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.     

In vitro biotransformation of atrazine by rat liver microsomal cytochrome P450 enzymes

We studied atrazine (ATZ) metabolism in male and female rat liver microsomes in vitro, and the major metabolite was deisopropylatrazine (DeiPr-ATZ) with deethylatrazine (DeEt-ATZ) and 1-hydroxyisopropylatrazine (iPrOH-ATZ) as minor metabolites in both sexes. The enzyme kinetics of ATZ biotransformation were examined by means of Eadie-Hofstee analyses. Although no remarkable sex difference of Michaelis Menten values for each pathway was observed, Cl(int)S (Vmax/Km) for DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ were slightly higher in female than in male rats. The formation of DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ from ATZ was substantially inhibited by SKF-525A, metyrapone, diallyl sulfide, 7-ethoxycoumarin, benzphetamine, nicotine, testosterone and lauric acid in both sexes. Cimetidine effectively inhibited the formation of all metabolites in male rats. On the other hand, the inhibition rates of the formation of DeiPr-ATZ and iPrOH-ATZ by cimetidine in female rats were lower than those in male rats, and DeEt-ATZ was hardly affected by the chemicals. In contrast with the results for cimetidine, the inhibition of ATZ biotransformation by bufuralol was more effective in female than in male rats. Anti-rat CYP2B1 and CYP2E1 antibodies effectively inhibited DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ formations in both sexes. Anti-rat CYP2C11 antibody also inhibited the three metabolites in both sexes, with the inhibition rates higher in male than in female rats, similar to cimetidine. In the case of anti-rat CYP2D1 antibody, the inhibitory effect on ATZ biotransformation in male rats was less than that in female rats. On the other hand, anti-rat CYP1A2, CYP3A2 and CYP4A1 antibodies did not affect the ATZ biotransformation in either sex. There was no significant correlation between the formation rate of ATZ metabolites and P450 isoform levels in either sex. These results may mean that CYP2B2, CYP2C11, CYP2D1 (only iPrOH-ATZ formation) and CYP2E1 in male rats, and CYP2B2, CYP2D1 and CYP2E1 in female rats are involved ATZ metabolism in liver, and that the substrate specificity of P450 isoforms for ATZ is broad.     

Modulatory effect of hyperthermia on hepatic microsomal cytochrome P450 in mice

It is widely known that the clearance of drugs is often compromised during episodes of infectious disease via a down-regulation of cytochrome P450 (P450) at a pre-translational step in enzyme synthesis. Etiocholanolone (ETC), a potent inflammatory agent, induces fever in humans and causes a decrease in the clearance of certain drugs that are metabolized by P450. On this basis it is widely believed that the fever per se rather than the immune modulation that occurs during infections may have a major role in depression of microsomal P450 enzymes during viral infections in humans. In the present study, we demonstrated that although ETC did not induce hyperthermia in mice, it still evoked a depression of the levels of P450 in hepatic microsomes. Ethoxyresorufin O-deethylase (EROD) was also inhibited significantly when hepatic microsomes were incubated with various concentrations of ETC in vitro. P450 levels and EROD activities remained unchanged following hyperthermia that was induced by a non-inflammatory procedure using 2,4-dinitrophenol. Provided the response in rodents is similar to humans, these results indicate that the depression of drug biotransformation by ETC in humans is more likely to be caused by the direct effects of this agent or other mechanisms rather than by the fever it produces. This may suggest that the loss of drug metabolism in humans during infections is due to the activation of host defence responses rather than to the febrile nature of the illness.     

Interactions of monoamine oxidase inhibitors, N-acetylenic analogues of tryptamine, with rat liver microsomal cytochrome P450

Interactions between some novel and potent monoamine oxidase inhibitors (MAOIs), acetylenic analogues of tryptamine, and rat liver microsomal cytochrome P450 (P450) as evidenced by visible spectra analysis were analysed. Compounds with a secondary aliphatic amine moiety throughout induced type II difference spectra and exhibited the highest affinity for P450, whereas tertiary amines induced type I spectral changes and showed diminished affinity. P450 dependent aniline hydroxylase activity was inhibited by all compounds in an irreversible time-dependent manner. Only tertiary aliphatic amines constituted the substrate for P450-dependent N-demethylase activity, with comparable kinetic parameters. The N-demethylated metabolites were identified by thin-layer chromatography and mass-spectrometric analyses. These findings describe the role of P450-dependent microsomal mono-oxygenase systems in the metabolism of some MAOI acetylenic tryptamine derivatives and the possible hepatic contribution to adverse interactions between MAOIs, endobiotics and sympathomimetic compounds.     

