Molecular modelling of the human glucocorticoid receptor (hGR) ligand-binding domain (LBD) by homology with the human estrogen receptor alpha (hERalpha) LBD: quantitative structure-activity relationships within a series of CYP3A4 inducers where induction is mediated via hGR involvement.

The results of homology modelling of the human glucorticoid receptor (hGR) ligand-binding domain (LBD) based on the ligand-bound domain of the human estrogen receptor alpha (hERalpha) are reported. It is shown that known hGR ligands which induce the human cytochrome P450 enzyme CYP3A4 are able to fit the putative ligand-binding site of the nuclear hormone receptor and form hydrogen bonds with key amino acid residues within the binding pocket. Quantitative structure-activity relationships (QSARs) have been derived for hGR-mediated CYP3A4 induction which involve certain molecular structural and physicochemical properties of the ligand themselves, yielding good correlations (R=0.96-0.98) with fold induction of CYP3A4 known to be mediated via hGR involvement.

Receptor-dependent transcriptional activation of cytochrome P4503A genes: induction mechanisms, species differences and interindividual variation in man.

1. The importance of CYP3A enzymes in drug metabolism and toxicology has yielded a wealth of information on the structure, function and regulation of this subfamily and recent research emphasis has been placed on the human forms, namely CYP3A4, CYP3A5, CYP3A7 and CYP3A43. 2. The current review will focus on the receptor-dependency of CYP3A regulation and includes consideration of the regulatory roles of the glucocorticoid (GR), pregnane X (PXR) and constitutive androstane (CAR) receptors. 3. Emphasis has been placed on the topics of expression and substrate specificity, assessment of induction, species differences in induction, CYP3A promoter sequences and regulation of gene expression, structural and functional aspects of receptor-mediated, CYP3A gene activation, receptor variants and interindividual variation in human CYP3A expression, the latter encompassing environmental, physiological and genetic aspects. 4. An outline of future research needs will be discussed in the context of receptor-mediated molecular mechanisms of CYP3A gene regulation and the impact on interindividual variations in CYP3A expression. 5. Taken collectively, this review highlights the importance of understanding the molecular mechanisms of CYP3A induction as a means of rationalizing human responses to many clinically used drugs, in addition to providing a mechanistically coherent platform to understand and predict interindividual variations in response and drug-drug interactions.

The transcription of the peroxisome proliferator-activated receptor alpha gene is regulated by protein kinase C.

The transcriptional regulation of peroxisome proliferator-activated receptor alpha (PPARalpha) by a variety of peroxisome proliferators was investigated. The treatment of primary cultures of rat hepatocytes with Wy14,643 or clofibrate increased mRNA steady state levels of both PPARalpha and acyl coenzyme A oxidase (ACOX). In contrast, fenofibrate and ciprofibrate increased the expression of ACOX without affecting that of PPARalpha. Inhibition of protein kinase C (PKC) activity using bisindolylmaleimide or calphostin C abolished the increased PPARalpha expression by the peroxisome proliferators whereas the expression of the ACOX gene remained unaffected. Phorbol-12-myristate-13-acetate increased PPARalpha mRNA levels without altering ACOX mRNA levels. It can thus be concluded that a number of peroxisome proliferators activate a PKC-dependent signalling pathway in addition to the PPARalpha pathway. The PKC signal transduction pathway contributes to the regulation of PPARalpha expression but does not influence the transcriptional activity of PPARalpha.

Use of a reporter gene assay to predict and rank the potency and efficacy of CYP3A4 inducers.

Regulation of the CYP3A4 gene has been studied using an in vitro reporter gene assay. The effect of 17 xenobiotics on approximately 1 kilobase of the CYP3A4 proximal promoter, upstream of a secretory placental alkaline phosphatase reporter gene was investigated following transfection into the HepG2 cell line. Transfections were carried out either in the basal system or with cotransfection of expression plasmids for the human pregnane X receptor (hPXR) and the human glucocorticoid receptor (hGR), two important receptors in the regulation of CYP3A4 gene expression. Compounds were tested at four concentrations, and the resulting data were used to calculate maximal induction (I(max)) and EC(50) values. An "overall inductive ability" (IA) was derived by dividing I(max) by EC(50). Of the compounds tested seven were established transcriptional inducers, all of which were positive in the in vitro assay. The remaining 10 compounds represented a group with preliminary evidence for CYP3A transcriptional activation. Nine of these compounds produced statistically significant inductions in vitro, with only pravastatin failing to activate the reporter gene. This is of potential interest in light of the high IA values observed with the other structurally and functionally similar statins tested. We conclude that a four-concentration-point, in vitro model is capable of identifying CYP3A4 transcriptional inducers and yields an IA value allowing the ranking of compounds for their overall ability to induce CYP3A4 transcription. In addition, the majority of the compounds tested showed increased IA values in the hPXR/hGR cotransfected system, underpinning the importance of these receptors in CYP3A4 gene transcriptional regulation.

