of almost all stage I CYP enzymes and for that reason have limited worth for prediction of hepatic biotransformation. Nevertheless, several methods to boost HepG2 liver organ function were referred to in the books [25, 26]. Included in these are genetic engineering from the cell range to improve CYP manifestation. Lately released data proven the effective era of a genetically modified CYP3A4-overexpressing HepG2 clone, showing considerable enzymatic activity for this specific CYP . A promising approach to obtain a more relevant surrogate of pHHs for biotransformation and toxicology studies is the use of primary-like hepatocytes such as HepaFH3 cells and Upcyte? hepatocytes, which are now available in the second generation [28, 29]. These cell strains are proliferation-competent by lentivirus-mediated transduction of defined proliferation genes (Upcyte? factors). They show some improved liver functions compared to cell lines such as HepG2.