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A homology model of rabbit CYP 2B4 was constructed on the basis of the crystallographic structure of truncated mammalian CYP 2C5/3 and bacterial soluble CYPs. To validate the CYP 2B4 homology model photoaffinity labeling was employed. Three probes (I-III) containing a photo-labile azido-group and an amino-group on opposite ends of the molecule were designed for photoaffinity labeling of the CYP 2B4 in increasing distance from the heme iron. Spectroscopic data proved probes I (the shortest) and II (a middle sized) to be coordinated with the heme iron via their amino-groups in the enzyme active center while the probe III (the longest) was not bound in this way. This binding orientation of probes I and II is in accordance with the model predicting ion-pairing of the negatively charged side chain of CYP 2B4 Asp 105 and a positively charged nitrogen located in an appropriate position in structures of probes I and II, only. The lack of heme binding of the probe III is clear from its docking into the CYP 2B4 model since no Asp 105 ion-pairing is possible. The target of photoactivated probe II, Arg 197, in a distance of about 16.5 A from the heme iron, exactly matches the position of that amino acid residue, predicted from the CYP 2B4 homology model. Moreover, using this technique, a substrate access channel has been identified. To assess the predicted substrate-binding pocket, an interaction of a specific CYP 2B4 substrate, diamantane, was examined. In "silico" docking revealed strong binding of diamantane in an orientation allowing experimentally observed C4-hydroxylation. Our homology model of CYP 2B4 is thus consistent with experimental metabolic and photoaffinity labeling data.

Lipopolysaccharide (LPS) treatment of rats suppresses CYP 4F4 and 4F5 expression by 50 and 40%, respectively, in a direct fashion occurring in the liver. This contention is borne out by essentially parallel dose-dependent changes observed upon treatment of rat hepatocyte cultures with LPS. An alternate avenue of triggering the inflammatory cascade is traumatic brain injury by controlled cortical impact. Such injury brings about a dramatic change in the expression of CYP 4F4 and 4F5 mRNA which reaches its greatest effect 24 h after impact compared with sham-operated but uninjured controls. At time points after 24 h the expression of both isoforms increases dramatically reaching highest levels at 2 weeks post-injury. These changes in mRNA expression are mirrored by changes in protein expression. The results are consistent with the notion that immediately after injury concentrations of leukotriene and prostaglandin mediators are elevated by decreased CYP 4F concentrations. As time after injury increases those conditions reverse. Increased CYP 4F expression leads to diminished concentrations of leukotriene and prostaglandin mediators and then to recovery and repair.

The health care system in the People's Republic of China (PRC) is undergoing a major transition that has made the government revise its approach to how medicine is taught and practiced. Family medicine, which provides a generalist approach to medical care, is at the forefront of this transition. This article reviews the recent history of medical education in the PRC, including the establishment of the discipline of family medicine in the mid 1980s, and factors promoting development of family medicine. These include the movement away from government-subsidized health care in hospital settings, the aging population, increased urbanization, increasing incidence of infectious diseases, and rising health care costs. We conclude from observations made in the PRC and from a review of secondary sources that family medicine in China is in its infancy. The value of understanding the role that family medicine plays within China's changing health care system is that we gain a broader perspective of the variety and growing international importance of family practice as a profession.

The metabolism of chlorpromazine by expressed recombinant cytochromes P450 4F4 and 4F5 cloned from rat brain was analyzed to characterize the individual activities of the isoforms. Both isoforms metabolized chlorpromazine to both the N-demethylated and the S-oxide products. When isoforms were incubated with chlorpromazine in the presence of increasing concentrations of imipramine, imipramine significantly inhibited both N-demethylation and S-oxidation of chlorpromazine. A dilution of the serum fraction of anti-4F antibody was also found to significantly inhibit both S-oxidation and N-demethylation of chlorpromazine by both 4F4 and 4F5.

