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ABSTRACT:In this study we describe a battery of fluorescence assays for rapid measurement in intact cells of the activity of nine cytochromes P450 (P450s) involved in drug metabolism. The assays are based on the direct incubation of monolayers of cells expressing individual P450 enzymes with a fluorogenic substrate followed by fluorimetric quantification of the product formed and released into incubation medium. For each individual P450 activity, different fluorescence probes were examined, and the one showing the best properties (highest metabolic rates, lowest background fluorescence) was selected: 3-cyano-7-ethoxycoumarin for CYP1A2 and CYP2C19, coumarin for CYP2A6, 7-ethoxy-4-trifluoromethylcoumarin for CYP2B6, dibenzylfluorescein for CYP2C8, 7-methoxy-4-trifluoromethylcoumarin ( Drug metabolism is one of the major determinants of drug clearance and the factor that is most frequently responsible for the interindividual differences in drug pharmacokinetics. Inappropriate pharmacokinetics result in an inadequate or variable clinical response of the drug that frequently compromises its therapeutic usage. Cytochrome P450 (P450) enzymes are major players in the oxidative metabolism of a wide range of structurally diverse xenobiotics including drugs. In the past two decades, thanks to the use of purified enzymes, the identification of selective substrates/ inhibitors, and the application of recombinant DNA technology to P450 genes, great progress has been made in the characterization of the role of human P450s in the metabolism of therapeutic agents. To speed up the selection of new drug candidates, pharmaceutical industries increasingly make use of different in vitro systems to investigate drug metabolism. As a result of this, it is now possible to identify the metabolic profile of drug candidates, potential drug interactions, or the role of polymorphic enzymes before clinical trials start, resulting in more cost-effective and ethically acceptable studies (Rodrigues, 1999).The large number of chemicals to be tested has created a need for high-throughput methods for screening of compounds for favorable metabolic properties in drug discovery. Among the different in vitro models developed for drug metabolism studies, recombinant P450 systems are now increasingly used (Masimirembwa et al., 1999;Rodrigues, 1999;Yoshitomi et al., 2001). P450 enzymes heterologously expressed in different cells show catalytic properties comparable with those of human liver microsomes (Gonzalez and Korzekwa, 1995;Masimirembwa et al., 1999). These enzymes can be produced in large amounts to meet the increasing demand of screening models for drug metabolism research. Metabolism of the new molecule can be easily examined after incubation with each P450-expressing system separately, helping to elucidate the metabolic pathways of the compound. The correct interpretation of kinetic data from cDNA-expression systems requires the confirmation of their metabolic competence. Therefore, measurement of individual P450 activities is needed for each ex...

Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation. INTRODUCTIONRecent observations from several laboratories indicate that membrane lipids regulate intracellular membrane transport, particularly in distal stages of the secretory pathway. Diacylglycerol (DAG) is a simple and small sized signal-transducing lipid which among other functions is necessary for protein transport from the Golgi complex to the cell surface both in yeast and in mammals. Thus, in budding yeast phosphatidylinositol (PI)-transfer Sec14p protein directly regulates DAG homeostasis in the Golgi complex and protein secretion (Bankaitis et al., 1990;Kearns et al., 1997;Huijbregts et al., 2000). In mammals, the reduction of DAG levels at the Golgi caused by the depletion of Nir2 (a peripheral Golgi protein containing a PI-transfer domain) inhibits post-Golgi protein transport (Litvak et al., 2005). DAG acts in the trans-Golgi network (TGN) as a membrane acceptor for specific proteins such as the protein kinase C (PKC) family member PKD/ PKC (Prestle et al., 1996;Liljedahl et al., 2001;Baron andMalhotra, 2002), and Hmun13 (Speight andSilverman, 2005).PKD together with PKC, the trimeric G-protein subunits ␤/␥ (Díaz Anel and Malhotra, 2005), and phosphatidylinositol-4 kinase III␤ (Hausser et al., 2005) directly participate in the post-Golgi transport of plasma membrane proteins containing basolateral sorting information (Yeaman et al., 2004). On the other hand, Hmun13, through the recruitment of Rab34, participates in the Golgi-lysosome protein trafficking (Speight and Silverman, 2005). DAG also promotes the Golgi membrane targeting and activation of other C1 domain-containing signaling molecules such as other PKC isoforms (PKC, PKC, and PKC␦;Maissel et al., 2006;Lehel et al., 1995; Wang et al., 1999, respectively) and Ras guanosine nucleotide-releasing proteins (RasGRPs;Caloca et al., 2003), whose potential involvement in Golgi-associated transport functions remains unexplored.The aforementioned Go...

ABSTRACT:Liver grafts discarded for transplantation because of macrosteatosis can constitute a valuable source of human hepatocytes for in vitro metabolic and pharmacotoxicological studies or for therapeutic applications. A condition for using hepatocyte suspensions for these purposes is the preservation of their metabolic competence and, particularly, drug-metabolizing enzymes. A reduction in microsomal cytochrome P450 (P450) activities was observed in fatty livers (>40% steatosis) with respect to normal tissue. Similarly, decreased levels of 7-ethoxycoumarin O-deethylation and testosterone metabolism were observed in human hepatocyte cultures prepared from steatotic liver tissue. To clarify the potential impact of lipid accumulation on human hepatic P450 enzymes, we have used an in vitro model of "cellular steatosis" by incubation of cultured hepatocytes with increasing concentrations (0.25-3 mM) of long-chain free fatty acids (FFA). A dose-dependent accumulation of lipids in the cytosol is induced by FFA mixture. Hepatocytes exposed to 1 mM FFA for 14 h showed lower activity values of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 enzymes than nontreated hepatocytes (about 45-65% reduction). This treatment also produced significant decreases in CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 mRNA to about 55 to 75% of mRNA levels in control cells. Our results suggest that although human hepatocytes isolated from steatotic liver show reduced P450 activities, they are metabolically competent and can be used for drug metabolism studies.

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