The involvement of cytochrome P450 peroxidase in the metabolic bioactivation of cumene hydroperoxide by isolated rat hepatocytes

Organic hydroperoxides are believed to be primarily detoxified in cells by the GSH peroxidase/GSSG reductase system and activated to cytotoxic radical species by non-heme iron. However, organic hydroperoxides seem to be bioactivated by cytochrome P450 (P450) in isolated hepatocytes as various P450 (particularly P450 2E1) inhibitors inhibited cumene hydroperoxide (CumOOH) metabolism and attenuated subsequent cytotoxic effects including antimycin A-resistant respiration, lipid peroxidation, iron mobilization, ATP depletion, and cell membrane disruption. CumOOH metabolism was also faster in P450 1A-induced hepatocytes and was inhibited by the P450 1A inhibitor alpha-naphthoflavone. The ferric chelator deferoxamine also prevented cytotoxicity even after CumOOH had been metabolized but had no effect on CumOOH metabolism. This emphasizes the toxicological significance of the iron released following hydroperoxide metabolic activation by cytochrome P450. The radical trap, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), had no effect on CumOOH metabolism but prevented CumOOH-induced antimycin A-resistant respiration, lipid peroxidation, iron mobilization, and loss of membrane integrity. These results suggest that CumOOH is metabolically activated by some P450 enzymes (e.g., P450 2E1) in hepatocytes to form reactive radical metabolites or oxidants that cause lipid peroxidation and cytotoxicity.     

Phytanic acid alpha-hydroxylation by bacterial cytochrome P450

The science of drug metabolism, like any other science, has advanced from simple beginnings (by today's standards) to its present state. One can examine the path that has been taken to understand the forces driving the direction of evolution of this science. The trends discovered can then be used to make reasonable extrapolations about the changes that might be expected in the future. That exercise is the subject of this article. The main focus will be on drug metabolism as practiced in the industrial environment, representing the author's main experience as well as the principal arena of practical applications of the science. The discussion will draw mainly on broad phenomena occurring in this application of drug metabolism to drug discovery and development.     

Metabolism of carteolol by cDNA-expressed human cytochrome P450

Staphylococcus aureus produces numerous bi-component toxins, e.g., Panton-Valentine leukocidin (Luk-PVL) and gamma-haemolysin, which consist of type S and F proteins. Previous studies showed that Luk-PVL induces inflammatory mediator release from human granulocytes that might reflect the in-vivo effects, e.g., dermonecrosis by Luk-PVL. Clinical isolates not only harbour the two genes coding for Luk-PVL (S-protein: LukS-PVL, F-protein: LukF-PVL) but also the three genes encoding gamma-haemolysin (S-protein: HlgA, HlgB; F-protein: HlgC). The interaction of all the possible potential toxins with human granulocytes was studied with regard to the generation of oxygen metabolites (chemiluminescence response), enzyme activity (beta-glucuronidase) and histamine release as well as interleukin (IL)-8 generation. The data clearly show that the individual subunits (S, F) differ in their activities. The following activities were obtained for the S components: LukS-PVL > HlgC > HlgA; the F components LukF-PVL and HlgB were similarly active. Thus, the toxins LukS-PVL/LukF-PVL and LukS-PVL/HlgB were the most potent inducers of inflammatory mediator release from human granulocytes, followed by HlgC/LukF-PVL and HlgC/HlgB and to a lesser degree by the toxins HlgA/LukF-PVL and HlgA/HlgB. The data indicate that class S components and class F components are interchangeable and give toxins with genuine biological activities.     

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.     

Effect of guaiazulene on some cytochrome P450 activities. Implication in the metabolic activation and hepatotoxicity of paracetamol

The influence of timing of surgery in relation to menstrual period on survival of breast cancer patients has been both advanced advocated and disputed. A meta-analysis on published series showed a statistically significant overall odds reduction when surgery is performed in the luteal phase. The records of 165 premenopausal M- breast cancer women, not on hormonal therapies, consecutively operated on from 1977 to 1991 were reviewed. All patients underwent modified radical mastectomies or quadrantectomies plus operative radiotherapy, Node-positive patients received standard adjuvant chemotherapy. Cox regression analysis was used to estimate the relative risk (RR) of death in three models including timing of surgery, age, histology, pathological T and N. In each model, patients were divided into two groups according to the criteria proposed by Badwe, Hrushesky, and Senie. Multivariate analysis showed a significant association between pT and pN and survival, whereas no association with survival was observed for timing of surgery according to Badwe or Hrushesky or Senie criteria (RR = 1.26, RR = 0.91, and RR = 0.88 respectively). Up-to-date agreement on the menstrual phase and relative expected better prognosis is still lacking.     

