Petr Pavek

Xenobiotic-induced transcriptional regulation of xenobiotic metabolizing enzymes of the cytochrome P450 superfamily in human extrahepatic tissues.

Numerous members of the cytochrome P450 (CYP) superfamily are induced after exposure to a variety of xenobiotics in human liver. We have gained considerable mechanistic insights into these processes in hepatocytes and multiple ligand-activated transcription factors have been identified over the past two decades. Families CYP1, CYP2 and CYP3 involved in xenobiotic metabolism are also expressed in a range of extrahepatic tissues (e.g. intestine, brain, kidney, placenta, lung, adrenal gland, pancreas, skin, mammary gland, uterus, ovary, testes and prostate). Since the expression of the majority of the isoforms appears to be very low in the extrahepatic tissues in comparison with predominant expression in adult liver, the role of the enzymes in overall biotransformation and total body clearance is minor. However, basal expression and up-regulation of extrahepatic CYP enzymes can significantly affect local disposition of xenobiotics or endogenous compounds in peripheral tissues and thus modify their pharmacological/toxicological effects or affect absorption of xenobiotics into systemic circulation. The goal of this review is to critically examine our current understanding of molecular mechanisms involved in induction of xenobiotic metabolizing CYP genes of human families CYP1, CYP2 and CYP3 by exogenous chemicals in extrahepatic tissues. We concentrate on organs such as the intestine, kidney, lung, placenta and skin, which are involved in drug distribution and clearance or are in direct contact with environmental xenobiotics. We also discuss single nucleotide polymorphisms (SNPs) of key CYPs, which at the level of transcription affect expression of the genes in the extrahepatic tissues or are associated with some pathophysiological stages or disorders in the organs.

Rifampicin Does not Significantly Affect the Expression of Small Heterodimer Partner in Primary Human Hepatocytes

The small/short heterodimer partner (SHP, NR0B2) is a nuclear receptor corepressor lacking a DNA binding domain. SHP is induced by bile acid-activated farnesoid X receptor (FXR) resulting in CYP7A1 gene suppression. In contrast, Pregnane X receptor (PXR) activation by its ligands was recently suggested to inhibit SHP gene transactivation to maximize the induction of PXR target genes. However, there are also conflicting reports in literature whether PXR or rodent Pxr activation down-regulates SHP/Shp expression. Moreover, the PXR-mediated regulation of the SHP gene has been studied only at the SHP mRNA and transactivation (gene reporter assay) levels. In this study, we studied the effect of rifampicin, a prototype PXR ligand, on SHP mRNA, and protein expression in three primary human hepatocyte cultures. We found that SHP mRNA is not systematically down-regulated in hepatocyte in culture after 24 h treatment with rifampicin. Consistently, we did not observe down-regulation of SHP protein in primary human hepatocytes after 24 and 48 h of incubation with rifampicin. We can conclude that although we observed slight down-regulation of SHP mRNA and protein in several hepatocyte preparations, the phenomenon is unlikely critical for PXR-mediated induction of its target genes.

Valproic Acid Induces CYP3A4 and MDR1 Gene Expression by Activation of Constitutive Androstane Receptor and Pregnane X Receptor Pathways

In our study, we tested the hypothesis whether valproic acid (VPA) in therapeutic concentrations has potential to affect expression of CYP3A4 and MDR1 via constitutive androstane receptor (CAR) and pregnane X receptor (PXR) pathways. Interaction of VPA with CAR and PXR nuclear receptors was studied using luciferase reporter assays, real-time reverse transcriptase polymerase chain reaction (RT-PCR), electrophoretic mobility shift assay (EMSA), and analysis of CYP3A4 catalytic activity. Using transient transfection reporter assays in HepG2 cells, VPA was recognized to activate CYP3A4 promoter via CAR and PXR pathways. By contrast, a significant effect of VPA on MDR1 promoter activation was observed only in CAR-cotransfected HepG2 cells. These data well correlated with up-regulation of CYP3A4 and MDR1 mRNAs analyzed by real-time RT-PCR in cells transfected with expression vectors encoding CAR or PXR and treated with VPA. In addition, VPA significantly up-regulated CYP3A4 mRNA in primary hepatocytes and augmented the effect of rifampicin. EMSA experiments showed VPA-mediated augmentation of CAR/retinoid X receptor α heterodimer binding to direct repeat 3 (DR3) and DR4 responsive elements of CYP3A4 and MDR1 genes, respectively. Finally, analysis of specific CYP3A4 catalytic activity revealed its significant increase in VPA-treated LS174T cells transfected with PXR. In conclusion, we provide novel insight into the mechanism by which VPA affects gene expression of CYP3A4 and MDR1 genes. Our results demonstrate that VPA has potential to up-regulate CYP3A4 and MDR1 through direct activation of CAR and/or PXR pathways. Furthermore, we suggest that VPA synergistically augments the effect of rifampicin in transactivation of CYP3A4 in primary human hepatocytes.

Endogenous and exogenous ligands of aryl hydrocarbon receptor: current state of art.