A study of hepatic mitochondrial respiration and microsomal cytochrome P450 content in mice infected with the liver fluke Fasciola hepatica

Previous studies of the effects of infection of Wistar rats with the common liver fluke, Fasciola hepatica, on liver bioenergetic and drug metabolism have demonstrated a loss of respiratory control in isolated mitochondria and reduced microsomal cytochrome P450 content, respectively, from 2 weeks post-infection throughout the acute phase of the infection. In the present study male Balb/c mice infected with F. hepatica showed a loss of respiratory control in isolated liver mitochondria only at 4 weeks post-infection. A similar time course was demonstrated for a reduction in hepatic microsomal cytochrome P450 content. Preparations from infected CBA mice showed similar changes to Balb/c mice but mitochondrial respiration in preparations from infected Swiss outbred mice was normal. A host difference between strains of mice and between mice and rats is therefore evident in the timing and extent of liver mitochondrial dysfunction and in the timing of the decrease in the cytochrome P450 content of hepatic microsomes. This difference between hosts may be related to the reported differences in cellular inflammatory responses to the migrating juvenile flukes in the livers of rats and mice.     

Amino acid residue 250 has a functional role in the assembly of rabbit liver microsomal cytochrome P450 2B4

Cytochrome P450 2B4 lacking amino acids 2-27, CYP2B4 (delta2-27), was mutated at position 250 and expressed in E. coli fused to glutathione S-transferase. Expression of the E250S variant (holo- plus apoenzyme) proceeded to an extent comparable with that of CYP2B4 (delta2-27), while the protein level of the E250P mutant averaged 42% that of the control pigment. Comparison of these data with the corresponding reduced CO difference spectra of the various CYP2B4 (delta2-27) forms revealed that, in the control and E250S preparations, about 90% and 44%, respectively, of the total amount of hemoprotein present existed in the form of holoenzyme, whereas the E250P derivative failed to produce a reduced carbonyl complex. Thus, replacement of the negatively charged E250 with an uncharged, polar serine residue substantially hampered assembly of CYP2B4 (delta2-27); introduction of an alpha-helix-disrupting proline completely blocked the formation of holoenzyme. These phenomena suggested that the negative charge of E250, residing in the putative G helix, underwent pairing with some positively charged group, possibly H285 located in the I helix. Deletion of the negative charge obviously perturbed the active-site geometry such as to affect both the incorporation and/or retention of the heme ligand and the spectral binding of substrates such as hexobarbital.     

Studies on the microsomal cytochrome P450s in the livers of carcinogen-resistant and sensitive rats during long-term administration of 3''-methyl-4-dimethylaminoazobenzene

The fluorescent indicator Fura-2 was used to characterize the store-operated Ca2+ entry in insulin-releasing pancreatic beta-cells. To avoid interference with voltage-dependent Ca2+ entry, the cells were hyperpolarized with 400 microM diazoxide and the channel blocker methoxyverapamil was also present in some experiments. The cytoplasmic Ca2+ concentration ([Ca2+]j) of hyperpolarized mouse beta-cells was strikingly resistant to changes in external Ca2+. In cells exposed to 20 mM glucose, stimulation with 100 microM carbachol induced an initial [Ca2+]j peak followed by a sustained increase due to store-operated influx of the cation. Store-operated influx was also induced by the intracellular Ca(2+)-ATPase inhibitor thapsigargin. In the presence of store-operated influx, [Ca2+]j became markedly sensitive to variations in external Ca2+, but this sensitivity was blocked by La3+. In beta-cells exposed to both Ca2+ and Mn2+ there was slow Mn2+ quenching of the Fura-2 fluorescence, which was accelerated upon stimulation of store-operated influx. This acceleration was reversed by glucose-stimulated filling of the internal Ca2+ stores. The store-operated Ca2+ entry increased markedly during culture of the beta-cells. Activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol-13 acetate, inhibition of serine/threonine phosphatase by okadaic acid and inhibition of tyrosine kinase by genistein had little effect on the store-operated influx of Ca2+. In beta-cells equilibrated in 5 mM Sr2+, carbachol exposure resulted in a pronounced cytoplasmic Sr2+ ([Sr2+]j) peak due to intracellular mobilization, but little or no sustained elevation. Moreover, after activating the store-operated pathway by exposure to thapsigargin, variations in extracellular Sr2+ between 0-2 mM had only marginal effects on [Sr2+]j. Although the store-operated influx apparently accounts for a minor fraction of the Ca2+ entry, its depolarizing influence may under certain conditions be up-regulated with resulting distortion of the beta-cell function.     