Inactivation of cytochrome P450 2E1 by tert-butylisothiocyanate

Several naturally occurring and synthethic isothiocyanates were evaluated for their ability to inactivate the major ethanol-inducible hepatic cytochrome P450 2E1. Of the compounds tested, tert-butylisothiocyanate (tBITC) was found to be the most selective inactivator of the 2E1 p-nitrophenol hydroxylation activity. tBITC was more specific for inactivating P450 2E1 activity than for rat P450 1A1, 1A2, 3A2, and 2B1, or the human cytochromes P450 3A4 and 2B6. The kinetics of inactivation of P450 2E1 by tBITC were characterized. P450 2E1, either in rat liver microsomes or in a purified reconstituted system containing the bacterially expressed rabbit cytochrome, was inactivated by tBITC in a mechanism-based manner. The loss of activity followed pseudo-first-order kinetics and was NADPH- and tBITC-dependent. The maximal rates for inactivation of P450 2E1 in microsomes or for the purified P450 2E1 at 30 degrees C were 0.72 and 0.27 min-1 and the apparent KI values were 11 and 7.6 microM, respectively. When cytochrome b5 was co-reconstituted with P450 2E1, the apparent KI for P450 2E1 inactivation by tBITC was similar to that seen in microsomes (14 microM). P450 2E1 T303A was also inactivated by tBITC with kinetic constants similar to that of the wild type enzyme. Co-incubations with an alternate substrate protected P450 2E1 from inactivation by tBITC. The extent of P450 2E1 inactivation by tBITC resulted in a comparable loss of the ability of the enzyme to form a reduced CO complex.     

Physiological and pathophysiological regulation of cytochrome P450

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the April 1998 Experimental Biology '98 meeting in San Francisco. The presentations focused on the mechanisms of regulation of cytochrome P450 gene expression by developmental factors and by hormones and cytokines, as well as on the interplay between physiological and chemical regulation. Approaches and systems used to address these questions included conditional gene knockouts in mice, primary hepatocyte cultures, immunofluorescence imaging of cells, and cell lines stably expressing reporter gene constructs.     

Metabolism of clozapine by cDNA-expressed human cytochrome P450 enzymes

The metabolism of clozapine was studied in vitro using cDNA-expressed human cytochrome P450 (CYP) enzymes 1A2, 3A4, 2C9, 2C19, 2D6, and 2E1. CYP1A2, 3A4, 2C9, 2C19, and 2D6 were able to N-demethylate clozapine. N-Oxide formation was exclusively catalyzed by CYP3A4. CYP2E1 did not metabolize clozapine. With regard to quantitative relationships, CYP1A2, 2C9, 2C19, and 2D6 displayed KM values ranging from 13 to 25 microM, whereas CYP3A4 had a 5-10 times higher KM value. CYP2C19 and 2D6 had the highest Vmax values (149-366 mol/hr/mol CYP). Taking into account the typical relative distribution of amounts of CYP enzymes in the liver, a simulation study suggested that at therapeutic concentrations CYP2C19 and CYP3A4 each accounted for about 35% of the metabolism. At toxic concentrations, the relative importance of CYP3A4 increased.     

Specificity of self-assembly of cytochrome P450

Under certain conditions, hexamers of microsomal cytochrome P450 can self-assemble from the subunits of different isoforms. However, the possibility for free choice results in recognition between identical subunits of each form of cytochrome P450 which provides preferential association of identical monomers into corresponding hexamers. The specificity of self-assembly suggests hexameric arrangement of cytochrome P450 in native membranes as we proposed earlier. In the present study, highly purified cytochrome P450 2B4 and cytochrome P450 1A2 (CYP 2B4 and CYP 1A2), including those immobilized by covalent attachment to an insoluble carrier of one protomer of each hexamer, were employed.     

