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.     

Identification of CYP4A11 as the major lauric acid omega-hydroxylase in human liver microsomes

Human liver microsomes are capable of oxidizing lauric acid (laurate), a model medium-chain fatty acid, at both the omega- and omega-1 positions to form 12- and 11-hydroxylaurate, respectively. These laurate hydroxylation reactions are apparently catalyzed by distinct P450 enzymes. While the P450 responsible for microsomal laurate omega-1 hydroxylation in human liver has been identified as CYP2E1, the enzyme catalyzing omega-hydroxylation remains poorly defined. To that end, we employed conventional purification and immunochemical techniques to characterize the major hepatic laurate omega-hydroxylase in humans. Western blotting with rat CYP4A1 antibodies was used to monitor a cross-reactive P450 protein (M(r) = 52 kDa) during its isolation from human liver microsomes. The purified enzyme (7.4 nmol P450/mg protein) had an NH2-terminal amino acid sequence identical to that predicted from the human CYP4A11 cDNA over the first 20 residues found. Upon reconstitution with P450 reductase and cytochrome b5, CYP4A11 proved to be a potent laurate omega-hydroxylase, exhibiting a turnover rate of 45.7 nmol 12-hydroxylaurate formed/min/nmol P450 (12-fold greater than intact microsomes), while catalyzing the omega-1 hydroxylation reaction at much lower rates (5.4 nmol 11-hydroxylaurate formed/min/nmol P450). Analysis of the laurate omega-hydroxylation reaction in human liver microsomes revealed kinetic parameters (a lone Km of 48.9 microM with a VMAX of 3.72 nmol 12-hydroxylaurate formed/min/nmol P450) consistent with catalysis by CYP4A11. In fact, incubation of human liver microsomes with antibodies raised to CYP4A11 resulted in nearly 85% inhibition of laurate omega-hydroxylase activity while omega-1 hydroxylase activity remained unaffected. Furthermore, a strong correlation (r = 0.89; P < 0.001) was found between immunochemically determined CYP4A11 content and laurate omega-hydroxylase activity in liver samples from 11 different subjects. From the foregoing, it appears that CYP4A11 is the principle laurate omega-hydroxylating enzyme expressed in human liver.     

A general strategy for the expression of recombinant human cytochrome P450s in Escherichia coli using bacterial signal peptides: expression of CYP3A4, CYP2A6, and CYP2E1

Heterologous expression of unmodified recombinant human cytochrome P450 enzymes (P450s) in Escherichia coli has proved to be extremely difficult. To date, high-level expression has only been achieved after altering the 5'-end of the native cDNA, resulting in amino acid changes within the P450 protein chain. We have devised a strategy whereby unmodified P450s can be expressed to high levels in E. coli, by making NH2-terminal translational fusions to bacterial leader sequences. Using this approach, we initially tested two leader sequences, pelB and ompA, fused to CYP3A4. These were compared with an expression construct producing a conventional NH2-terminally modified CYP3A4 (17alpha-3A4). Both leader constructs produced spectrally active, functional protein. Furthermore, the ompA-3A4 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane fractions, when compared with 17alpha-3A4. We then tested the ompA leader with CYP2A6 and CYP2E1, again comparing with the conventional (17alpha-) approach. As before, the leader construct produced active enzyme, and, for CYP2E1 at least, gave a higher level of expression than the 17alpha-construct. The ompA fusion strategy thus appears to represent a significant advance for the expression of P450s in E. coli, circumventing the previous need for individual optimization of P450 sequences for expression.     

