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The gel-retardation, band-shift, or electromobility shift assay is used to investigate protein—DNA interactions. Nuclear protein from the desired source is incubated with a radiolabeled DNA fragment. The products of the reaction are then analyzed by electrophoresls through a nondenaturing polyacrylamide gel. Free DNA will migrate rapidly through the gel, but DNA bound to protein will move more slowly, i.e., the migration of bound DNA ts retarded. The protein-DNA complexes formed can be detected by autoradiography. [ABSTRACT FROM AUTHOR]

Aspects of cytochrome P450 (CYP) nomenclature are addressed. The rules for naming a P450 are outlined, though individuals should not name their own genes. The nomenclature is presented as a unifying principle to enhance communication across disciplines. Because of the historical nature of gene sequencing, sometimes names have to be changed, but this is kept to a bare minimum to avoid confusion in the literature. CYP names have now reached four digits owing to proliferation of CYP families in the fungi and lower eukaryotes. For example, CYP5034A1 is from Ustilago maydis. P450 sequence motifs are described that are useful in making global alignments. CYP clans are defined as clusters of CYP families. The clan names are useful in describing higher-order evolution of the gene superfamily. The nomenclature of orthologs and pseudogenes is also discussed. [ABSTRACT FROM AUTHOR]

Mimicking the in vivo microenvironment is one of the current strategies to maintain liver-specific functionality in primary cultured hepatocytes for long periods. Freshly isolated hepatocytes entrapped in collagen gel type I (collagen gel immobilization culture) or sandwiched between two layers of hydrated collagen type I (collagen gel sandwich culture) are known to display liver-specific functions (e.g., biotransformation capacity) for more than 6 wk. We describe how to set up both types of organotypical hepatocyte culture systems. Besides a detailed protocol, we give some practical tips, taken from our own experience with long-term hepatocyte culture. [ABSTRACT FROM AUTHOR]

Transcriptional activation of CYP gene expression by xenobiotics may have fundamental effects on body physiology. It may result in the altered pharmacokinetics of other chemicals in the body, both xenobiotic and endogenous substrates, potentially altering their effects. This may often result in no observable clinical effect, but in a significant number of cases these interactions lead to altered physiology or failure of a therapeutic drug. It is therefore important to be able to screen novel chemical entities for their ability to activate CYP gene expression. In addition, through mechanistic studies of how such transcriptional activation occurs, the ability to predict and avoid such potential interactions is improved. Reporter gene assays provide a simple, high-throughput methodology for examining the transcriptional activation of CYP gene expression by xenobiotics. They are suitable for use in screening as well as mechanistic studies and are of use in both the drug discovery/development and research arenas. [ABSTRACT FROM AUTHOR]

Methods are described for the injection of mouse embryonic stem cells, in which Fmo genes have been targeted to disrupt gene function, into 3.5-d-old blastocysts and the implantation of these into foster mothers. Successful injection and implantation of blastocysts will produce mice of mixed coat color (the chimera). Also described are methods to establish the success of blastocyst injection and implantation of germ-line transmission of the knockout (KO) mutation. Breeding strategies to produce congenic and isogenic KO mouse lines are outlined. Simple methods for the isolation of tail DNA, the tagging of mice, and record keeping of the line are also given. [ABSTRACT FROM AUTHOR]

The manipulation of genes in mouse embryonic stem (ES) cells can result in chromosome abnormalities. This chapter describes methods for karyotyping of the manipulated ES cell line before injection into blastocysts and the use of fluorescence in situ hybridization to confirm the deletion of a targeted gene. The method is illustrated by describing how an ES cell line targeted for the deletion of Fmo genes was characterized. [ABSTRACT FROM AUTHOR]

The steps required to delete genes from the mouse genome are illustrated by showing how a cluster of three flavin-containing monooxygenase (Fmo) genes (Fmo1, Fmo2, and Fmo4) were deleted from mouse chromosome 1. Such large deletions are accomplished using loxP/Cre recombinase technology. Genomic clones corresponding to the genes to be deleted are first isolated, and then appropriate genomic fragments are cloned into vectors containing a loxP site. This produces targeting vectors, which are electroporated into mouse embryonic stem (ES) cells to allow a homologous recombination event to take place between the mouse genomic fragment, present within the vector, and the homologous sequences in the ES cell genome. Screening of ES cells for recombinants in which loxP sites have been inserted on either side of the gene cluster to be deleted is described. Recombination by Cre recombinase to produce ES cell lines carrying the deletion on chromosome 1 is also described. [ABSTRACT FROM AUTHOR]

Methods are described for the cellular localization of expression of flavin-containing monooxygenase (FMO) genes in various mouse tissues by in situ hybridization. These include the production of digoxigenin (DIG)-labeled antisense and sense RNA probes by transcription from FMO cDNA templates, the preparation of paraffin wax-embedded and cryostat tissue sections, the hybridization of RNA probes to tissue sections, and the specific detection of hybridized probes using an antibody to DIG. [ABSTRACT FROM AUTHOR]

