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Adult stem cells, especially isolated from bone marrow, have been extensively investigated in recent years. Studies focus on their multiple plasticity of transdifferentiating into various cell lineages and on their potential in cellular therapy in regenerative medicine. In many cases, there is the need for tissue engineering manipulation. Among the different approaches of stem cells tissue engineering, microencapsulation can immobilize stem cells to provide a favorable microenvironment for stem cells survival and functioning. Furthermore, microencapsulated stem cells are immunoisolated after transplantation. We show that one intraperitoneal injection of microencapsulated bone marrow stem cells can prolong the survival of liver failure rat models with 90% of the liver removed surgically. In addition to transdifferentiation, bone marrow stem cells can act as feeder cells. For example, when coencapsulated with hepatocytes, stem cells can increase the viability and function of the hepatocytes in vitro and in vivo. [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]

Methods to microencapsulate enzymes and cells including recombinant enzymes, stem cells, and genetically engineered cells have been described in this chapter. More specific examples of enzyme encapsulation include the microencapsulation of xanthine oxidase for Lesch Nyhan disease, phenylalanine ammonia lyase for phenylketonuria, and microencapsulation of multienzyme systems with cofactor recycling for multistep enzyme conversions. Methods for cell encapsulation include the details for encapsulating hepatocytes for liver failure and for gene therapy. This also includes the details of a novel two-step method for encapsulation of high concentrations of smaller cells. The recent co-encapsulation of bone marrow stem cells with hepatocytes is also included. Another new approach is the detailed method for encapsulation of genetically engineered Escherichia coli DH5 cells and other microorganisms for the removal or conversion metabolites. Another new approach is the nanoencapsulation of hemoglobin and enzymes to form 150-nm diameter nanocapsules; the details are available elsewhere. [ABSTRACT FROM AUTHOR]

Microencapsulation of biologically active material in the form of artificial cell was reported as early as 1964 (1-4). However, it is only in the past 10 yr that many centers have extensively developed this (5). More recently, we have concentrated on three areas of artificial cells for blood substitutes, enzyme therapy, and cell therapy. Space allows only a few examples to be given here. [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]

Chapters 10-20 describe assay methods for determining enzyme activities that are frequently employed as markers for specific cytochrome P450 (P450) enzymes in human tissues. However, under certain circumstances, e.g., when the objective is to verify that individual cDNA-expressed P450 enzymes are expressed in catalytically active form, it is often advantageous to assay the panel of enzymes using a "general" P450 substrate, one that is metabolized by multiple P450 enzymes. An example of a general P4.50 substrate is 7-ethoxycoumarin. Enzyme kinetic analysis with human liver microsomes indicates that 7-ethoxycoumarin is metabolized by at least two P450 enzymes or by two groups of P450 enzymes that are kinetically distmguishable (1, 2). The apparent Km and maximal specific activity (i.e., specific activity at saturating substrate concentration) values for 7-ethoxycoumarin O-deethylation by human liver microsomes are 11-27 µM and 0.05-0.21 nmol/min/mg microsoma1 protein, respectively, for the low Km component. By contrast, for the high Km component, the apparent Km is approx 150 µM and the maximal specific activity is 0.35-0.95 nmol/min/mg microsomal protein (2). Experiments with recombinant human P450s have shown that multiple enzymes in the human CYP1, CYP2, and CYP3 families are active catalysts of 7-ethoxycoumarin O-deethylation (3). Consequently, the 7-ethoxycoumarin O-deethylase assay has been used to verify the catalytic activity of a panel of recombinant human P450s in the CYPl, CYP2 and CYP3 families (4-6). [ABSTRACT FROM AUTHOR]