Chemical modification of Tyr34 and Tyr129 in rabbit liver microsomal cytochrome b5 affects interaction with cytochrome P-450 2B4

Rabbit liver microsomal cytochrome b5 was allowed to react with tetranitromethane. Up to three tyrosine residues in each cytochrome b5 molecule were found to be accessible to the nitrating agent. Co-modification of tryptophan and histidine residues could be disregarded. CD-spectral measurements disproved gross changes in cytochrome b5 structure as a consequence of derivatization. Introduction of 1.6 nitro groups/polypeptide chain resulted in a fivefold increase in binding affinity for cytochrome P-450 2B4 (P-450 2B4), whereas spectral interaction with cytochrome c remained unaffected. Furthermore, the capacity of nitrated cytochrome b5 to shift the spin equilibrium to the high-spin conformer of P-4502B4 was diminished by 44% compared with the control. This corresponded with the partial disruption of NADH-dependent electron flow to ferric P-450 2B4. Changes in the redox potential of cytochrome b5 could be discounted as being responsible for this effect. The overall oxidative turnover of 4-nitroanisole did not respond to cytochrome b5 modification. MS analysis and sequencing of peptide fragments produced by tryptic digestion of modified cytochrome b5 permitted the detection of three nitrated tyrosine residues located at positions 11, 34 and 129. Derivatization of cytochrome b5 in the presence of a protective amount of P-450 2B4 provided evidence of the involvement of Tyr34 and Tyr129 in complexation of the two hemoproteins. It is proposed that Tyr129 might control docking of cytochrome b5 to P-450 2B4, whereas Tyr34 could be of functional importance in electron transfer.     

Model calculation of the crystal-field magnetostriction and its temperature dependence in the itinerant uniaxial ferromagnet Y2Fe17

A stimulatory effect of increased salt content on the metabolism of benzphetamine, 7-ethoxycoumarin, and coumarin by rabbit liver microsomes, CYP2B4 and rabbit CYP1A2, was seen, indicating that the effect was not specific for either substrate or form of cytochrome P450. The stimulation was not due to an action on the cytochrome P450 itself as increased salt concentration minimally affected the substrate turnover when cumene hydroperoxide was used as the source of active oxygen. The elevation of ionic strength increased the coupling efficiency of the monooxygenase reaction with benzphetamine as substrate. Cytochrome b5 also can increase the monooxygenase coupling efficiency. At low ionic strength cytochrome b5 did not much influence the reduction of P450, but the rate constant of the cytochrome b5 reduction was increased about 15-fold by its binding to cytochrome P450. A stimulatory effect of cytochrome b5 on benzphetamine oxidation was seen at low ionic strength, but it was lost at elevated ionic strength as the binding of cytochrome b5 to cytochrome P450 was weakened. At the higher ionic strength cytochrome b5 competes with cytochrome P450 for the reductase, an action that slows cytochrome P450 reduction. Based upon these observations, plus those in the literature, a scheme is suggested that proposes the stimulatory effect of cytochrome b5 on the cytochrome P450-mediated monooxygenation reaction is due to an increase in the efficiency of the electron transfer reaction: With cytochrome b5 bound to cytochrome P450, two electrons can be provided from the reductase to the P450-b5 complex in a single interaction, obviating the need for a second interaction with the reductase.     

Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel pathway for the biogenesis of hepatic mitochondrial P450MT2

Cytochrome P4501A1 is a hepatic, microsomal membrane-bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from beta-naphthoflavone-induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30-amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33-44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.     

Major cytochrome P450 enzyme responsible for oxidation of secondary alcohols to the corresponding ketones in mouse hepatic microsomes. Oxidation of 7-hydroxy-delta8-tetrahydrocannabinol to 7-oxo-delta8-tetrahydrocannabinol

The oxidative activities of 7alpha- and 7beta-hydroxy-Delta8-tetrahydrocannabinol (7alpha- and 7beta-hydroxy-Delta8-THC) to 7-oxo-Delta8-THC in hepatic microsomes of mice were significantly increased by the treatment of mice with dexamethasone or phenobarbital. A cytochrome P450 enzyme, named P450MDX-B, was purified from hepatic microsomes of dexamethasone-treated mice, and its apparent molecular mass was estimated to be 51,000. The NH2-terminal amino acid sequence of P450MDX-B was the same as that of CYP3A11. The oxidative activities of 7alpha- and 7beta-hydroxy-Delta8-THC were 2.55 and 4.92 nmol/min/nmol P450, respectively. The antibody against P450MDX-B almost completely inhibited the oxidative activities of 7alpha- and 7beta-hydroxy-Delta8-THC in mice. These results indicate that P450MDX-B (CYP3A11) is a major enzyme responsible for the oxidation of 7alpha- and 7beta-hydroxy-Delta8-THC to 7-oxo-Delta8-THC in mouse liver.     