Primary culture of human hepatocytes is an in vitro model widely used to investigate numerous aspects of liver physiology and pathology. The technique used to isolate human hepatocytes is based on two-step collagenase perfusion. Originally performed in situ for obtaining hepatocytes from the adult rat, this technique has been adapted to the ex vivo treatment of human liver from organ donors or from lobectomy resection for medical purposes. This chapter describes experimental protocols for the isolation of hepatocytes from human liver tissue and for the preparation of short- and long-term cultures in which cells retain a differentiated phenotype for at least 1 mo. The various aspects emphasized here include the conditions for obtaining tissue, quality control of tissue for efficient perfusion, collagenase perfusion parameters, solutions for perfusion and culture media, cell substrate, cell plating, specific equipment, and safety conditions. [ABSTRACT FROM AUTHOR]

Five different transfection reagents—calcium phosphate, TransFast™ Transfection Reagent, Superfect™ Transfection Reagent, Effectene™ Transfection Reagent, and Tfx™-20—were compared for their ability to effectively transfect primary cultures of male rat hepatocytes. Hepatocytes were isolated by the collagenase perfusion method and then cultured on Matrigel-coated plates for 24 h before transfection. The cells were transfected with either pGL3-Control or pGL3-Basic plasmids. The efficiency of transfection of each reagent was monitored using the dual luciferase reporter gene assay system. Superfect Transfection Reagent, Effectene Transfection Reagent and Tfx-20 were the most effective for the transfection of primary hepatocytes and gave comparable transfection efficiencies. Calcium phosphate was found to be the least effective transfection reagent and gave the most variable transfection results. Tfx-20 gave the least variable transfection results when different hepatocyte preparations were compared. [ABSTRACT FROM AUTHOR]

Stem cell research has become an important field of study for molecular, cellular, and clinical biology as well as pharmaco-toxicology. Indeed, stem cells have a strong proliferative and unlimited self-renewal potential and are multipotent. In vivo as well as in vitro studies have confirmed the differentiation of adult bone marrow stem cells into muscle cells, adipocytes, cardiomyocytes, neuroectodermal cells, osteoblasts, chondroblasts, and so on. Recently, it has been shown that, under appropriate culture conditions, adult bone marrow stem cells may also differentiate into hepatocyte-like cells. Because of their extensive proliferative capacity and pluripotency, adult bone marrow stem cells could serve in the future as an unlimited source of hepatocytes for pharmaco-toxicological research and testing. We describe a protocol for isolation of mononuclear cells from adult rat bone marrow. [ABSTRACT FROM AUTHOR]

Isolated hepatocytes are a physiologically relevant in vitro model exhibiting intact subcellular organelles, xenobiotic transport, and integrated phase I and phase II biotransformation. They represent the "gold standard" for investigating xenobiotic biotransformation and metabolic bioactivation. When used in suspension, they provide an easy-to-handle and relatively cheap in vitro system that can be used for up to 4 h. The use of animal- and human-derived hepatocytes allows interspecies comparisons of metabolic properties. In contrast with microsomes, which are easily prepared from human liver tissue and can be stored in liquid nitrogen with minimal loss of functionality, cryopreservation of isolated human hepatocytes has been shown to be more difficult: after thawing losses of cell viability and biotransformation capacity occur. We provide general recommendations for the appropriate use of hepatocytes in suspension for pharmaco-toxicological studies. We also provide protocols for the cryopreservation of freshly isolated hepatocytes and their handling on thawing. [ABSTRACT FROM AUTHOR]

In vitro models, based on liver cells or tissues, are indispensable in the early preclinical phase of drug development. An important breakthrough in establishing cell models has been the successful high-yield preparation of intact hepatocytes. In this chapter, the practical aspects of the two-step collagenase perfusion method, modified from the original procedure of Seglen, are outlined. Although applicable to the liver of various species, including human, the practical aspects of the method are explained here for rat liver. Critical parameters for the successful isolation of primary rat hepatocytes are highlighted and a troubleshooting guide is provided. In addition, a new development based on the inhibition of histone deacetylase activity is presented. This approach allows inhibition of cell-cycle reentry during hepatocyte isolation, a process known to underlie the dedifferentiation process of cultured hepatocytes. [ABSTRACT FROM AUTHOR]

Primary cultures of hepatocytes are useful tools for both short- and long-term pharmacotoxicological research. Under conventional conditions, isolated hepatocytes form a monolayer and survive for about 1 wk but lose some liver-specific functions, including xenobiotic biotransformation. In comparison with the conventional monolayer culture model, cocultures with rat liver epithelial cells (RLECs) have an extended life-span and better maintain their drug-metabolizing capacity, owing to the presence of cell-cell interactions. In this chapter, techniques for setting up conventional monolayer cultures and cocultures of hepatocytes with RLECs (including isolation, culture, and cryopreservation of RLECs) are described in detail. In addition, comments derived from our own experience are given for successfully culturing primary hepatocytes. [ABSTRACT FROM AUTHOR]

