The CYP2 family: models, mutants and interactions

1. The construction of three-dimensional models of mammalian cytochromes P450 from the CYP2 family is reported based on protein sequence alignment with CYP102, a bacterial P450 of known crystal structure. 2. The homology models of CYP2 family enzymes appear to show self-consistency with the currently accumulated information from site-directed mutagenesis and chemical modification of amino acid residues known to affect redox partner interactions. 3. The generation of these models from the recently reported crystal structure of substrate-bound CYP102 enables the exploration of likely active site contacts with specific substrates of CYP2 family isozymes.     

Molecular modelling of mammalian CYP2B isoforms and their interaction with substrates, inhibitors and redox partners

1. The construction of three-dimensional models of CYP2B isozymes from rat (CYP2B1), rabbit (CYP2B4) and man (CYP2B6), based on a multiple sequence alignment with CYP102, a unique eukaryotic-like bacterial P450 (in terms of possessing an NADPH-dependent FAD- and FMN-containing oxidoreductase redox partner) of known crystal structure, is reported. 2. The enzyme models described are shown to be consistent with experimental evidence from site-directed mutagenesis studies, antibody recognition sites and amino acid residues identified as being associated with redox partner interactions, together with the location of a key serine residue (Ser-128) likely to be involved in protein kinaseA-mediated phosphorylation. 3. A substantial number of known substrates and inhibitors of CYP2B isozymes are shown to fit the putative active sites of the enzyme models in agreement with their reported position of metabolism or mode of inhibition respectively. In particular, there is complementarity between the characteristic non-planar geometries of CYP2B substrates and key groups in the enzymes' active sites. 4. Molecular modelling of CYP2B isozymes appears to rationalize a number of the reported findings from quantitative structure-activity relationship investigations on series of CYP2B substrates and inhibitors.     

Identification of human liver cytochrome P450 isoforms involved in the in vitro metabolism of cyclobenzaprine

Cyclobenzaprine (Flexeril) is a muscle relaxant, possessing a tricyclic structure. Numerous therapeutic agents containing this structure are known to be metabolized by polymorphic cytochrome P4502D6. The aim of this study was to determine if cytochrome P4502D6 and other isoforms are involved in the metabolism of cyclobenzaprine in human liver microsomes. Selective cytochrome P450 inhibitors for CYP1A1/2 (furafylline and 7,8-benzoflavone) and CYP3A4 (troleandomycin, gestodene, and ketoconazole) inhibited the formation of desmethylcyclobenzaprine, a major metabolite of cyclobenzaprine, in human liver microsomes. Antibodies directed against CYP1A1/2 and CYP3A4 inhibited the demethylation reaction whereas anti-human CYP2C9/10, CYP2C19, and CYP2E1 antibodies did not show any inhibitory effects. When a panel of microsomes prepared from human B-lymphoblastoid cells that expressed specific human cytochrome P450 isoforms were used, only microsomes containing cytochromes P4501A2, 2D6, and 3A4 catalyzed N-demethylation. In addition, demethylation catalyzed by these recombinant cytochromes P450 can be completely inhibited with selective inhibitors at concentrations as low as 1 to 20 microM. Interestingly, cyclobenzaprine N-demethylation was significantly correlated with caffeine 3-demethylation (1A2) and testosterone 6 beta-hydroxylation (3A4) but not with dextromethorphan O-demethylation (2D6) in human liver microsomes. To further determine the involvement of cytochrome P4502D6 in cyclobenzaprine metabolism, liver microsomes from a human that lacked CYP2D6 enzyme activities was included in this study. The data showed that cyclobenzaprine N-demethylation still occurred in the incubation with this microsome. These results suggested that cytochrome P4502D6 plays only a minor role in cyclobenzaprine N-demethylation whereas 3A4 and 1A2 are primarily responsible for cyclobenzaprine metabolism in human liver microsomes. Due to the minimum involvement of CYP2D6 in the vitro metabolism of cyclobenzaprine, the polymorphism of cytochrome P4502D6 in man should not be of muci concern in the clinical use of cyclobenzaprine.     

