bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for Functional Interactions between 5‐HT1Aand 5‐HT2AReceptors in Rat Thermoregulatory Mechanisms
Authors: Salmi Peter , Ahlenius Sven
Published: March 2009

Abstract:The present study was designed to examine 1) functional interactions between 5‐HT1Aand 5‐HT2A/Creceptors in thermoregulation in rats and 2) the specific involvement of 5‐HT2Aand 5‐HT2Creceptors in such interactions. The 5‐HT2A/Creceptor agonist DOI (0.025 1.6 mg kg−1, subcutaneously) produced a dose‐dependent hyperthermia in rats, which was enhanced by addition of either of two 5‐HT1Areceptor antagonists, (‐)‐pindolol (0.5‐1.0 mg kg−1, subcutaneously) or WAY‐100,635 (0.1‐0.4 mg kg−1, subcutaneously). Furthermore, the DOI‐induced hyperthermia was counteracted by pretreatment with the 5‐HT1Areceptor agonist 8‐OH‐DPAT (0.05 mg kg−1, subcutaneously). The hyperthermia produced by DOI, alone or in combination with WAY‐100,635, was fully antagonized by pretreatment with the 5‐HT2A/Creceptor antagonist ritanserin (1.0 mg kg−1, subcutaneously), as well as with the selective 5‐HT2Areceptor antagonist amperozide (2.0 mg kg−1, subcutaneously). The present results provide evidence for functional interactions between 5‐HT1Aand 5‐HT2Areceptors in temperature regulation in rats, and also suggest an important role for postsynaptic 5‐HT2Areceptors in the mediation of DOI‐induced hyperthermia.
Excitatory Amino Acid Antagonists Alleviate Convulsive and Toxic Properties of Lindane in Mice
Authors: Blaszczak Piotr , Turski Waldemar A.
Published: March 2009

Abstract:Pesticides acting at GABAAreceptors may induce convulsions in man and animals, but the mechanisms responsible for their convulsant activity are not fully explained. The following excitatory amino acid antagonists were studied for their protective action in mice intoxicated with chlorinated hydrocarbon insecticide lindane (δ‐hexachlorocyclohexane): the competitive NMDA antagonist: 3‐(2‐carboxypiperazine‐4‐yl)propenyl‐1‐phosphonic acid (D‐CPPene, 20 mg/kg), the non‐competitive NMDA antagonist: dizocilpine (MK‐801, 0.4 mg/kg), the glycine site antagonist of NMDA receptor: 2‐phenyl‐l,3‐propane‐diol dicarbamate (felbamate, 400 mg/kg) and the competitive AMPA antagonist: 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoyl‐benzo(F)quinoxaline (NBQX, 100 mg/kg). Systemic administration of an antagonist prior to lindane resulted in a strong anticonvulsant effect. D‐CPPene, MK‐801 and NBQX produced a marked increase of CD50 values of lindane for clonic convulsions. All the antagonists protected animals against tonic convulsions. Toxicity of lindane was potently reduced, as assessed 2, 24 and 120 hr after administration of the pesticide. Our results demonstrate that excitatory amino acid antagonists reduce convulsant properties and toxicity of lindane, suggesting that excitatory amino acid neurotransmission may be involved in its central action.
Optimization and Application of the Head Space Liver S9 Equilibration Technique for Metabolic Studies of Organic Solvents
Authors: Mortensen Bjarte , Nilsen Odd G.
Published: March 2009

Abstract: A head space liver S9 vial equilibration technique for qualitative and quantitative metabolism of volatile organic solvents in vitro is described and investigated for optimal experimental conditions with toluene and n‐hexane as model substrates. The method was also used for studying the metabolic interaction between acetone and styrene. NADP+ was a critical cofaclor in the system as concentrations above 2.5 mM resulted in inhibition of metabolism. Induction of cytochrome P450 by phenobarbital did not alter the observed cofactor dependency, and addition of vehicles such as rat blood did not enhance metabolism. Acetone‐induced liver S9 increased the primary oxidation of styrene and also the formation of styrene oxide, but not significantly that of mandelic acid. Acetone as such had no effect on the metabolic elimination of styrene, indicating that the interaction is mainly on the level of enzyme induction occurring after long‐term exposure to acetone. As earlier reports on styrene metabolism in vivo has not been able to detect styrene oxide in toxicokinetic studies with a sufficient degree of accuracy, the head space liver S9 vial equilibration technique may offer a fast and sensitive tool for screening of metabolic interactions between organic solvents when optimized for metabolic activity.
Cytochrome P450 Specificity of Metabolism and Interactions of Oxybutynin in Human Liver Microsomes
Authors: Lukkari Eeva , Taavitsainen Päivi , Juhakoskr Auni , Pelkonen Olavi
Published: March 2009

Abstract:Oxybutynin has an extensive first pass metabolism after oral administration, the main active metabolite being N–desethyloxybutynin. The purpose of this study was to investigate the CYP isoform specificity of oxybutynin N–deethylation and possible interactions. Oxybutynin N–deethylation in human liver microsomesin vitrowas potently inhibited by ketoconazole (IC504.5 uM), less and variably by itraconazole and not by quinidine or several other reference inhibitors, suggesting that CYP3A enzymes are predominant catalysts of the reaction. Recombinant CYP3A5 enzyme had higher activity in oxybutynin N–deethylation than recombinant CYP3A4. Ketoconazole inhibited oxybutynin N–deethylation by the recombinant CYP3A4 and CYP3A5 almost completely, whereas itraconazole inhibited the activity of CYP3A4 more potently than that of CYP3A5. Oxybutynin inhibited CYP3A4– and CYP2D6– associated activities (testosterone 6β–hydroxylase and dextromethorphan O– demethylase, respectively) in human liver microsomes. CYP1A1/2–, CYP2A6–, CYP2C9– and CYP2E1–associated activities were inhibited less potently or not at all by oxybutynin when compared with reference inhibitors. Although the reasons for the weak and variable inhibition by itraconazole remain to be studied, it seems that oxybutynin is predominantly metabolized by CYP3A4 and CYP3A5 but not by CYP2D6. However, it seems to have some affinity also to the latter enzyme.
Inhibitory Effect of Vanadium Compounds on Glutamate Dehydrogenase Activity in Mitochondria and Hepatocytes Isolated from Rabbit Liver
Authors: Kiersztan Anna , Jarzyna Robert , Bryla Jadwiga
Published: March 2009