Oxidative metabolism of zolpidem by human liver cytochrome P450S

The aim of this study was to identify the form(s) of cytochrome P450 (CYP) responsible for the biotransformation of zolpidem to its alcohol derivatives which, after rapid conversion to carboxylic acids, represents the main way of metabolism in humans. In human liver microsomes, zolpidem was converted to alcohol derivatives. Production of these correlated with the level of CYP3A4 and with cyclosporin oxidation and erythromycin N-demethylation activities, but not with the level of CYP1A2 nor with ethoxyresorufin O-deethylation or S-mephenytoin 4'-hydroxylation activities. Liver microsomes from CYP2D6-deficient patients exhibited normal activity. Production of alcohol derivatives was significantly inhibited by anti-CYP3A antibodies and by ketoconazole. Antibodies directed against other CYP forms (including CYP1A1, CYP1A2, CYP2A6, CYP2B4, and CYP2C8), and CYP-specific substrates or inhibitors (including propranolol, coumarin, mephenytoin, sulfaphenazole, quinidine, aniline, and lauric acid) produced a moderate or no inhibitory effect. cDNA-expressed CYP3A4 and CYP1A2 generated significant amounts of one of the alcohol derivatives, whereas CYP2D6 generated both of them in similar amounts. In human hepatocytes in primary culture, zolpidem was extensively and almost exclusively converted to one of the carboxylic acid derivatives, the main species identified in vivo. Treatment of cells with inducers of CYP1A (beta-naphthoflavone) and CYP3A (rifampicin and phenobarbital) greatly increased the rate of production of this metabolite. We conclude that the formation of alcohol derivatives of zolpidem is rate-limiting and principally mediated by CYP3A4. Both CYP1A2 and CYP2D6 participate in alcohol formation; but, because of their low relative level of expression in the human liver, their contribution is minor.     

Topogenesis of a microsomal cytochrome P450 and induction of endoplasmic reticulum membrane proliferation in Saccharomyces cerevisiae

Cytochrome P450 52A3 (P450Cm1) is one of the membrane proteins known to trigger by its high-level expression a marked proliferation of the endoplasmic reticulum, (ER). To gain insight into the relationship between the expression of a membrane protein and the induction of ER proliferation we have characterized the membrane topology of P450Cm1 and identified the structural determinants required for ER targeting, formation of correct membrane orientation, and ER retention. We show that all these features are interrelated and determined by sequence elements within the NH2-terminal region of P450Cm1. Using several approaches--a protease protection assay followed by probing with peptide-specific antibodies, immunolabeling of the intact membrane-bound P450 protein, and expression of fusion proteins in Saccharomyces cerevisiae--membrane topology was defined as follows: residues 2-16 are located in the ER lumen, only the first hydrophobic segment (residues 17-34) spans the membrane, a second hydrophobic segment (48-66) is exposed at the cytoplasmic side, and the remaining part (67-523) forms a large cytosolic domain. Fused to a cytosolic reporter protein, the first 44-amino-acid sequence of P450Cm1 was sufficient to mediate ER targeting, wild-type membrane orientation, and retention in the ER. Similar to wild-type P450Cm1, various fusion proteins were able to induce distinctly organized structures of proliferated ER provided that they were either permanently retained in the ER or accumulated in this compartment due to a delay in further transportation. Thus, we conclude that membrane insertion of the first hydrophobic segment is sufficient to deliver a signal for increased membrane formation.     