Female exposure to high G: chronic adaptations of cardiovascular functions

The substrate structure-activity relationships described for the major human drug-metabolizing cytochrome P450 (P450 or CYP) enzymes suggest that the H1 receptor antagonist terfenadine could interact with CYP2D6 either as a substrate or as an inhibitor, in addition to its known ability to act as a substrate for CYP3A4. Based on this substrate structure-activity relationship, computer modeling studies were undertaken to explore the likely interactions of terfenadine with CYP2D6. An overlay of terfenadine and dextromethorphan, a known substrate of CYP2D6, showed that it was possible to superimpose the site of hydroxylation (t-butyl group) and the nitrogen atom of terfenadine with similar regions in dextromethorphan. These observations were substantiated by the ease of docking of terfenadine into a protein model of CYP2D6. Experimentally, terfenadine inhibited CYP2D6 activity in human liver microsomes with an IC50 of 14-27 microM, depending on the CYP2D6 substrate used. The inhibition of CYP2D6 was further defined by determining the Ki for terfenadine against bufuralol 1'-hydroxylase activity in four human livers. Terfenadine inhibited bufuralol 1'-hydroxylase activity with a Ki of approximately 3.6 microM. The formation of the hydroxylated metabolite (hydroxyterfenadine) in microsomes prepared from human liver and specific P450 cDNA-transfected B lymphoblastoid cells indicated that only CYP2D6 and CYP3A4 were involved in this transformation. As expected, the rate of formation was greatest with CYP3A4 (Vmax = 1257 pmol/min/nmol of P450), with CYP2D6 forming the metabolite at a 6-fold lower rate (Vmax = 206 pmol/min/nmol of P450). The two enzymes had similar KM values (9 and 13 microM, respectively). These data indicate that, as predicted from modeling studies, terfenadine has the structural features necessary for interaction with CYP2D6.     

Exercise-induced changes in protein metabolism

Mono-specific antibodies against the human cytochrome P450 (P450) enzymes CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP4A11 and an antibody that binds to CYP2C8, CYP2C9 and CYP2C19 have been produced by immunising rabbits with synthetic peptides representing small regions of each of these P450 enzymes. The specificity of the antibodies was confirmed by immunoblotting using recombinant P450 enzymes and samples of human hepatic microsomal fraction. Each of the antibodies bound only to their respective target P450 enzyme(s). The relative intensity of immunoreactive bands was compared with a variety of P450 activities and correlations were found between CYP1A2 and phenacetin O-deethylase activity, CYP2A6 and coumarin 7-hydroxylase activity, CYP2C9 and tolbutamide 4-hydroxylase activity, CYP2C19 and S-mephenytoin 4-hydroxylase activity, CYP2D6 and debrisoquine 4-hydroxylase activity, CYP2E1 and chlorzoxazone 6-hydroxylase activity, CYP3A4 and midazolam 1'-hydroxylase activity, and CYP4A11 and lauric acid 12-hydroxylase activity. A proportion of the 30 liver samples examined lacked CYP2A6 (7%), CYP2C19 (10%) or CYP2D6 (13%), consistent with the polymorphic expression of these P450 enzymes in human liver. Although CYP3A5 was detected in most individuals (97%), expression was polymorphic with 20% containing substantially higher levels. CYP2B6 was expressed in 20% of the human liver samples, with one sample containing a particularly high level. No immunodetectable CYP1A1 or CYP1B1 was found, consistent with the low level of expression of these P450 enzymes in human liver. The results demonstrate the utility of the antipeptide approach for producing specific antibodies against human P450 enzymes, enabling a comprehensive panel of antibodies against human P450 enzymes to be produced.     