Cytochromes P450, P420 & mixed-function oxidases as biomarkers of polychlorinated biphenyl (PCB) exposure in harbour seals (Phoca vitulina)

Hepatic microsomal cytochrome P450, EROD and ECOD activity were investigated as biomarkers of PCB exposure in harbour seals (Phoca vitulina). Due to the difficulty of obtaining undegraded seal liver samples, standard spectrophotometric methodology was adapted to investigate P420 (degraded P450) as a PCB biomarker with partially degraded samples. Total PCB burdens in both blubber and liver had positive correlations with P450, P420 and MFO activity levels. The use of P420 biomarkers in this study supports the inclusion of samples from by-caught marine mammals for future biomonitoring studies. P450 isozymes CYP1A (P4501A) and CYP2B (P4502B) in conjunction with MFO activity were investigated as "specific" biomarkers of PCB exposure. They were found to reliably reflect levels of [MC] and [PB]-type PCB exposure in harbour seal liver.     

Suppression of constitutive and inducible cytochrome P450 gene expression by alpha-hederin in mice

The effects of alpha-Hederin, a triterpenoid saponin which exists in some oriental herbs, on the expression of liver cytochrome P450s were examined in mice. The administration of alpha-Hederin to mice significantly decreased the hepatic content of P450 and the activities of microsomal ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, and aniline hydroxylase, representative activities of cytochrome-P4501A1, -P4501A2, and -P4502E1, respectively, in a dose- and time-dependent manner. However, pentoxyresorufin O-dealkylase, a representative activity of cytochrome P4502B1/2, was decreased to a lesser extent. alpha-Hederin also decreased inducible monooxygenase activities in the same manner. Suppressions of P450 isozyme expression occurred in alpha-Hederin treated hepatic microsomes, as determined by immunoblot analysis in a manner consistent with that of the enzyme activity levels. Levels of mRNA of P4501A1/2 and P4502B1/2 were also decreased by alpha-Hederin as shown by Northern blot analysis. In contrast, the level of P4502E1 mRNA in the liver of alpha-Hederin treated mice was unchanged. These results suggest that alpha-Hederin may act as a more specific suppressor for P4501A and P4502E1 than P4502B and that the suppression involves decreases in mRNA levels except in the case of P4502E1.     

Role of human liver P450s and cytochrome b5 in the reductive metabolism of 3'-azido-3'-deoxythymidine (AZT) to 3'-amino-3'-deoxythymidine

Our laboratory has shown that human liver microsomes metabolize the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) via a P450-type reductive reaction to a toxic metabolite 3'-amino-3'-deoxythymidine (AMT). In the present study, we examined the role of specific human P450s and other microsomal enzymes in AZT reduction. Under anaerobic conditions in the presence of NADPH, human liver microsomes converted AZT to AMT with kinetics indicative of two enzymatic components, one with a low Km (58-74 microM) and Vmax (107-142 pmol AMT formed/min/mg protein) and the other with a high Km (4.33-5.88 mM) and Vmax (1804-2607 pmol AMT formed/min/mg). Involvement of a specific P450 enzyme in AZT reduction was not detected by using human P450 substrates and inhibitors. Antibodies to human CYP2E1, CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2A6 were also without effect on this reaction. NADH was as effective as NADPH in promoting microsomal AZT reduction, raising the possibility of cytochrome b5 (b5) involvement. Indeed, AZT reduction among six human liver samples correlated strongly with microsomal b5 content (r2 = 0.96) as well as with aggregate P450 content (r2 = 0.97). Upon reconstitution, human liver b5 plus NADH:b5 reductase and CYP2C9 plus NADPH:P450 reductase were both effective catalysts of AZT reduction, which was also supported when CYP2A6 or CYP2E1 was substituted for CYP2C9. Kinetic analysis revealed an AZT Km of 54 microM and Vmax of 301 pmol/min for b5 plus NADH:b5 reductase and an AZT Km of 103 microM and Vmax of 397 pmol/min for CYP2C9 plus NADPH:P450 reductase. Our results indicate that AZT reduction to AMT by human liver microsomes involves both b5 and P450 enzymes plus their corresponding reductases. The capacity of these proteins and b5 to reduce AZT may be a function of their heme prothestic groups.     

