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Risks of ovulation-induction therapy in systemic lupus erythematosus.

We report on four women with systemic lupus erythematosus who developed two types of complications after ovulation-induction therapy for primary or secondary infertility. Primary infertility was associated with endometriosis in one patient. Three had previously known systemic lupus erythematosus. All had inactive disease at onset of ovulation-induction therapy. Three patients developed symptoms consistent with moderate lupus flare a few weeks after the onset of ovulation-induction therapy. One patient developed inferior vena cava and unilateral left renal vein thrombosis. No patient became pregnant. A high oestrogen level induced by ovulation-induction therapy may explain the occurrence of lupus flare in patients with prior inactive lupus. All our patients had prior asymptomatic antiphospholipid antibodies. One patient developed a major thrombotic event. The presence of antiphospholipid antibodies increases the thrombotic risk related to ovulation-induction therapy. We conclude that ovulation-induction therapy should be restricted to patients with long-standing inactive systemic lupus erythematosus. A preventive increase of the corticosteroid dosage should be proposed in addition to heparin or antiaggregant therapy for those with prior asymptomatic antiphospholipid antibodies, or with heparin therapy for those with prior antiphospholipid antibody-related events.

Adult↗

Induction of CYP1A1 in primary rat hepatocytes by 3,3',4,4',5-pentachlorobiphenyl: evidence for a switch circuit element.

In vivo induction of CYP1A1 in hepatocytes by aryl hydrocarbon receptor agonists is heterogeneous. Using immunohistochemistry, cells appear to be either induced or not induced as if the response of an individual cell is better represented as a switch. We have examined induction of CYP1A1 in vitro in primary rat hepatocytes to distinguish the responses of populations of cells and responses of individual cells. Cells were treated with various concentrations of the aryl hydrocarbon receptor agonist, 3,3',4,4',5-pentachlorobiphenyl. Concentration-response and time-course responses were determined for the population of cells by Western blotting for CYP1A1 protein and by real-time RT-PCR for CYP1A1 mRNA. Individual cell responses were visualized by immunocytochemistry (ICC) for protein and by in situ hybridization (ISH) for mRNA. CYP1A1 mRNA was quantified by frequency distribution analysis of grains observed on the ISH slides. Population responses showed time- and concentration-related increases in induction. Single cell responses appeared as all-or-none in the field, with cells appearing to be induced and others appearing to be not induced. Even at the highest concentrations (2.5 x 10(-7) M), some hepatocytes remained unresponsive. Distribution frequencies of single cell induction were more consistent with a switch with variable levels of induction in cells depending on treatment concentration. Combined with the reports from in vivo studies, our results support a switch with rheostat behavior for individual hepatocytes. Mechanistic studies in liver cell lines that are confirmed to exhibit switch-like induction of single cells will be necessary to assess the molecular pathways of this circuit element.

Animals↗

HO-1 induction restores c-AMP-dependent lung epithelial fluid transport following severe hemorrhage in rats.

Inhibition of cAMP-dependent stimulation of the vectorial fluid transport across the lung epithelium following hemorrhagic shock is mediated by NO released within the airspaces of the lung. We tested here the hypothesis that prior induction of HO-1 would attenuate the release of NO in the airspaces, thus preventing the inhibition of the c-AMP stimulation of alveolar fluid clearance (ALC) in rats. Indeed, HO-1 induction restored the cAMP-mediated up-regulation of ALC after hemorrhage by decreasing NO released within the airspaces of the lung. In vitro studies demonstrated that HO-1 induction significantly reduced the iNOS-mediated release of NO by alveolar macrophages stimulated with endotoxin for 24 h. This effect is explained in part by a HO-1-dependent attenuation of the LPS-mediated nuclear translocation of NF-kappaB. In addition, HO-1 induction also significantly reduced the iNOS-mediated release of NO by MH-S cells that were stimulated with interferon-gamma by decreasing the phosphorylation of STAT 1, another transcription factor important for the activation of the iNOS promoter. In contrast, HO-1 induction did not affect the production of NO by rat alveolar epithelial type II cells that were stimulated with cytomix (a mixture of TNF-alpha, IL-1beta, and IFN-gamma) for 24 h. In summary, these results provide the first in vivo evidence that the induction of HO-1 in the lung restores a normal fluid transport capacity of the alveolar epithelium following hemorrhagic shock by inhibiting the iNOS-mediated release of NO by alveolar macrophages.