Effects of propofol on human hepatic microsomal cytochrome P450 activities

1. The potential of propofol to inhibit the activity of major human cytochrome P450 enzymes has been examined in vitro using human liver microsomes. Propofol produced inhibition of CYP1A2 (phenacetin O-deethylation), CYP2C9 (tolbutamide 4'-hydroxylation), CYP2D6 (dextromethorphan O-demethylation) and CYP3A4 (testosterone 6beta-hydroxylation) activities with IC50 = 40, 49, 213 and 32 microM respectively. Ki for propofol against all of these enzymes with the exception of CYP2D6, where propofol showed little inhibitory activity, was 30, 30 and 19 microM respectively for CYPs 1A2, 2C9 and 3A4. 2. Furafylline, sulphaphenazole, quinidine and ketoconazole, known selective inhibitors of CYPs 1A2, 2C9, 2D6 and 3A4 respectively, were much more potent than propofol having IC50 = 0.8, 0.5, 0.2 and 0.1 microM; furafylline and sulphaphenazole yielded Ki = 0.6 and 0.7 microM respectively. 3. The therapeutic blood concentration of propofol (20 microM; 3-4 microg/ml) together with the in vitro Ki estimates for each of the major human P450 enzymes have been used to estimate the extent of cytochrome P450 inhibition, which may be produced in vivo by propofol. This in vitro-in vivo extrapolation indicates that the degree of inhibition of CYP1A2, 2C9 and 3A4 activity which could theoretically be produced in vivo by propofol is relatively low (40-51%); this is considered unlikely to have any pronounced clinical significance. 4. Although propofol has now been used in > 190 million people since its launch in 1986, there are only single reports of possible drug interactions between propofol and either alfentanil or warfarin. Consequently, it is difficult to conclude from either the published literature or the ZENECA safety database whether there is any evidence to indicate that propofol produces clinically significant drug interactions through inhibition of cytochrome P450-related drug metabolism.     

Interactions of a bicyclic analog of colchicine with beta-tubulin isoforms alphabeta(II), alphabeta(III) and alphabeta(IV)

In this study we have investigated the occurrence of cytochrome P450 isoforms and of related cytochrome P450 reductase in human hepatic stellate cells (hHSC), a type of cell having relevant roles in physiopathological conditions of the liver. By performing immunoblotting of hHSC microsomes and immunofluorescence analysis associated to confocal laser microscopy we detected only P450 enzymes belonging to the cytochrome P450 3A subfamily (CYP3A) as well as cytochrome P450 reductase. The presence of CYP3A was further indicated by detection of testosterone 6beta-hydroxylase activity in hHSC microsomes. Other important human P450 forms were either undetectable (CYP1A2, CYP2E1, CYP2C8/9/19 and CYP4A) or bearly detectable (CYP1A1) in hHSC. This is the first study showing existence of active cytochrome P450 isoforms in human HSC.     

Detection and localization of CYP1A1/CYP1A2 in murine liver: electrophoresis, immunoblotting and immunocytochemistry

Multiple forms of cytochrome P450 exist some of which are selectively inducible by exposure of the organism to a variety of foreign compounds. In this study, a monoclonal antibody specific for 3-methyl-cholanthrene-inducible cytochrome P450, Mab 1-7-1, was used to detect, localize and quantify CYP1A1/CYP1A2 in livers of C57BL/6 mice. Mab 1-7-1 recognized a faint band in the range between 45-66 Kd in Western immunoblots of liver microsomes from control mice, and a strong band in the same range, in liver microsomes from beta-naphthoflavone-treated mice. Microsome from control liver contained minimal levels of CYP1A1/CYP1A2; pretreatment with beta-naphthoflavone caused an increase in their expression. Immunoelectron microscopy was used to demonstrate the cellular localization and quantification of these isozymes in the liver. The immunolabeling procedure confirmed the endoplasmic reticulum as the primary site of CYP1A1/CYP1A2 induction in hepatocytes. This organelle showed the highest labeling density after treatment with beta-naphthoflavone. Increase in CYP1A1/CYP1A2 was 33.4-fold by morphometric analysis in induced hepatocytes in comparison to non-induced cells. In conclusion, CYP1A1/CYP1A2 is highly induced by beta-naphthoflavone in C57BL/6 mouse liver, and the cellular site of expression is the endoplasmic reticulum.     