When a new chemical entity is tested for its safety for humans and their environment, in vivo experiments on living animals are usually conducted. However, in the early preclinical stage of drug development, in vitro techniques, and more specifically hepatocyte-based in vitro models, are currently being applied. A major problem, however, related to the use of hepatocytes and their cultures is their limited viability, which is associated with the loss of phase I and phase II biotransformation capacity. Therefore, in order to keep the hepatocytes functional for a longer period, the in vivo microenvironment is mimicked in vitro as closely as possible through the addition of soluble medium components, coculture with helper cells, or culture on an extracellular matrix (sandwich culture). We discuss the advantages and disadvantages of current short- and long-term hepatocyte culture systems are discussed as well as their proper use in toxicological research and testing. [ABSTRACT FROM AUTHOR]

Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and (*4 alleles), CYP2C8 ((*3 and (*4 alleles), CYP2C9 ((*2, (*3, and (*11 alleles), CYP2C19 ((*2 and (*3 alleles), CYP2D6 ((*3, (*4, (*5, and (*6 alleles), CYP2E1 ((*5A, (*5B, and (*6 alleles), and CYP3A5 ((*3 allele). [ABSTRACT FROM AUTHOR]

Interindividual variability in xenobiotic metabolism and drug response is extensive. Genetic factors are predicted to account for 15-30% of this variability in general, but for certain drugs the genetic factor is the major determinant for outcome of drug therapy. Of particular importance for drug metabolism, drug response, and adverse drug reactions are the cytochrome P450 (CYP) enzymes, many of which are polymorphic. An essential basis for research and applications regarding interindividual variability in xenobiotic metabolism and toxicity by polymorphic CYPs is to have a common nomenclature for genetic variants and a system that allows researchers to be rapidly updated within the field. Since 1999 this has been achieved by the operation of the Human Cytochrome P450 Allele Nomenclature Committee Web site (http://www.imm.ki.se/CYPalleles/), where novel allelic variants are published after peer review. Currently, this Web site covers the nomenclature for polymorphic alleles of 22 CYP isoforms including more than 200 functionally different variants. Each CYP has its own Web page, which lists the alleles with their nucleotide changes, their functional consequences, and links to publications where the allele has been identified and characterized. The CYP allele Web site offers a rapid on-line publication of new alleles, provides an overview of peer-reviewed data, and serves as a form of quality control on research on new alleles. [ABSTRACT FROM AUTHOR]

An approach to raising antibodies is described that can be applied to the majority of cytochrome P450 (P450) enzymes. Its application is limited only by the availability of suitable protein sequence information. The method is based on immunizing animals with synthetic peptides that mimic small regions of target proteins. In practice, this has proved to be a relatively simple, rapid, and effective method of producing antibodies. The antibodies are particularly suited to immunoblotting (Western blotting) and immunocytochemistry. Unlike antibodies produced by other techniques, the epitopes of these antibodies are predetermined, allowing them to be directed toward specific regions of P450. Detailed practical information is described on the selection of regions to target, the synthesis and conjugation of peptides to carrier protein, immunization, and the assessment of the resultant antisera. The approach is illustrated using examples of antibodies targeted against rat and human P450. [ABSTRACT FROM AUTHOR]

Asymmetric aryl alkyl sulfides (R-S-R′) are metabolized by flavin-containing monooxygenase (FMO) and cytochrome P450 enzymes to enantiomerically enriched sulfoxide products (R-SO-R′) that are readily analyzed with a host of commercially available chiral stationary phases. Prochiral sulfoxidation of probe compounds based on p-tolyl methyl sulfide is a particularly useful method for discriminating among FMO1, FMO3, and FMO5, because the stereochemistry of the resulting products is isoform dependent, but apparently species independent. If studies are performed with crude tissue microsomal preparations, the cytochrome P450 component must be quenched to unmask catalysis specifically by the FMO component of the tissue. This chapter details experimental protocols for stereochemical analysis of sulfoxides generated from methyl, ethyl, n-propyl, and n-butyl p-tolyl sulfide by purified FMO isoforms and tissue microsomal preparations. [ABSTRACT FROM AUTHOR]

Benzydamine is a nonsteroidal anti-inflammatory drug that undergoes flavin-containing monooxygenase (FMO)-dependent metabolism to a stable N-oxide. This metabolite can be quantified with high specificity and sensitivity by using a simple reverse-phase high-performance liquid chromatography (HPLC) assay with fluorescence detection. Studies with recombinant FMO enzymes demonstrate that FMO1 and FMO3 are the primary catalysts of benzydamine N-oxygenation, with minimal contributions from cytochrome P450 enzymes. Investigations conducted with human liver microsomes confirm that FMO3, in large part, is responsible for benzydamine N-oxide formation in this tissue. These features render benzydamine a useful in vitro probe for FMO activity in a wide range of tissues and cell types. In addition, benzydamine appears to be a suitable in vivo probe for human liver FMO3. This chapter provides a detailed account of the experimental protocol for determining rates of formation of benzydamine N-oxide by FMO-containing enzyme fractions. [ABSTRACT FROM AUTHOR]