Changes in rat liver cytochrome P450 enzymes by atrazine and simazine treatment

1. We examined the effect of two chloro-s-triazines (atrazine and simazine) on hepatic microsomal cytochrome P450 enzymes in rat. Rats were treated intraperitoneally with atrazine or simazine daily for 3 days with 100, 200 and 400 mumol/kg. 2. Among the P450-dependent monooxygenase activities, testosterone 2 alpha-hydroxylase (T2AH) activity in rat, which is associated with CYP2C11, was significantly decreased at all doses of atrazine and simazine. The levels relative to control activities were 59-46 and 60-32% respectively. Similarly, oestradiol 2-hydroxylase (ED2H) activity was also significantly decreased by 28-51% by atrazine and simazine at all doses. However, no change in CYP2C11 protein level by either chloro-s-triazine was observed. K(m) for T2AH was significantly increased only by simazine (200 mumol/kg), whereas the Vmax and Cl(int) for T2AH were significantly decreased by atrazine and simazine at all doses. 3. 7-Ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD) and 7-pentoxyresorufin O-depentylase (PROD) activities were significantly increased by 1.4-1.6-, 1.7-3.2- and 1.5-2.2-fold respectively, by both chloro-s-triazines at 200 or 400 mumol/kg. Lauric acid omega-hydroxylase (LAOH) was also increased by 1.4-fold by simazine at 200 and 400 mumol/kg. Immunoblotting showed that only simazine induces CYP1A2 and CYP4A1/2 protein expression. 4. The activities of 7-ethoxycoumarin O-deethylase (ECOD), bufuralol 1'-hydroxylase (BF1'H), chlorzoxazone 6-hydroxylase (CZ6H), testosterone 6 beta-hydroxylase (T6BH) and testosterone 7 alpha-hydroxylase (T7AH) were not affected by either chloro-s-triazine. 5. These results suggest that the pattern of changes in P450 isoforms by chloro-s-triazines differs between atrazine and simazine, that these herbicides change the constitutive and/or male specific P450 isoform(s) in rat liver, and that these changes closely relate to the toxicity of chloro-s-triazines.     

Activation of the anti-cancer drug ifosphamide by rat liver microsomal P450 enzymes

The NADPH-dependent metabolism of ifosphamide catalyzed by rat liver microsomes was investigated in order to identify individual P450 enzymes that activate this anti-cancer drug and to ascertain their relationship to the P450 enzymes that activate the isomeric drug cyclophosphamide. Pretreatment of rats with phenobarbital or clofibrate increased by up to 8-fold the activation of both ifosphamide and cyclophosphamide catalyzed by isolated liver microsomes. Studies using P450 form-selective inhibitory antibodies demonstrated that constitutively expressed P450s belonging to subfamily 2C (forms 2C11/2C6) make significant contributions to the activation of both oxazaphosphorines in uninduced male rat liver microsomes, while the phenobarbital-inducible P450 2B1 was shown to be a major catalyst of these activations in phenobarbital-induced microsomes. Pretreatment of rats with dexamethasone increased liver microsomal activation of ifosphamide approximately 6-fold without a corresponding effect on cyclophosphamide activation rates. Ifosphamide activation catalyzed by dexamethasone-induced liver microsomes was minimally inhibited by anti-P450 2B or anti-P450 2C antibodies, but was selectively inhibited by anti-P450 3A antibodies. Selective inhibition of liver microsomal ifosphamide activation was also effected by the macrolide antibiotic triacetyloleandomycin, an inhibitor of several dexamethasone-inducible 3A P450s. These studies establish that a dexamethasone-inducible family 3A P450 can make an important contribution to rat liver microsomal ifosphamide activation, and suggest that dexamethasone pretreatment might provide a useful approach for modulation of ifosphamide metabolism in order to improve its therapeutic efficacy in cancer patients.     

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.     