Modulation of snake hepatic cytochrome P450 by 3-methylcholanthrene and phenobarbital

Ninety-four GB virus C/hepatitis G virus (GBV-C/ HGV) RNA-positive serum samples were obtained from all over the world. We found that all 15 GBV-C/HGV isolates from the Pygmies and the Bantu in the Central African region had a 12-amino acid indel (i.e. insertion or deletion) in the non-structural protein (NS) 5A region. Phylogenetic analyses of the NS5A region, using GBV-A as an outgroup, showed that these 15 isolates had diverged from the common ancestor much earlier than the remaining isolates, indicating an African origin of GBV-C/HGV.     

Design of a novel P450: a functional bacterial-human cytochrome P450 chimera

We report the construction of a functional chimera from approximately 50% bacterial (cytosolic) cytochrome P450cam and 50% mammalian (membrane-bound) cytochrome P450 2C9. The chimeric protein shows a reduced CO-difference spectrum absorption at 446 nm, and circular dichroism spectra indicate that the protein is globular. The protein is soluble and catalyzes the oxidation of 4-chlorotoluene using molecular oxygen and reducing equivalents from bacterial putidaredoxin and putidaredoxin reductase. This chimera provides a novel method for addressing structure-function issues and may prove useful in the design of oxidants for benign and stereospecific synthesis, as well as catalysts for bioremediation of polluted areas. Furthermore, these results provide the first evidence that bacterial P450 enzymes and mammalian P450 enzymes are likely to share a common tertiary structure.     

Interactions of substrate and product with cytochrome P450: P4502B4 versus P450cam

OBJECTIVE: To determine the relative abilities of somatostatin receptor scintigraphy (SRS) and conventional imaging studies (computed tomography, magnetic resonance imaging, ultrasound, angiography) to localize gastrinomas before surgery in patients with Zollinger-Ellison syndrome (ZES) subsequently found at surgery, and to determine the effect of SRS on the disease-free rate. SUMMARY BACKGROUND DATA: Recent studies demonstrate that SRS is the most sensitive imaging modality for localizing neuroendocrine tumors such as gastrinomas. Because of conflicting results in small series, it is unclear in ZES whether SRS will alter the disease-free rate, which gastrinomas are not detected, what factors contribute to failure to detect a gastrinoma, or whether the SRS result should be used to determine operability in patients without hepatic metastases, as recently recommended by some investigators. METHODS: Thirty-five consecutive patients with ZES undergoing 37 exploratory laparotomies for possible cure were prospectively studied. All had SRS and conventional imaging studies before surgery. Imaging results were determined by an independent investigator depending on surgical findings. All patients underwent an identical surgical protocol (palpation after an extensive Kocher maneuver, ultrasound during surgery, duodenal transillumination, and 3 cm duodenotomy) and postoperative assessment of disease status (fasting gastrin, secretin test imaging within 2 weeks, at 3 to 6 months, and yearly), as used in pre-SRS studies previously. RESULTS: Gastrinomas were detected in all patients at each surgery. Seventy-four gastrinomas were found: 22 duodenal, 8 pancreatic, 3 primaries in other sites, and 41 lymph node metastases. The relative detection order on a per-patient or per-lesion basis was SRS > angiography, magnetic resonance imaging, computed tomography > ultrasound. On a per-lesion basis, SRS had greater sensitivity than all conventional studies combined. SRS missed one third of all lesions found at surgery. SRS detected 30% of gastrinomas < or =1.1 cm, 64% of those 1.1 to 2 cm, and 96% of those >2 cm and missed primarily small duodenal tumors. Tumor size correlated closely with SRS rate of detection. SRS did not increase the disease-free rate immediately after surgery or at 2 years mean follow-up. CONCLUSIONS: SRS is the most sensitive preoperative imaging study for extrahepatic gastrinomas in patients with ZES and should replace conventional imaging studies as the preoperative study of choice. Negative results of SRS for localizing extrahepatic gastrinomas should not be used to decide operability, because a surgical procedure will detect 33% more gastrinomas than SRS. SRS does not increase the disease-free rate. In the future, more sensitive methods to detect small gastrinomas, especially in the duodenum and in periduodenal lymph nodes, or more extensive surgery will be needed to improve the postoperative disease-free rate in ZES.     