Animals↗

Cyclosporine and rapamycin affect protein kinase C induction of the intracellular activation signal, activator of DNA replication.

Immunosuppressive agents may initiate their pharmacologic action by disrupting phosphorylation cascades critical to T lymphocyte intracellular signaling after alloantigen recognition. Activator of DNA Replication (ADR), a transduction signal sensitive to CsA as well as rapamycin (RAPA) immunosuppression, seemed a likely candidate for phosphoregulation. This communication reports the development of a cell-free assay wherein CsA and RAPA inhibit ADR induction by protein kinase C (PKC). ADR and PKC are inactive in the cytosol of resting cells. Endogenous PKC activity in quiescent lymphocytes was triggered with the tumor promoter PMA, leading to the appearance of ADR, an event subsequently quantitated by the ability of ADR to trigger [3H]thymidine triphosphate incorporation into isolated nuclei. After PKC induction, ADR activity is increased (890 +/- 120 vs. 3910 +/- 345 cpm, P < 0.001). In the presence of the PKC inhibitor H7, ADR activity fails to increase (100 +/- 50 vs. 3910 +/- 345 cpm, P < 0.001). The high levels of ADR found in PHA-stimulated cells is marginally affected by in situ PKC induction, although the sustained impact of H7 throughout the cell cycle suggests that ADR is constantly being phosphorylated. Titration of CsA or RAPA into the cell-free system inhibited ADR induction in a dose-dependent fashion. The utility of ADR induction as a marker for immunosuppression was investigated by comparing ADR inducibility in resting cells that had been preloaded with CsA or RAPA with the proliferative index of cells cultivated with CsA or RAPA. ADR induction directly correlated with CsA or RAPA proliferative inhibition. The results suggested that ADR is a constituent of the PKC phosphoregulatory cascade essential for cell cycle progression. The correlation between cell-free ADR inducibility and proliferative inhibition by CsA or RAPA suggest that this procedure may be useful for in vitro prediction of allografted patient immunologic response.

Cell-Free System↗

Comparative induction of nitrate reductase by nitrate and nitrite in barley leaves.

The comparative induction of nitrate reductase (NR) by ambient NO3- and NO2- as a function of influx, reduction (as NR was induced) and accumulation in detached leaves of 8-day-old barley (Hordeum vulgare L.) seedlings was determined. The dynamic interaction of NO3- influx, reduction and accumulation on NR induction was shown. The activity of NR, as it was induced, influenced its further induction by affecting the internal concentration of NO3-. As the ambient concentration of NO3- increased, the relative influences imposed by influx and reduction on NO3- accumulation changed with influx becoming a more predominant regulant. Significant levels of NO3- accumulated in NO2(-)-fed leaves. When the leaves were supplied cycloheximide or tungstate along with NO2-, about 60% more NO3- accumulated in the leaves than in the absence of the inhibitors. In NO3(-)-supplied leaves NR induction was observed at an ambient concentration of as low as 0.02 mM. No NR induction occurred in leaves supplied with NO2- until the ambient NO2- concentration was 0.5 mM. In fact, NR induction from NO2- solutions was not seen until NO3- was detected in the leaves. The amount of NO3- accumulating in NO2(-)-fed leaves induced similar levels of NR as did equivalent amounts of NO3- accumulating from NO3(-)-fed leaves. In all cases the internal concentration of NO3-, but not NO2-, was highly correlated with the amount of NR induced. The evidence indicated that NO3- was a more likely inducer of NR than was NO2-.

Cycloheximide↗

Differential effects of partial hepatectomy and carbon tetrachloride administration on induction of liver cell foci in a model for detection of initiation activity.