Characterization of the effects of musk ketone on mouse hepatic cytochrome P450 enzymes

Nitroaromatic musks, including musk ketone (MK; 2,6-dimethyl-3,5-dinitro-4-t-butylacetophenone), are chemicals used as perfume ingredients in household products, cosmetics, and toiletries. Musk xylene (MX; 1,3,5-trinitro-2-t-butylxylene), another nitromusk, is not genotoxic but has been reported to produce mouse liver tumors in a chronic bioassay. In addition, MX has been shown to both induce and inhibit mouse liver cytochrome P450 2B (CYP2B) isozymes. The ability of MX to inhibit CYP2B enzyme activity is attributable to inactivation of the enzyme by a specific amine metabolite. MK is structurally similar to MX, but lacks the nitro substitution that is reduced to the inactivating amine metabolite. Therefore, we hypothesized that MK would induce, but not inhibit, CYP2B isozymes. To test this hypothesis, and to evaluate the effects of MK on mouse liver cytochrome P450 enzymes, two sets of experiments were performed. To evaluate the ability of MK to induce cytochromes P450, mice were dosed daily by oral gavage at dosages ranging from 5 to 500 mg/ kg MK for 7 days. This treatment resulted in a pleiotropic response in mouse liver, including increased liver weight, increased total microsomal protein, and centrilobular hepatocellular hypertrophy. At the highest dose tested, MK caused a 28-fold increase in CYP2B enzyme activity and a small (approximately 2-fold) increase in both cytochromes P450 1A and 3A (CYP1A and CYP3A) enzyme activities over control levels. Protein and mRNA analyses confirmed the relative levels of induction for CYP2B, CYP1A, and CYP3A. In addition, the no-observable-effect level (NOEL) for CYP2B induction by MK was 20 mg/kg. To evaluate the ability of MK to inhibit phenobarbital-induced CYP2B activity, mice were given 500 ppm phenobarbital (PB) in the drinking water for 5 days to induce CYP2B isozymes, followed by a single equimolar (0.67 mmol/kg) oral gavage dose of either MK (198 mg/kg) or MX (200 mg/kg), and microsomes were prepared 18 h later. While MX inhibited more than 90% of the PB-induced CYP2B activity in the microsomes, MK caused only a small (about 20%) reduction in PB-induced CYP2B enzyme activity. These results indicate that, like MX. MK is a PB-type inducer of mouse liver CYP2B isozymes, but unlike MX, MK does not effectively inhibit PB-induced CYP2B enzyme activity.     

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.     

