NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies

Abstract

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

Design and Characterization of the First Selective and Potent Mechanism-Based Inhibitor of Cytochrome P450 4Z1

“…A mammalian enzyme source was preferred, and, historically, HepG2 cells have been suitable for heterologous expression of CYPs due to their very low basal CYP levels and catalytically sufficient amounts of the co-enzymes cytochrome P450 reductase and cytochrome b 5 . , Therefore, a CYP4Z1-expressing HepG2 cell line was generated via lentiviral transduction (Figure S1A) and membranes prepared from these cells were used as the primary enzyme source for screening. This expression system provided an adequate amount of CYP4Z1, with no detectable enzyme in a control vector cell line, as analyzed by western blot (Figure S1B).…”

Inhibition of phase-1 biotransformation and cytostatic effects of diphenyleneiodonium on hepatoblastoma cell line HepG2 and a CYP3A4-overexpressing HepG2 cell clone

“…We were interested to evaluate the potential of diphenyleneiodonium (DPI) for the targeted modification of phase-1 monooxygenase activity in cell-based in vitro systems based on previous results from other groups [13,15,23,39]. HepG2 cells as well as recombinant CYP3A4-overexpressing HepG2 cells were used as hepatocyte model systems for functional and toxicological studies [17,[46][47][48][49][50]. HepG2 exhibit in vitro low basal CYP activity and are therefore well suited for recombinant modification with specific CYP activities [44,51].…”

“…For the prediction of the pharmacokinetics of new drug candidates, including relevant metabolites and hepatotoxicity, a clear understanding of the enzymatic phase-1 and -2 reactions interplay in the liver is crucial. In this context, preclinical drug screening with regard to biotransformation and toxicology is mostly based on physiologically relevant sensitive, reliable and in particular adaptable in vitro metabolism models of human hepatocytes [17][18][19][20]. Research into specific scientific issues also involves the availability of substances for targeted modulation.…”

Preclinical models of idiosyncratic drug-induced liver injury (iDILI): Moving towards prediction

“…However, as with any other cell model, results must be taken cautiously due to differences in protein expression compared to in vivo tissues. For instance, Upcyte® hepatocytes have a significantly lower NADPH-cytochrome P450 reductase activity 66 , and low expression of proteins of sinusoidal solute carrier transporters (except for NTCP and OATP2B1). In contrast, Upcyte® cells have a well-preserved expression of canalicular efflux pumps proteins, and two-dimensional sandwich configuration preserves the expression of organic anion-transporting polypeptide OATP1B1/SLCO1B1, OATP2B1/SLCO2B1, NTCP/SLC10A1, and OCT1/SLC22A1 67 .…”