7-Ethoxycoumarin is metabolized by many cytochrome P450 enzymes active in foreign compound metabolism and has been used as a prototypic substrate to monitor P450 (P450) activity in both hepatic and extrahepatic tissues. A spectrofluorometric method is described for determination of P450-catalyzed 7-ethoxycoumarin O-deethylation. Following acidification of the incubation mixture, the enzymatic product, 7-hydroxycoumarin, is recovered by a double-extraction procedure and measured at an excitation wavelength of 370 nm and an emission wavelength of 450 nm. This method is applicable to enzymatic studies to determine the catalytic activity of cDNA-expressed human enzymes in the CYP1, CYP2, and CYP3 families, and 7-ethoxycoumarin O-deethylation activity in microsomes isolated from liver and other tissues. [ABSTRACT FROM AUTHOR]

A normal-phase, isocratic high-performance liquid chromatography assay is described for cholesterol 7α-hydroxylation catalyzed by CYP7A1, which corresponds to the first and rate-limiting step in the conversion of cholesterol into bile acids. This method is based on the conversion of the primary cytochrome P450 metabolite, 7α-hydroxycholesterol, into 7α-hydroxy-4-cholesten-3-one in a reaction catalyzed by exogenous cholesterol oxidase, followed by chromatographic separation with monitoring at 254 nm. This technique is applicable to enzymatic studies for determination of cholesterol 7α- hydroxylation activity catalyzed by cDNA-expressed CYP7A1 and animal or human liver microsomes. [ABSTRACT FROM AUTHOR]

Lauric acid serves as an endogenous substrate for the cytochrome P450 enzyme CYP4A11. A reverse-phase, high-performance liquid chromatography method is described for the quantification of 12-hydroxylauric acid formed enzymatically by incubation of 14C-labeled lauric acid with cDNA-expressed CYP4A11 or human liver microsomes. Analytical separation is achieved using a C18 column and a gradient of 30% acetonitrile and 2 mM perchloric acid to 100% methanol, using a detection scintillation counter. This method is applicable to enzymatic studies for determination of lauric acid 12-hydroxylation activity. [ABSTRACT FROM AUTHOR]

Testosterone and other steroid hormones have been studied as prototypic examples of endogenous substrates for hepatic cytochrome P450 (P450) enzymes. CYP3A enzymes from various species, including human, metabolize testosterone by a 6β-hydroxylation reaction, which is unique to this P450 subfamily. A thin-layer chromatographic method is described for the determination of 6β-hydroxytestosterone formed enzymatically by incubation of [14C]-testosterone with cDNA-expressed CYP3A enzymes or liver microsomes. 14C-labeled enzymatic products are applied to silica gel thin-layer plates, which are developed sequentially with methylene chloride:acetone (80:20) followed by chloroform, ethyl acetate, and absolute ethanol (80:20:14). Metabolite quantification is performed by autoradiography and liquid scintillation counting. This method is applicable to enzymatic studies for the determination of CYP3A-dependent testosterone 6β- hydroxylation activity in both human and animal liver microsomes. [ABSTRACT FROM AUTHOR]

The cytochrome P450 enzyme CYP2E1 catalyzes the oxidative metabolism of many solvents and other small organic molecules. A spectrophotometric method is described for determination of CYP2E1 activity by monitoring the formation of p-nitrocatechol from p-nitrophenol by cDNA-expressed CYP2E1 or isolated liver microsomes. The enzymatic product, p-nitrocatechol, is assayed at 535 nm after acidification of the reaction mixture with trichloroacetic acid followed by neutralization using 2 M NaOH. This method is applicable to enzymatic studies for determination of P450-catalyzed p-nitrophenol hydroxylation activity. [ABSTRACT FROM AUTHOR]

A reverse-phase, high-performance liquid chromatography method is described for the quantification of 1′-hydroxybufuralol formed enzymatically by the incubation of bufuralol with cDNA-expressed CYP2D6 or human liver microsomes. Analytical separation is achieved using a C18 column and a mobile phase consisting of 30% acetonitrile and 2 mM perchloric acid, with detection by fluorescence using an excitation wavelength of 252 nm and an emission wavelength of 302 nm. This method is applicable to enzymatic studies for determination of CYP2D6-catalyzed bufuralol 1′-hydroxylation activity. [ABSTRACT FROM AUTHOR]

A reverse-phase, high-performance liquid chromatography method is described for the quantification of 4′-hydroxymephenytoin formed enzymatically from 14C-labeled (S)-mephenytoin following incubation with cDNA-expressed CYP2C19 or human liver microsomes. Analytical separation is achieved using a C18 column developed with a gradient from 10 to 100% methanol, with detection using a scintillation detector. This method is applicable to enzymatic studies for determination of CYP2C19-catalyzed (S)-mephenytoin 4′-hydroxylation activity. [ABSTRACT FROM AUTHOR]

A reverse-phase, high-performance liquid chromatography method is described for quantification of diclofenac 4′-hydroxylation catalyzed by human liver microsomes or cDNA-expressed CYP2C9. Analytical separation is achieved using a C18 column developed with a gradient of 30% acetonitrile and 2 mM perchloric acid in water to 100% methanol, with detection at 280 nm. This method is applicable to enzymatic studies for determination of CYP2C9-catalyzed diclofenac 4′-hydroxylation activity. [ABSTRACT FROM AUTHOR]

