Inducers of the cytochrome P-450 superfamily as promoters of carcinogenesis

This review summarizes data on the mechanisms of tumor-promoting activity of non-genotoxic compounds that are inducers of cytochrome P-450 isoforms. Their promoting activity is analyzed in term of synthesis of new cytochrome P-450 isoforms. Active oxygen species formed by cytochrome P-450 isoforms can simultaneously act as stimulators of proliferation and inhibitors of intercellular communications. Promoter effects can be associated with changes in the ratio of cellular signaling non-protein molecules induced by newly synthesized cytochrome P-450 isoforms. Data on induction of cytochrome P-450 isoforms of family 1 indicate that inducer interaction with its receptor causes cellular events resulting in the stimulation of cell proliferation.     

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.     

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.     

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.     

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.     

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.     

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.     

Inhibition and induction of cytochrome P450 and the clinical implications

The cytochrome P450s (CYPs) constitute a superfamily of isoforms that play an important role in the oxidative metabolism of drugs. Each CYP isoform possesses a characteristic broad spectrum of catalytic activities of substrates. Whenever 2 or more drugs are administered concurrently, the possibility of drug interactions exists. The ability of a single CYP to metabolise multiple substrates is responsible for a large number of documented drug interactions associated with CYP inhibition. In addition, drug interactions can also occur as a result of the induction of several human CYPs following long term drug treatment. The mechanisms of CYP inhibition can be divided into 3 categories: (a) reversible inhibition; (b) quasi-irreversible inhibition; and (c) irreversible inhibition. In mechanistic terms, reversible interactions arise as a result of competition at the CYP active site and probably involve only the first step of the CYP catalytic cycle. On the other hand, drugs that act during and subsequent to the oxygen transfer step are generally irreversible or quasi-irreversible inhibitors. Irreversible and quasi-irreversible inhibition require at least one cycle of the CYP catalytic process. Because human liver samples and recombinant human CYPs are now readily available, in vitro systems have been used as screening tools to predict the potential for in vivo drug interaction. Although it is easy to determine in vitro metabolic drug interactions, the proper interpretation and extrapolation of in vitro interaction data to in vivo situations require a good understanding of pharmacokinetic principles. From the viewpoint of drug therapy, to avoid potential drug-drug interactions, it is desirable to develop a new drug candidate that is not a potent CYP inhibitor or inducer and the metabolism of which is not readily inhibited by other drugs. In reality, drug interaction by mutual inhibition between drugs is almost inevitable, because CYP-mediated metabolism represents a major route of elimination of many drugs, which can compete for the same CYP enzyme. The clinical significance of a metabolic drug interaction depends on the magnitude of the change in the concentration of active species (parent drug and/or active metabolites) at the site of pharmacological action and the therapeutic index of the drug. The smaller the difference between toxic and effective concentration, the greater the likelihood that a drug interaction will have serious clinical consequences. Thus, careful evaluation of potential drug interactions of a new drug candidate during the early stage of drug development is essential.     

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.     

Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites

Some cytochrome P450 catalyzed reactions show atypical kinetics, and these kinetic processes can be grouped into five categories: activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. A two-site model in which the enzyme can bind two substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 and naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as dapsone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and naproxen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics suggestive of a low Km, low Vmax component for the first substrate molecule and a high Km, high Vmax component for the second substrate molecule. 7, 8-Benzoflavone activation of phenanthrene metabolism by CYP3A4 and dapsone activation of flurbiprofen and naproxen metabolism by CYP2C9 were also observed. Furthermore, partial inhibition of 7, 8-benzoflavone metabolism by phenanthrene was observed. These results demonstrate that various P450 isoforms may exhibit atypical enzyme kinetics depending on the substrate(s) employed and that these results may be explained by a model which includes simultaneous binding of two substrate molecules in the active site.     

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.     