Abstract:The effect of orthovanadate, vanadyl sulphate and vanadyl acetylacetonate on glutamate dehydrogenase activity was studied in liver mitochondria and isolated hepatocytes of rabbit. In pcrmeabilized mitochondria with free access of substrates and drugs to glutamate dehydrogenase, orthovanadate and vanadyl sulphate at 200 μM concentrations decreased both glutamate synthesis and glutamate deamination by 80 and 50 %, respectively, while vanadyl acetylacetonate was less potent. In view of kinetic data obtained at various ammonium concentrations, orthovanadate appeared to be a competitive inhibitor (Ki= 40 × 3 μM), while vanadyl sulphate was a non–competitive one (Ki= 147 × 10 μM). In contrast to orthovanadate, vanadyl sulphate augmented the inhibitory action of increased above 0.5 mM 2–oxoglutarate concentrations. All these effects on the enzyme activity were partially reversed in the presence of L–leucine and ADR which are allosteric activators of glutamate dehydrogenase. Moreover, all compounds studied suppressed both glutamate formation and glutamate deamination in isolated hepatocytes incubated under various metabolic conditions, as concluded from decreased rates of glutamate and urea synthesis, respectively. In view of these observations it seems likely that vanadiumcontaining compounds may be potent inhibitors of glutamate metabolism in liver.
Allometric Species Comparison of Toluene andn–Hexane Metabolism: Prediction of Hepatic Clearance in Man from Experiments with Rodent Liver S9 in a Head Space Vial Equilibration System
Authors: Mortensen Bjarte , Nilsen Odd G.
Published: March 2009

Abstract:The liver S9 head space vial equilibration technique is anin vitroalternative that holds promises for a satisfactoryin vivoextrapolation of liver metabolism of volatile organic chemicals. The aim of this study was to investigate the suitability of this methodology for the extrapolation ofin vitrometabolic data from rodent to man by allometry with the two highly metabolized organic solvents toluene andn–hexane as model substances. The calculated hepatic clearance of toluene in man from rodent liver S9 in this study was equal to the reported total body clearance of toluene in man, suggesting insignificant extrahepatic clearance of toluene in humans. The calculated hepatic clearance ofn–hexane was less than the reported values of total body clearance ofn–hexane in man, indicating an about 80% extrahepatic clearance ofn–hexane in humans. Both results are in line with our present knowledge of the metabolism of the two organic solvents in man. Allometric scaling from rodent liver S9 head space incubations toin vivometabolism of toluene andn–hexane in man thus seems promising and could be a method of choice for scaling of organic solvent metabolism in general.
Adenosine A1Receptor Activation Inhibits Basal Accumulation of Inositol Phosphates in Rat Hippocampus
Authors: Cascalheira Jose F. , Sebastiäo Ana M.
Published: March 2009

Abstract:The ability of the adenosine A1receptor selective agonist, N6–cyclopentyladenosine, to modify basal accumulation of inositol phosphates in rat hippocampal slices, was investigated. Cyclopentyladenosine (10–300 nM) inhibited the basal accumulation of total [3H]inositol phosphates, with an EC50of 10 nM and an Emaxof 24%. This effect of cyclopentyladenosine was prevented by the adenosine A1receptor selective antagonist, l,3–dipropyl–8–cyclopentylxanthine (30 nM). Cyclopentyladenosine (100 nM) also inhibited histamine (300 μM)–stimulated accumulation of [3H]inositol phosphates, this effect being quantitatively similar to that observed on basal [3H]inositol phosphates accumulation. The results suggest that adenosine A1receptor activation is able,per se, to inhibit the formation of phosphatidylinositol–derived second messengers in hippocampus.
Mitochondrial Cytochrome c Oxidase Inhibition during Acute Carbon Monoxide Poisoning
Authors: Miró Òscar , Casademont Jordi , Barrientos Antoni , Urbano–Márquez Álvaro , Cardellach Francesc
Published: March 2009

Abstract: Clinical symptoms of acute carbon monoxide (CO) poisoning are mainly related to the capability of haemoglobin to bind CO. However, the persistence of some clinical alterations after carboxyhaemoglobin normalization suggests that other heme containing proteins, like cytochrome c oxidase, could play a role in its pathogenesis. We studied mitochondrial enzyme activities of lymphocytes from three patients suffering from acute CO poisoning. HbCO levels were 11.6%, 19.6% and 22.3% in the acute phase, 2.3%, 2.4% and 1.5% on day 3 after admission, and 1.2%, 3.3% and 1.1% on day 12. Complex II, III and glycerol–3–phosphate dehydrogenase activities remained normal along the study, while cytochrome coxidase (complex IV) activity showed a 76% inhibition compared to controls during acute poisoning (P < 0.01) and 48% at day 3 (P < 0.05). The activity was normal already on day 12 after the complete disappearance of symptomatology. Our results suggest that mitochondrial cytochrome c oxidase is also a target site in human acute CO poisoning, and its extended and generalized inhibition could explain the persistence of different symptoms after the normalization of HbCO levels.
Effect of Chronic Administration of Magnesium Sulfate on 1–Methyl–4–phenyl–1,2,3,6–tetrahydropyridine–Induced Neurotoxicity in Mice
Authors: Tariq Mohammad , Khan Haseeb Ahmed , Moutaery Khalaf Al , Deeb Saleh M. Al
Published: March 2009

