Thermodynamics of Ligand Binding to P450 2B4 and P450eryF Studied by Isothermal Titration Calorimetry

Structural plasticity and cooperativity in ligand recognition are two key aspects of the catalytic diversity of cytochrome P450 enzymes. As more mammalian P450 crystal structures have emerged, computational modeling has become a major tool to predict drug metabolism and interactions. There is a need for real solution thermodynamic data to support modeling and crystallographic observations. Using isothermal titration calorimetry (ITC) we successfully evaluated the conformational flexibility of P450 2B4 in binding imidazole inhibitors of different size and chemistry and dissected the stoichiometry and energetics of ligand binding allostery in P450eryF. Thermodynamic signatures obtained by ITC nicely correlated with structural and modeling results. Thus, ITC is a powerful tool to study structure-function relationships in P450s.     

Development and validation of an in silico P450 profiler based on pharmacophore models

In today's drug discovery process, the very early consideration of ADME properties is aimed at a reduction of drug candidate drop out rate in later development stages. Apart from in vitro testing, in silico methods are evaluated as complementary screening tools for compounds with unfavorable ADME attributes. Especially members of the cytochrome P450 (P450) enzyme superfamily-- e.g. P450 1A2, P450 2C9, P450 2C19, P450 2D6, and P450 3A4-- contribute to xenobiotic metabolism, and compound interaction with one of these enzymes is therefore critically evaluated. Pharmacophore models are widely used to identify common features amongst ligands for any target. In this study, both structure-based and ligand-based models for prominent drug-metabolizing members of the P450 family were generated employing the software packages LigandScout and Catalyst. Essential chemical ligand features for substrate and inhibitor activity for all five P450 enzymes investigated were determined and analyzed. Finally, a collection of 11 pharmacophores for substrates and inhibitors was evaluated as an in silico P450 profiling tool that could be used for early ADME estimation of new chemical entities.     

Analysis of mammalian cytochrome P450 structure and function by site-directed mutagenesis

Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and/or bioremediation.     

P450 structures and oxidative metabolism of xenobiotics

This review focuses on the structural models for cytochrome P450, which are improving our knowledge and understanding of the P450 catalytic cycle, and the way in which substrates bind to the enzyme leading to catalytic conversion and subsequent formation of mono-oxygenated metabolites. Various stages in the P450 reaction cycle have now been investigated using X-ray crystallography and electronic structure calculations, whereas homology modeling of mammalian P450s is currently revealing important aspects of pharmaceutical and other xenobiotic metabolism mediated by P450 involvement. These features are explored in the current review on P450-based catalysis, which emphasizes the importance of structural modeling to our understanding of this enzyme's function. In addition, the results of various quantitative structure-activity relationships (QSAR) analyses on series of chemicals, which are metabolized via P450 enzymes, are presented such that the importance of electronic and other structural factors in explaining variations in rates of metabolism can be appreciated. As an important example of biocatalysis, the P450 system has a major future as an enzyme for use in many biotechnological applications, including biodegradation and bioremediation.     

Practical application of cytochrome P450

Recent progress on the application of cytochrome P450 (P450) to bioconversion processes, biosensors, and bioremediation were reviewed. Because regio- and enantioselective hydroxylation makes chemical synthesis difficult, a bioconversion process using P450 would be quite attractive. One of the most successful industrial applications of P450 may be the bioconversion process for pravastatin formation using a Streptomyces carbophilus CYP105A3. Unfortunately, practical application of P450s in the bioconversion process is limited because of their low stability, low activity and co-factor dependency. However, directed evolution is expected to generate useful P450 biocatalysts for a wide range of substrates. Shunt pathways of CYP152A1, CYP152A2, and CYP152B1 are notably promising for practical application, because these P450s require neither reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) nor electron donor proteins, and efficiently catalyze using hydrogen peroxide. A P450 biosensor using biochip technology is expected to become a tool for rapidly determining drugs and endogenous substances in plasma at a low cost. Bioremediation of dioxins and polychlorinated biphenyls (PCBs) by the CYP1 family appears to be possible by using suicidal, genetically engineered microorganisms. The P450 superfamily has tremendous potential for practical applications in various fields.     