Differential effects of partial hepatectomy (PH) and carbon tetrachloride (CCl(4)) administration on induction of glutathione S-transferase placental form (GST-P)-positive foci were investigated in a model for detection of initiation activity. Firstly, we surveyed cell proliferation kinetics and fluctuation in cytochrome P450 (CYP) mRNA levels by means of relative-quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and CYP 2E1 apoprotein amount by immunoblotting (experiment I) after PH or CCl(4) administration. Next, to assess the interrelationships among cell proliferation, fluctuation of CYPs after PH or CCl(4) administration and induction of liver cell foci, the non-hepatocarcinogen, 1,2-dimethylhydrazine (DMH) was administered to 7-week-old male F344 rats and initiated populations were selected using the resistant hepatocyte model (experiment II). In experiment I, the values of all CYP isozyme mRNAs after PH or CCl(4) administration were drastically decreased at the 12-h time point. From 72 h, mRNAs for all CYP isozymes began increasing, with complete recovery after 7 days. The CYP 2E1 apoprotein content in the PH group fluctuated weakly, whereas in the CCl(4) group it had decreased rapidly after 12 h and was still low at the 48 h point. In experiment II, induction of GST-P-positive foci was related to cell kinetics in the PH group, with about a 6-h time lag between time for carcinogen administration giving greatest induction of GST-P-positive foci and peaks in bromodeoxyuridine (BrdU) labeling, presumably due to the necessity for bioactivation of DMH. With CCl(4) administration, induction of foci appeared dependent on the recovery of CYP 2E1. In conclusion, PH was able to induce cell proliferation with maintenance of CYP 2E1, therefore being advantageous for induction of liver cell foci in models to detect initiation activity.

Animals↗

The inflammatory and proliferative response of normal skin in a model for acute chemical injury: ornithine decarboxylase induction as a common feature in various models for acute skin injury.

Application of sodium dodecyl sulphate (SDS) on the skin of healthy volunteers was used as a model for acute chemical injury. The time course of the response with respect to cell proliferation was studied using ornithine decarboxylase (ODC) activity. Erythema, polymorphonuclear leucocyte (PMN) infiltration, and the induction of epidermal antiproteinase activity (SKALP/elafin) were used as markers for the inflammatory response. ODC induction was similar to that in other models of acute skin injury, such as tape-stripping and ultraviolet light radiation. The amount of PMN infiltration correlated with erythema, but not with ODC induction. In contrast with findings in the tape-stripping model, no induction of SKALP/elafin activity was found after SDS application. We conclude that cell proliferation as measured by ODC induction is a common feature in the various models for skin injury. Both the kinetics and the intensity of the inflammatory response, and the induction of epidermal antiproteinase activity, appear to vary, depending on the specific model.

Acute Disease↗

Multicentre phase III trial on fludarabine, cytarabine (Ara-C), and idarubicin versus idarubicin, Ara-C and etoposide for induction treatment of younger, newly diagnosed acute myeloid leukaemia patients.

Fludarabine plus cytarabine (Ara-C) and idarubicin (FLAI) is an effective and well-tolerated induction regimen for the treatment of acute myeloid leukaemia (AML). This phase III trial compared the efficacy and toxicity of FLAI versus idarubicin plus Ara-C and etoposide (ICE) in 112 newly diagnosed AML patients <60 years. Fifty-seven patients received FLAI, as the first induction-remission course, and 55 patients received ICE. Post-induction treatment consisted of high-dose Ara-C (HDAC). After HDAC, patients in complete remission (CR) received a second consolidation course (mitoxantrone, etoposide, Ara-C) and autologous stem cell transplantation (auto-SCT) or allogeneic (allo)-SCT, according to the age, disease risk and donor availability. After a single induction course, CR rate was 74% in the FLAI arm and 51% in the ICE arm (P = 0.01), while death during induction was 2% and 9% respectively. Both haematological (P = 0.002) and non-haematological (P = 0.0001) toxicities, especially gastrointestinal (i.e. nausea, vomiting, mucositis and diarrhoea), were significantly lower in FLAI arm. In both arms, relapses were more frequent in patients who were not submitted to allo-SCT. After a median follow-up of 17 months, 30% and 38% of the patients are in continuous CR in FLAI and ICE arm respectively. Our prospective randomised study confirmed the anti-leukaemic effect and the low toxic profile of FLAI as induction treatment for newly diagnosed AML patients.