Abstract:This investigation was aimed to study the effect of magnesium on 1–methyl–4–phenyl–1,2,3,6–tetrahydropyridine (MPTP)–induced neurotoxicity in mice. Four groups of mice were given magnesium sulfate (MgSO4.7H2O) in drinking water at four different concentrations of 0.0 g/l (control), 2.5 g/l (low), 5.0 g/l (medium) and 10.0 g/l (high) respectively for a period of 16 weeks; these animals also received MPTP (30 mg/kg, intraperitoneally daily) during the last five days of Mg treatment. Other four groups of mice were given similar dose regimen of MgSO4but received injections of saline instead of MPTP. Seventy–two hr after the last dose of MPTP, neurobehavioural studies including locomotor activity, pole climbing test and heat nociception test were performed and striata were collected for the analysis of dopamine. The results of this study show that treatment of mice with MgSO4or MPTP individually has no effect on their behaviour. Concomitant administration of low dose of MgSO4(2.5 g/l) along with MPTP produced increase in motor activity and latency to heat stimuli; whereas medium and high doses of MgSO4in combination with MPTP produced opposite (as compared to low dose) effects resulting in a decrease in motor activity and latency to heal stimuli and increase in pole climbing time. However, MgSO4dose–dependently exacerbated MPTP–induced depletion of striatal dopamine. The mortality was drastically increased (30–55%) in the animals receiving combined treatments of MPTP and MgSO4as compared to the mice treated with MPTP alone (12%). This study clearly points towards the ability of MgSO4to modify MPTP–induced neurotoxicity.
Cadmium Uptake from Apical Membrane of LLC–PK Cells via Inorganic Anion Exchanger
Authors: Endo Tetsuya , Kimura Osamu , Sakata Masakatsu
Published: March 2009

Abstract:Effects of Na+, Cl–, OH–and HCO3–, and an inorganic anion exchange inhibitor (HCO3–/Cl–), 4,4′–diisothiocyanostilbcne–2,2′–disulfonic acid, on Cd uptake from the apical membrane of LLC–PK1cells were investigated to elucidate the mechanism of Cd uptake by these cells. Furthermore, the initial uptake of Cd incubated at different temperatures was analyzed by using the Arrhenius plot to test if Cd is taken up by the cells via the carrier–mediated process. The cells were incubated with 1 μM CdCl2for 30 min. at 37° in phosphate buffer containing NaCl at pH 5.5 or 7.4. Cd accumulation by the cells at pH 7.4 was about 5 times higher than at pH 5.5. Replacement of NaCl in the phosphate buffer with KC1 or mannilol decreased the Cd accumulation at pH 7.4 about 80%, but had no effect at pH 5.5. The addition of 2 mM NaHCO3to the pH 7.4 buffer containing NaCl significantly increased the Cd accumulation, and pretreatment of the cells with the inorganic anion exchange inhibitor abolished this effect of NaHCO3on Cd accumulation. The cells were incubated for 10 min. at different temperatures with 1 μM CdCl2in the phosphate buffer containing NaCl at pH 7.4 in the presence or absence of 2 mM NaHCO3or at pH 5.5 in the absence of NaHCO3. In all cases, the Arrhenius plots of Cd accumulation were nonlinear. The breakpoint was observed at about 30° in the Cd accumulation at pH 7.4 in the presence of NaHCO3, suggesting the involvement of a carrier–mediated process. This breakpoint was not as clear in the Cd accumulation at pH 7.4 in the absence of NaHCO3and not observed at all at pH 5.5. These findings suggested that Cd is partially taken up from the apical membrane of LLC–PK1cells via the inorganic anion exchanger and the uptake of Cd is more efficient at alkaline pH and in the presence of Na+, Cl–and HCO3.
Short–Term Effects of Nicotine on Bone and Calciotropic Hormones in Adult Female Rats
Authors: Fung Yiu K. , Mendlik Mark G. , Haven Mary C. , Akhter Mohammed P. , Kimmel Donald B.
Published: March 2009

Abstract:This study assessed the effects of two months nicotine treatment on bone formation and resorption end–points in adult, female rats. In addition, the concentrations of calciotropic hormones which included parathyroid hormone, calcitonin, 25–hydroxyvitamin D and 1,25–dihydroxyvitamin D in rats (7 months old) were determined. All animals received either saline (n = 7/group). nicotine (3.0 mg/kg/day) (n = 7/group) or nicotine (4.5 mg/kg/day) (n = 7/group) via subcutaneous implantation of osmotic minipumps containing either saline or nicotine for a period of two months. Serum, right tibia, left femur and lumbar vertebra (3–5) were collected for determination of hormonal concentrations as well as various parameters, including histomorphometry, bone mineral density, bone mineral content and vertebral strength. Although nicotine–treated rats showed a lower level of 25–hydroxyvitamin D [54.4 × 3.1 ng/ml for the 3.0 mg/kg/day and 55.8 × 2.8 for the 4.5 mg/kg/day group] (mean × S.E.M.) as compared to controls (74.8 × 2.8 ng/ml) (P < 0.01, Newman–Keuls test), no significant difference could be detected for the levels of the remaining hormones. Similarly, no statistical differences were detected on histomorphometric end–points, bone mineral density, bone mineral content and vertebral strength of rats. We conclude that, in spite of lowering serum 25–hydroxyvitamin D by about 30%, nicotine administration of two months duration does not alter bone mass, strength or formation and resorption end–points.
Amlodipine Dynamic Effects and Myocardial Pharmacokinetics in the Isolated and Perfused Guinea–Pig Heart
Authors: Jeppesen Peter , Bruun Jens , Nielscn–Kudsk Folmer
Published: March 2009