Prediction of drug metabolism: the case of cytochrome P450 2D6

Cytochromes P450 (Cyt P450s) constitute the most important biotransformation enzymes involved in the biotransformation of drugs and other xenobiotics. Because drug metabolism by Cyt P450s plays such an important role in the disposition and in the pharmacological and toxicological effects of drugs, early consideration of ADME-properties is increasingly seen as essential for the discovery and the development of new drugs and drug candidates. The primary aim of this paper is to present various computational approaches used to rationalize and predict the activity and substrate selectivity of Cyt P450s, as well as the possibilities and limitations of these approaches, now and in the future. Attention is also paid to the experimental validation of these approaches by using high-throughput screening (HTS) of affinities to drug-drug interactions at the level of Cyt P450-isoenzymes. Since human Cyt P450 2D6 is one of the most important drug metabolizing enzymes and since in this regard much pioneering work has been done with this Cyt P450, Cyt P450 2D6 is chosen as a model for this discussion. Apart from early mechanism-based ab initio calculations on substrates of Cyt P450 2D6, pharmacophore modeling of ligands (i.e. both substrates and inhibitors) of Cyt P450 2D6 and protein homology modeling have been used successfully for the rationalisation and prediction of metabolite formation by this Cyt P450 isoenzyme. Significant protein structure-related species differences have been reported recently. It is concluded that not one computational approach is capable of rationalizing and reliably predicting metabolite formation by Cyt P450 2D6, but that it is rather the combination of the various complimentary approaches. It is moreover concluded, that experimental validation of the computational models and predictions is often still lacking. With the advent of novel, easily and well applicable in vitro based high throughput assays for ligand binding and turnover this limitation could be overcome soon, however. When effective links with other new and recent developments, such as bioinformatics, neural network computing, genomics and proteomics can be created, in silico rationalisation and prediction of drug metabolism by Cyt P450s is likely to become one of the key technologies in early drug discovery and development processes.     

Structural insights into understudied human cytochrome P450 enzymes

Human cytochrome P450 (CYP) enzymes are widely known for their pivotal role in the metabolism of drugs and other xenobiotics as well as of endogenous chemicals. In addition, CYPs are involved in numerous pathophysiological pathways and, hence, are therapeutically relevant. Remarkably, a portion of promising CYP targets is still understudied and, as a consequence, untargeted, despite their huge therapeutic potential. An increasing number of X-ray and cryo-electron microscopy (EM) structures for CYPs have recently provided new insights into the structural basis of CYP function and potential ligand binding. This structural knowledge of CYP functionality is essential for both understanding metabolism and exploiting understudied CYPs as drug targets. In this review, we summarize and highlight structural knowledge about this enzyme class, with a focus on understudied CYPs and resulting opportunities for structure-based drug design.Teaser: This review summarizes recent structural insights into understudied cytochrome P450 enzymes. We highlight the impact of molecular modeling for mechanistically explaining pathophysiological effects establishing understudied CYPs as promising drug targets.     

The first structure of a microsomal P450--implications for drug discovery

Although numerous microbial, soluble P450 structures have been known for some time, it is only recently that the first crystal structure of a microsomal, membrane-associated P450 has been determined. The structure gives an increased understanding of the membrane, substrate and reductase binding surfaces, and has enabled the modeling of other P450s with greater accuracy. Other milestones include the first determination of a true P450 drug target, CYP51, the determination of a thermophilic P450 and the structures of cryogenically trapped intermediates of the P450 reaction cycle.     