CYP7A1, which is the only CYP7A subfamily P450 tdentified to date (1), catalyzes cholesterol 7α-hydroxylation, the first, and rate-limiting, step in the converston of cholesterol to bile acids. CYP7Al has been Isolated from human liver and purified to apparent homogeneity (2). Rodent-model studies have established that CYP7A1 is highly regulated by physiological factors that influence hepatic-bile-acid biosynthesis, including cholesterol feedmg, dmrnal factors, and bile acids, whtch feedback-inhibit the overall btosynthetlc pathway in large part at the level of CYP7A1 gene expression (3). Relatrvely little is known about the regulation of human CYP7A1, although liver biopsy analyses have shown that hepatic CYP7A1 protem content is elevated in patients treated with the bile-acid sequesterant cholestyramme (4). Purified CYP7A1 is catalytically active in cholesterol 7α-hydroxylation (2) and immunoinhibition experiments with antihuman CYP7A1 antibodies have shown that CYP7A1 accounts for most of the cholesterol 7a-hydroxylase acttvtty in human liver microsomes (4). Thus, mlcrosomal cholesterol 7α-hydroxylase activity can be used as a marker for human liver CYP7A1. Several analytical methods have been developed to quantify hepatrc mrcrosomal cholesterol 7α-hydroxylation, including reversed-phase high-performance hquid chromatography (RPHPLC) (5), isotope dilution-mass spectrometry (6) and thin-layer chromatography (TLC) (7). This chapter describes a normal-phase, isocratic HPLC assay for the determination of cholesterol 7α-hydroxylase acttvity based on the conversion by cholesterol oxidase of the primary P450 metabohte 7α-hydroxycholesterol to 7α-hydroxy-4-cholesten-3-one, which can be detected at 254 nm. [ABSTRACT FROM AUTHOR]

CYP2D6 is an enzyme that is subject to genetic polymorphism 1. This P450 is absent in individuals with the poor metabolizer CYP2D6 phenotype 2, 3), resulting in a substantially reduced capacity to metabolize a large number of clinically useful drugs, including metoprolol, propafenone, haloperrdol, dextromethorphan and codeine (1). Quinidine competrtively inhibits CYP2D6, with a K1 of 3-30 mM (3-5). Because of its preferential inhibition of CYP2D6 (6 7), quinidine is frequently used as an inhibitory chemical probe of this polymorphically expressed P450. Diagnostic catalytic monitors of human hepatic CYP2D6 include debrisoquine 4-hydroxylase (4), dextromethorphan Odemethylase (8), and bufuralol l'-hydroxylase activities (2-4). Advantages of using bufuralol as a CYP2D6-selective substrate include the high sensitivity of the assay owing to the highly fluorescent l'-hydroxybufuralol metabohte and the fact that the use of radiolabeled substrate is not required. This chapter describes a modification of a reversed-phase ion-pair high-performance liquid chromatographic (HPLC) assay (9) with fluorescence detection for the determmation of bufuralol l'-hydroxylation activity. Methods for other CYP2D6 assays such as debrisoquine 4-hydroxylase (9) and dextromethorphan O-demethylase (9, 10) can be found in the cited references. [ABSTRACT FROM AUTHOR]

CYP2C 19 is one of at least three CYP2C enzymes expressed in human hver (1, 2), although the abundance of this P450 is generally less than that of etther CYP2C8 or CYP2C9 (3). Considerable interindividual differences occur in hepatic CYP2C19 content (1, 2), primarily owmg to a genetic polymorphism in this enzyme (4). Approximately 15-20% of Orientals and 3% of Caucasians exhibit the CYP2C19 poor-metabolizer phenotype, which is associated with at least two mutant alleles, designated CYP2C 19ml and CYP2C 19m2 (4). Studies with cDNA-expressed CYP2C 19 have identified several drugs as substrates for this polymorphically expressed P450, including omeprazole (5, 6), diazepam (7), cyclophosphamlde (3), and lfosfamide (3). However, the precise role of human hepatic CYP2C19 in drug biotransformation is poorly understood because mhibitory monospecific antiCYP2C19 antibodies are not available and a CYP2C19-specific chemical inhibitor has yet to be identified. Recent correlational analyses have led to the conclusion that CYP2C19 is a major P450 responsible for (S)-mephenytoin 4'-hydroxylation activity in human liver microsomes (1, 2). This is supported by the finding that cDNA-expressed CYP2C19 has a 50-to lOO-fold greater turnover number than CYP2C8, CYP2C9, or CYP2C 18 for (S)-mephenytoin 4−hydroxylatlon (2). As a result, (S)-mephenytom 4'-hydroxylase activity is frequently used as a catalytic monitor for human hepatic CYP2C19. In addmon, (S)-mephenytom 4'-hydroxylation is a useful indicator of the catalytic activity of cDNA-expressed CYP2C 19 (2). Varrous analytical methods have been developed to measure the enzymatic formation of 4'-hydroxymephenytoin, including isocratrc, reversed-phase hrgh performance hqutd chromatography (8). This chapter describes a method for the determination of (S)-mephenytoin 4'-hydroxylation by gradient, reversedphase high performance llqurd chromatography using 14C-labeled substrate and radiometrrc detection. [ABSTRACT FROM AUTHOR]