Regulation of the CYP3A4 gene by hydrocortisone and xenobiotics: role of the glucocorticoid and pregnane X receptors.

The molecular mechanisms of regulation of the CYP3A4 gene have been examined in an in vitro reporter gene system, containing -1 kb of the CYP3A4 promoter, in a HepG2 cell line. This system allows for the separate and combined transfection of expression plasmids encoding the human glucocorticoid receptor (hGR) and the human pregnane X receptor (hPXR), and, therefore, the opportunity to assess the role of these receptors in the induction process. Hydrocortisone produces a dose-dependent increase in CYP3A4 activation, a response that is increased in the presence of either receptor. Moreover, transfection of the hPXR decreased the EC(50) for hydrocortisone-dependent induction by a factor of 3.3, a response that was not changed by simultaneous cotransfection of the hGR. In addition, the hydrocortisone dose-response curve falls within the physiological blood level concentration of this steroid, implicating a regulatory role for hydrocortisone in the basal level of CYP3A4 expression. Although the responses to dexamethasone and rifampicin were both increased by both receptors, dexamethasone activation of CYP3A4 was similar for both the hGR and the hPXR, whereas rifampicin-dependent activation favored the hPXR. We conclude that regulation of the CYP3A4 gene is receptor-dependent and that hydrocortisone may function as a regulator of basal expression via the hPXR and the hGR. The implications of this latter conclusion for possible regulatory interactions between hydrocortisone and xenobiotic inducers remain to be clarified.

Use of suppression-PCR subtractive hybridisation to identify genes that demonstrate altered expression in male rat and guinea pig livers following exposure to Wy-14,643, a peroxisome proliferator and non-genotoxic hepatocarcinogen.

Understanding the genetic profile of a cell at all stages of normal and carcinogenic development should provide an essential aid to developing new strategies for the prevention, early detection, diagnosis and treatment of cancers. We have attempted to identify some of the genes that may be involved in peroxisome-proliferator (PP)-induced non-genotoxic hepatocarcinogenesis using suppression PCR subtractive hybridisation (SSH). Wistar rats (male) were chosen as a representative susceptible species and Duncan-Hartley guinea pigs (male) as a resistant species to the hepatocarcinogenic effects of the PP, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643). In each case, groups of four test animals were administered a single dose of Wy-14,643 (250 mg/kg per day in corn oil) by gastric intubation for 3 consecutive days. The control animals received corn oil only. On the fourth day the animals were killed and liver mRNA extracted. SSH was carried out using mRNA extracted from the rat and guinea pig livers, and used to isolate genes that were up and downregulated following Wy-14,643 treatment. These genes included some predictable (and hence positive control) species such as CYP4A1 and CYP2C11 (upregulated and downregulated in rat liver, respectively). Several genes that may be implicated in hepatocarcinogenesis have also been identified, as have some unidentified species. This work thus provides a starting point for developing a molecular profile of the early effects of a non-genotoxic carcinogen in sensitive and resistant species that could ultimately lead to a short-term assay for this type of toxicity.

Control and statistical analysis of in vitro reporter gene assays.

The use of in vitro gene reporter assays is becoming increasingly widespread in biology and particularly in drug metabolism, where the need for rapid screening of novel compounds is a driving factor. There is, however, little standardization of technique in the control of such assays, nor in the interpretation of results. This leads to confusion in the literature, with the possibility of a single piece of data being interpreted by several different methods, potentially giving vastly differing results. We have developed a reporter gene assay methodology that controls for many biological and experimental variables in the system and allows the application of a mathematical model to determine statistical significance between groups. Use of this methodology, we feel, allows an accurate and reproducible method of analyzing in vitro reporter gene assay data and increases its value as a biological tool.

Differential gene expression in drug metabolism and toxicology: practicalities, problems and potential.

