Petr Nachtigal

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.

Dexrazoxane-afforded protection against chronic anthracycline cardiotoxicity in vivo: effective rescue of cardiomyocytes from apoptotic cell death

Background:Dexrazoxane (DEX, ICRF-187) is the only clinically approved cardioprotectant against anthracycline cardiotoxicity. It has been traditionally postulated to undergo hydrolysis to iron-chelating agent ADR-925 and to prevent anthracycline-induced oxidative stress, progressive cardiomyocyte degeneration and subsequent non-programmed cell death. However, the additional capability of DEX to protect cardiomyocytes from apoptosis has remained unsubstantiated under clinically relevant in vivo conditions.Methods:Chronic anthracycline cardiotoxicity was induced in rabbits by repeated daunorubicin (DAU) administrations (3 mg kg−1 weekly for 10 weeks). Cardiomyocyte apoptosis was evaluated using TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling) assay and activities of caspases 3/7, 8, 9 and 12. Lipoperoxidation was assayed using HPLC determination of myocardial malondialdehyde and 4-hydroxynonenal immunodetection.Results:Dexrazoxane (60 mg kg−1) co-treatment was capable of overcoming DAU-induced mortality, left ventricular dysfunction, profound structural damage of the myocardium and release of cardiac troponin T and I to circulation. Moreover, for the first time, it has been shown that DEX affords significant and nearly complete cardioprotection against anthracycline-induced apoptosis in vivo and effectively suppresses the complex apoptotic signalling triggered by DAU. In individual animals, the severity of apoptotic parameters significantly correlated with cardiac function. However, this effective cardioprotection occurred without a significant decrease in anthracycline-induced lipoperoxidation.Conclusion:This study identifies inhibition of apoptosis as an important target for effective cardioprotection against chronic anthracycline cardiotoxicity and suggests that lipoperoxidation-independent mechanisms are involved in the cardioprotective action of DEX.

Atorvastatin has hypolipidemic and anti-inflammatory effects in apoE/LDL receptor-double-knockout mice

Statins are first-line pharmacotherapeutic agents for hypercholesterolemia treatment in humans. However the effects of statins in animal models of atherosclerosis are not very consistent. Thus we wanted to evaluate whether atorvastatin possesses hypolipidemic and anti-inflammatory effects in mice lacking apolipoprotein E/low-density lipoprotein receptor (apoE/LDLR-deficient mice). Two-month-old female apoE/LDLR-deficient mice (n=24) were randomly subdivided into 3 groups. The control group of animals (n=8) was fed with the western type diet (atherogenic diet) and in other two groups atorvastatin was added to the atherogenic diet at the dosage of either 10 mg/kg or 100 mg/kg per day for a period of 2 months. Biochemical analysis of lipids, ELISA analysis of monocyte chemotactic protein-1 (MCP-1) in blood, quantification of lesion size and expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) in the atherosclerotic lesion by means of immunohistochemistry and Western blot analysis were performed. The biochemical analysis showed that administration of atorvastatin (100 mg/kg/day) significantly decreased level of total cholesterol, lipoproteins (VLDL and LDL), triacylglycerol, and moreover significantly increased level of HDL. ELISA analysis showed that atorvastatin significantly decreased levels of MCP-1 in blood and immunohistochemical and Western blot analysis showed significant reduction of VCAM-1 and ICAM-1 expression in the vessel wall after atorvastatin treatment (100 mg/kg/day). In conclusion, we demonstrated here for the first time strong hypolipidemic and anti-inflammatory effects of atorvastatin in apoE/LDLR-deficient mice. Thus, we propose that apoE/LDLR-deficient mice might be a good animal model for the study of statin effects on potential novel markers involved in atherogenesis and for the testing of potential combination treatment of new hypolipidemic substances with statins.

Aryl Hydrocarbon Receptor and Aryl Hydrocarbon Nuclear Translocator Expression in Human and Rat Placentas and Transcription Activity in Human Trophoblast Cultures