Aryl hydrocarbon receptor (AhR) is an important transcriptional regulator of drug metabolizing enzymes that dominantly controls the expression of cytochrome P450 CYP1 family genes and some phase II enzymes. AhR also has many endogenous functions including cell cycle control, immune response, and cell differentiation. In addition, AhR is well-known to be involved in chemically-induced carcinogenesis. AhR is activated by a variety of endogenous and exogenous ligands. While exogenous activation of AhR has deleterious effects on human organism, sustained activation of AhR by endogenous ligands is indispensable for proper cell functions. Therefore, the effects of exogenous and endogenous ligands on AhR resemble the Dr. Jekyll and Mr. Hyde story. The aim of the current paper is to summarize and update the knowledge on exogenous and endogenous AhR ligands.

Variation of Drug Kinetics in Pregnancy

Significant changes in the physiological and biotransformation processes that govern pharmacokinetics occur during pregnancy. Consequently, the disposition of many medications is altered in gestation and the efficacy and toxicity of drugs used by pregnant women can be difficult to predict or can lead to serious side effects. Gastrointestinal absorption and bioavailability of drugs vary due to changes in gastric secretion and small intestine motility. Various pregnancy-related hemodynamic changes such as an increase in cardiac output, blood volume, the volume of distribution (Vd), renal perfusion and glomerular filtration may affect drug disposition and elimination, and can cause increase or decrease in the terminal elimination half-life of drugs. Changes in maternal drug biotransformation activity also contribute to alterations in pharmacokinetics of drugs taken in pregnancy. Therefore, pregnant women may require different dosing regimens or their adjustment than both men and non-pregnant women. In addition, the prenatal pharmacotherapy is unique due to the presence of feto-placental unit. Considerations regarding transplacental pharmacokinetics and safety for the developing fetus are thus essential aspects of medication in pregnancy. The aim of this review is to summarize major physiological and biotransformation changes associated with pregnancy that affect pharmacokinetics in pregnant women. In addition, we point out the most important examples of altered kinetics of drugs administered in pregnancy with mechanistic explanation of the phenomena based on maternal adaptation in pregnancy.

Expression and Transport Activity of Breast Cancer Resistance Protein (Bcrp/Abcg2) in Dually Perfused Rat Placenta and HRP-1 Cell Line

Breast cancer resistance protein (BCRP/ABCG2) is a member of the ATP-binding cassette transporter family that recognizes a variety of chemically unrelated compounds. Its expression has been revealed in many mammal tissues, including placenta. The purpose of this study was to describe its role in transplacental pharmacokinetics using rat placental HRP-1 cell line and dually perfused rat placenta. In HRP-1 cells, expression of Bcrp, but not P-glycoprotein, was revealed at mRNA and protein levels. Cell accumulation studies confirmed Bcrp-dependent uptake of BODIPY FL prazosin. In the placental perfusion studies, a pharmacokinetic model was applied to distinguish between passive and Bcrp-mediated transplacental passage of cimetidine as a model substrate. Bcrp was shown to act in a concentration-dependent manner and to hinder maternal-to-fetal transport of the drug. Fetal-to-maternal clearance of cimetidine was found to be 25 times higher than that in the opposite direction; this asymmetry was partly eliminated by BCRP inhibitors fumitremorgin C (2 μM) or N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918; 2 μM) and abolished at high cimetidine concentrations (1000 μM). When fetal perfusate was recirculated, Bcrp was found to actively remove cimetidine from the fetal compartment to the maternal compartment even against a concentration gradient and to establish a 2-fold maternal-to-fetal concentration ratio. Based on our results, we propose a two-level defensive role of Bcrp in the rat placenta in which the transporter 1) reduces passage of its substrates from mother to fetus but also 2) removes the drug already present in the fetal circulation.

EXPRESSION AND FUNCTIONAL ACTIVITY OF BREAST CANCER RESISTANCE PROTEIN (BCRP, ABCG2) TRANSPORTER IN THE HUMAN CHORIOCARCINOMA CELL LINE BEWO

1 Breast cancer resistance protein (BCRP, ABCG2) is a drug efflux transporter that is believed to affect the drug disposition of several drugs and xenobiotics. In the present study, we evaluated the localization and functional expression of BCRP in the human choriocarcinoma cell line BeWo, an in vitro model of the human trophoblast, and compared it with the expression of P‐glycoprotein (MDR1, ABCB1) as the most widely studied placental transporter. In addition, the expression of BCRP at the mRNA level was compared with that of MDR1 in the human term placenta. 2 Western blotting analysis revealed high endogenous expression of BCRP protein in BeWo cells. Using indirect immunofluorescence microscopy, we found that the BCRP transporter appears to be localized predominantly at the apical plasma membrane. Functional studies showed a significant effect of the BCRP inhibitors GF120918 (5 mmol/L) and Ko143 (1 mmol/L) on mitoxantrone accumulation and, thus, confirmed efflux activity of BCRP in BeWo cells. 3 Using absolute mRNA quantification with real‐time reverse transcription–polymerase chain reaction, we found high expression of BCRP in BeWo cells, whereas no transcript of MDR1 (P‐glycoprotein), the most extensively studied drug transporter, was detected. 4 In the human placenta, BCRP was localized predominantly in the syncytiotrophoblast layer; however, immunopositivity for the BXP‐21 antibody was also observed in fetal vessels of the chorionic villi. The number of BCRP transcripts in the human term placenta was found to be more than 10‐fold higher compared with the expression of MDR1. 5 In conclusion, we suggest that BeWo cells could serve as a suitable in vitro model to study trans‐trophoblast transport of BCRP substrates and that placental BCRP can play an important role in preventing the accumulation of potentially toxic xenobiotics in the trophoblast cells.