1. An important feature of the work of many molecular biologists is identifying which genes are switched on and off in a cell under different environmental conditions or subsequent to xenobiotic challenge. Such information has many uses, including the deciphering of molecular pathways and facilitating the development of new experimental and diagnostic procedures. However, the student of gene hunting should be forgiven for perhaps becoming confused by the mountain of information available as there appears to be almost as many methods of discovering differentially expressed genes as there are research groups using the technique. 2. The aim of this review was to clarify the main methods of differential gene expression analysis and the mechanistic principles underlying them. Also included is a discussion on some of the practical aspects of using this technique. Emphasis is placed on the so-called 'open' systems, which require no prior knowledge of the genes contained within the study model. Whilst these will eventually be replaced by 'closed' systems in the study of human, mouse and other commonly studied laboratory animals, they will remain a powerful tool for those examining less fashionable models. 3. The use of suppression-PCR subtractive hybridization is exemplified in the identification of up- and down-regulated genes in rat liver following exposure to phenobarbital, a well-known inducer of the drug metabolizing enzymes. 4. Differential gene display provides a coherent platform for building libraries and microchip arrays of 'gene fingerprints' characteristic of known enzyme inducers and xenobiotic toxicants, which may be interrogated subsequently for the identification and characterization of xenobiotics of unknown biological properties.

A reporter gene assay to assess the molecular mechanisms of xenobiotic-dependent induction of the human CYP3A4 gene in vitro.

1. A plasmid containing 1 kb of the CYP3A4 regulatory (promoter) region coupled to a reporter gene for secretary placental alkaline phosphatase (SPAP) was transfected into HepG2 cells. Transfected cells were dosed with several known inducers of CYP3A4 and the levels of SPAP were measured. The effect of co-transfecting a plasmid encoding the human glucocorticoid receptor on reporter gene activity was also examined. 2. Dexamethasone induced CYP3A4-dependent reporter gene expression in a concentration-dependent manner and induction was approximately doubled in the presence of the glucocorticoid receptor. Dexamethasone-dependent induction was blocked by RU-486 (a glucocorticoid receptor antagonist), in the presence of the co-transfected glucocorticoid receptor. 3. Induction of CYP3A4-dependent reporter gene expression and enhancement of the induction by the glucocorticoid receptor was also observed with pregnenolone-16alpha-carbonitrile (PCN), rifampicin, phenytoin, carbamazepine, phenylbutazone and phenobarbitone, all known in vivo inducers of CYP3A4 in man. 4. Metyrapone and sulfinpyrazone induced CYP3A4-dependent reporter gene expression, but induction was not enhanced by the glucocorticoid receptor. 5. Clotrimazole, erythromycin and triacetyloleandomycin (TAO) did not induce CYP3A4-dependent reporter gene expression, consistent with the observation that these inducers act through post-transcriptional mechanisms. 6. These results highlight differences in the molecular mechanisms of induction of CYP3A4 by the xenobiotics studied and indicate that the glucocorticoid receptor is involved in the induction of the CYP3A4 gene by some, but not all, CYP3A4 inducers. 7. We propose that the approach described here provides a useful in vitro approach for the identification of transcriptional regulators of the CYP3A4 gene.

Ascorbic acid deficiency decreases the expression of CYP4A1 in liver microsomes of guinea pigs.

The cytochrome P-450 monooxygenase system plays a central role in the oxidation of a wide variety of structurally unrelated compounds. Its contribution is affected by nutritional and several other factors. Ascorbic acid (AA) deficiency decreases the content of cytochrome P-450 in liver microsomes of guinea pigs (GPs). Included in the group of cytochromes P-450 are the phenobarbital and 3-methylcholanthrene inducible moieties. In the present study the effect of AA status on another specific cytochrome P-450, CYP4A1, laurate omega-hydroxylase was investigated. Ascorbic acid may selectively increase or decrease certain forms of cytochromes. For four weeks adult male Hartley GPs were fed a diet containing 2.5 (Group I), 0.1 (Group II) and 0% (Group III) AA. The liver microsomes were isolated at this stage and cytochrome P-450 content was determined. Group III showed a significant decrease in cytochrome P-450 compared to groups I and II. They also showed a marked decrease in aminopyrine N-demethylase activity. The expression of CYP4A1 was evaluated using Western blot and anti-CYP4A1 antibody. Group III GPs showed a marked decrease in CYP4A1 expression. Groups I and II showed similar expression. This study demonstrates that CYP4A1, a specific cytochrome induced by hypolipidemic agents, is decreased by AA deficiency.