Nutritional status modulates rat liver cytochrome P450 arachidonic acid metabolism

Alterations in nutritional status affect hepatic cytochrome P450 levels. Since cytochromes P450 participate in the metabolism of arachidonic acid, we hypothesized that changes in liver P450 arachidonic acid metabolism occur during fasting and refeeding. Male Fisher 344 rats were either fed, fasted 48 hr (F48), fasted 48 hr and then refed 6 hr (F48/R6), or fasted 48 hr and then refed 24 hr (F48/R24). F48 rats had reduced body weight, increased plasma beta-hydroxybutyrate, and reduced plasma insulin compared with the other groups. Although there was no significant change in total liver P450 content, there was a significant 20%, 48%, and 24% reduction in total hepatic microsomal arachidonic acid metabolism in F48, F48/R6, and F48/R24 rats, respectively, compared with fed rats. Epoxygenase activity decreased by 28%, 51%, and 26% in F48, F48/R6, and F48/R24 rats, respectively. In contrast, omega-1 hydroxylase activity increased by 126% in F48 rats compared with fed rats. Immunoblotting revealed that levels of CYP2C11 protein were markedly reduced, whereas levels of CYP2E1 protein were markedly increased in the F48 and F48/R6 groups. In contrast, levels of CYP1A1, CYP1A2, CYP2B1, CYP2J3, CYP4A1, and CYP4A3 were unchanged with fasting/refeeding. Northern blots revealed that levels of CYP2C11 mRNAs were decreased, whereas CYP2E1 mRNAs were increased in F48 and F48/R6 rats. Recombinant CYP2C11 metabolized arachidonic acid primarily to epoxides with preference for the 14(S),15(R)-, 11(R), 12(S)-, and 8(S),9(R)- epoxyeicosatrienoic acid enantiomers. We conclude that (1) nutritional status affects hepatic microsomal arachidonic acid metabolism, (2) reduced epoxygenase activity in F48 and F48/R6 rats is accompanied by decreased levels of CYP2C11, (3) increased omega-1 hydroxylase activity is accompanied by augmented levels of CYP2E1, and (4) the effects of fasting on CYP2C11 and CYP2E1 expression occur at the pretranslational level.     

Suppression of male-specific cytochrome P450 isoforms by bisphenol A in rat liver

IBD results from the interaction of genetic and environmental factors (e.g., smoking). Clinical suspicion is the key to diagnosis, which then rests on colonoscopy, histopathological examination of multiple biopsy specimens, small bowel barium radiology and faecal examination. The primary goal of treatment is remission--histological in ulcerative colitis and symptomatic in Crohn's disease. Treating active disease and maintaining remission require different approaches. For active disease, short term corticosteroids are the mainstay of treatment, while immunosuppressive drugs are important in chronically active disease. For maintenance, mesalazine-delivering drugs and immunosuppressive agents are efficacious in both ulcerative colitis and Crohn's disease; patients with Crohn's disease should not smoke.     

Portal vein ligation selectively lowers hepatic cytochrome P450 levels in rats

In rats, surgical creation of a portacaval shunt leads to hepatic atrophy and lowered levels of cytochrome P450, the key component of liver enzymes involved with drug metabolism. These effects are largely attributable to diversion of portal blood away from the liver and not to decreased hepatic blood flow. The present study has established a simpler model of portal blood diversion in order to examine the role of portal blood constituents in the regulation of hepatic cytochrome P450. Portal vein ligation was performed on male Wistar rats in which portasystemic anastomoses had been produced by subcutaneous transposition of the spleen. Portal vein ligation resulted in portal hypertension, as evidenced by splenomegaly, and in hepatic atrophy. In liver of rats with portal vein ligation, microsomal cytochrome P450 levels were significantly less than in sham-operated control rats, but cytochrome b5, NADPH-cytochrome c reductase, and glucose-6-phosphatase were unaltered. The activities of four mixed function oxidases also were reduced significantly in the liver of rats with portal vein ligation, the changes being greatest for ethylmorphine N-demethylase, a prototype substrate for the phenobarbital-inducible isoenzyme of cytochrome P450. In contrast, the activity of microsomal heme oxygenase, the rate-limiting step in catabolism of heme to bilirubin, was enhanced after portal vein ligation. Experiments in pair-fed rats showed that the changes observed in liver from rats with portal vein ligation could not be attributed to caloric deprivation. Administration of phenobarbital increased liver mass, cytochrome P450 levels, and mixed function oxidase activities both in rats with portal vein ligation and in controls, indicating that the liver of the ligated rats retained considerable protein synthetic capacity. It appears that hepatic atrophy and lowering of cytochrome P450 levels that follow portal vein ligation are consequences of altered exposure of the liver to factors normally present in portal blood, and that the same alterations may also enhance heme oxygenase activity.     