High catalytic activity of human cytochrome P450 co-expressed with human NADPH-cytochrome P450 reductase in Escherichia coli

Forms of human cytochrome P450 (P450 or CYP), such as CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, were expressed or co-expressed together with human NADPH-P450 reductase in Escherichia coli. When P450 was expressed alone in E. coli, the expression level of holo-P450 ranged from 310 to 1620 nmol/L of culture. The expression level of holo-P450 decreased by co-expression with the reductase, and the level ranged from 66 to 381 nmol/L of culture. The expression level of the reductase varied depending on the forms of P450 co-expressed, and ranged from 204 to 937 U/L of culture. We assayed the catalytic activity of P450 using E. coli cells disrupted by freeze-thaw. When co-expressed with the reductase, human P450 catalyzed the oxidation of representative substrates at efficient rates. The rates appeared comparable to the reported activities of P450 in a reconstituted system containing purified preparations of P450 and the reductase.     

Evaluation of triacetyloleandomycin, alpha-naphthoflavone and diethyldithiocarbamate as selective chemical probes for inhibition of human cytochromes P450

A variety of chemicals, including triacetyloleandomycin (TAO), alpha-naphthoflavone (ANF), and diethyldithiocarbamate (DDC), are widely used as inhibitory probes for select individual human cytochrome P450 (CYP) enzymes, despite the fact that the selectivity of these inhibitors has not been rigorously evaluated. In the present study we take advantage of recent advances in cDNA-directed human P450 expression to evaluate directly the P450 form selectivity of TAO, ANF, and DDC, using a panel of 10 individual cDNA-expressed human P450s. Under experimental conditions known to yield maximal TAO complexation with P450 hemoproteins, TAO (20 microM) inhibited the catalytic activity of expressed CYPs 3A3, 3A4, and 3A5, whereas it did not affect CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, or 2E1 activity. ANF inhibited not only CYPs 1A1 and 1A2 (IC50 = 0.4-0.5 microM), but it was also similarly effective against CYPs 2C8 and 2C9. Increasing the concentration of ANF to 10 microM led to inhibition of CYP2A6 and CYP2B6. Although a previous study suggested that DDC is a selective inhibitor of CYP2E1, the present investigation shows that at concentrations required to inhibit CYP2E1 (IC50 approximately 125 microM when preincubated with NADPH), DDC also inhibited CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 3A3, and 3A4. Decreasing the concentration of DDC to 10 microM, however, led to inhibition of CYP2A6 (65% inhibition) and CYP2B6 (50% inhibition), but none of the other P450s examined, including CYP2E1. Overall, these results establish that (a) TAO is a selective inhibitor of the human CYP3A subfamily; (b) ANF potently inhibits CYP2C8 and CYP2C9, in addition to CYPs 1A1 and 1A2; and (c) DDC cannot be employed as a diagnostic inhibitory probe for CYP2E1.     

Regional hepatic CYP1A1 and CYP1A2 induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin evaluated with a multicompartment geometric model of hepatic zonation