Abstract:Myocardial dynamic effects and pharmacokinetics of amlodipine were studied in the isolated retrogradely perfused and spontaneously beating guinea–pig heart. Pharmacokinetic analysis of drug accumulation showed one–compartment characteristics with an half–life of 76 min. whereas disposition exhibited two–compartment characteristics with phasic half–lives of 25 and 174 min., respectively. Myocardial drug accumulation was increased by 600 times at steady–state compared to the perfusion liquid. Dynamic effect parameters were studied during increasing amlodipine concentrations from 0.16 to 220 nM. Dynamic steady–states developed within 20 min. Coronary flow–rate increased with an Emaxof 119% and an EC50of 1.2 10–8M. Amlodipine produced inhibitory effects on contraction amplitude and velocities of contraction and relaxation. Observed Emax–values and curve–fitted EC50–values were: 97, 97 and 94% and 1 10–8, 7.7 10–9and 2.1 10–8M, respectively. Heart frequency was not changed. Oxygen consumption increased markedly to a maximum of 44%, at 3 10–8M amlodipine and then decreased to nearly initial values. The frequency–corrected QT–interval decreased to a maximal extent of 20% at the three highest concentrations. Myocardial efficiency expressed as the ratio of contraction velocity times frequency to oxygen consumption exhibited a progressive decline to about 2% of initial values. The PQ–interval was not changed and the QRS–interval showed only a small but significant decrease at the highest amlodipine concentration. No arrythmogenic effects were observed. The study demonstrated a very slow accumulation and disposition of amlodipine in the guinea–pig heart with a steady–state myocardial drug concentrating accumulation of 600 times. Marked increase in coronary flow–rale and oxygen consumption accompanied by a progressive negative inotropic effect were observed.
Paraquat: A Useful Tool for thein vivoStudy of Mechanisms of Neuronal Cell Death
Authors: Corasaniti Maria Tiziana , Strongoli Maria Concetta , Rotiroti Domenicantonio , Bagetta Giacinto , Nisticò Giuseppe
Published: March 2009

Abstract:The present article reviews the results of experimental studies on paraquat neurotoxicity, started by our group several years ago – when clinical and experimental reports had increased the interest for the possibility that environmental chemicals, including paraquat, may be related to the development of Parkinson's disease –, and which are still continuing since paraquat appears to be a promising tool to study the mechanisms of neuronal cell deathin vivo. Our observations have demonstrated that paraquat causes evident neurotoxic effects after intracerebroventricular or intracerebral injection in experimental animals; however, it seems that the herbicide does not exibit a selective neurotoxicity towards the dopaminergic nigro‐striatal system since potent behavioural and electrocortical changes are induced by paraquat after injection in brain areas other than the substantia nigra and caudate nucleus. By studying the mechanisms through which paraquat induces neurotoxic effectsin vivo, it was shown that either free radical production and activation of cholinergic and glutamatergic transmission may be regarded as related events which play a crucial role in paraquat‐induced neurotoxicity. In addition, it was observed that in rats paraquat penetrates the blood‐brain barrier following systemic administration to give rise to a differential brain regional distribution; the latter observation rises some concern over the hazard of paraquat as a potential environmental neurotoxin. Indeed, paraquat, administered systemically in rats produces behavioural excitation and brain damage. The brain damage appears to be selective for the pyriform cortex and this does not seem to be strictly related to the high concentrations reached by the herbicide in this area but to the higher vulnerability of this cortical area to the enhanced cholinergic transmission. The recent observation that paraquat, injected into the rat hippocampus, induces the expression of apoptotic neuronal cell death, appears of valuable interest also with a view to paraquat as an useful experimental model in the development of neuroprotective drugs able to block the molecular events which, once activated, are responsible for the induction of neuronal cell death.
Effects of ATP‐Sensitive Potassium Channel Opener on Potassium Transport and Alveolar Fluid Clearance in the Resected Human Lung
Authors: Sakuma Tsutomu , Takahashi Keiji , Ohya Nobuo , Nakada Tasuku , Matthay Michael A.
Published: March 2009

Abstract:Since the effect of an ATP‐sensitive potassium channel (KATPchannel) opener on the function of alveolar epithelial cells is unknown, the effect of YM934, a newly synthesized KATPchannel opener, on potassium influx into the alveolar spaces and alveolar fluid clearance was determined in the resected human lung. An isosmolar albumin solution with a low potassium concentration was instilled into the distal airspaces of resected human lungs. Alveolar fluid clearance was measured by the progressive increase in alveolar protein concentration. Net potassium transport was measured by the change in potassium concentration and alveolar fluid volume. YM934 (10–4M) increased net influx of potassium by 140% into the alveolar spaces and also increased alveolar fluid clearance by 60% in the experiments with a potassium concentration of 0.3 mEq/1. Glibenclamide (10–4M), a KATPchannel blocker, inhibited the YM934–increased influx of potassium transport and the increase in alveolar fluid clearance. Also amiloride (10–5M), an inhibitor of apical sodium uptake, blocked the YM934 stimulated increase in net alveolar fluid clearance. These results indicate that a KATPchannel opener can effect potassium transport and net vectorial fluid movement across the human alveolar epithelium.
Effect of Concomitant β‐Adrenoceptor Stimulation on α1‐Adrenoceptor‐Mediated Increase of Inositol‐1, 4, 5‐trisphosphate Mass in Adult Rat Cardiomyocytes
Authors: Viko Håvard , Sandnes Dagny , Skomedal Tor , Osnes Jan‐Bjørn
Published: March 2009

Abstract:The aim of the present study was to investigate the accumulation of inositol‐1, 4, 5‐trisphosphate (IP3) in isolated adult rat ventricular cardiomyocytes after α1and β‐adrenoceptor stimulation, separate and in combination, in order to elucidate a possible influence of concomitant β‐adrenoceptor stimulation on the α1‐adrenoceptor stimulated response. IP3was measured by a radioligand binding assay based on an (1, 4, 5)IP3‐specific binding protein from bovine adrenal cortex. The basal IP3content was 4.06 + 0.31 pmol/mg protein (N=56). α1‐Adrenoceptor stimulation resulted in a rapid increase in the IP3level, which reached a plateau, 50–80% above basal level, at 10–30 sec. The plateau lasted at least up to 120 sec, while at 300 sec. there was no significant difference between control values and values after α1‐adrenoceptor stimulation. Li+did not affect either the basal IP3level, or the magnitude or time course of α1‐adrenoceptor‐stimulated IP3accumulation. Combined adrenoceptor stimulation gave a similar response as separate α1‐adrenoceptor stimulation, whereas there was no significant change in the IP3level after β‐adrenoceptor stimulation. No inhibitory influence of simultaneous β‐adrenoceptor stimulation on the α1‐adrenoceptor‐stimulated increase of IP3mass was revealed.
Glucose Tolerance and Serum Insulin Levels in an Animal Model of Obesity Induced by the Antipsychotic Drug, Sulpiride
Authors: Baptista Trino , LaCruz Anny , Hernández Luis
Published: March 2009