Acute Disease↗

Glutamine synthetase induction in embryonic neural retina. Interactions of receptor-hydrocortisone complexes with cell nuclei.

In the neural retina of the chick embryo, hydrocortisone (HC) elicits differential gene expression resulting in the induction of glutamine synthetase (GS), which is an enzyme marker of differentiation in the retina. The relationship between nuclear binding of receptor-hydrocortisone (R-HC) complexes and GS induction was investigated in cultures of retina tissue from 12-day chick embryos. The number of HC binding sites in the cytoplasm was estimated as 1650+/-200 per retina cell; there are approximately 1500+/-100 acceptor sites for R-HC per retina nucleus. GS induction in the retina became detectable only after R-HC bound to more than 40% of the nuclear acceptors sites; increased binding coincided with higher induction levels, until complete site saturation was attained; Proflavine, which blocks preferentially and completely GS induction in the retina by interfering in the nucleus with the enzyme-inducing action of the hormone, reduced nuclear binding of R-HC by only 20%; thus, only part of the R-HC that binds in the nucleus appears to be directly involved in eliciting the induction of GS. Within one hour after exposure of the retina to an inducing dose of HC, there was translocation of HC and HC-receptors (as R-HC complexes) from the cytoplasm into the nucleus and saturation of nuclear accepegan to decline; in 12 h, it was reduced to 50% of the initial saturation level. Since, during this time, the enzyme activity to increase, persistence of the induced state depends on association of the hormone with only a portion of the sites in the nucleus to which it can bind. The decrease in the amount of bound HC in the nuclei of induced cells was accompanied by an increase in the level of HC receptors in the cytoplasm. About 50% of this increase could be prevented by cycloheximide; this suggests that the reappearance of HC receptors in the cell cytoplasm may be due, at least in part, to de novo synthesis of HC receptors.

Animals↗

Transfilter lens induction in avian embryo.

The directive influence of the optic vesicle during lens induction of 2-day chick embryos was studied in vitro. Trunk ectoderm was chosen for the responding tissue. This uncommitted ectoderm formed distinct lentoid bodies when grown together with the optic vesicle. Crystallin synthesis was demonstrated in the lentoids with fluorescein labelled antiserum. By interposing filters of different thicknesses and pore sizes between the interacting tissues, three questions were put forward: (1) Whether the intimate contact between the interactants was essential for induction, (2) how far the inductive influence of the optic vesicle extended, and (3) what was the smallest pore through which the inductive influence could penetrate. The inductive influence reached across a Millipore filter with a thickness of 100 micrometer. It also penetrated a dialyzer membrane, allowing passage of molecules with molecular weights (MW) of less than 12,000 daltons. It was concluded that the directive, inductive signal(s) passing from the optic vesicle, diffused in the extracellular space as far as 100 micrometer and had a molecular weight of less than 12,000 daltons.

Animals↗

Induction of ornithine decarboxylase in guinea-pig lymphocytes. Synergistic effect of diacylglycerol and calcium.

Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5) and trifluoperazine inhibited ornithine decarboxylase induction in lymphocytes activated with phytohemagglutinin or inophore A23187. W-7, a more potent calmodulin antagonist than W-5, suppressed ornithine decarboxylase induction in a higher extent than did W-5. These results suggest that calmodulin may play an important role in ornithine decarboxylase induction in the activated lymphocytes. However, the extent of ornithine decarboxylase induction was greater in cells pretreated with Clostridium phospholipase C and then incubated with ionophore A23187 than in cells incubated with ionophore A23187 without the pretreatment. Moreover, combined treatment of cells with ionophore A23187 and tumor promotor, phorbol 12-myristate 13-acetate, caused synergistic induction of ornithine decarboxylase activity. These results, taken together, suggest that both activations of Ca2+-activated phospholipid-dependent protein kinase by diacylglycerol and of calmodulin-dependent function resulted from an elevation of cytosolic Ca2+ concentration may operate in the induction of ornithine decarboxylase in the activated lymphocytes.

Animals↗

Enhancing effect of a phorbol ester and of retinoic acid on glucocorticoid induction of chenodeoxycholate hydroxylation in hepatoma cultures.