A physiologically based pharmacokinetic (PBPK) model for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was combined with a five-compartment geometric model of hepatic zonation to predict both total and regional induction of CYP450 proteins within the liver. Three literature studies on TCDD pharmacokinetics and protein induction in female rats were analyzed. In simulating low-dose behavior for mRNA in whole liver and, particularly, in representing immunohistochemical observations, the five-compartment model was more successful than conventional homogeneous one-compartment liver models. The five-compartment liver model was used with the affinity of TCDD for the Ah receptor (AhR) held constant across all the liver (Kb = 0.2 nM). The presumed affinities of the AhR-TCDD complex for TCDD responsive elements in the CYP1A1 (Kd1) and CYP1A2 (Kd2) genes varied between adjacent compartments by a factor of 3. This parameterization leads to predicted 81-fold differences in affinities between the centrilobular and the periportal regions. The affinities used for AhR-TCDD complex binding to TCDD response elements for CYP1A2 in compartment 3 (the midzonal area) ranged from 0.08 to 1.0 nM in the three studies modeled. For CYP1A1 the corresponding dissociation constant in compartment 3 varied from 0.6 to 2.0 nM. In each compartment, the Hill coefficient for induction had to be 4 or greater to match the immunohistochemical results. This multi-compartment liver model is consistent with data on protein and mRNA induction throughout the liver and on the regional distribution of these proteins. No previous model has incorporated regional variations in induction. The PBPK analysis based on the multicompartment liver model suggests that the low-dose behavior for hepatic CYP1A1/CYP1A2 induction by TCDD is highly non-linear.     

Purification and characterization of heterologously expressed mouse CYP2A5 and CYP2G1: role in metabolic activation of acetaminophen and 2,6-dichlorobenzonitrile in mouse olfactory mucosal microsomes

The metabolic activation of two known olfactory mucosal (OM) toxicants, acetaminophen (AP) and 2,6-dichlorobenzonitrile (DCBN), was examined with mouse liver and OM microsomes and purified, heterologously expressed mouse CYP2A5 and CYP2G1. In reconstituted systems, both isoforms were active in metabolizing DCBN and AP to metabolites that formed protein adducts. The formation of DCBN- or AP-protein adducts and other AP metabolites, including 3-hydroxy-AP and, in the presence of glutathione, AP-glutathione conjugate, was also detected in OM microsomal reactions and to a much greater extent than in liver microsomes. Evidence was obtained that CYP2A5 and CYP2G1 play major roles in mouse OM microsomal metabolic activation of DCBN and AP. Immunoblot analysis indicated that CYP2A5 and CYP2G1 are abundant P450 isoforms in OM microsomes. OM microsomal AP and DCBN metabolic activation was inhibited by 5- and 8-methoxsalen, which inhibit both CYP2A5 and CYP2G1, and by an inhibitory anti-CYP2A5 antibody that also inhibits CYP2G1. In addition, the roles of CYP1A2 and CYP2E1 in the OM bioactivation of AP and DCBN were ruled out by comparing activities of acetone-treated mice or Cyp1a2(-/-) mice with those of control mice. Thus, CYP2A5 and CYP2G1 may both contribute to the known OM-selective toxicity of AP and DCBN. Further analysis of the kinetics of AP and DCBN metabolism by the purified P450s suggested that CYP2A5 may play a greater role in OM microsomal metabolism of AP, whereas their relative roles in DCBN metabolism may be dose dependent, with CYP2G1 playing more important roles at low substrate concentrations.     

Induction, suppression and inhibition of multiple hepatic cytochrome P450 isozymes in the male rat and bobwhite quail (Colinus virginianus) by ergosterol biosynthesis inhibiting fungicides (EBIFs)