Abstract:To assess the role of insulin in the development of obesity induced by antipsychotic drugs, a glucose tolerance test was conducted in 40 female rats during the peak of sulpiride‐induced weight gain and in 40 vehicle‐treated animals. The glucose area under the curve did not differ between the groups (P=0.24), however, the area under the insulin curve was significantly decreased by sulpiride (55.2+2.8 versus 115.6+18.9, P=0.007). The results suggest that insulin resistance and hyperinsulinaemia are not involved in the excessive weight gain observed in this animal model of drug‐induced obesity. Alternatively, the insulin‐dampened response observed in the sulpiride‐treated rats may be related to increased insulin sensitivity, which may promote weight gain as proposed by Ravussin (1995).
Adriamycin Induces Protein Oxidation in Erythrocyte Membranes
Authors: DeAtley S. Michael , Aksenov Michael Y. , Aksenova Marina V. , Carney John M. , Butterfield D. Allan
Published: March 2009

Abstract:Adriamycin is an anthracycline antineoplastic agent whose clinical effectiveness is limited by severe side effects, including cardiotoxicity. A current hypothesis for adriamycin cardiotoxicity involves free radical oxidative stress. To investigate this hypothesis in a model system, we applied the technique of immunochemical detection of protein carbonyls, known to be increased in oxidized proteins, to study the effect of adriamycin on rat erythrocyte membranes. Erythrocytes obtained from adriamycin‐treated rats demonstrated an increase of carbonyl formation in their membrane proteins. Yet, in separate experiments when adriamycin was incubated with rat erythrocyte ghosts, there was no significant increase of membrane protein carbonyls detected. In contrast, isolated erythrocytes incubated with an adriamycin‐Fe3+complex exhibited a robust carbonyl incorporation into their membrane proteins in a time‐dependent manner. The level of carbonyl formation was dependent upon the concentration of Fe3+known to form the adriamycin‐Fe3+complex. When the time course between protein carbonyl formation and lipid peroxidation was compared, protein carbonyl detection occurred earlier than lipid peroxidation as assayed by thiobarbituric acid reactive substances formation. These results are consistent with the notion that oxidative modification of membrane proteins may contribute to the development of the acute adriamycin‐mediated toxicity.
Detection of Styrene and Styrene Oxide‐Induced DNA Damage in Various Organs of Mice Using the Comet Assay
Authors: Vaghef Hamid , Hellman Björn
Published: March 2009

Abstract: Styrene (100–500 mg/kg b. wt.) and styrene oxide (50–200 mg/kg b. wt.) were given as a single intraperitoneal injection to female mice (C57BL/6) at various time intervals before sacrifice. Primary DNA damage in various organs was studied using alkaline single cell gel electrophoresis (comet) assay. Both substances induced significant DNA damage in lymphocytes, liver, bone marrow and kidney after 4 hr. The lymphocytes and liver cells were found to be the most sensitive cells to the DNA damaging effects of both agents. With the exception of bone marrow cells, the degree of DNA damage in all other cell types was decreased from 4 hr to 16 hr after the administration of both compounds. A strong sublinear dose‐response relationship was observed in the lymphocytes, liver and bone marrow cells, possibly indicating a saturation of the detoxifying enzyme systems in these organs. The present work suggests that the comet assay can be used for detection of primary DNA damage induced by styrene and styrene oxide in vivo and for comparing the sensitivity of various target organs.
Variations of Arterial Responsesin vitroin Different Sections of Rat Main Superior Mesenteric Artery
Authors: Tolvanen Jari‐Petteri , Sallinen Kirsimarja , Wu Xiumin , Kähönen Mika , Arvola Pertti , Pörsti Ilkka
Published: March 2009

Abstract:We examined the control of vascular tone in rat main superior mesenteric artery. Three standard rings (3 mm in length) of the mesenteric artery were cut, beginning 5 mm, 13 mm and 21 mm distally from the mesenteric arteryaorta junction. In noradrenaline‐precontracted rings, relaxations to acetylcholine in the absence and presence of the cyclooxygenase inhibitor diclofenac, did not differ in the studied sections. However, the nitric oxide synthase inhibitor, NG‐nitro‐L‐arginine methyl ester, attenuated the diclofenac‐resistant responses to acetylcholine more effectively in the proximal than the distal section. Glibenclamide, an inhibitor of ATP‐sensitive K+channels, diminished relaxations evoked by acetylcholine only in the distal section, whereas the inhibitor of Ca2+activated K+channels, apamin, attenuated the responses in all sections. Furthermore, relaxation sensitivity to nitroprusside and isoprenaline was lower in the proximal than distal section. Arterial contractile sensitivity to noradrenaline and potassium chloride was higher, while the maximal contractile force generation was lower in the proximal than the distal part. In conclusion, in different sections of rat main superior mesenteric artery considerable variability was observed in vasoconstrictor and vasodilator responses, as well as in the contribution of endothelial nitric oxide and endothelium‐mediated hyperpolarization to vasodilation. Therefore, the present results emphasize the fact that only corresponding vessel segments should be used when investigating the control of arterial tone.
δ‐Aminolevulinate Dehydratase Inhibition by 2, 3‐Dimercaptopropanol is Mediated by Chelation of Zinc from a Site Involved in Maintaining Cysteinyl Residues in a Reduced State*
Authors: Emanuelli Tatiana , Rocha João Batista T. , Pereira Maria Ester , Nascimento Paulo C. , Souza Diogo O. G. , Beber Flávio A.
Published: March 2009