A putative binding region for cumene hydroperoxide in the active site of cytochrome P4501A1 was identified using photoaffinity labeling. Thr501 was determined as the most likely site of modification by azidocumene used as the photoaffinity label (T. Cvrk and H. W. Strobel, (1998) Arch. Biochem. Biophys. 349, 95-104). To evaluate further the role of this amino acid residue a site-directed mutagenesis approach was employed. P4501A1 wild type and two mutants, P4501A1Glu501 and P4501A1Phe501, were expressed in and purified from Escherichia coli and used for kinetic analysis to confirm the role of Thr501 residue in cumene hydroperoxide binding. The mutation resulted in a two- to fourfold decrease in the rate of heme degradation in the presence of 0.5 mM cumene hydroperoxide. The mutations do not prevent or significantly alter binding of the tested substrates; however, binding of 2-phenyl-2-propanol (product generated from cumene hydroperoxide) to P4501A1Glu501 and P4501A1Phe501 exhibited four- and eightfold decreases, respectively, suggesting that the mutations strongly affected the affinity of cumene hydroperoxide for the P4501A1 active site. The kinetic analysis of cumene hydroperoxide-supported reactions showed that both mutants exhibit increased Km and decreased VMax values for all tested substrates. Furthermore, the mutations affected product distribution in testosterone hydroxylation. On the basis of P4501A1Glu501 and P4501A1Phe501 characterization, it can be concluded that Thr501 plays an important role in cumene hydroperoxide/P4501A1 interaction.

Cytochrome P450 4F isoforms catalyze the hydroxylation of eicosanoids such as leukotriene B(4), prostaglandins, and lipoxins as well as hydroxyeicosatetraenoic acids. In the present study, we report the molecular cloning of two novel mouse CYP4F isoforms, CYP4F15 and CYP4F16. Sequence comparison showed that CYP4F15 has 93.5% homology to CYP4F4 and CYP4F16 has 90.8% homology to CYP4F5, therefore they are the orthologs for rat CYP4F4 and CYP4F5, respectively. Both isoforms are expressed in liver and also in extrahepatic tissues but the patterns of expression are slightly different. To elucidate further the regulation and regulatory mechanism of the two isoforms, renal and hepatic CYP4F15 and CYP4F16 expression were analyzed using wild-type (SV/129) mice and peroxisome proliferator-activated receptor (PPAR) alpha null mice with or without challenge by bacterial endotoxin (LPS) or clofibrate. Renal expression of CYP4F15 was induced by LPS and clofibrate in (+/+) mice, and these effects were absent in the (-/-) mice. Renal expression of CYP4F16 was not affected by LPS or clofibrate in (+/+) or (-/-) mice. In contrast, hepatic expression of CYP4F15 and CYP4F16 was significantly reduced by LPS-treatment in (+/+) mice. A lesser reduction was also seen in the (-/-) mice, suggesting that PPARalpha is partially responsible for this down-regulation. Clofibrate treatment caused the reduction of hepatic CYP4F16 expression and this effect was not dependent on PPARalpha. Clofibrate treatment had no effect on hepatic CYP4F15 expression. Together, our data indicate that CYP4Fs are regulated in an isoform-specific, tissue-specific, and species-specific manner.

It has been proposed that negatively charged amino acids on the surface of reductase and positively charged amino acids on the surface of P450 mediate the binding of both proteins through electrostatic interactions. In this study, we used a site-directed mutagenesis approach to determine a role for two lysine residues (Lys271 and Lys279) of cytochrome P4501A1 in the interaction of P4501A1 with reductase. We prepared two mutants P4501A1Ile271 and P4501A1Ile279 with a mutation of the lysine at positions 271 and 279, respectively. We observed a strong inhibition (>80%) of the 7-ethoxycoumarin and ethoxyresorufin deethylation activity in the reductase-supported system for both mutants. In the cumene hydroperoxide-supported system, P4501A1Ile279 exhibited wild-type activity, but the P4501A1Ile271 mutant activity remained low. The CD spectrum and substrate-binding assay indicated that the secondary structure of P4501A1Ile271 is perturbed. To evaluate further the involvement of these P4501A1 lysine residues in reductase binding, we measured the KM of reductase for wild type and mutants. Both wild type and P4501A1Ile271 reached saturation in the range of reductase concentrations tested with KM values 5.1 and 11.2 pM, respectively. The calculated KM value for P4501A1Ile279 increased 9-fold, 44.4 pM, suggesting that the mutation affected binding of reductase to P4501A1. Stopped-flow spectroscopy was employed to evaluate the effect of mutations on electron transfer from reductase to heme iron. Both wild type and P450Ile279 showed biphasic kinetics with a approximately 40% participation of the fast step in the total activity. On the other hand, only single-phase kinetics for iron reduction was observed for P450Ile271, suggesting that the low activity of this mutant can be attributed not only to major structural changes but also to a disturbance in the electron transport.