CYP2C9 is a major CYP2C enzyme expressed in human liver (1-3). Recent experiments with primary cultures of human hepatocytes have suggested that CYP2C9 protein expression can be increased in cells treated with a P450 inducer such as phenobarbital, dexamethasone, or rlfampin (rifampicin) (3). This is consistent with the clmical finding that rlfampin increases the total body clearance of tolbutamide (4), which is metabolized almost entirely by hepatic CYP2C9 (5). Other substrates for CYP2C9 include phenytom (5, 6), warfarin (7) and diclofenac (8). Sulfaphenazole is a CYP2C9-selective chemical mhlbitor (9-12) and inhibition experiments with this sulfur-containing compound have suggested that human liver microsomal diclofenac 4′-hydroxylatlon IS selectively catalyzed by CYP2C9 (8). This chapter describes a high-performance liquid chromatographic (HPLC) assay for the determination of diclofenac 4′-hydroxylase activity. Methods for other CYP2C9 assays such as tolbutamide methylhydroxylase (13) and (S)-warfarin 7-hydroxylase (14, 15) can be found in the cited references. [ABSTRACT FROM AUTHOR]

CYP2C8 is a major CYP2C protein expressed in human liver 1-4. Considerable interindividual differences (-20-fold) have been observed in hepatic CYP2C8 content (5) and a blmodal dlstrlbutlon in CYP2C8 protein amounts m a panel of human liver mlcrosomes has been reported (6). Experiments with primary cultures of human hepatocytes have indicated that CYP2C8 is subject to inductton by phenobarbital, dexamethasone, and rifampin (rifampicm) (4). Little is known about the function of CYP2C8, although studies with lmmunologically purified or cDNA-expressed CYP2C8 have indicated that this P450 catalyzes the metabohsm of retmol(6), retinolc acid (6), arachldonic acid (7, 8), carbamazepine (9) and paclitaxel (10-12). Oxldatlon of the anticancer drug paclitaxel to 6a-hydroxypachtaxel appears to be selectively catalyzed by CYP2C8 because cDNA-expressed human CYP2C8 is active in this reaction, whereas CYPlA2,2A6, 2B6,2C8, 2C9-Ile359, 2C9-Cys'44, 2C18, 2C19, 2D6, 2EI,3A3,3A4 and 3A5 are inactive (10-12). Paclitaxel Ga-hydroxylase actlvlty may therefore be a potentially useful dlagnostlc catalytic marker for human hepatic CYP2C8 see Note 1. This chapter describes a high-performance liquid chromatographic (HPLC) assay for the determination of paclltaxel 6α-hydroxylase activity. [ABSTRACT FROM AUTHOR]

CYP2B6 protein has been isolated and purified from human liver (1, 2). In a panel of 60 individual human liver microsome samples, this P450 accounts for

CYP2A6 has been isolated and purified to apparent homogeneity from human liver (1, 2). The level of CYP2A6 protein expressed in liver is low (∼4% of total hepatic P450 content) (3), although substantial intermdividual variation exists (3-5). This P450 is primarily a hepatic protein, as suggested by its absence from several extrahepatic tissues, including adult human lung, colon, breast, kidney, and placenta microsomes (2). According to experiments with primary cultures of human hepatocytes, CYP2A6 is mducible by phenobarbital, dexamethasone, and rifampm (rifampicin) (6). Experiments with individual cDNA-expressed human P450 forms have indicated that CYP2A6 is a major catalyst of coumarm 7-hydroxylation (7). Among the other recombinant human P450s examined, CYPlAl, lA2, 2C8, 2C9, 2D6, 2E1, 3A3, 3A4, and 3A5 are catalytically inactive with respect to coumarm 7-hydroxylation, whereas CYP2B6 is active at only ∼5% the rate of CYP2A6 (7). Immunoinhibition studies have validated the use of coumarm 7-hydroxylase activity as a diagnostic catalytic marker for human hepatic CYP2A6. Antihuman CYP2A6 IgG inhibits coumarm 7-hydroxylase activity by >90% in human liver microsomes (2), indicating that CYP2A6 is the principal and perhaps the sole catalyst of human liver microsomal coumarin 7-hydroxylase activity. This activity can be inhibited by a variety of compounds, includmg 8-methoxypsoralen (8), tranylcypromme (8), α-naphthoflavone (8-11) and diethyldithiocarbamate (8, 10, 11). This chapter describes a modification (12) of a spectrofluorometric method (13) for the determination of coumarm 7-hydroxylase activity. [ABSTRACT FROM AUTHOR]