Abstract: The mechanisms underlying mouse δ‐aminolevulinate dehydratase (ALA‐D) inhibition by a chelating agent used in the treatment of heavy metal poisoning, 2, 3‐dimercaptopropanol (British Anti‐Lewisite), were investigated. ALA‐D inhibition by 2, 3‐dimercaptopropanol was totally reversed by 25–100 μM Zn2+, indicating that inhibition was due to chelation of zinc by 2, 3‐dimercaptopropanol. Our data suggested that zinc bound to a labile site (displaced by 25–40 μM EDTA or 500 μM 2, 3‐dimercaptopropanol) is involved in maintaining the sulfhydryl groups of ALA‐D in a reduced state (essential for enzyme activity), since inhibition by these compounds was reversed by 10 mM dithiotreitol (a reducing agent). On the other hand, 10 mM dithiotreitol did not reverse ALA‐D inhibition by a higher concentration of EDTA (100 μM). Accordingly, 2, 3‐dimercaptopropanol appears to inhibit ALA‐D through a mechanism similar to that of low EDTA concentrations. Neither oxidized 2, 3‐dimercaptopropanol nor reactive oxygen species appeared to contribute for ALA‐D inhibition by reduced 2, 3‐dimercaptopropanol. Taken together, these results suggest that 2, 3‐dimercaptopropanol inhibits ALA‐D by chelating Zn2+ from a labile site that is involved in maintaining enzyme sulfhydryl groups in a reduced state. This site is compatible with the ZnB or Znβ previously described in mammalian and bacterial ALA‐D.
Accumulation of Bisphosphonates in Human Artery and their Effects on Human and Rat Arterial Functionin vitro
Authors: Ylitalo Ritva , Kalliovalkama Jarkko , Wu Xiumin , Kankaanranta Hannu , Salenius Juha‐Pekka , Sisto Tero , Lähteenmäki Tuula , Ylitalo Pauli , Pörsti Ilkka
Published: March 2009

Abstract:Clodronate, etidronate and pamidronate are bisphosphonates introduced in the treatment of hypercalcaemia and osteoporosis. Interestingly, they also inhibit development of experimental atherosclerosis and affect smooth muscle tone of isolated rat tail artery. We have studiedin vitrowhether these hydrophilic compounds 1) accumulate in the wall of the human artery, 2) influence human arterial tone, and 3) interfere with the vascular action of L‐type Ca2+antagonists. Human internal mammary artery rings were incubated with14C‐labelled bisphosphonates. After a 2‐hr incubation, the ratios of artery‐to‐incubate concentrations with 4 and 40 μmol/1 of clodronate were, respectively, 3.0+0.5 (mean+S.E.M.) and 1.3+0.2, with 4 and 40 μmol/1 of etidronate 7.4+0.9 and 3.2+0.4, and with 0.4 and 4 μmol/1 of pamidronate 4.7+0.7 and 3.9+0.8. Both tested bisphosphonates, clodronate and pamidronate, reduced the arterial contractile force induced by α‐adrenergic stimulation with noradrenaline and membrane depolarization with high concentration of KCl. Clodronate also decreased the arterial contraction induced by cumulative addition of Ca2+with KCl as the agonist, and had an additive inhibitory effect on this response with the L‐type Ca2+‐channel blocker nifedipine. The results demostrate that 1) bisphosphonates accumulate markedly in human artery, 2) clodronate and pamidronate reduce human arterial contactile force to α‐adrenergic and depolarizing stimuli, and 3) as shown with clodronate, bisphosphonates may exert an additive inhibitory effect on human arterial contractions with an L‐type Ca2+‐channel blocker.
The Effect of Dexamethasone on the Pharmacokinetics of Triazolam
Authors: Villikka Kirsti , Kivistö Kari T. , Neuvonen Pertti J.
Published: March 2009

Abstract:The effects of short‐term use of a small dose of dexamethasone on the pharmacokinetics and pharmacodynamics of the CYP3A4 substrate, triazolam, were examined. In a randomized, double‐blind cross‐over study with two phases, ten healthy volunteers were given either 1.5 mg dexamethasone or placebo once a day for 4 days. On the 5th day, 0.5 mg triazolam was administered orally. Plasma triazolam concentrations and effects of triazolam were measured for 10 hr. Dexamethasone did not have statistically significant effects on the pharmacokinetics of triazolam. The mean total area under the plasma triazolam concentration‐time curve was, however, 19% smaller during the dexamethasone phase than during the placebo phase (11.4 + 5.7 ng ml–1hr versus 14.1 + 8.8 ng ml–1hr (mean + S.D); P=0.09). The four psychomotor tests employed did not show significant differences in the effects of triazolam between the phases. Although dexamethasone had only small effects on the pharmacokinetics and pharmacodynamics of triazolam in the present study, higher doses or prolonged use of dexamethasone might cause a more pronounced induction of CYP3A4. Further studies on the effects of dexamethasone on the pharmacokinetics of CYP3A4 substrates in man are needed.
Placental Expression of Arylamine N‐Acetyltransferases: Evidence for Linkage Disequilibrium betweenNAT1*10andNAT2*4Alleles of the Two Human Arylamine N‐Acetyltransferase LociNAT1andNAT2
Authors: Smelt Valerie A. , Mardon Helen J. , Sim Edith
Published: March 2009