Possible role of cytochrome P450 in inactivation of testosterone in immortalized hippocampal neurons

The hippocampus as part of the limbic system is sensitive to gonadal hormones. The time-dependent expression of steroid receptors and the testosterone converting enzyme aromatase (CYP19) is well studied. In contrast, little is known about other cytochrome P450 enzymes in hippocampus which inactivate the gonadal hormones. For investigation of the total cytochrome P450 content and the expression of testosterone degrading CYP2B10 we used embryonic (E18) in comparison to postnatal (P21) immortalized hippocampal neurons. These embryonic neurons were demonstrated to react to hormones according a 'critical period' of sexual differentiation: testosterone treatment (1 microM to 5 microM in the culture medium) resulted in a decrease of beta-tubulin, as showed by immunocytochemistry and Western blotting. Measurements with reduced CO-difference spectrum elucidated that the P450 concentration in the embryonic neurons (10.2 pmol/mg protein; S.D. +/- 1.9) was twice as high as in the postnatal ones (5.2 pmol/mg protein; S.D. +/- 1.0). Correspondingly, a high value of the mitochondrial subfraction of approx. 141 pmol P450/mg protein was found in the embryonic neurons relative to the mitochondrial value of 37.7 pmol P450/mg protein in the postnatal neurons. Our results suggest a differential expression of cytochrome P450 during development. CYP2B10 was proved by electron microscopy and hormone degrading activity.     

Reductive activation of 1,1-dichloro-1-fluoroethane (HCFC-141b) by phenobarbital- and pyridine-induced rat liver microsomal cytochrome P450

1. During anaerobic reductive incubation of liver microsomes, from either the pyridine- or phenobarbital-treated rat, with 1,1-dichloro-1-fluoroethane (HCFC-141b) in the presence of a NADPH-regenerating system, a time- and dose-dependent formation of reactive metabolites was detected as indicated by a depletion of added exogenous glutathione. 2. A statistically significant, dose-dependent loss of both cytochrome P450 and microsomal haem was also observed under these experimental conditions. Furthermore, a statistically significant decrease of p-nitrophenol hydroxylase and pentoxyresorufin O-depentylase activity was measured in microsomes from the pyridine- and phenobarbital-induced rat, respectively indicating that both P4502E1 and P4502B undergo substrate-dependent inactivation. 3. Both reactive metabolite formation and P450 inactivation were almost completely inhibited by previous bubbling of the incubation mixture with carbon monoxide, indicating that interaction of the substrate with a free and reduced P450 haem iron is required for substrate bioactivation and enzyme loss. 4. The presence in the incubation mixture of the spin-trap N-t-butyl-alpha-phenylnitrone (PBN) and the carbene trap 2,3-dimethyl-2-butene (DMB) largely prevented both glutathione depletion and P450 loss. This suggests that free radical and carbene intermediates formed by the metabolic activation of the substrate are involved in the inactivation of P450 and the loss of its prosthetic haem group.     

Effects of smoking cessation on maternal airway function and birth weight

Recombinant human neutrophil leukotriene B4 (LTB4) omega-hydroxylase (cytochrome P450 4F3) has been purified to a specific content of 14. 8 nmol of P450/mg of protein from yeast cells. The purified enzyme was homogenous as judged from the SDS-PAGE, with an apparent molecular weight of 55 kDa. The enzyme catalyzed the omega-hydroxylation of LTB4 with a Km of 0.64 microM and Vmax of 34 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b5. Furthermore, various eicosanoids such as 20-hydroxy-LTB4, 6-trans-LTB4, lipoxin A4, lipoxin B4, 5-HETE and 12-HETE, and 12-hydroxy-stearate and 12-hydroxy-oleate were efficiently omega-hydroxylated, although their Km values were much higher than that of LTB4. In contrast, no activity was detected toward laurate, palmitate, arachidonate, 15-HETE, prostaglandin A1, and prostaglandin E1, all of which are excellent substrates for the CYP4A fatty acid omega-hydroxylases. This is the first time human neutrophil LTB4 omega-hydroxylase has been isolated in a highly purified state and characterized especially with respect to its substrate specificity.     