Validation of model of cytochrome P450 2D6: an in silico tool for predicting metabolism and inhibition

There has been much interest in the development of a predictive model of cytochrome P450 2D6 particularly because this enzyme is involved in the oxidation of at least 50 drugs. Previously we have described the combined use of homology modeling and molecular docking to correctly position a range of substrates in the CYP2D6 active site with the known sites of metabolism above the heme. Here, our approach identifies correctly the site of metabolism of the atypical (no basic nitrogen) cytochrome P450 2D6 substrate, spirosulfonamide. The same method is used to screen a small compound database for cytochrome P450 2D6 inhibition. A database containing 33 compounds from the National Cancer Institute database was docked into our cytochrome P450 2D6 homology model using the program GOLDv2.0. Experimental IC50 values for the 33 compounds were determined; comparison with the corresponding docked scores revealed a correlation with a regression coefficient of r2 = 0.61 (q2 = 0.59). The method was able to discriminate between tight and weak binding compounds and correctly identified several novel inhibitors. The results therefore suggest that our approach, which combines homology modeling with molecular docking, has produced a useful predictive in silico tool for cytochrome P450 2D6 inhibition, which is best used as one filter in a multifilter database screen.     

The inducibility and catalytic activity of cytochromes P450c (P450IA1) and P450d (P450IA2) in rat tissues

The metabolism of phenacetin is primarily by cytochrome P450-dependent O-deethylation to paracetamol (POD activity). In untreated rats, microsomal POD activity is detectable in both the liver and lung, but not in the small intestine or the kidney. POD activity is highly induced in both hepatic and extrahepatic tissues of the rat following treatment with polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (MC). Only cytochrome P450c (P450IA1) is inducible in rat extrahepatic tissues by MC or isosafrole, whereas in the liver both cytochromes P450c and P450d (P450IA2) are inducible by these compounds. Specific antibodies to cytochromes P450c and P450d were used to study the expression and function of these two related isoenzymes in rat liver and extrahepatic tissues before and after induction with MC. Whereas cytochrome P450d is responsible for all of the high affinity POD activity in hepatic microsomal fractions of both untreated and MC treated rats, this activity is mediated only by P450c in microsomal fractions from extrahepatic tissues following MC treatment. POD activity of microsomal fractions from lung of untreated rats was not mediated by either cytochrome P450c or P450d.     

Induction of cytochrome P450IA1 in mouse hepatoma cells by several chemicals. Phenobarbital and TCDD induce the same form of cytochrome P450

The mouse hepatoma cell line Hepa-1 was studied for aryl hydrocarbon hydroxylase (AHH) inducibility by sixteen compounds known to be inducers of cytochrome P450 of different "classes". Both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and sodium phenobarbital induced AHH activity. A cytochrome P450IA1-specific (P1-450) mouse cDNA probe was used to quantitate mRNA induction. There was a good correlation between the amount of cytochrome P450IA1 mRNA induced and AHH activity. Immunoblots with monoclonal antibody 1-7-1, which recognizes rat liver P450IA1 and P450IA2 (P450c and P450d, respectively), showed that both phenobarbital and TCDD increase the amount of a P450 isozyme immunorelated to P450IA1 in this cell line. Hepa-1 mutants with no AHH inducibility (no functional P450IA1 structural gene; no Ah receptor; no nuclear translocation of the inducer-receptor complex; and presence of dominant repressor) did not respond to phenobarbital. The cytosolic receptor for TCDD (Ah receptor) was characterized to see if phenobarbital induced cytochrome P450IA1 mRNA and the hydroxylase enzyme through the same mechanism as TCDD. 20 mM Phenobarbital almost completely abolished the binding of 3H-TCDD to the cytosolic receptor. These data indicate that phenobarbital can be a weak ligand for the Ah receptor and thus induce cytochrome P450IA1 and AHH activity. The observation increases the list of different P450 forms inducible by phenobarbital.     