Abstract:The study of placental xenobiotic metabolism is important for the determination of foetal exposure to environmental chemicals as placental metabolism influences the nature of chemicals reaching the foetus from its mother's blood. Arylamine N‐acetyltransferases are drug metabolizing enzymes which N‐acetylate hydrazines and arylamines, including carcinogenic arylamines and sulphonamide drugs. The two human arylamine N‐acetyltransferase isoenzymes, NAT1 and NAT2, are encoded at multi‐allelic loci. Here, we have determined N‐acetyltransferase (NAT) activity in term placentas from normal, uncomplicated pregnancies. Both NAT1 and NAT2 enzyme activities were detectable. Placental NAT1 activity was at least 1000 fold greater than NAT2 activity. There was a 6 fold inter‐placental variation in NAT1 activity. Mean placental NAT1 specific activity was 1.42 nmolespara‐aminobenzoic acid N‐acetylated × min–1× mg protein–1, which is comparable to NAT1 specific activities which have been measured in adult tissues. The NAT1, but not the NAT2, protein was detectable in placentas by Western blotting. Maternal and foetalNATgenotypes were determined from placenta, using placental blood clots and cord blood respectively, allowingNAThaplotype determination. There appeared to be linkage disequilbrium betweenNAT1*andNAT2*alleles such that the combinationNAT1*10/NAT2*4was found 3.5 times more frequently than would be expected.
Characterization of Guanoxabenz Reducing Activity in Rat Brain
Authors: Dambrova Maija , Uhlén Staffan , Wikberg Jarl E. S.
Published: March 2009

Abstract: Guanoxabenz (1‐(2, 6–dichlorobenzylidene‐amino)‐3–hydroxyguanidine) and guanabenz (1‐(2, 6–dichlorobenzylidene‐amino)‐3–guanidine) are both known as centrally active antihypertensive drugs. We have previously shown that enzymatic activity in the rat spleen can induce N‐reduction of guanoxabenz, leading to high affinity α2–adrenoceptor binding, due to the formation of the α2–adrenoceptor active drug, guanabenz. The spleen activity appears to reside in xanthine oxidase as it is activated by xanthine and blocked by allopurinol. We report that high affinity guanoxabenz binding is also induced in rat brain membranes after addition of NADH or NADPH cofactors. However, the brain process was clearly different from that of the spleen, as the formation of high affinity binding in the brain was not blocked by allopurinol. Moreover the NADH/NADPH activated mechanism of the brain membranes was not blocked by carbon monoxide and SKF525A, thus the activity appears not to reside in cytochrome P450 enzymes. Instead the activity was blocked by menadione and dicumarol. We conclude that the rat cerebral cortex contains an enzymatic activity that may activate guanoxabenz leading to formation of a metabolite showing high affinity for α2–adrenoceptors. We also conclude that the rat brain activity is clearly distinct from that of the rat spleen.
Streptozotocin‐ and Alloxan‐Induced Diabetes Modifies Total Plasma and Lipoprotein Lipid Concentration and Composition without Altering Cholesteryl Ester Transfer Activity
Authors: Wasan Kishor M. , Ng Samson P. , Wong Wesley , Rodrigues Brian B.
Published: March 2009

Abstract:The objectives of this study were to determine the total plasma and lipoprotein lipid concentration and composition and cholesteryl ester transfer activity in two diabetic animal models (alloxan‐induced diabetes in rabbits and streptozotocin‐induced diabetes in rats). Furthermore, we wanted to determine if the severity of diabetes influences lipoprotein lipid profiles and cholesteryl ester transfer activity. Rats and rabbits were randomly divided into non‐diabetic and diabetic groups. Rats were administered either 55 mg/kg or 100 mg/kg of streptozotocin intravenously through the tail vein, while rabbits were administered 100 mg/kg or 200 mg/kg of alloxan intravenously through the marginal ear vein under light anesthesia. Hyperglycaemia was tested for at 48 hr following the doses. Total and lipoprotein cholesterol and triglyceride concentrations using enzymatic kits and cholesteryl ester transfer activity from low‐density lipoproteins to high‐density lipoproteins using3H‐cholesteryl ester incorporated into low‐density lipoproteins were determined. Elevations in both total and lipoprotein cholesterol and triglyceride concentrations and alterations in lipoprotein lipid composition are observed following the onset of drug‐induced diabetes in rats and rabbits compared to non‐diabetics. However, these findings were observed only in animals administered the higher streptozotocin and alloxan dose. Furthermore, cholesteryl ester transfer from low‐density lipoproteins to high‐density lipoproteins is not significantly different in drug‐induced diabetic compared to non‐diabetic rats and rabbits, regardless of which streptozotocin and alloxan dose was used. These findings suggest that difference in lipoprotein lipid concentration and composition as a result of drug‐induced diabetes is independent of cholesteryl ester transfer activity in both rats and rabbits. Furthermore, diabetic severity may influence lipoprotein metabolism in these animal models.
Expression ofCYP2E1in Human Lung and Kidney during Development and in Full‐Term Placenta: A Differential Methylation of the Gene is Involved in the Regulation Process
Authors: Vieira Isabel , Pasanen Markku , Raunio Hannu , Cresteil Thierry
Published: March 2009

Abstract:Methylation of dinucleotide CG residues located in the 5′end of theCYP2E1gene has been demonstrated to play a role in the control of gene expression in the human developing liver. This study was undertaken to examine the CYP2E1 RNA content of human lung, kidney and full‐term placenta and to determine whether the expression ofCYP2E1was controlled by its methylation status in these tissues.CYP2E1was expressed at a very low level in the lung and kidney at whatever age, and at a variable level in full‐term placentas. The restriction profile of genomic DNA was identical in lung and kidney and corresponded to a heavy methylation of Hpall/Mspl sites located within the promoter, the first exon and first intron of theCYP2E1gene. A different pattern of methylation was obtained in full‐term placentas, indicating that CpG residues located in the 5′end of the gene were predominantly but not fully demethylated. However, the variable level of CYP2E1 RNA in full‐term placentas suggests the involvement of other elements in the regulation process ofCYP2E1in this tissue.
Muscarinic Receptor Binding Sites of the M4Subtype in Porcine Lung Parenchyma
Authors: Chelala Jean L. , Kilani Ahmad , Miller Martha J. , Martin Richard J. , Ernsberger Paul
Published: March 2009