The CYP3A subfamily is the most abundant of the human hepatic cytochrome P450 enzymes. They mediate the biotransformation of many drugs, including a number of psychotropic, cardiac, analgesic, hormonal, immunosuppressant, antineoplastic, and antihistaminic agents. We studied diet/ethanol interactions using total enteral nutrition in adult male Sprague-Dawley rats with diets containing 16% protein, ethanol (13 g/kg), corn oil (fat; 25-45%), and carbohydrate (CHO; 1-21%). Using this model, chronic ethanol feeding decreased CYP3A activity (testosterone 6 beta-hydroxylation) and apoprotein levels (Western blot) (P <.05) and these effects were independent of the dietary CHO/fat ratio. The CYP3A2 mRNA levels decreased (P <.05) in the rats fed ethanol-containing diets by 73 to 83% compared with rats fed control diets, regardless of the CHO/fat ratio. In contrast, ethanol induced CYP3A9 mRNA levels (P <.05) and this effect was greater (P <.05) in the high-CHO/low-fat group (11.3-fold) than in the low-CHO/high-fat group (2.6-fold). Purified recombinant rat P450 3A9 had a chlorzoxazone 6-hydroxylase activity with a turnover number 1.3 nmol/min/nmol of P450. These results indicate that 1) ethanol differentially affects the expression of CYP3A gene family and this regulation appears to be modulated by dietary CHO/fat ratio; 2) the decrease in testosterone 6 beta-hydroxylase activity and CYP3A apoprotein levels are most likely due to the ethanol-induced decrease in CYP3A2 mRNA levels; and 3) CYP3A9 is induced by ethanol and is a low-affinity, high-K(m) chlorzoxazone hydroxylase.

The catalytic requirements and the role of P450 3A9, a female-specific isoform of CYP3A from rat brain, in the metabolism of several steroid hormones were studied using recombinant P450 3A9 protein. The optimal steroid hormone hydroxylase activities of P450 3A9 required cholate but not cytochrome b5. P450 3A9 was active in the hydroxylation reactions of testosterone, androstenedione, progesterone and dehydroepiandrosterone (DHEA). No activity of P450 3A9 toward cortisol was detectable under our reconstitution conditions. Among all the steroid hormones examined, female-specific P450 3A9 seemed to catalyze most efficiently the metabolism of progesterone, one of the major female hormones, to form three mono-hydroxylated products, 6beta-, 16alpha-, and 21-hydroxyprogesterone. Our data also showed that P450 3A9 can catalyze the formation of a dihydroxy product, 4-pregnen-6beta, 21-diol-3, 20-dione, from progesterone with a turnover number, 1.3 nmol/min/nmol P450. Based on the Vmax/Km values for P450 3A9 using either 21-hydroxprogesterone or 6beta-hydroxyprogesterone as a substrate, 4-pregnen-6beta, 21-diol-3, 20-dione may be formed either by 6beta-hydroxylation of 21-hydroxprogesterone or 21-hydroxylation of 6beta-hydroxyprogesterone. As a major isoform of CYP3A expressed in rat brain, the activities of P450 3A9 toward two major neurosteroids, progesterone and DHEA suggested a possible role for P450 3A9 in the metabolism of neurosteroids.