Cloning and heterologous expression of P450Um-1, a novel bacterial P450 gene, for hydroxylation of immunosuppressive agent AS1387392

Biotransformation technology involving enzymatic modification of original substrates by organisms such as microbes is a valuable tool in improving pharmacokinetics or physicochemical properties of the base compounds. The fungal metabolite AS1387392 is a histone deacetylase inhibitor with potential as a therapeutic immunosuppressant. However, its paucity of functional groups, essential to synthesizing derivatives, is a drawback. Amycolatopsis azurea JCM-3275 catalyzed hydroxylation of AS1387392 to AS1429716, which may facilitate the synthesis of more derivatives by the additional hydroxyl moiety present in AS1429716. This reaction was inhibited by cytochrome P450 inhibitor metyrapone, indicating that cytochrome P450 may be responsible for the transformation. Degenerate PCR primers were subsequently constructed and used to clone genes encoding cytochrome P450 from the genomic DNA of A. azurea JCM-3275. We cloned an entire novel P450 gene (1209?bp) and named it P450Um-1. Its deduced amino acid sequence was homologous with that of the CYP105 subfamily. Further cloning of the upstream region, which may contain the native promoter site, was followed by insertion of the open reading frame with the upstream area into Streptomycetes high copy vector pIJ702, giving the expression plasmid pNUm-1. P450Um-1 was specifically expressed in Streptomyces lividans TK24, and this recombinant strain converted AS1387392 to AS1429716 without any redox partners. These results show that P450Um-1, a novel bacterial P450, catalyzed hydroxylation of AS1387392 to AS1429716. This resultant recombinant strain is expected to be an efficient biocatalyst with application to more suitable redox systems than those tested here.     

Visible spectra of type II cytochrome P450-drug complexes: evidence that "incomplete" heme coordination is common.

The visible spectrum of a ligand-bound cytochrome P450 is often used to determine the nature of the interaction between the ligand and the P450. One particularly characteristic form of spectra arises from the coordination of nitrogen-containing ligands to the P450 heme iron. These type II ligands tend to be inhibitors because they stabilize the low reduction potential P450 and prevent oxygen binding to the heme. Yet, several type II ligands containing aniline, imidazole, and triazole moieties are also known to be substrates of P450, although P450 binding spectra are not often scrutinized to make this distinction. Therefore, the three nitrogenous ligands aniline, imidazole, and triazole were used as binding spectra standards with purified human CYP3A4 and CYP2C9, because their small size should not present any steric limitations in their accessing the heme prosthetic group. Next, the spectra of P450 with drugs containing the three nitrogenous groups were collected for comparison. The absolute spectra demonstrated that the red-shift of the low-spin Soret band is mostly dependent on the electronic properties of the nitrogen ligand since they tended to match their respective standards, aniline, imidazole, and triazole. On the other hand, difference spectra seemed to be more sensitive to the steric properties of the ligand because they facilitated comparison of the spectral amplitudes achieved with the drugs versus those with the standard nitrogen ligands. Therefore, difference spectra may help reveal "weak" coordination to the heme that results from suboptimal orientation or ligand binding to more remote locations within the P450 active sites.     

Differential oxidation of two thiophene-containing regioisomers to reactive metabolites by cytochrome P450 2C9