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.     

Reconstruction of mitochondrial cytochrome P450-dependent steroid hydroxylases in artificial phospholipid membranes: studies of cytochrome P450scc topology by limited proteolysis

Highly purified cytochrome P450scc from bovine adrenal cortex mitochondria was inserted in artificial phospholipid membranes prepared from phosphatidylcholine to study the main principles of its membrane organization in the model system. Topology of the cytochrome P450scc polypeptide chain in proteoliposomes was studied by limited proteolysis with trypsin or chymotrypsin followed by immunochemical identification of the products of proteolysis products of the membrane-bound heme protein. It is shown that limited proteolysis of cytochrome P450scc in proteoliposomes results in a significant decrease of Vmax for the reaction of cholesterol hydroxylation to pregnenolone in the reconstituted system in the presence of exogenously added adrenodoxin-reductase and adrenodoxin. However, after proteolytic modification of cytochrome P450scc with trypsin and chymotrypsin the affinity of the heme protein to adrenodoxin is increased. Different models of membrane organization as well as functional specificity of cytochrome P450scc in artificial membranes are discussed.     

Escherichia coli expression and substrate specificities of canine cytochrome P450 3A12 and rabbit cytochrome P450 3A6

High level Escherichia coli expression of cytochromes P450 3A12 and 3A6 has facilitated the characterization of proteins which exhibit limited activity as purified hepatic enzymes in reconstituted systems. Three 3A12 and two 3A6 constructs modified at the 5'-end to encode the bovine 17 alpha-sequence (Barnes et al., Proc. Natl. Acad. Sci. U.S.A. 88: 5597-5601, 1991), or related sequences, exhibited expression levels ranging from 2 to 89 nmol of cytochrome P450 liter-1. Recombinant canine 3A12 catalyzed steroid 6 beta-hydroxylation and erythromycin demethylation at rates comparable to those obtained in phenobarbital-induced canine liver microsomes. In contrast, 3A12 troleandomycin demethylase activity (2.5 nmol/min/nmol) was significantly lower than that of canine phenobarbital-induced liver microsomes (6.6 nmol/min/nmol). This difference in activity suggests that at least two 3A forms, which may differ functionally, are present within the canine liver. Purification of recombinant rabbit 3A6 revealed that homogeneous and E. coli-solubilized membrane preparations of 3A6 exhibit similar metabolic rates and identical substrate specificities; 3A activity was modulated by 25 microM alpha-naphthoflavone, which stimulated an unidentified progesterone metabolite 9-fold in 3A6 reconstituted systems in contrast to the 4-fold stimulation of 3A12. Furthermore, 25 microM alpha-naphthoflavone inhibited erythromycin demethylation 64 and 33% by purified recombinant 3A6- or 3A6-solubilized membrane fractions, respectively; 3A12-mediated erythromycin demethylation in solubilized membrane fractions was resistant to flavonoid inhibition. These results indicate that, although 3A substrate specificities are highly conserved between species, functional differences exist between canine 3A12 and rabbit 3A6, which may be utilized to better understand 3A structure-function relationships.     

The substrate specificity of cytochrome P450cam

The catalytic turnover of xenobiotics by cytochrome P450cam results in both the formation of organic metabolites and the uncoupled production of H2O2, and H2O. Previous studies have shown that a receptor-constrained three-dimensional screening program (DOCK) can be used to identify potential ligands (ergo substrates) for the enzyme (De Voss, J. J.; Sibbesen, O.; Zhang, Z.; Ortiz de Montellano, P. R. J. Am. Chem. Soc. 1997, 119, 5489). A new set of 10 compounds has now been examined to further test the substrate specificity of P450cam and the ability of DOCK to identify substrates for this enzyme. The results expand the known specificity of P450cam and define limitations in the use of DOCK to predict its substrate specificity.