In cultures of the differentiated clones Faza 967, Fao and HF, derived from Reuber hepatoma, physiological doses of glucocorticoid induce chenodeoxycholate 6 beta-hydroxylation, a microsomal cytochrome-P-450-mediated activity (enhanced in liver by phenobarbital and not by benzo[a]anthracene). Whereas 12-O-tetradecanoylphorbol 13-acetate (TPA) alone has no effect the tumor promoter, when added to dexamethasone, enhances this induction. This enhancement, half-maximum with 10 ng/ml TPA, is a function of the dose between 1 ng/ml and 50 ng/ml; 50 ng/ml (80 nM) increase 4-7-fold the induction rate (as measured in cultures by the amount of bile acid hydroxylated per 10(6) cells in 24 h, and in homogenates from treated cells) and 2.5-fold the maximum activity attained by the third day of induction. When added to cultures of the dedifferentiated clone H5, treated with benzo[a]anthracene, TPA does not influence benzo[a]pyrene hydroxylase induction, as shown by the total and relative amounts of the various hydrosoluble benzo[a]pyrene metabolites. TPA does not affect tyrosine aminotransferase induction in dexamethasone-treated Fao cultures. The enhancement is not suppressed by indomethacin, an inhibitor of prostaglandin synthesis. After dexamethasone removal from induced Faza 967 cultures, addition of TPA to the medium does not affect the decay rate of the chenodeoxycholate-hydroxylating activity. Retinoic acid similarly enhances the induction by dexamethasone of chenodeoxycholate hydroxylation, both in treated Faza 967 cultures and in homogenates from treated cultures. The effects of TPA and retinoic acid are additive. These results suggest a possible cooperation at the transcriptional level between transactive factors, involving TPA-mediated alterations, retinoic acid and glucocorticoid receptors. The system described might provide a convenient experimental approach in the study of its mechanism.

Animals↗

Differential tissue-specific expression and induction of cytochrome P450IVA1 and acyl-CoA oxidase.

We have examined the tissue-specific expression and inducibility of acyl-CoA oxidase and cytochrome P450IVA1 (P450IVA1) RNA in rats. Groups of three rats were dosed daily by gavage with methylclofenapate at 25 mg/kg in 5 ml/kg corn oil for nine weeks, or were administered a vehicle control. P450IVA1 and acyl-CoA oxidase RNA were detected using an RNase protection assay. Similar levels of acyl-CoA oxidase RNA were present in control liver and kidney, but the level of this RNA in lung, muscle and testis was 6-11%, and in pancreas was 0.13%, of that in liver. Treatment of rats with methylclofenapate led to an 11-fold induction of acyl-CoA oxidase RNA in liver and also produced a significant induction of this RNA in kidney, lung, muscle and testis of 1.7-fold, 1.3-fold, 2-fold and 1.7-fold, respectively. Acyl-CoA oxidase RNA was not induced in pancreas. P450IVA1 RNA was present in control liver and also in kidney of control rats at 28% of the level in liver. In contrast to acyl-CoA oxidase RNA, P450IVA1 RNA was not detected in lung, pancreas or testis. Methylclofenapate treatment of rats led to an 18-fold induction of P450IVA1 RNA in liver, and a sevenfold induction in kidney. Induction of P450IVA1 was not detected in any of the other tissues examined. Quantification of the relative amounts of acyl-CoA oxidase and P450IVA1 RNA in control liver revealed that acyl-CoA oxidase RNA was present in a 17.5-fold molar excess over P450IVA1 RNA. Western blotting with an anti-P450IVA IgG revealed two bands of similar apparent molecular mass in liver and kidney microsomes, but not in microsomes from the testis of control rats. Methylclofenapate treatment of rats caused an increase in the intensity of these bands in microsomes from liver, but no induction was obvious in kidney. Immunocytochemical staining for both the microsomal P450IVA and peroxisomal acyl-CoA oxidase proteins was restricted to the proximal convoluted tubule in the kidney cortex, with staining being most intense in the S3 region.

Acyl-CoA Oxidase↗

The Ets transcription factor ESE-1 mediates induction of the COX-2 gene by LPS in monocytes.

Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins that are major inflammatory agents. COX-2 production is triggered by exposure to various cytokines and to bacterial endotoxins. We present here a novel role for the Ets transcription factor ESE-1 in regulating the COX-2 gene in response to endotoxin and other pro-inflammatory stimuli. We report that the induction of COX-2 expression by lipopolysaccharide (LPS) and pro-inflammatory cytokines correlates with ESE-1 induction in monocyte/macrophages. ESE-1, in turn, binds to several E26 transformation specific (Ets) sites on the COX-2 promoter. In vitro analysis demonstrates that ESE-1 binds to and activates the COX-2 promoter to levels comparable to LPS-mediated induction. Moreover, we provide results showing that the induction of COX-2 by LPS may require ESE-1, as the mutation of the Ets sites in the COX-2 promoter or overexpression of a dominant-negative form of ESE-1 inhibits LPS-mediated COX-2 induction. The effect of ESE-1 on the COX-2 promoter is further enhanced by cooperation with other transcription factors such as nuclear factor-kappa B and nuclear factor of activated T cells. Neutralization of COX-2 is the goal of many anti-inflammatory drugs. As an activator of COX-2 induction, ESE-1 may become a target for such therapeutics as well. Together with our previous reports of the role of ESE-1 as an inducer of nitric oxide synthase in endothelial cells and as a mediator of pro-inflammatory cytokines in vascular and connective tissue cells, these results establish ESE-1 as an important player in the regulation of inflammation.

Animals↗

The role of pregnane X receptor in 2-acetylaminofluorene-mediated induction of drug transport and -metabolizing enzymes in mice.

Activation of the pregnane X receptor (PXR) mediates the induction of several drug transporters and -metabolizing enzymes. In vitro studies have reported that several of these genes are induced after exposure to the hepatocarcinogen, 2-acetylaminofluorene (2-AAF). Thus, we hypothesized that PXR may play a role in the in vivo induction of gene expression by 2-AAF. We examined the expression of the drug-metabolizing enzymes CYP1A2 and CYP3A11 and the drug transporters breast cancer resistance protein (BCRP), MRP2, and OATP2. Wild-type (PXR+/+) and PXR-null (PXR-/-) C57BL/6 mice were injected daily for 7 days with 150 or 300 mg/kg 2-AAF suspended in corn oil (i.p.), whereas the control group received corn oil vehicle. Levels of mRNA isolated from liver were measured by reverse transcription-polymerase chain reaction and normalized to beta-actin. Treatment of PXR+/+ mice resulted in a dose-dependent 2- to 4-fold induction (p<0.001) of MRP2, OATP2, BCRP, CYP3A11, and CYP1A2, but no induction was observed in PXR-/- mice. Induction of PXR mRNA was observed in the 2-AAF-treated PXR+/+ mice. Furthermore, a dose-dependent increase in CYP3A4 promoter construct activity was observed in HepG2 cells cotransfected with human or rat PXR, indicating that 2-AAF does indeed activate PXR. These results suggest that PXR is responsible for 2-AAF-mediated induction of drug efflux transporters and biotransformation enzymes in the liver. Moreover, novel findings demonstrate that PXR plays a role in regulation of the drug efflux transporter, BCRP, in mice.

2-Acetylaminofluorene↗

Utility of long-term cultured human hepatocytes as an in vitro model for cytochrome p450 induction.

Cytochrome P450 (P450) induction may have considerable implications for drug therapy. Therefore, understanding the induction potential of a new chemical entity at an early stage in discovery is crucial to reduce the risk of failure in the clinic and help the identification of noninducing chemical structures. Availability of human viable tissue often limits evaluation of induction potential in human hepatocytes. A solution is to increase the time period during which the hepatocytes remain viable. In this study we have investigated the induction of several P450 isozymes in long-term cultured hepatocytes compared with short-term cultured hepatocytes from the same individuals. Short- and long-term cultured primary hepatocytes isolated from each individual were cultured in a 96-well format and treated for 24 h with a range of prototypical P450 inducers and Merck Research Laboratories compounds. CYP3A4, 1A1, 1A2, 2B6, and 2C9 mRNA levels were measured using quantitative real-time reverse transcriptase-polymerase chain reaction (TaqMan) from the same cultured hepatocyte wells. CYP3A4, 1A1, 1A2, 2B6, and 2C9 were shown to be inducible in long-term cultured hepatocytes. The -fold induction varied between donors, and between short- and long-term cultured hepatocytes from the same donor. However, this variability can be controlled by normalizing data from each hepatocyte preparation to a positive control. The use of long-term cultured hepatocytes on 96-well plates has proven to be sensitive, robust, and convenient for assessing P450 induction potential of new compound entities during the drug discovery process.