Xenobiotics and xenoestrogens in fish: modulation of cytochrome P450 and carcinogenesis

The periaqueductal gray matter (PAG) serves as the midbrain link between forebrain emotional processing systems and motor pathways used in the defense reaction. Part of this response depends upon PAG efferent pathways that modulate cardiovascular-related sympathetic outflow systems, including those that regulate the heart. While it is known that the PAG projects to vagal preganglionic neurons, including possibly cardiovagal motoneurons, no information exists on the PAG circuits that may affect sympathetically mediated cardiac functions and, thus, the purpose of this study was to use neuroanatomical methods to identify these pathways. First, viral transneuronal retrograde tracing experiments were performed in which pseudorabies virus (PRV) was injected into the stellate ganglion of rats. After 4 days survival, five PAG regions contained transynaptically infected neurons; these included the dorsomedial, lateral and ventrolateral PAG columns as well as the Edinger-Westphal and precommissural nuclei. Second, the descending efferent PAG projections were studied with the anterograde axonal marker Phaseolus vulgaris leuco-agglutinin (PHA-L) with a particular focus on determining whether the PAG projects to the intermediolateral cell column (IML). Almost no axonal labeling was found throughout the thoracic IML suggesting that the PAG modulates sympathetic functions by indirect pathways involving synaptic relays through sympathetic premotor cell groups, especially those found in the medulla oblongata. This possibility was examined by a double tracing study. PHA-L was first injected into either the lateral or ventrolateral PAG and after 6 days, PRV was injected into the ipsilateral stellate ganglion. After an additional 4 days survival, a double immunohistochemical procedure for co-visualization of PRV and PHA-L was used to identify the sympathetic premotor regions that receive an input from the PAG. The PAG innervated specific groups of sympathetic premotor neurons in the hypothalamus, pons, and medulla as well as providing reciprocal intercolumnar connections within the PAG itself (Jansen et al., Brain Res. 784 (1998) 329-336). The major route terminates in the ventral medulla, especially within the medial region which contains sympathetic premotor neurons lying within the raphe magnus and gigantocellular reticular nucleus, pars alpha. Both serotonergic and non-serotonergic sympathetic premotor neurons in these two regions receive inputs from the PAG. Weak PAG projections to sympathetic premotor neurons were found in the rostral ventrolateral medulla (including to C1 adrenergic neurons), locus coeruleus, A5 cell group, paraventricular and lateral hypothalamic nuclei. In summary, both the lateral and ventrolateral PAG columns appear to be capable of modulating cardiac sympathetic functions via a series of indirect pathways involving sympathetic premotor neurons found in selected sites in the hypothalamus, midbrain, pons, and medulla oblongata, with the major outflow terminating in bulbospinal regions of the rostral ventromedial medulla.     

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.     

Expression of CYP71B7, a cytochrome P450 expressed sequence Tag from Arabidopsis thaliana

c-Yes was purified 322-fold from a rat liver plasma membrane fraction to a single 60-kDa band on SDS-PAGE. The purified protein contained essentially no phosphotyrosine residues and was autophosphorylated with Mg2+. ATP exclusively at tyrosine residues with a concomitant increase in the protein-tyrosine kinase activity. The autophosphorylated c-Yes was extensively digested by trypsin and the resultant two major phosphopeptides, peptides I and II, were purified by HPLC on a reversed-phase C-18 column. The amino acid sequence of peptide I was determined to be LIEDNEYTAR, which is identical with the sequence from Leu-418 through Arg-427 of mouse c-Yes, indicating that one of the autophosphorylation sites corresponds to Tyr-424 of the mouse c-Yes. After partial determination of the N-terminal sequence of 10 amino acid residues of peptide II, the 230 bp sequence of rat cDNA that encodes the N-terminal 76 amino acid residues of c-Yes covering peptide II, was determined. From the predicted amino acid sequence, the sequence of peptide II was assumed to be from Tyr-16 through Lys-46, YTPENPTEPVNTSAGHYGVEHATAATTSSTK. The purified c-Yes phosphorylated the tyrosine residue of synthetic peptides covering Tyr-32 and its surrounding sequence but did not phosphorylate peptides covering Tyr-16 and its surrounding sequence, suggesting that the other autophosphorylation site is Tyr-32.