The uricosuric diuretic agent tienilic acid (TA) is a thiophene-containing compound that is metabolized by P450 2C9 to 5-OH-TA. A reactive metabolite of TA also forms a covalent adduct to P450 2C9 that inactivates the enzyme and initiates immune-mediated hepatic injury in humans, purportedly through a thiophene-S-oxide intermediate. The 3-thenoyl regioisomer of TA, tienilic acid isomer (TAI), is chemically very similar and is reported to be oxidized by P450 2C9 to a thiophene-S-oxide, yet it is not a mechanism-based inactivator (MBI) of P450 2C9 and is reported to be an intrinsic hepatotoxin in rats. The goal of the work presented in this article was to identify the reactive metabolites of TA and TAI by the characterization of products derived from P450 2C9-mediated oxidation. In addition, in silico approaches were used to better understand both the mechanisms of oxidation of TA and TAI and/or the structural rearrangements of oxidized thiophene compounds. Incubation of TA with P450 2C9 and NADPH yielded the well-characterized 5-OH-TA metabolite as the major product. However, contrary to previous reports, it was found that TAI was oxidized to two different types of reactive intermediates that ultimately lead to two types of products, a pair of hydroxythiophene/thiolactone tautomers and an S-oxide dimer. Both TA and TAI incorporated 1?O from 1?O? into their respective hydroxythiophene/thiolactone metabolites indicating that these products are derived from an arene oxide pathway. Intrinsic reaction coordinate calculations of the rearrangement reactions of the model compound 2-acetylthiophene-S-oxide showed that a 1,5-oxygen migration mechanism is energetically unfavorable and does not yield the 5-OH product but instead yields a six-membered oxathiine ring. Therefore, arene oxide formation and subsequent NIH-shift rearrangement remains the favored mechanism for formation of 5-OH-TA. This also implicates the arene oxide as the initiating factor in TA induced liver injury via covalent modification of P450 2C9. Finally, in silico modeling of P450 2C9 active site ligand interactions with TA using the catalytically active iron-oxo species revealed significant differences in the orientations of TA and TAI in the active site, which correlated well with experimental results showing that TA was oxidized only to a ring carbon hydroxylated product, whereas TAI formed both ring carbon hydroxylated products and an S-oxide.     

Synthesis and Evaluation of Azole-Substituted Tetrahydronaphthalenes as Inhibitors of P450 arom,P450 17, and P450 TxA2

In search of potential drugs for the treatment of estrogen- and androgen-dependent cancer as well as the prophylaxis of metastases, tetralones, tetralins, and dihydronaphthalenes bearing a OCH₃ substituent at the benzene nucleus and an imidazol-4-yl, imidazol-1-yl, or 1,2,4-triazol-1-yl substituent in 2-position were synthesized with and without C₁-spacer between the rings (compounds 2 – 26 ). The compounds were tested in vitro for inhibition of the three target enzymes P450 arom (human placental microsomes), P450 17 (rat testicular microsomes), and P450 TxA₂ (citrated human whole blood). To examine selectivity, some compounds were further tested in vitro for inhibition of P450 18 (bovine adrenal mitochondria), P450 see (bovine adrenal mitochondria) and corticoid formation (aldosterone, corticosterone; ACTH stimulated rat adrenal tissue). In vivo, selected compounds were examined in Sprague Dawley rats regarding P450 TxA₂ inhibition, reduction of plasma testosterone concentration, antiuterotrophic activity (inhibition of the uterotrophic activity of androstenedione), reduction of plasma estradiol concentration (pregnant mares' serum gonadotropin-primed rats), and mammary tumor inhibiting activity (dimethylbenzanthracene-induced tumor; pre- and postmenopausal model). In the series of imidazol-4-yl compounds, which represent a novelty in the field of azole inhibitors of steroidogenic P450 enzymes, strong inhibitors of P450 arom and/or P450 17 were found: 7-OCH₃-2-(imidazol-4-ylmethylene)-1-tetralone ( 4 ) and 7-OCH₃-2-(imidazol-4-ylmethyl)-tetralin ( 12 ) are among the most potent inhibitors of P450 arom in vitro known so far. Compound 4 is a selective inhibitor, whereas 12 shows in addition strong inhibition of P450 17. In contrast to 12 , the 6-OCH₃ derivative (compound 11 ) is a selective inhibitor of P450 17, being 50 times more potent than ketoconazole. Some imidazol-1-yl compounds show a marked inhibition of P450 TxA₂: 2-(imidazol-1-ylmethyl)-1-tetralone ( 13 ) is a selective inhibitor of P450 TxA₂, whereas 7-OCH₃-2-(imidazol-1-ylmethyl)-tetralin ( 17 ) as well as 2-(imidazol-1-ylmethyl)-tetralin ( 16 ) and 7-OCH₃-2-imidazol-1-yl-3,4-dihydronaphthalene ( 25 ) additionally show strong inhibition of P450 arom and P450 17. Regarding the other steroidogenic P450 enzymes as well as corticosterone formation, the compounds show only little inhibitory activity. Aldosterone formation, however, is inhibited at low concentrations. Nevertheless, 4 and 12 are more selective, i.e. inhibit aldosterone synthesis less than the well known inhibitor of P450 arom fadrozole. The compounds show activity in the aforementioned in vivo tests.     