The function of cytochrome P450 1A1 enzyme (CYP1A1) and aryl hydrocarbon receptor (AhR) in the placenta.

CYP1A1, an enzyme of the cytochrome P450 superfamily, is the most important xenobiotic-metabolizing enzyme of the placenta for which relevant inducible activity has been demonstrated throughout pregnancy. CYP1A1 metabolizes several drugs and compounds widely used in pharmacotherapy or present in diets. At the same time, this enzyme plays a key role in the bioactivation of procarcinogens and proteratogens, such as arylamines and polycyclic aromatic hydrocarbons (PAHs), which bind to placental and foetal DNA as DNA-adducts. The expression of CYP1A1 is transcriptionally up-regulated through the ligand-activated aryl hydrocarbon receptor (AhR). AhR plays an important role as mediator of an adaptive response to xenobiotics, as well as in normal physiology and embryonic development. Several exogenous AhR ligands, such as PAHs, polychlorinated biphenyls and halogenated dioxins, can be found in the constituents of numerous commercial products, including insulators and flame retardants, or as products of combustion processes, including chimney soot, charbroiled foods and cigarette smoke, or as the product of waste incineration. Exposure to these compounds subsequently affects cellular growth and differentiation, homeostasis, level of growth factors, reproduction function and hormonal regulation. Importantly, elevated CYP1A1 activity through activated AhR in placentas of women smokers has been associated with pregnancy complications, such as premature birth, intrauterine growth retardation (IUGR), structural abnormalities, foetal death or placenta abruption, risk of low birth weight, low birth length and low head circumference. We summarize the recent findings related to toxicological consequences of AhR activation and CYP1A1 induction in the human placenta during pregnancy.

Dexamethasone controls aryl hydrocarbon receptor (AhR)-mediated CYP1A1 and CYP1A2 expression and activity in primary cultures of human hepatocytes.

CYP1A1 and CYP1A2 genes encode members of the cytochrome P450 superfamily of enzymes primarily involved in xenobiotic and drug metabolism. In this paper we examined the effects of synthetic glucocorticoid dexamethasone (DEX) on aryl hydrocarbon receptor (AhR)-mediated regulation of CYP1A1 and CYP1A2 genes and their enzymatic activity in primary cultures of human hepatocytes obtained from 17 donors and prepared in 3 countries. Dexamethasone significantly reduced both basal and inducible CYP1A1/2 ethoxyresorufin-O-deethylase (EROD) activities by more than 75 and 50%, respectively. Glucocorticoid receptor (GR) antagonist RU486 abolished this effect suggesting the involvement of GR in the process. In contrast, dexamethasone significantly augmented transcriptional activation of CYP1A2 mRNA but not CYP1A1 gene by prototype AhR ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3MC). Dexamethasone had no effect on basal and TCDD-inducible levels of CYP1As proteins; however, it reduced the levels of AhR and GRalpha mRNAs and AhR protein levels. In addition, using RT(2) Profiler PCR Array, we found the effect of dexamethasone on the expression of several co-activators of AhR and GR nuclear receptors in the primary human hepatocytes. We conclude that dexamethasone controls CYP1A1 and CYP1A2 expression and activity in human hepatocytes via multiple mechanisms, which remain to be elucidated.

Lack of Interactions between Breast Cancer Resistance Protein (BCRP/ABCG2) and Selected Antiepileptic Agents

Summary:  Purpose: Recent studies have indicated constitutive expression of efflux transporter, breast cancer resistance protein (BCRP, ABCG2), in endothelial cells of the blood–brain barrier (BBB). In epileptogenic brain tumors such as ganglioma, astrocytoma, anaplastic astrocytomas, or glioma multiforme, strong expression of BCRP in the microvasculature of the BBB was observed. Therefore it was hypothesized that this phenomenon could critically influence the bioavailability of drugs in these tumors and potentially contribute to the failure of antiepileptic treatment. The aim of this study was to test whether some commonly used antiepileptic drugs (AEDs) are substrates transported by human BCRP. In particular, we focused on phenobarbital, phenytoin, ethosuximide, primidone, valproate, carbamazepine, clonazepam, and lamotrigine. Furthermore, the inhibitory potency of these AEDs to BCRP was examined.