Aryl hydrocarbon receptor (AHR) and its heterodimer aryl hydrocarbon nuclear translocator (ARNT) form a ligand-activated transcription complex that regulates expression of the AHR battery of target genes that includes the most important placental biotransformation enzyme cytochrome CYP1A1. Expression, placental localization, and ontogeny of AHR/Ahr and ARNT/Arnt have not been systematically studied in either human or rat placentas. Moreover, induction of such AHR target genes as CYP1A1, CYP1A2, CYP1B1, UGT1A1, and breast cancer resistance protein (BCRP), as well as of AHR, ARNT, and aryl hydrocarbon receptor repressor (AHRR) genes, after exposure to AHR ligands have not been studied in human placental trophoblast cultures. In this article, we show that only CYP1A1 messenger RNA (mRNA), but not CYP1A2, CYP1B1, UGT1A1, BCRP, AHR, ARNT, and AHRR mRNAs, is significantly induced in human term placental trophoblast cultures after exposure to prototype AHR ligands/activators 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, omeprazole, and β-naphthoflavone. We localized AHR/Ahr and ARNT/Arnt in rat placental trophoblasts throughout gestation and in first trimester and term human placental trophoblast, which comprise the crucial component of the maternal-fetal barrier. We demonstrate that rat Ahr and Cyp1a1 reached highest expression during gestation days 15 and 18, which might indicate different response to Ahr ligands in placental Cyp1a1 induction during rat gestation. We also propose the JEG3 choriocarcinoma cell line as a cellular model for human trophoblast induction studies through AHR. In conclusion, we describe expression and ontogeny of AHR/Ahr and ARNT/Arnt and systematically characterize induction of major AHR target genes in human placental trophoblast forming the placental maternal-fetal morphological and metabolic barrier.

Organic Cation Transporter 3 (OCT3/SLC22A3) and Multidrug and Toxin Extrusion 1 (MATE1/SLC47A1) Transporter in the Placenta and Fetal Tissues: Expression Profile and Fetus Protective Role at Different Stages of Gestation

ABSTRACT In our previous study, we described synchronized activity of organic cation transporter 3 (OCT3/SLC22A3) and multidrug and toxin extrusion 1 (MATE1/SLC47A1) transporter in the passage of organic cations across the rat placenta and the role of these transporters in fetal defense; in this study, we hypothesized that changes in placental levels of OCT3 and MATE1 throughout gestation might affect the fetal protection and detoxification. Using quantitative RT-PCR, Western blot analysis, and immunohistochemistry, we were able to detect Oct3/OCT3 and Mate1/MATE1 expression in the rat placenta as early as on Gestation Day (gd) 12 with increasing tendency toward the end of pregnancy. Comparing first versus third trimester human placenta, we observed stable expression of OCT1 and decreasing expression of OCT2 and OCT3 isoforms. Contrary to the current literature, we were able to detect also MATE1/MATE2 isoforms in the human placenta, however, with considerable inter- and intraindividual variability. Using infusion of 1-methyl-4-phenylpyridinium (MPP+), a substrate of OCT and MATE transporters, into pregnant dams, we investigated the protective function of the placenta against organic cations at different gds. The highest amount of MPP+ reached the fetus on gd 12 while from gd 15 onward, maternal-to-fetal transport of MPP+ decreased significantly. We conclude that increased expression of placental OCT3 and MATE1 along with general maturation of the placental tissues results in significantly lower transport of MPP+ from mother to fetus. In contrast, decreasing expression of OCT3 and MATE1 in human placenta indicates these transporters may play a role in fetal protection preferentially at earlier stages of gestation.

The changes in the endothelial expression of cell adhesion molecules and iNOS in the vessel wall after the short-term administration of simvastatin in rabbit model of atherosclerosis

Cell adhesion molecules P‐selectin, VCAM‐1 and ICAM‐1 play an important role in the pathogenesis of atherosclerosis. High levels of nitric oxide (NO) produced by inducible NO synthase (iNOS) have been associated with atherosclerotic processes. Simvastatin is an HMG‐CoA reductase inhibitor responsible for many clinical benefits. The aim of this study was to detect and quantify changes in endothelial expression of P‐selectin, VCAM‐1, ICAM‐1 and iNOS in the vessel wall after the short‐term administration of simvastatin in a rabbit model of atherosclerosis. Eighteen New Zealand White rabbits were randomly divided into three groups (n = 6). In the cholesterol group, rabbits consumed an atherogenic diet (0.4% cholesterol) for eight weeks. In the simvastatin group, rabbits consumed an atherogenic diet for six weeks and then consumed an atherogenic diet supplemented with simvastatin (10 mg kg−1) for two weeks. Biochemical analysis showed that administration of simvastatin led to an almost two‐fold lowering of the total serum cholesterol, VLDL, LDL and HDL, but not triglycerides, compared with the cholesterol‐fed rabbits only. Stereological analysis of the immuno‐histochemical staining revealed that administration of simvastatin (10 mg kg−1 daily) in an atherogenic diet decreased the endothelial expression of P‐selectin, ICAM‐1 and iNOS in both aortic arch and carotid artery compared with the cholesterol fed‐rabbits only. We conclude that simvastatin has beneficial effects on endothelial function by decreasing expression of P‐selectin, ICAM‐1 and iNOS in endothelial cells in the very early stages of atherogenesis.