Modelling cytochromes P450 binding modes to predict P450 inhibition,metabolic stability and isoform selectivity

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Highlights► P450 Binding Mode (BM) is how the ligand ‘approaches’ the heme. ► BMs are ranked according to the accessibility of corresponding atoms/groups. ► Metabolism and inhibition come from competition between BMs. ► Metabolism occurs when a BM is productive. ► P450 inhibition might be due to type II, time-dependent, productive or nonproductive BMs.     

Small Intestinal Cytochromes P450

Abstract

Small intestinal cytochromes P450 (P450) provide the principal, initial source of biotransformation of ingested xenobiotics. The consequences of such biotransformation are detoxification by facilitating excretion, or toxification by bioactivation. P450s occur at highest concentrations in the duodenum, near the pylorus, and at decreasing concentrations distally — being lowest in the ileum. Highest concentrations occur from midvillus to villous tip, with little or none occurring in the crypts of Lieberkuehn. Microsomal P4503A, 2C8-10, and 2D6 forms have been identified in human small intestine, and P450s 2B1, possibly 2B2, 2A1, and 3A1/2 were located in endoplasmic reticulum of rodent small intestine, while P4502B4 has been purified to electrophoretic homogeneity from rabbit intestine. Some evidence indicates a differential distribution of P450 forms along the length of the small intestine and even along the villus. Rat intestinal P450s are inducible by xenobiotics — with phenobarbital (PB) inducing P4502B1, 3-methylcholanthrene (3-MC) inducing P4501A1, and dexamethasone inducing two forms of P4503A. Induction is most effectively achieved by oral administration of the agents, and is rapid — aryl hydrocarbon hydroxylase (AHH) was increased within 1 h of administration of, for example, 3-MC. AHH, 7-ethoxycoumarin O-deethylase (ECOD), and 7-ethoxyresorufin O-deethylase (EROD) have been used most frequently as substrates to characterize intestinal P450s. Dietary factors affect intestinal P450s markedly — iron restriction rapidly decreased intestinal P450 to beneath detectable values; selenium deficiency acted similarly but was less effective; Brussels sprouts increased intestinal AHH activity 9.8-fold, ECOD activity 3.2-fold, and P450 1.9-fold; fried meat and dietary fat significantly increased intestinal EROD activity; a vitamin A-deficient diet increased, and a vitamin A-rich diet decreased intestinal P450 activities; and excess cholesterol in the diet increased intestinal P450 activity. The role of intestinal P450 in toxifying or detoxifying specific xenobiotics has been clearly demonstrated to only a limited extent. However, elevated intestinal P450 levels have been indirectly linked to gastrointestinal cancer. Intestinal metabolism of 2,2,2-trifluoroethanol produces intestinal lesions with consequent systemic bacterial infection.     

The mechanism of halothane binding to microsomal cytochrome P450

Summary The unusual difference spectrum obtained with halothane and reduced rat liver microsomal cytochrome P450 can be simulated by addition of trifluoro diazoethane to dithionite reduced microsomes. Chemical evidence and model reactions suggest that in both cases a trifluoromethyl carbene complex is formed with the reduced hemoprotein. The spectral dissociation constants of the two species are similar as are their competitive reactions with carbon monoxide. The partial destruction of the carbenoid-cytochrome P450 complex characterizes the ligand as a highly reactive species. It is assumed that under anaerobic conditions this complex is formed by a two electron reduction of halothane and that covalent binding to microsomal proteins proceeds by this carbenoid species. A possible relationship to the hepatotoxicity of polyhalogenated compounds and anesthetics is discussed.     