Subacute cocaine treatment changes expression of mouse liver cytochrome P450 isoforms

Acute administration of single high doses of cocaine (50 or 60 mg/kg) produces liver injury in mice that have been pretreated with inducers of mixed function oxidases. Multiple low doses of cocaine (10-30 mg/kg) will produce hepatotoxicity without prior induction. To establish whether cocaine can induce its own activation, mice were given three daily injections of cocaine. Total cytochrome P450 content of the liver did not change. After 3 days the amount of cytochrome P450 2B10, as measured by pentoxy resorufin-O-dealkylase activity and immunoblotting, increased 3-fold. Cytochrome P450 2A5-catalyzed coumarin 7-hydroxylase activity and immunoreactive protein increased by about 50%. Enzyme activities and Western blotting of isoforms 1A, 2E, and 3A showed no change during this time. Chronic cocaine increased N-hydroxylation of norcocaine. Immunoinhibition studies showed that cytochrome P450 2A5 was the major isoform responsible for norcocaine N-hydroxylation. These results demonstrate that chronic cocaine can induce its own metabolism. Similar increases were also observed in mice not susceptible to liver injury from chronic cocaine.     

Studies on cytochrome P450 responsible for oxidative metabolism of imipramine in human liver microsomes

The activity of imipramine 2-hydroxylase highly correlated with that of desipramine 2-hydroxylase but not with that of desipramine N-demethylase. The correlation was also found between N-demethylation and 2-hydroxylation when imipramine was used as a substrate, whereas no correlation was observed between them when desipramine was used in place of imipramine. Both activities of desipramine and imipramine 2-hydroxylase were markedly inhibited by quinidine but not by quinine. Although the activity of imipramine N-demethylase was slightly inhibited by both quinidine and quinine, the activity of desipramine N-demethylase was unaffected under the same conditions. The activity of imipramine N-demethylase was roughly correlated with the amounts of P450 3A4 immunochemically determined and the activities of testosterone 6 beta-hydroxylase in human liver microsomes. The P450 3A4 catalyzed imipramine N-demethylation much more efficiently than 2-hydroxylation in a reconstituted system, whereas neither N-demethylation nor 2-hydroxylation of desipramine was catalyzed by P450 3A4. The activity of imipramine N-demethylase was inhibited, to various extents, by anti-P450 3A4 antibodies in human liver microsomes. Taking together these and other results, it is suggested that P450 3A4, other than P450 2Cmp, also partly contributes to N-demethylation of imipramine, depending on human liver microsomes.     

Avian forms of cytochrome P450

Despite the importance of avian P450 forms in modulation of the toxicity of pesticides and other environmental chemicals, relatively little work has been done upon them, and very few forms have been fully characterised. An avian form that appears to belong to family 1A is readily inducible by planar molecules (e.g. coplanar PCB's, PCDD and certain PAHs) and has been the basis of a biomarker assay used in field studies. Although it is recognised by antibodies for mammalian P450 1A1, it evidently differs from the mammalian forms of the enzyme in catalytic properties. Phenobarbitone induces two forms of P450 in the domestic fowl (2H1 and 2H2) which have been purified, and these resemble P450 2B1 and P450 2B2 of the rat respectively. Two further phenobarbitone inducible forms, PB-A and PB-B have been partially purified. Also there is an acetone inducible form that resembles rodent P450 2E. In field studies evidence has been produced for the induction of P450s recognised by antibodies to mammalian forms of P450 1A1 and P450 2B in avian liver (adults and embryos), in response to environmental levels of PCBs. Fungicides which act as ergosterol biosynthesis inhibitors (EBI fungicides) such as prochloraz and propiconazole potentiate the toxicity of certain phosphorothionates to birds.