The function of cytochrome P450 (P450) in the mammalian brain is not well understood. In an effort to further this understanding, this study identifies two endogenous substrates for P450 2D18. Previous reports have shown that this isoform is expressed in the rat brain, and the recombinant enzyme catalyzes the N-demethylation of the antidepressants imipramine and desipramine. By further examining the substrate profile of P450 2D18, inferences can be made as to potential endogenous P450 substrates. Herein we demonstrate the metabolism of the central nervous system-acting compounds chlorpromazine and chlorzoxazone with turnover numbers of 1.8 and 0. 9 nmol/min/nmol, respectively. Because the four aforementioned pharmaceutical substrates work by binding to neurotransmitter receptors, binding assays and oxidation reactions were performed to test whether dopamine is a substrate for P450 2D18. These data indicate a K(S) value of 678 microM and that P450 2D18 can support the oxidation of dopamine to aminochrome through a peroxide-shunt mechanism. We also report the P450 2D18-mediated omega-hydroxylation and epoxygenation of arachidonic acid, primarily leading to the formation of 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids, compounds that have been shown to have vasoactive properties in brain, kidney, and heart tissues. The data presented herein suggest a possible role for P450 involvement in membrane and receptor regulation via epoxyeicosatrienoic acid formation and a potential involvement of P450 in the oxidation of dopamine to reactive oxygen species under aberrant physiological conditions where the sequestering of dopamine becomes compromised, such as in Parkinson's disease.

By a combination of cDNA library screening and rapid amplification of cDNA ends analysis, a novel human cytochrome P450 4F isoform has been cloned and sequenced. The new 4F isoform is designated CYP4F11 and contains 1765 nucleotides. The coding region encodes 524 amino acid residues, and the heme-binding region is highly conserved. The CYP4F11 amino acid sequence has 80.0, 82.3, and 79.2% identity to CYP4F2, CYP4F3, and CYP4F8 amino acid sequences, respectively. In vitro translation shows the molecular mass of CYP4F11 is approximately 57 kDa, consistent with the calculated molecular mass. CYP4F11 is expressed mainly in human liver, followed by kidney, heart, and skeletal muscle. The genomic structure of CYP4F11 was solved by database searching and computer analysis. The coding region of CYP4F11 has 12 exons. The CYP4F11 gene is located 16 kb upstream of the CYP4F2 gene on chromosome 19. This is consistent with the notion that the human cytochrome P450 4F genes form a cluster on chromosome 19., (Copyright 2000 Academic Press.)

Three heterobifunctional photoaffinity probes, N-(p-azidobenzyl)-N-methyl-p-aminobenzylamine (I), N-(p-azidobenzyl)-N-methyl-p-aminophenethylamine (II), and N-(p-azidophenethyl)-N-methyl-p-aminophenethylamine (III), were synthesized and characterized. These probes, containing a photolabile azido-group and an amino-group on opposite sides of the molecule, were designed for photoaffinty labeling of the cytochrome P450 (CYP) 2B active site cavity differing in distance from the heme iron. Spectroscopic studies proved that probes I and II coordinated with the heme iron via their amino-group in the enzyme active center, whereas probe III did not. This result in conjunction with data from kinetic studies suggests probes I and II are appropriate for photoaffinity labeling of the CYP 2B active center. Thus, probe II was used to identify amino acid residues within a distance of the probe length (about 16.5 A) from the heme. Analysis of a Lys-C digest of the probe II-labeled CYP 2B4 revealed a single labeled hexapeptide corresponding to position 192-197 of the CYP 2B4 sequence. Using postsource decay/matrix-assisted laser desorption ionization-time of flight, Arg197 was identified as a probe II target. The location of the labeled site in three-dimensional structures of bacterial CYPs and in CYP 2B homology models is discussed., (Copyright 1999 Academic Press.)

Drug metabolizing enzymes are known to be present and active in most extrahepatic tissues. In this review, evidence is presented that expression and activity of several extrahepatic drug-metabolizing enzymes are regulated in unique ways, which may be associated with tissue functions and activities. In several instances evidence is offered that hormonal effects may be regulated through tissue specific distribution and/or responses of transcription factors.