Ergosterol biosynthesis inhibiting fungicides (EBIFs) have complex effects on the hepatic microsomal monooxygenase systems of vertebrate species, having been described as mixed inducers and inhibitors of cytochrome P450. In the current study, we examined the effects of two EBIFs in clinical use, clotrimazole and ketoconazole, and two agricultural EBIFs, propiconazole and vinclozolin, on hepatic monooxygenase activities and P450 apoprotein expression in the male Sprague-Dawley rat and the male bobwhite quail. EBIFs produced Type II binding spectra with hepatic microsomes from both species and were effective inhibitors of methoxyresorufin O-demethylase, an activity selective for P450 isozymes in gene family 1. However, the EBIFs varied widely in their effectiveness as inducers of P450 isozymes in gene families 1, 2, 3 and 4, both within the same species and between species. In the rat, clotrimazole was the most effective inducer, increasing expression of CYP 3A isozymes over 450-fold, CYP 2B1/2 30-fold and CYP 1A1/2 12-fold and suppressing expression of CYP 2C11 nearly 70%. By contrast, in the quail, clotrimazole was the least effective inducer. In quail, vinclozolin and propiconazole elevated total P450 content 10- and 7-fold, respectively. The induction response also appeared to be mixed, but in this case consisted of a 5-fold induction of P450s in gene family 1A, a 3-fold induction of P450s in gene family 3A and 4A, and induction of protein(s) from gene family 2, cross-reactive with antisera against rat CYP 2C11 and CYP 2A1. A protein that was cross-reactive with antibodies raised against rat CYP 2B1 was decreased with EBIF treatment. In conclusion, EBIFs have complex patterns of induction, suppression and inhibition of cytochrome P450 isozymes in both mammals and birds, which vary according to both the fungicide and the species.     

Regulation of cytochrome P450 expression by inhibitors of hydroxymethylglutaryl-coenzyme A reductase in primary cultured rat hepatocytes and in rat liver

It was previously demonstrated that treatment of primary cultured rat hepatocytes with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, induced the mRNAs for several cytochromes P450 (P450s), including CYP2B1/2, CYP3A1/2, and CYP4A. In this study, we have compared the effects of lovastatin with those of three additional HMG-CoA reductase inhibitors (simvastatin, pravastatin, and the structurally dissimilar drug fluvastatin) on P450 expression in primary cultured rat hepatocytes, and we have also characterized the effects of in vivo treatment with fluvastatin on P450 expression in rat liver. Treatment of cultured hepatocytes with lovastatin, simvastatin, or fluvastatin increased CYP2B1/2, CYP3A1/2, and CYP4A mRNA and immunoreactive protein levels over the dose range (3 x 10(-6) to 3 x 10(-5) M) required to increase the amount of HMG-CoA reductase mRNA. The increases in CYP2B1/2 levels produced by 3 x 10(-5) M fluvastatin treatment were larger than those produced by lovastatin or simvastatin treatment or by treatment with 10(-4) M phenobarbital. In contrast, treatment of cultured hepatocytes with 3 x 10(-5) M lovastatin, simvastatin, or fluvastatin increased CYP3A1/2 and CYP4A mRNA and immunoreactive protein to lower levels than those produced by treatment with 10(-5) M dexamethasone or 10(-4) M ciprofibrate. Treatment of cultured hepatocytes with pravastatin had little or no effect on the amount of any of the P450s examined, although this drug induced HMG-CoA reductase mRNA as effectively as did fluvastatin. Incubation of hepatocytes with 10(-4) M fluvastatin increased CYP1A1 mRNA to 67% of the level induced by treatment with 10(-5) M beta-naphthoflavone. Doses of 50 or 100 mg/ kg/day fluvastatin administered for 3 days to rats increased the hepatic levels of CYP2B1/2 and CYP4A mRNA and immunoreactive protein, although to much lower levels than those produced by treatment with phenobarbital or ciprofibrate, respectively. Treatment of rats with fluvastatin had no effect on hepatic levels of CYP3A1/2 mRNA or immunoreactive protein. However, treatment with 50 mg/kg/day fluvastatin induced CYP1A1 mRNA and protein. The effects of fluvastatin treatment on P450 expression seen in primary cultured rat hepatocytes thus largely recapitulated the effects seen in vivo. The differences in effects among the HMG-CoA reductase inhibitors suggest that simple inhibition of HMG-CoA reductase cannot explain all of the effects of these drugs on P450 expression.     