Aryl Hydrocarbon Hydroxylases↗

Induction of CYP3A expression by dehydroepiandrosterone: involvement of the pregnane X receptor.

Dehydroepiandrosterone (DHEA) is a steroid produced by the human adrenal gland. Administration of pharmacological doses of DHEA to rats changes expression of many genes, including the cytochrome P450 family members CYP4A1 and CYP3A23. It is known that induction of CYP4A expression by DHEA requires the peroxisome proliferator-activated receptor alpha (PPAR(alpha)). In the current study, PPAR(alpha)-null mice were used to examine the role of PPAR(alpha) in expression of CYP3A. In wild-type mice, 150 mg/kg DHEA-sulfate induced Cyp4a and Cyp3a11 mRNAs by 5- and 2-fold, respectively. Induction of Cyp4a expression by DHEA-sulfate was not observed in PPAR(alpha)-null mice, whereas induction of Cyp3a11 expression by DHEA-sulfate was similar between genotypes. This suggests that PPAR(alpha) is not involved in induction of Cyp3a11 expression by DHEA. Because expression of CYP3A family members can be induced by activation of another member of the nuclear receptor superfamily, the pregnane X receptor (PXR), we examined the ability of DHEA to activate PXR. In transient transfection assays, DHEA and its metabolites androst-5-ene-3beta,17beta-diol (ADIOL), androst-5-ene-3,17-dione, and androst-4-ene-3,17-dione were activators of PXR. Maximal induction of a PXR-responsive reporter gene of approximately 3-fold was observed at concentrations of 50 to 100 microM, indicating that these steroids are relatively weak activators of PXR. Human and murine PXR exhibited different specificities for DHEA and its metabolites. ADIOL activated reporter gene expression in the presence of murine but not human PXR. Results of these studies suggest that the induction of rodent CYP3A expression upon treatment with high doses of DHEA occurs through activation of PXR.

Androstenediol↗

Induction of cytochrome P450 3A by paclitaxel in mice: pivotal role of the nuclear xenobiotic receptor, pregnane X receptor.

Paclitaxel, a taxane anti-microtubule agent, is known to induce CYP3A in rat and human hepatocytes. Recent studies suggest that a member of the nuclear receptor family, pregnane X Receptor (PXR), is a key regulator of the expression of CYP3A in different species. We investigated the role of PXR activation, in vitro and in vivo, in mediating Cyp3a induction by paclitaxel. Pregnenolone 16 alpha-carbonitrile (PCN), an antiglucocorticoid, was employed as a positive control for mouse PXR (mPXR) activation in vitro, and Cyp3a induction in vivo. In cell based reporter gene assays paclitaxel and PCN activated mPXR with an EC(50) of 5.6 and 0.27 microM, respectively. Employing PXR wild-type and transgenic mice lacking functional PXR (-/-), we evaluated the expression and activity of CYP3A following treatment with paclitaxel and PCN. Paclitaxel significantly induced CYP3A11 mRNA and immunoreactive CYP3A protein in PXR wild-type mice. Consistent with kinetics of CYP3A induction, the V(max) of testosterone 6 beta-hydroxylation in microsomal fraction increased 15- and 30-fold in paclitaxel- and PCN-treated mice, respectively. The Cyp3a induction response was completely abolished in paclitaxel- and PCN-treated PXR-null mice. This suggests that paclitaxel-mediated CYP3A induction in vivo requires an intact PXR-signaling mechanism. Our study validates the use of PXR activation assays in screening newer taxanes for potential drug interactions that may be related to PXR-target gene induction.

Animals↗