The role of endoglin in atherosclerosis

Endoglin (CD 105, TGF-β receptor III) is a homodimeric transmembrane glycoprotein that plays a regulatory role in TGF-β signaling. Its functional role in the context of atherosclerosis has yet to be defined and should be stated here. Therefore, we focused on the role of endoglin in atherosclerosis in both humans and experimental animals. Endoglin expression was demonstrated in atherosclerotic vessels predominantly in endothelial cells and smooth muscle cells in various types of blood vessels in mice and humans, suggesting its participation in atherogenesis. Endoglin expression was also related to the expression of eNOS in endothelium, repair of the vessel wall, plaque neoangiogenesis, production of collagen and stabilization of atherosclerotic lesions. In addition, increased levels of soluble endoglin were associated with hypercholesterolemia, atherosclerosis, acute myocardial infarction and were related to inhibition of TGF-β signaling in the vessel wall. Moreover, soluble endoglin levels were significantly lowered after a series of extracorporeal eliminations in patients with familial hypercholesterolemia. Additionally, statin treatment decreased levels of soluble endoglin and increased its expression in aorta, which was related to reduced atherosclerosis in mice. In conclusion, we propose that measurement of soluble endoglin might give information about progression of the atherosclerotic process or the efficacy of therapeutic interventions, which is the task that must be answered in clinical trials.

Synchronized Activity of Organic Cation Transporter 3 (Oct3/Slc22a3) and Multidrug and Toxin Extrusion 1 (Mate1/Slc47a1) Transporter in Transplacental Passage of MPP+ in Rat

The aim of the present study was to investigate the expression, localization, and function of organic cation transporter 3 (Oct3, Slc22a3) and multidrug and toxin extrusion protein 1 (Mate1, Slc47a1) in the rat placenta. Using qRT-PCR and Western blotting techniques, we demonstrated abundant Oct3 and Mate1 mRNA and protein expression achieving significantly higher levels than those in the maternal kidney (positive control). Immunohistochemical visualization revealed preferential localization of Oct3 on the basolateral, i.e., fetus facing side of the placenta, whereas Mate1 positivity was located in the labyrinth area predominantly on the apical, i.e., maternal side of the placenta. To investigate the role of these transporters in the transplacental pharmacokinetics, the in situ method of dually perfused rat term placenta was employed in open- and closed-circuit arrangements; 1-methyl-4-phenylpyridinium (MPP(+)) was used as a model substrate of both Oct3 and Mate1. We provide evidence that Oct3 and Mate1 cause considerable asymmetry between maternal-to-fetal and fetal-to-maternal transport of MPP(+) in favor of fetomaternal direction. Using closed-circuit experimental setup, we further describe the capacity of Oct3 and Mate1 to transport their substrate from fetus to mother even against a concentration gradient. We conclude that Oct3, in a concentration-dependent manner, takes up MPP(+) from the fetal circulation into the placenta, whereas Mate1, on the other side of the barrier, is responsible for MPP(+) efflux from placenta to the maternal circulation. These two transport proteins, thus, form an efficient transplacental eliminatory pathway and play an important role in fetal protection and detoxication.

Atorvastatin slows down the deterioration of inner ear function with age in mice

Statins have revolutionized the treatment of hypercholesterolemia due to their ability to inhibit cholesterol biosynthesis. Their immunomodulatory and anti-inflammatory effects and positive effects on the treatment of atherosclerosis and its complications are well known. Here, we describe the effects of statins on the treatment of presbycusis in C57BL/6J mice. In this strain with accelerated aging, we demonstrate that animals treated with atorvastatin (10mg/kg per day in chow diet) for 2 months showed larger amplitudes of distortion product otoacoustic emissions (DPOAE) than did the non-treated control group. This finding indicates a better survival of outer hair cell function in the inner ear of C57BL/6J mice. The observed decreased expression of intercellular and vascular adhesion molecules in the aortic wall of atorvastatin-treated animals suggests that reducing endothelial inflammatory effects may contribute to the positive effect of atorvastatin on the amplitudes of DPOAE by influencing the blood supply to the inner ear. No such beneficial effect of statins was found in apoE(-/-) mice treated with atorvastatin under the same conditions. Our results suggest that statins could also slow down the age-related deterioration of hearing in man.