Development of an in vitro reporter gene assay to assess xenobiotic induction of the human CYP3A4 gene.

The current work concerns the development and validation of an in vitro reporter gene assay system for the assessment of induction of human CYP3A4. A plasmid containing approximately 1 kb of the CYP3A4 regulatory region (which contains several recognised regulatory elements including glucocorticoid responsive elements) coupled to the reporter gene for human secreted placental alkaline phosphatase (SPAP) was transfected into the human hepatoblastoma cell line HepG2. Calcium phosphate precipitation was the method of choice for transfection. The transfected cells were dosed with known inducers of CYP3A4 and the levels of SPAP in the medium were subsequently measured using a chemiluminescent assay, as an indirect measure of CYP3A4 induction. The inducers used in this study included dexamethasone, phenytoin, triacetyloleandomycin (TAO), rifampicin, carbamazepine, phenylbutazone and sulfinpyrazone. These compounds activated CYP3A4 by between 1.5-4.5-fold thus representing a major advance in assessing the induction of human CYP genes in vitro.

CYP4A1 gene transfection studies and the peroxisome proliferator-activated receptor: development of a high-throughput assay to detect peroxisome proliferators.

An in vitro reporter gene assay has been established to examine cytochrome P4504A1 (CYP4A1) induction. A response element from the upstream region of the rat CYP4A1 gene containing a peroxisome proliferator response element (PPRE) has been linked to the chloramphenicol acetyl-transferase (CAT) gene in a reporter vector (1). This CYP4A1 reporter construct has been co-transfected into human HepG2 cells in the presence and absence of expression vectors encoding the transcription factors PPAR alpha and RXR alpha. The assay employs calcium phosphate-DNA co-precipitate mediated transfection. Reporter gene products have been quantitated using chemiluminescent based assays. We have shown that, in the presence of PPAR alpha, the above CYP4A1 construct is transcriptionally activated by a range of structurally different peroxisome proliferators including Wy-14,643, ciprofibrate, clofibric acid and nafenopin. Our future efforts will focus on the establishment of a high-throughput assay for the detection of peroxisome proliferators. Such an assay would provide an invaluable in vitro test for the screening of developmental drug candidates prior to in vivo studies.

Use of competitive RT-PCR in the molecular analysis of peroxisome proliferation.

The technique of quantitative competitive RT-PCR to determine the levels of mRNA expression of genes encoding peroxisome proliferator-activated receptor alpha (PPAR alpha), acyl coenzyme-A (ACOX) and cytochrome P450 4A1 (CYP4A1) in primary rat hepatocyte cultures is described. This technique is based on the co-amplification of an internal standard (PCR MIMIC) and target DNA sequence with one set of primers. Following total RNA extraction and reverse transcription, competitive PCR was carried out by mixing various dilutions of known concentrations of PCR MIMIC with constant amounts of cDNA. Densitometry was then carried out on the DNA bands obtained following gel electrophoresis and, after correcting for size differences between the target DNA and MIMIC, the concentration of target DNA was calculated and expressed as attomoles (10-18 moles) per microgram total RNA. Constitutive levels of PPAR alpha, ACOX and CYP4A1 obtained were 0.037 +/- 0.003, 1.858 +/- 0.470m and 0.035 +/- 0.007 attomoles/microgram RNA, respectively. Following 24 h culture of rat primary hepatocytes in the presence of sodium clofibrate (a peroxisome proliferator), the levels of PPAR alpha, ACOX and CYP4A1 were increased by 2.1-, 3.3- and 12.8-fold, respectively. Thus the technique described in this study has high sensitivity and can be used to accurately measure the mRNA steady state levels in cell cultures.

An epitope-tagging system for studying regulation of the peroxisome proliferator activated receptor alpha (PPAR alpha).