Induction and inhibition of cytochrome P450 isoforms by imazalil, a food contaminant, in mouse small intestine and liver

1. The effects of imazalil, a food contaminant used as a fungicide, were investigated on the expression and activity of cytochrome P450 in the small intestinal mucosa and liver of mice. Imazalil was orally administered to mice daily at 1 or 10 mg/kg for 3 days. 2. Imazalil enhanced cytochrome P450-catalysed ethoxyresorufin O-deethylase and pentoxyresorufin O-depentylase (PROD) activities in both tissue microsomes at the 10 mg/kg/day dose level, indicating the induction of cytochrome P450 subfamilies CYP1A and CYP2B. In addition, immunochemical analyses also demonstrated an enhanced expression of CYP2B, CYP2C and CYP3A subfamilies in both tissues. 3. Imazalil was a potent inhibitor of cytochrome P450-dependent monooxygenase activities (PROD, aminopyrine N-demethylase and erythromycin demethylase) in in vitro assays using both small intestinal and liver microsomes. 4. From these findings, imazalil has been demonstrated to have not only a potent inhibitory activity but also a significant inducing ability of P450 isoforms in the small intestine. Prolonged ingestion of such a food contaminant may modulate the xenobiotic-metabolizing enzyme system at the site of a primary portal of xenobiotic entry to the systemic circulation.     

Use of condoms among the population of the Czech Republic: results of a national study]

beta-Arteether (AE) is an endoperoxide sesquiterpene lactone derivative currently being developed for the treatment of severe, complicated malaria caused by multidrug-resistant Plasmodium falciparum. Studies were undertaken to determine which form(s) of human cytochrome P-450 catalyze the conversion of beta-arteether to its deethylated metabolite, dihydroqinghaosu (DQHS), itself a potent antimalarial compound. In human liver microsomes, AE was metabolized to DQHS with a Km of 53.7 +/- 29.5 microM and a Vmax of 1.64 +/- 1. 78 nmol DQHS/min/mg protein. AE biotransformation to DQHS was inhibited by ketoconazole and troleandomycin. Ketoconazole was a competitive inhibitor, with an apparent Ki of 0.33 +/- 0.11 microM. Because AE is being developed for patients who fail primary treatment, it is possible that AE may be involved in life-threatening drug-drug interactions, such as the associated cardiotoxicity of mefloquine and quinidine. Coincubation of AE with other antimalarials showed mefloquine and quinidine to be competitive inhibitors with a mean Ki of 41 and 111 microM, respectively. Metabolism of AE using human recombinant P450s provided evidence that cytochrome P450s 2B6, 3A4, and 3A5 were the primary isozymes responsible for its deethylation. CYP3A4 metabolized AE to dihydroqinghaosu at a rate approximately 10 times that of CYP2B6 and approximately 4.5-fold greater than that of CYP3A5. These results demonstrate that CYP3A4 is the primary isozyme involved in the metabolism of AE to its active metabolite, DQHS, with secondary contributions by CYP2B6 and -3A5.     

Metabolism of MPTP by cytochrome P4502D6 and the demonstration of 2D6 mRNA in human foetal and adult brain by in situ hybridization

1. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a thermal breakdown product of synthetic 'street' heroin, is known to cause Parkinson's Disease-like symptoms in man. 2. The mechanism of action of this neurotoxin is thought to involve activation by the monoamine oxidase B system and subsequent toxicity by inhibition of neuronal mitochondrial respiration. The manifestation of toxicity will be a balance between the rate of activation of this compound versus its rate of inactivation through metabolism by enzymes such as the cytochrome P450-dependent monooxygenases. 3. In this report we demonstrate that MPTP N-demethylation, a detoxification pathway, is catalysed by cytochrome P450 CYP2D6 and up to 40% of the hepatic metabolism is mediated by this enzyme. 4. Perhaps more importantly we also demonstrate by in situ hybridization that CYP2D6 is localized in the pigmented neurons of the substantia nigra indicating that 2D6-mediated detoxification will occur in target cells. 5. These data present evidence that CYP2D6 will be a factor in susceptibility to MPTP neuronal toxicity and provide a biochemical rationale for the genetic observations linking a polymorphism at the CYP2D6 locus with susceptibility to Parkinson's.