Abstract:Muscarinic acetylcholine receptors regulate distal airway resistance and secretion. The subtype expressed in the lung in different species remains uncertain. It has recently become possible to identify the M4subtype by careful comparison of antagonist affinities. We characterized the binding of [3H]quinuclidinyl benzilate ([3H]QNB) to muscarinic receptors in cell membranes from lung parenchyma of 2–8 week old pigs in comparison to cloned human M3and M4receptors expressed in COS cells, to M2in rat atria and to M4in bovine adrenal medulla. In porcine lung, [3H]QNB bound with high affinity (Kd=95±9pM) to a single homogeneous population of muscarinic receptor sites (Bmax=340±10 fmol/mg protein). Competition studies showed that the affinity (expressed as pKi) of 3 selective blockers was in close agreement between pig lung and cloned human m4(r=0.996). A series of 10 blockers showed affinities closely matching reported values for M4receptors of the adrenal medulla (r=0.965). Conversely, affinity values in porcine lung differed significantly (P< 0.05, t‐test) from those determined in parallel with either human cloned M3or with rat atria expressing the M2subtype. We conclude that pig lung muscarinic receptor binding sites most closely resemble the M4subtype, in contrast to the M3subtype typical of large airways in this species.
Transport of Paraquat by Isolated Renal Proximal Tubular Segments from Rabbits
Authors: Mølck Anne‐Marie , Friis Christian
Published: March 2009

Abstract:Paraquat is a non‐selective herbicide, which induces lung, liver and kidney damage in mammalian species. Because paraquat is mainly eliminated by the kidneys, the induced kidney damage may suppress excretion and enhance toxicity of paraquat in other organs. Since proximal tubules appear to be the target segment of the nephron, this study focuses on transport of paraquat by isolated proximal tubular segments from rabbits. Proximal tubules were isolated using a combined magnetic iron perfusion and collagenase method. Incubations were carried out at 25° under 100% oxygen or nitrogen for varying times at different concentrations of paraquat. Proximal tubules accumulated paraquat by a slow process, which was non‐saturable in the concentration range (0.1–5 μM) examined. Tubular excretion of cations involves transport across both basolateral and luminal membranes of the cell. The basolateral uptake of paraquat was inhibited by low temperature, low medium pH and quinine. In contrast to quinine, tetraethylammonium enhanced paraquat accumulation probably by trans‐stimulating the basolateral uptake. Incubation under nitrogen enhanced paraquat accumulation possibly by reducing the transport out of the cell at the luminal membrane. Thus, this study shows that proximal tubules accumulate paraquat by an active process related to the cation transport mechanism.
NADH: Ubiquinone Oxidoreductase Inhibitors Block Induction of Ornithine Decarboxylase Activity in MCF‐7 Human Breast Cancer Cells*
Authors: Rowlands J. Craig , Casida John E.
Published: March 2009

Abstract:Rotenone is the classical inhibitor of NADH: ubiquinone oxidoreductase and its analogue deguelin is a potent inhibitor of 12‐O‐tetradecanoylphorbol 13‐acetate (TPA)‐induced ornithine decarboxylase mRNA steady state level and enzyme activity in mouse 308 cells (Gerhäuseret al.1995). In MCF‐7 human breast cancer cells, rotenone, deguelin and two structurally‐unrelated miticides (pyridaben and fenazaquin) inhibit not only NADH: ubiquinone oxidoreductase but also induced ornithine decarboxylase activity with IC50values of <1 to 70 nM. Rotenone inhibits ornithine decarboxylase activity equally well as induced by TPA, insulin‐like growth factor I and 17β‐oestradiol. Pyridaben is the most potent of the four inhibitors not only for NADH: ubiquinone oxidoreductase activity (bovine heart enzyme) and TPA‐induced ornithine decarboxylase activity and mRNA steady state level but also for TPA‐induced reactive oxygen species. It is therefore proposed that NADH: ubiquinone oxidoreductase inhibitors block multiple and possibly reactive oxygen species‐modulated pathways which regulate ornithine decarboxylase activity.
Structure‐Activity Relationship for Inhibition of CYP11B1–Dependent Glucocorticoid Synthesis in Y1 Cells by Aryl Methyl Sulfones
Authors: Johansson Maria , Larsson Christina , Bergman Åke , Lund Bert‐Ove
Published: March 2009

Abstract: The effects of xenobiotics on CYP11B1‐dependent corticosterone synthesis (11β‐hydroxylase) in mouse adrenocortical Y1 cells were studied. 3‐Methylsulfonyl‐2, 2‐bis(4‐chlorophenyl)‐1, 1‐dichloroethene (MeSO2‐DDE) and some methylsulfonyl polychlorinated biphenyls (MeSO2‐PCB) inhibited the corticosterone synthesis, whereas PCBs or DDE did not. This indicates a crucial role of the methyl sulfone group for this inhibitory effect. Kinetic analyses of MeSO2‐DDE and the two most potent MeSO2‐PCBs were conducted using Lineweaver‐Burk double‐reciprocal plots. The data showed a competitive inhibition of CYP11B1 by the compounds, with apparent inhibitory constants (Ki) of 1.6, 4.6, and 6.7 μM for MeSO2‐DDF., 4‐MeSO2‐2, 3, 6, 4′‐tetrachlorobiphenyl, and 4‐MeSO2‐2, 3, 6, 3,’4′‐pentachlorobiphenyl, respectively. For comparison, the substrate Km was 3.5 μM in the cells, and metyrapone and ketoconazole had apparent Revalues of 0.8 and 0.04 μM, respectively. In contrast to all previously known inhibitors of CYP11B1, the aryl methyl sulfones are the first examples of CYP11B1 inhibitors not being heterocyclic amines or steroids. The aryl methyl sulfones are widespread environmental pollutants and their inhibition of CYP11B1 constitutes another potential mechanism for endocrine disruption. Their influence on the synthesis of adrenocortical hormones thus merits further interest.