Paclitaxel (Taxol) is a naturally occurring member of the taxane family of antitumor drugs, which act by stabilizing microtubules. Paclitaxel is inactivated in human liver by a cytochrome P450 (P450)-catalyzed 6α-hydroxylation reaction. A reverse-phase, high-performance liquid chromatographic assay is described for the analysis of paclitaxel 6α-hydroxylation catalyzed by human liver microsomes or cDNA-expressed P450 enzyme CYP2C8. Analytical separations are achieved using a C18 column with a linear gradient of 10-100% methanol, with detection at 230 nm. This method is applicable to enzymatic studies for determination of CYP2C8-catalyzed paclitaxel 6α-hydroxylation activity. [ABSTRACT FROM AUTHOR]

The cytochrome P450 enzyme CYP2B6 plays an important role in the metabolism of structurally diverse drugs, including the anticancer drug cyclophosphamide, and may be an important determinant of clinical responses to these agents. A spectrofluorometric method is described for the determination of CYP2B6-catalyzed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity in human liver microsomes. The specificity of this method for CYP2B6 is increased by the use of inhibitory antibodies to CYP1A2, CYP2C, and CYP2E1, which block the contributions of these higher-Km enzymes to human liver microsomal metabolism of 7-ethoxy-4-trifluoromethylcoumarin. The enzymatic product, 7-hydroxy-4-trifluoromethylcoumarin, is monitored by fluorescence using an excitation wavelength of 410 nm and an emission wavelength of 510 nm. This approach can be modified to assay the catalytic activity of cDNA-expressed CYP2B6. [ABSTRACT FROM AUTHOR]

A spectrofluorometric method is described for the determination of CYP2A6-catalyzed coumarin 7-hydroxylation. Following acidification of the reaction mixture, the enzymatic product, 7-hydroxycoumarin, is recovered by a double-extraction procedure and assayed using an excitation wavelength of 370 nm and an emission wavelength of 450 nm. This assay is applicable to enzymatic studies of cDNA-expressed CYP2A6 and can be used to monitor coumarin 7-hydroxylation activity in microsomes prepared from liver and other tissues and in isolated hepatocytes and cultured cells that express this cytochrome P450 activity. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (P450) enzymes belonging to the CYP1 family are highly inducible by polycyclic aromatic hydrocarbons and other environmental chemicals and play a major role in the metabolism of many foreign chemicals and endogenous substances. We describe a spectrofluorometric method for determining 7-ethoxyresorufin O-dealkylation catalyzed by CYP1A1, CYP1A2, and CYP1B1. The formation of the enzymatic product, resorufin, is monitored continuously by fluorescence using an excitation wavelength of 530 nm and an emission wavelength of 580 nm. This method can be applied to assay P450-catalyzed formation of resorufin from other alkoxyresorufins, such as 7-methoxyresorufin, 7-benzyloxyresorufin, and 7-pentoxyresorufin. It can also be used to assay 7-ethoxyresorufin O-dealkylation activity in isolated hepatocytes and cultured cells that express this P450 activity. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (P450) is a superfamily of individual monooxygenase enzymes that metabolize structurally diverse xenochemicals, including many clinically useful drugs and foreign chemicals widespread in the environment. P450 substrates that can be used to selectively monitor individual P450 enzymes or P450 subfamilies have been identified through studies using P450 enzyme-selective inhibitory antibodies and chemical inhibitors in conjunction with experiments utilizing individual cDNA-expressed P450 enzymes. This chapter describes P450 form-selective substrates that can be used to monitor the activities of human P450 enzymes CYP1A, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A, CYP4A11, and CYP7A1. Cautions that need to be exercised when using these substrates to probe for individual P450 activities in human liver and other tissues are discussed. [ABSTRACT FROM AUTHOR]

Human liver microsomes contain multiple forms of cytochrome P450 (CYP or P450) that catalyze oxidation of a number of xenobiotic and endobiotic chemicals. Individual P450 forms have unique, but overlapping, substrate specificities. It is necessary to determine which P450s play more important roles in the oxidation of these chemicals. A good way of studying the roles of P450s in the metabolism of these chemicals is to reconstitute the activities by mixing purified P450s and nicotinamide adenine dinucleotide phosphate-cytochrome P450 reductase in the membranes of phospholipid vesicles. However, our studies have suggested that the conditions for reconstitution of activities vary depending on the P450 enzymes used. For example, some reactions catalyzed by P450s require cytochrome-b5 and a particular phospholipid environment for exerting their full catalytic activities. In this chapter, we describe optimal conditions that have been determined in our laboratories for the reconstitution of drug oxidation activities catalyzed by purified human CYP1A2, 2C9, 2E1, and 3A4. [ABSTRACT FROM AUTHOR]

The baculovirus/insect cell heterologous expression system provides an important tool for investigating the catalytic activity of individual drug-metabolizing enzymes toward a particular substrate. In this chapter we describe a baculovirus/insect cell system that we have used for the expression of human and mouse flavin-containing monooxygenases. Methods are described for the generation of recombinant baculoviral DNAs, via both site-specific transposition in Escherichia coli and site-specific recombination in vitro; adaptation of Spodoptera frugiperda (Sf) 9 cells to shaking culture and to serum-free medium; cryopreservation and transfection of Sf9 cells; amplification of baculovirus and determination of viral titer; analysis of baculoviral DNA; and expression and analysis of recombinant proteins. [ABSTRACT FROM AUTHOR]