Enhanced expression of cytochrome P450 in stomach cancer

The cytochromes P450 have a central role in the oxidative activation and detoxification of a wide range of xenobiotics, including many carcinogens and several anti-cancer drugs. Thus the cytochrome P450 enzyme system has important roles in both tumour development and influencing the response of tumours to chemotherapy. Stomach cancer is one of the commonest tumours of the alimentary tract and environmental factors, including dietary factors, have been implicated in the development of this tumour. This type of tumour has a poor prognosis and responds poorly to current therapies. In this study, the presence and cellular localization of several major forms of P450, CYP1A, CYP2E1 and CYP3A have been investigated in stomach cancer and compared with their expression in normal stomach. There was enhanced expression of CYP1A and CYP3A in stomach cancer with CYP1A present in 51% and CYP3A present in 28% of cases. In contrast, no P450 was identified in normal stomach. The presence of CYP1A and CYP3A in stomach cancer provides further evidence for the enhanced expression of specific forms of cytochrome P450 in tumours and may be important therapeutically for the development of anti-cancer drugs that are activated by these forms of P450.     

Spatially distinct expression of two new cytochrome P450s in leaves of Nepeta racemosa: identification of a trichome-specific isoform

Using a PCR-based approach, two novel cytochrome P450 cDNAs were isolated from a catmint (Nepeta racemosa) leaf cDNA library. The cDNAs (pBSK3C7 and pBSK4C3) were 76.9% identical in their nucleotide sequences, indicating that they are the products of two closely-related genes. A comparison of the sequence of these cDNAs with database sequences indicated that they represent new members of the CYP71 gene family of plant cytochrome P450s. Clone pBSK3C7 contains the full-length coding sequence of a cytochrome P450, whilst pBSK4C3 lacks ca. 6 codons at the 5' end. The cytochromes P450 encoded by these clones were designated CYP71A5 and CYP71A6 (pBSK3C7 and pBSK4C3, respectively). Southern blot analysis indicated that the corresponding genes were present as single copies in the genome of N. racemosa. Northern blot analysis showed that a gene homologous with CYP71A5 was expressed in the related species N. cataria, but no homologue of CYP71A6 was detected in this species. Expression of CYP71A5 in N. racemosa was maximal in flowers, tissues within the apical bud, and young expanded leaves. That of CYP71A6 was maximal in older leaves. Expression of CYP71A5 occurred exclusively in trichomes present on the leaf surfaces, in contrast to that of CYP71A6, which occurred predominantly within the leaf blade tissues.     

Identification and characterization of human cytochrome P450 isoforms interacting with pimozide

Using human liver microsomes (HLMs) and recombinant human cytochrome P450 (CYP450) isoforms, we identified the major route of pimozide metabolism, the CYP450 isoforms involved, and documented the inhibitory effect of pimozide on CYP450 isoforms. Pimozide was predominantly N-dealkylated to 1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one (DHPBI). The formation rate of DHPBI showed biphasic kinetics in HLMs, which suggests the participation of at least two activities. These were characterized as high-affinity (K(m1) and Vmax1) and low-affinity (K(m2) and Vmax2) components. The ratio of Vmax1 (14 pmol/min/mg protein)/K(m1) (0.73 microM) was 5.2 times higher than the ratio of Vmax2 (244 pmol/min/mg protein)/K(m2) (34 microM). K(m2) was 91 times higher than K(m1). The formation rate of DHPBI from 25 microM pimozide in nine human livers correlated significantly with the catalytic activity of CYP3A (Spearman r = 0.79, P = .028), but not with other isoforms. Potent inhibition of DHPBI formation from 10 microM pimozide was observed with ketoconazole (88%), troleandomycin (79%), furafylline (48%) and a combination of furafylline and ketoconazole (96%). Recombinant human CYP3A4 catalyzed DHPBI formation from 10 microM pimozide at the highest rate (V = 2.2 +/- 0.89 pmol/min/pmol P450) followed by CYP1A2 (V = 0.23 +/- 0.08 pmol/min/pmol P450), but other isoforms tested did not. The K(m) values derived with recombinant CYP3A4 and CYP1A2 were 5.7 microM and 36.1 microM, respectively. Pimozide itself was a potent inhibitor of CYP2D6 in HLMs when preincubated for 15 min (Ki = 0.75 +/- 0.98 microM) and a moderate inhibitor of CYP3A (Ki = 76.7 +/- 34.5 microM), with no significant effect on other isoforms tested. Our results suggest that pimozide metabolism is catalyzed mainly by CYP3A, but CYP1A2 also contributes. Pimozide metabolism is likely to be subject to interindividual variability in CYP3A and CYP1A2 expression and to drug interactions involving these isoforms. Pimozide itself may inhibit the metabolism of drugs that are substrates of CYP2D6.     