Peroxisome proliferators (PPs) are a group of compounds which cause peroxisome proliferation and hepatocellular carcinomas in rodents, and form a class of non-genotoxic carcinogens. It is thought that PPs act via a receptor similar to members of the nuclear hormone superfamily termed the peroxisome proliferator activated receptor (PPAR). Multiple subtypes (alpha, beta, delta and gamma) of the receptor exist and are differentially expressed between tissues and species. PPAR alpha has been shown to activate transcription by binding to response elements upstream of peroxisome proliferator responsive genes. However, despite the isolation of transcriptionally active human subtypes of the receptor, hPPAR alpha and hNUC1, humans are thought to be non-responsive to PPs. This is possibly due to regulation of PPAR, and it has been recently reported that PPAR alpha is a phosphoprotein in vivo and insulin regulates its phosphorylation. A system employing epitope-tagged receptors has been developed to study this further, with the aim of establishing stably transfected cell lines expressing high levels of epitope-tagged mouse and human PPAR alpha. Our experiments clearly demonstrate that an epitope-tagged mPPAR alpha receptor has an equal ability to modulate transcription as the native receptor in transactivation assays and will be further used to examine the molecular mechanisms of peroxisome proliferation.

Molecular profiling of non-genotoxic hepatocarcinogenesis using differential display reverse transcription-polymerase chain reaction (ddRT-PCR).

The technique of differential display reverse transcription-polymerase chain reaction (ddRT-PCR) has been used to produce unique profiles of up-regulated and down-regulated gene expression in the liver of male Wistar rats following short term exposure to the non-genotoxic hepatocarcinogens, phenobarbital and WY-14,643. Animals were treated for 3 days, whereupon their livers were extracted and snap frozen. mRNA was prepared from the livers and used for ddRT-PCR. Individual bands from the differential displays were extracted and cloned. False positives were eliminated by dotblot screening and true positives then sequenced and identified.

Developmental modulation of cysteine conjugate beta-lyase/glutamine transaminase K/kynurenine aminotransferase mRNA in rat brain.

Cysteine conjugate beta-lyase/glutamine transaminase K/kynurenine aminotransferase (CS-lyase/GTK/KAT) is a tri-functional enzyme found in several organs, including the brain. Kynurenine aminotransferase is important in tryptophan metabolism in the CNS, producing kynurenic acid, a NMDA receptor antagonist and neuroprotective. Tryptophan not metabolised via kynurenine aminotransferase may form quinolinic acid, a NMDA receptor agonist and neurotoxin. Kynurenic acid co-treatment blocks quinolinic acid induced lesions in the CNS in rat. In many conditions exhibiting neurodegeneration (i.e. Huntington's, Parkinsonism, Down's syndrome) quinolinic acid and/or kynurenic acid concentrations are altered, suggesting the ratio of these chemicals may be important in neurodegeneration. We have investigated the developmental modulation of CS-lyase/GTK/KAT mRNA in rat brain. CS-lyase/GTK/KAT mRNA was measured in 14, 21, 28, 35, 42 day post-natal and adult rats. While many regions demonstrated a steady increase to adult levels, two other profiles were seen. Five regions rapidly reached adult levels of the mRNA, while two peaked above the adult level before falling back. This provides evidence that expression of the CS-lyase/GTK/KAT gene is physiologically modulated, and provides the basis for further investigation into the mechanism of control. Artificial modulation could possibly be used to alter levels of the neuroprotectant kynurenic acid in neurodegeneration.

Bifonazole, but not the structurally-related clotrimazole, induces both peroxisome proliferation and members of the cytochrome P4504A sub-family in rat liver.

Male Wistar rats were treated with a low (150 mumol/kg) and a high (750 mumol/kg) dose of either clotrimazole of bifonazole. Bifonazole, but not clotrimazole, exhibited the characteristics of a peroxisome proliferator including hepatomegaly (increase in liver:body weight ratio), up to a 4-fold induction of lauric acid omega-hydroxylase activity and an 8-fold induction of palmitoyl-CoA oxidation by rat liver peroxisomes. This induction of enzyme activities was paralleled by increased protein levels as determined by immunochemical analysis for both liver microsomal cytochrome P4504A1 and the peroxisomal trifunctional protein of the beta-oxidation spiral. In contrast, clotrimazole did not increase protein levels of either cytochrome P4504A or the trifunctional protein. Western blot analyses demonstrated that bifonazole also induced P4502B1/2B2, P4503A and P4501A1, but not P4502E1. Clotrimazole induced a similar spectrum of P450s as determined by Western blotting with the exception that this azole was a marginal P4501A1 inducer under the conditions studied. Taken collectively, our data provides evidence that bifonazole is one of the increasingly recognised, non-carboxylate containing xenobiotics that induce both peroxisome proliferation and the cytochrome P4504A sub-family in rat liver.