A general procedure for the solubilization of cytochrome P450 (P450) from bacterial membranes specifically for a human P450 expressed heterologously in the host Escherichia coli is described. The example involves the use of a P450 (3A4) with a C-terminal oligohistidine tag and includes sequential DEAE and metal affinity chromatography. [ABSTRACT FROM AUTHOR]

Cytochromes P450 (CYP) have been expressed in a variety of systems such as mammalian cells, yeast, and bacteria. The bacterial system is technically the least demanding and provides large amounts of catalytically active P450s for metabolic and structural studies relating to preclinical drug development. This chapter provides a detailed technical description of the processes that allow the coexpression of various CYP isoforms together with CYP reductase in Escherichia coli and gives some examples of the results that can be achieved for the expression of human P450s. [ABSTRACT FROM AUTHOR]

UV/Vis spectroscopy is the major means of identifying intact holocytochrome P450. The carbon monoxide complex of the intact ferrous hemoprotein exhibits a characteristic spectrum between 448 and 452 nm, considerably distinct from the usual Soret absorption peaks of hemoproteins. Methods are described for identification and quantitation of cytochrome P450 (CYP) in membranes, in tissue homogenates, and in purified form, using difference spectroscopy and absolute spectroscopy. CYP are b-type cytochromes, containing protoporphyrin IX as the prosthetic group. Methods are also provided, using alkali and pyridine, for quantitation of the hemoprotein by this prosthetic group. In its oxidized, or ferric state, CYP exists as an equilibrium mixture of high- and low-spin configurations, each with distinctive UV/Vis absorption peaks. Substrate binding causes shifts in the spin equilibrium, and methods are shown for using these shifts for quantitation of substrate binding to CYP. [ABSTRACT FROM AUTHOR]

CYP2E1 is expressed in adult (1, 2) and fetal (3) human liver in addition to extrahepatic tissues such as lung and placenta (4). Treatment of primary cultures of human hepatocytes with ethanol induces CYP2E1 protein (5) and this is consistent with the finding that hepatic CYP2El protein (6) and mRNA (7) amounts are increased in alcoholics. Although only a few drugs (e.g., acetaminophen [8]) have been identified as substrates for CYP2E1, many low molecular weight procarcinogens are activated by this P450 (9). Chlorzoxazone 6-hydroxylation (19-12), N-nitrosodimethylamine N-demethylation (11, 13, 14), and p-nitrophenol hydroxylation (12, 14) reactions can be used to measure the catalytic activity of cDNA-expressed CYP2El. Each of these activities is also frequently used as an enzyme-selective catalytic monitor for human hepatic CYP2El (see Notes 1 and 2). Experiments with inhibitory antiCYP2E1 antibodies and CYP2E1-selective chemical inhibitors suggest that CYP2E1 contributes extensively to these activities in human liver microsomes (9, 15-18). Recently, lauric acid 11 -hydroxylation was identified as an alternative probe for human hepatic CY2El (19). However, an advantage of using p-nitrophenol hydroxylation to measure CYP2E1 activity is that the formation of p-nitrocatechol can be measured by a simple spectrophotometric assay, although high-performance liquid chromatographic (HPLC) assays have also been developed to quantify thep-nitrocatechol metabolite (20, 21). This chapter describes a modification of a spectrophotometric method (22) for the determination of p-nitrophenol hydroxylase activity. [ABSTRACT FROM AUTHOR]

At least two cytochromes P450 belonging to the CYP3A subfamily may be expressed in adult human liver (1), CYP3A4 and CYP3A5. A third enzyme, CYP3A7, is expressed in human fetal liver (2). The CYP3A enzymes account for an estimated ~30% of total human cytochrome P450 content in adult liver (3), although large inter-individual differences exist in hepatic CYP3A content. CYP3A4 is present in all adult human livers and is inducible by drugs such as rifampin (rifampicin) and dexamethasone (4-6). By contrast, CYP3A5 is expressed in only ~ 10-30% of liver samples (7) and does not respond to typical CYP3A inducers (5, 6). Triacetyloleandomycin (8, 9) and gestodene (9) are CYP3A-selective chemical inhibitors. Many commonly used drugs are substrates for CYP3A, including erythromycin (10), infedipine (11) and midazolam (12). Immunoinhibition experiments with CYP3A subfamily-specific antibodies have established several microsomal enzyme activities, including nifedipine oxidase (11, 13) and testosterone 6β-hydroxylase (14, 15), as useful catalytic monitors for hepatic CYP3A In a recent study, an inhibitory antipeptide antibody against CYP3A4, which did not cross-react with cDNA-expressed CYP3A5 as judged by Western-blot analysis and did not inhibit cDNA-expressed CYP3A5-catalyzed testosterone 6β-hydroxylation, was found to inhibit virtually all of the testosterone 6β-hydroxylase activity in human liver microsomes (16), suggesting that this activity has a high specificity for hepatic CYP3A4. [ABSTRACT FROM AUTHOR]