The human CYP1 family consists of at least three proteins, CYP1A1, CYP1A2, and CYP1B1 (1), CYP1A1 is absent or present at very low levels in human liver (2, 3), but its expression is readily detectable in lung (4, 5). By contrast, CYP1A2 is constitutively expressed in human liver (2, 3) and is absent in lung (4, 5). CYP1B1 is primarily an extrahepatic P450, as suggested by the findmg that CYP1B1 mRNA is present in much greater abundance in tissues such as kidney, prostate, and breast than in liver (6). Exposure to polycyclic aromatic hydrocarbons such as those found in cigarette smoke induces the expression of both CYP1A1 and CYP1A2 (3, 7). CYP1B1 is also inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as demonstrated by cell-culture experiments (8, 9). The chemical inhibitor a-naphthoflavone, which is a potent inhibitor of CYP1A1 and CYP1A2 (10), also inhibits CYP1B1 and with semilar efficacy and potency (11). cDNA-expressed CYP1A1, CYP1A2 and CYP1B1 are each active in the oxidation of theophylline (11), caffeine (11, 12), estradiol (13, 14), benzo[a]pyrene (11) and 7-ethoxyresorufin (11). With 7-ethoxyresorufin as substrate, the rank order of specific activity (pmol/min/nmol P450) is CYP1A1 > CYP1B1 > CYP1A2 (11). 7-Ethoxyresorufin O-deethylation activity can be a useful and convenient catalytic monitor when conducting a comparative study using a panel of these three recombinant CYP1 enzymes (seeNote 1). Immunoinhibition experiments with inhibitory CYP1A-selective antibodies have suggested that CYP1A2 is a major contributor to 7-ethoxyresorufin O-deethylase activity in human liver microsomes (15, 16). [ABSTRACT FROM AUTHOR]

Cytochrome P450 (P450) enzymes catalyze the blotransformatlon of a broad range of structurally diverse foreign chemicals and endogenous substances. Many P450 enzymes have been isolated and identified in liver and other tlssues, including kidney, lung, intestines, and brain. Although these enzymes have a high degree of similarity in their ammo-acid sequences, many of them are subject to differential regulation and have distinct catalytic functions With the development of P450 form-specific (or P450 subfamlly-specific) inhlbltory antibodies, the discovery of enzyme-selective chemical inhibitors, and the general availability of catalytically active, mdivldual P450 cDNA-expressed proteins, several substrates, including drugs and endogenous substances, have been identified as useful catalytic monitors for specific P450 enzymes or subfamilies (see Table 1). These catalytic monitors can serve as useful expenmental tools in a variety of studies. These include studies designed: 1 To identify individual P45Os that catalyze the actlvatlon or detoxification of a particular compound, 2 To quantify the expression and catalytic activity of an mdlvrdual P450 in tissue samples and In cultured cells, 3. To analyze the enzyme kinetics of a particular P450; and 4 To eluctdate the mechanism of interaction between xenoblotlcs and mdlvldual P450 enzymes [ABSTRACT FROM AUTHOR]

Enzymes, proteins, cells, microorganisms, adsorbents, magnetic materials and other biologically active materials can be encapsulated within artificial cells with artificial polymer membranes (1-9). This type of artificial cells are not liposomes. The artificial cells protect the encapsulated biological materials from immunological reactions. At the same time, the enclosed materials continue to act in the immunologically isolated environment. The use of artificial cells containing adsorbent for hemoperfusion is a routine procedure used in patients for the removal of toxic substances or unwanted metabolites. Clinical studies are ongoing in patients using artificial cells as red blood cell substitutes, for enzyme therapy, and for delivery of biotechnological agents. Chang first developed a drop method for the bioencapsulation of cells and proposed its use in cell therapy (4,5). "Micro encapsulation of intact cells … the enclosed material might be protected from destruction and from participation in immunological processes, whereas the enclosing membrane would be permeable to small molecules of specific cellular product which could then enter the general extracellular compartment of the recipient. … The situation is comparable to that of a graft placed in an immunologically favorable site" (4). This was not explored by others for sometime. However, with increasing interests in biotechnology, many groups are now actively studying this for bioencapsulation of cells or genetically engineered microorganisms in cell and gene therapy. This chapter describes only two examples: bioencapsulation of hepatocytes or genetically engineered microorganisms. These examples are used to demonstrate the methods of preparations and their potential applications in cell and gene therapy. [ABSTRACT FROM AUTHOR]