Cholesterol-metabolizing cytochromes P450.

By catalyzing the first steps in different pathways of cholesterol degradation, cytochromes P450 (P450s) 7A1, 27A1, 11A1, and 46A1 play key roles in cholesterol homeostasis. CYP7A1 is a microsomal liver-specific enzyme that converts cholesterol to 7alpha-hydroxycholesterol. CYP27A1 is a ubiquitously expressed mitochondrial P450 that metabolizes cholesterol to 27-hydroxycholesterol. CYP11A1 also resides in mitochondria but is expressed mainly in steroidogenic tissues, where it catalyzes the conversion of cholesterol to pregnenolone. Finally, CYP46A1 is a brain-selective microsomal monooxygenase producing 24S-hydroxycholesterol from cholesterol. Catalytic efficiencies of cholesterol-metabolizing P450s vary significantly and probably reflect physiological requirements of different organs for the rate of cholesterol turnover. P450s 7A1, 27A1, 11A1, and 46A1 represent a unique system for elucidation of how different enzymes have adapted to fit their specific roles in cholesterol elimination. Studies of cholesterol-metabolizing P450s suggest that their activities could be modulated post-translationally and that they should also be considered as targets for regulation of cholesterol homeostasis.     

Small intestinal cytochromes P450.

Small intestinal cytochromes P450 (P450) provide the principal, initial source of biotransformation of ingested xenobiotics. The consequences of such biotransformation are detoxification by facilitating excretion, or toxification by bioactivation. P450s occur at highest concentrations in the duodenum, near the pylorus, and at decreasing concentrations distally--being lowest in the ileum. Highest concentrations occur from midvillus to villous tip, with little or none occurring in the crypts of Lieberkuehn. Microsomal P4503A, 2C8-10, and 2D6 forms have been identified in human small intestine, and P450s 2B1, possibly 2B2, 2A1, and 3A1/2 were located in endoplasmic reticulum of rodent small intestine, while P4502B4 has been purified to electrophoretic homogeneity from rabbit intestine. Some evidence indicates a differential distribution of P450 forms along the length of the small intestine and even along the villus. Rat intestinal P450s are inducible by xenobiotics--with phenobarbital (PB) inducing P4502B1, 3-methylcholanthrene (3-MC) inducing P4501A1, and dexamethasone inducing two forms of P4503A. Induction is most effectively achieved by oral administration of the agents, and is rapid--aryl hydrocarbon hydroxylase (AHH) was increased within 1 h of administration of, for example, 3-MC. AHH, 7-ethoxycoumarin O-deethylase (ECOD), and 7-ethoxyresorufin O-deethylase (EROD) have been used most frequently as substrates to characterize intestinal P450s. Dietary factors affect intestinal P450s markedly--iron restriction rapidly decreased intestinal P450 to beneath detectable values; selenium deficiency acted similarly but was less effective; Brussels sprouts increased intestinal AHH activity 9.8-fold, ECOD activity 3.2-fold, and P450 1.9-fold; fried meat and dietary fat significantly increased intestinal EROD activity; a vitamin A-deficient diet increased, and a vitamin A-rich diet decreased intestinal P450 activities; and excess cholesterol in the diet increased intestinal P450 activity. The role of intestinal P450 in toxifying or detoxifying specific xenobiotics has been clearly demonstrated to only a limited extent. However, elevated intestinal P450 levels have been indirectly linked to gastrointestinal cancer. Intestinal metabolism of 2,2,2-trifluoroethanol produces intestinal lesions with consequent systemic bacterial infection.