The human CYP4.411 gene encodes a P450 protein that has been isolated from human kidney (1, 2) and liver (3) and purified to apparent homogeneity. CYP4All mRNA is present in greater abundance in kidney than in liver, whereas it is absent in lung (3). Various fatty acids, including lauric acid (2-4), are substrates for recombinant CYP4Al l. Experiments with a panel of individual cDNA-expressed P450 enzymes indicate that CYP4Al l is a major lauric acid 12-hydroxylase (5). Immunoinhibition experiments with heterologous and antihuman CYP4Al l antibodies have further established that CYP4Al l contributes a major fraction of the lauric acid 12-hydroxylase activity in human liver microsomes (3, 5, 6). Accordingly, microsomal lauric acid 12-hydroxylase activity is used as a marker for human hepatic CYP4A11. Although CYP4A11 is a major lauric acid 12-hydroxylase in human liver, lauric acid 11 -hydroxylation in this tissue is catalyzed by CYP2E1 and not CYP4A11 (7, 8). Several analytical methods are available for the quantification of 12-hydroxylauric acid, including the use of nonradioactive lauric acid as the substrate by a highperformance liquid chromatographic (HPLC) assay with fluorescence detection (9, 10). This chapter describes a radiometric HPLC assay for the determination of lauric acid 12-hydroxylase activity. [ABSTRACT FROM AUTHOR]

The ability to manipulate the murine genome via the use of gene targeting in pluripotent embryonic stem cells is arguably one of the most important scientific achievements of the last decade (1-4). This technology holds the potential to produce murine strains and cell-culture systems that harbor an allelic series of mutations at almost any given locus, As a result, models can now be generated in complex eukaryotic systems in which a gene of interest is no longer expressed ("null" allele) or in which some aspect of its function or expression has been altered. The techniques commonly used in the generation of null alleles have been the focus of many excellent review articles, books, and laboratory manuals (5-9). Therefore, our goal in preparing this chapter was to draw attention to gene-targeting strategies that provide mechanistic insight when null alleles provide incomplete information. Additionally, we discuss some examples of how allelic series can sometimes yield surprising results that arise when different laboratories use different approaches to create a null mutation at the same locus. We would like to discuss how gene targeting has proven to be a useful technique for pharmacologists. However, owing to the nascent stage of the technology, few examples are available in this area. Therefore, our presentation will be more general in nature. First, we provide an overvrew of the most popular gene-targeting approaches in use today. Second, we describe gene-targeting experiments at the CFTR locus, arguably one of the largest allelic series to date. Third, we provide a brief review of targeting experiments at loci encoding cytochrome P450 structural genes and their regulators. We hope that the chapter will help the reader appreciate how gene targeting can be used to generate novel mouse models that are useful in drug design and for the investigation of metabolic pathways and the mechanism of action of toxicants. [ABSTRACT FROM AUTHOR]

Inducible proteins are attractive candidates for biomarkers-i.e. biological responses that reflect exposure to, or effects of, environmental pollutants in an organism (1). Blomarkers are increasingly regarded as powerful and informative tools in ecotoxicology and environmental management (1). [ABSTRACT FROM AUTHOR]

A number of common functtonally significant genetic polymorphisms in genes encoding certain cytochromes P450 have been described and characterized (for review, seerefs. 1 and 2). Individuals may lack certain enzyme activities or have higher or lower than normal activities owing to the presence of certain variant alleles. These individuals may be at altered risk of developing adverse drug reactions or drseases associated wtth xenobiotrc exposure, including cancer. Variant alleles can be detected by the direct approach of genotyping where the individual's DNA is directly examined usually by an assay involving use of the polymerase chain reactton (PCR), or by phenotypmg, where the individual's pattern of metabolites produced from a probe drug is examined. This chapter describes the detection of common polymorphisms in cytochrome P450 genes using both genotyping and phenotypmg approaches. Genotyping is now generally preferred to phenotyping because it is more convenient, requiring only a single blood sample, which can be taken at any time. It can also be used on stored blood or other tissue samples. Phenotyping, however, is useful when investigating drug interactrons in viva and in situations where the molecular basis of a polymorphism is unclear. In the sections of this chapter covering genotyping, we describe general methods for PCR and analysts of PCR products that have been used to genotype for pharmacogenetic polymorphisms, together with the specific conditions used to detect a range of known polymorphisms in cytochrome P450 genes. Methods for phenotypic detection of polymorphisms in CYP2D6 and CYP2A6 are also described. [ABSTRACT FROM AUTHOR]

Most mechanistic analyses of gene regulation are performed in contexts different from the physiological conditions of the intact cell. For example, transcription factors are often studied according to their ability to modulate the expression of plasmid-based reporter genes rather than endogenous chromosomal genes; DNA regulatory elements are usually analyzed as oligonucleotides in vitro or as components of recombinant plasmids, rather than in their native chromosomal setting. Such studies, though informative, may not faithfully reflect gene regulation in vivo. In addition, many studies ignore the regulatory effects of chromatin structure on gene expression. In studies that do address chromatin structure, reconstituted chromatin is often analyzed in vitro; in such situations, the stoichiometric relationship between DNA and transcription factors may not resemble those found in vivo, and other important regulatory macromolecules may be inactive or missing. Therefore, it is important to develop approaches for studying gene regulation under conditions similar to those that exist in VIVO. [ABSTRACT FROM AUTHOR]