Asymptomatic hypertension in the ED

(R)-(+)-Menthofuran is a potent, mechanism-based inactivator of human liver cytochrome P450 (CYP or P450) 2A6. Menthofuran caused a time- and concentration-dependent loss of CYP2A6 activity. The inactivation of CYP2A6 was characterized by a Ki of 2.5 microM and a kinact of 0.22 min-1 for human liver microsomes and a Ki of 0.84 microM and a kinact of 0.25 min-1 for purified expressed CYP2A6. Addition of various nucleophiles, a chelator of iron, or scavengers of reactive oxygen species or extensive dialysis failed to protect CYP2A6 from inactivation. An antibody to metallothionein conjugates of a suspected reactive metabolite of menthofuran was used to detect reactive menthofuran metabolite adducts with CYP2A6. These adducts were formed only in the presence of NADPH-P450 reductase and NADPH. Glutathione, methoxylamine, and semicarbazide did not prevent adduction of reactive menthofuran metabolites to CYP2A6, however. The menthofuran metabolite formation/CYP2A6 inactivation partition ratio was determined to be 3.5 +/- 0.6 nmol/nmol of P450. Menthofuran was unable to inactivate CYP1A2, CYP2D6, CYP2E1, or CYP3A4 in a time- and concentration-dependent manner.     

Rapid tolerance and crosstolerance to motor impairment effects of benzodiazepines, barbiturates, and ethanol

Susceptibility to develop Parkinson's disease has been linked to abnormalities of P450 enzyme function. Multiple P450 enzymes are expressed in brain but the relationship of these to Parkinson's disease is unknown. We have investigated the expression of P450 enzymes in the rat substantia nigra and their co-localization in tyrosine hydroxylase-positive neurons and astrocytes. Immunohistochemistry was performed using anti-peptide antisera against the following P450 enzymes: CYP1A1, CYP1A2, CYP2B1/2, CYP2C12, CYP2C13/2C6, CYP2D1, CYP2D4, CYP2E1, CYP3A1, CYP3A2 and NADPH-P450 oxidoreductase. Immunoreactivity in nigral cells was found only for CYP2E1 and CYP2C13/2C6. CYP2E1 immunoreactivity was localized to many midbrain nuclei including the substantia nigra pars compacta but not the substantia nigra pars reticulata while immunoreactivity to CYP2C13/2C6 was found in the substantia nigra pars compacta, substantia nigra pars reticulata and many other midbrain nuclei. Sections of rat midbrain double labelled for either CYP2E1 or CYP2C13/2C6 and tyrosine hydroxylase or glial fibrillary acidic protein were examined for co-localization by confocal laser scanning microscopy. CYP2E1 and CYP2C13/2C6 immunoreactivity was found in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta but not in glial cells. CYP2C13/2C6, but not CYP2E1, was also found in non-glial, non-tyrosine hydroxylase-expressing cells in the substantia nigra pars reticulata. Isoniazid induction increased CYP2E1 fluorescence signal intensity from nigral dopaminergic neurons. At least two P450 enzymes are found in nigral dopamine containing cells and one, namely CYP2E1, is selectively localized to this cell population. CYP2E1 is a potent generator of free radicals which may contribute to nigral pathology in Parkinson's disease. The expression of CYP2E1 in dopaminergic neurons in substantia nigra raises the possibility of a causal association with Parkinson's disease.