Functional analysis of the 5′-flanking region of a gene has become a routme procedure for identifying the cis-acting DNA elements involved in the control of the transcriptional activity of the gene (1). This experimental approach requires the transfection of reporter plasmids, harboring various fragments of the 5′-flanking region of the gene, into appropriate cultured cells and the subsequent measurement of the activity of the reporter gene, either constitutively or in response to regulatory stimuli. Continuous cell lines derived from mahgnant tissues have been extensively used for this purpose because they can be easily cultured and transfected. However, these cell lines have two major drawbacks: they are dedifferentiated with respect to the normal tissue from which they originate; that is, the tissue-specific transcription factors are generally expressed at a low level, if at all; and their culture requires the use of serum, the chemical and hormonal composition of which is not fully defined. In fact, it would be preferable to transfect normal cells in a primary culture in which the phenotype of the tissue of origin is maintained. The problem with this approach is that the methods currently used to transfect cell lines, including electroporation and calcmm phosphate-or diethylaminoethyl (DEAE) dextran-DNA coprecipitation, are generally not convenient for primary cultures because of their toxicity (2-4). [ABSTRACT FROM AUTHOR]

DNA transfection has become an important technique in molecular biology. The fusion of putative gene-regulatory sequences to reporter genes and their introduction into a eukaryotic cell can be used for a detailed analysis of cisacting DNA regulatory sequences (1). DNA regulatory elements such as those responsive to CAMP (2) or to glucocorticoids and antiglucocorticoids (3) as well as to drugs and xenobiotics such as phenobarbital (4, 5) can be identified through the use of approprtate reporter-gene constructs. Cotransfection experi-ments with vectors expressing specific transcription factors can be used to iden-tify the role of these transcription factors in the expression of specific genes (6) and in addition, the developmental activation of a promoter can be examined (7). The expression of functional protein in transfected cells can be useful for studies on genetic toxicology (8) or biochemical pathways (9). By the intro-duction of mutated DNAs that produce modified protems in the transfected cell, the functional significance of protein domains (10) and individual aminoacid residues (11, 12) can be elucidated, and the transfection of cDNAs isolated from patients has already proved useful for the analysis of molecular defects that result in genetic disorders (13). [ABSTRACT FROM AUTHOR]

Bell et al. (1) showed that a dermal equivalent can be obtained by mixing living fibroblasts with a solution of collagen that will gel and further contract into a firm lattice by extrusion of water. Epidermal keratmocytes can be seeded on top of these lattices, giving rise to a simplified skin (2, 3). [ABSTRACT FROM AUTHOR]

Primary culture of hepatocytes is an in vitro model widely used to investigate various aspects of liver physiology and pathology (1). In particular, such cultures have been extensively used for assessing the expression and function of drug-metabolizing enzymes including cytochromes P450 (CUP), drug metabolism, drug-drug interactions, and the mechanisms of cytotoxtcity and genotoxicity. Most of these studies have been carried out with rodent hepatocytes. However, because of species-specificity in both the regulation and activity of drug-metabolizing enzymes, extrapolation from animals to humans is not generally possible. For this reason, several groups have developed human hepatocyte culture systems (2-8). The technique used to isolate human hepatocytes is based on the two-step collagenase perfusion first mtroduced by Berry and Friend (9) and modified by Seglen (10). Originally performed in situ for obtaining hepatocytes from the adult rat, this technique of perfusion has been adapted to the ex vivo treatment of human liver tissue. The aim of this chapter is to describe the authors′ experience in the isolation of hepatocytes from human liver tissue and the preparation of short- and long-term cultures [ABSTRACT FROM AUTHOR]

Cultivation of cells in Petri dishes or culture flasks is usually performed by changing the medium after certain periods of time, e.g., 24 h (stationary cultivation). Clearly, the concentration of nutrients and other components as well as of added hormones will decrease over this period because of the metabolic activity of the cultured cells Conversely, metabolic end products such as lactate, ammonium ions and even bile salts will accumulate. In order to replace the sequential changes of the culture medium by a continuous supply, several systems for perifusion or circumfusion of the cells with medium have been designed. Usage of one such approach, reported for cultivation of hepatocytes (1), indicated that perifusion was beneficial to the cultured hepatocytes, resulting in a prolonged life-span and an enhanced metabolic performance In particular, it was found that the hormonal response to dexamethasone and glucagon was increased (2), partly because steady-state concentrations of hormones are established even at low initial concentrations. Since these early studies, the technique for perifusion has improved and it has been found that oxygen tension within the culture medium is a very critical parameter; surprismgly, in contrast to previous assumptions that oxygen supply in normal stationary cultures is insufficient (3), higher oxygen tensions, in conjunction with elevated flow rates, proved to be cytotoxic under perifusion conditions. If this and other special features and requirements are considered, perifusion cultivation has considerable advantages over conventional culture techniques, particularly for studies on biotransformation and drug toxicity (4, 5). [ABSTRACT FROM AUTHOR]