Faculty of Health Sciences University Fernando Pessoa Rua Carlos da Maia, 296 4200-150 Porto Portugal
mcarv@ufp.edu.pt
Assistant Professor of Toxicology in the Faculty of Health Sciences, University Fernando Pessoa, Porto, Portugal. Graduated in Pharmaceutical Sciences from the Faculty of Pharmacy, University of Porto. She also obtained a post-graduated Master degree in Quality Control - Scientific Area in Drug Substances and Medicinal Plants at the Faculty of Pharmacy, University of Porto, and awarded with the degree of PhD in Toxicology at the Faculty of Pharmacy, University of Porto. Investigator at the Associated Laboratory REQUIMTE.
Present research interests 1. Toxicological studies using human normal and cancer renal primary cell cultures from surgical specimens. 2.Studies on the mechanisms underlying the toxicity of drugs of abuse using in vitro cellular systems and in vivo studies. 3. Evaluation of in vitro antioxidant and antitumoral activity of plant extracts. 4. Studies of oxidative stress mechanisms associated with the Ischemic Cardiac Disease.
To the present date, she authored or co-authored more than 40 international publications. Reviewer for several national and international journals related to the field of Toxicology and Cancer. Collaboration in Funded Projects FCT-POCTI/FCB/36099, FCT-POCTI/SAU-OBS/55849/2004 and FCT-PTDC/SAU-OSM/101437/2008.
Abstract: The present study reports the antioxidant properties of Arbutus unedo L. leaf and fruit extracts using different in vitro assays including (i) reducing power, (ii) scavenging effect on DPPH free radicals, and (iii) inhibitory effect on AAPH-induced hemolysis and lipid peroxidation in human erythrocytes. All assays demonstrated antioxidant efficiency for A. unedo L. aqueous extracts, being consistently higher in the leaf. EC(50) values for reducing power and DPPH radical scavenging activities were, respectively, 0.318±0.007 and 0.087±0.007mg/mL for leaf, and 2.894±0.049 and 0.790±0.016mg/mL for fruit extracts. Under the oxidative action of AAPH, A. unedo leaf and fruit extracts protected the erythrocyte membrane from hemolysis (IC(50) of 0.062±0.002 and 0.430±0.091mg/mL, respectively) and decreased the levels of malondialdehyde, a breakdown product of lipid peroxidation (IC(50) of 0.075±0.014 and 0.732±0.452mg/mL, respectively). In accordance with antioxidant activity, phenolic content was found to be significantly higher in leaf extract. To our knowledge, this is the first time that the antioxidant activity of A. unedo species is evaluated using human biological membranes. Overall, our results suggest that A. unedo leaves are a promising source of natural antioxidants with potential application in diseases mediated by free radicals.
Abstract: This study reports for the first time the biological properties of Portuguese propolis. The antioxidant potential of propolis samples from Bornes (Northeast) and Fundão (Centre) regions of Portugal was evaluated by their ability to inhibit the 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis and lipid peroxidation in human erythrocytes. Bornes and Fundão propolis strongly protected the erythrocyte membrane from hemolysis (IC(50) of 6.3±0.7 and 10.4±2.7 μg/ml, respectively), in a time- and concentration-dependent manner. This effect was found to be significantly higher than that presented by ascorbic acid (IC(50) of 31.0±5.6 μg/ml). In addition, human erythrocytes treated with propolis extracts showed concentration-dependent decrease in levels of malondialdehyde, a breakdown product of lipid peroxidation. Propolis extracts were also assayed for their anticancer properties on human renal cell carcinoma (RCC). Primary cultures of normal and cancerous renal cells derived from RCC patients, in addition to A-498 cell line, were treated with propolis extracts (0-100 μg/ml). Cytotoxic and antiproliferative effects were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Propolis extracts exhibited selective toxicity against malignant cells compared to normal cells. In vitro RCC growth was strongly inhibited by Bornes and Fundão propolis in a concentration-dependent manner. Our results indicate that Portuguese propolis constitutes an excellent source of effective natural antioxidant and chemopreventive agents.
Abstract: The kidney is a target organ for the toxicity of several xenobiotics and is also highly susceptible to the development of malignant tumors. In both cases, in vitro studies provide insight to cellular damage, and represent adequate models to study either the mechanisms underlying the toxic effects of several nephrotoxicants or therapeutic approaches in renal cancer. The development of efficient methods for the establishment of human normal and tumor renal cell models is hence crucial. In this study, a technically simple and rapid protocol for the isolation and culture of human proximal tubular epithelial cells and human renal tumor cells from surgical specimens is presented. Tumor and normal tissues were processed by using the same methodology, based on mechanical disaggregation of tissue followed by enzymatic digestion and cell purification by sequential sieving. The overall procedure takes roughly one hour. The resulting cell preparations have excellent viabilities and yield. Establishment of primary cultures from all specimens was achieved successfully. The origin of primary cultured cells was established through morphological evaluation. Normal cells purity was confirmed by immunofluorescent staining and reverse transcription-polymerase chain reaction analysis for expression of specific markers.
Abstract: Pathologic heart conditions, particularly heart failure (HF) and ischemia-reperfusion (I/R) injury, are characterized by sustained elevation of plasma and interstitial catecholamine levels, as well as by the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Despite the continuous and extensive research on catecholamines since the early years of the XX(th) century, the mechanisms underlying catecholamine-induced cardiotoxicity are still not fully elucidated. The role of catecholamines in HF, stress cardiomyopathy, I/R injury, ageing, stress, and pheochromocytoma will be thoroughly discussed. Furthermore and although the noxious effects resulting from catecholamine excess have traditionally been linked to adrenoceptors, in fact, several evidences indicate that oxidative stress and the oxidation of catecholamines can have important roles in catecholamine-induced cardiotoxicity. Accordingly, the reactive intermediates formed during catecholamine oxidation have been associated with cardiac toxicity, both in in vitro and in vivo studies. An insight into the influence of ROS, RNS, and catecholamine oxidation products on several heart diseases and their clinical course will be provided. In addition, the source and type of oxidant species formed in some heart pathologies will be referred. In this review a special focus will be given to the research of cardiac pathologies where catecholamines and oxidative stress are involved. An integrated vision of these matters is required and will be provided along this review, namely how the concomitant surge of catecholamines and ROS occurs and how they can be interconnected. The concomitant presence of these factors can elicit peculiar and not fully characterized responses on the heart. We will approach the existing data with new perspectives as they can help explaining several controversial results regarding cardiovascular diseases and the redox ability of catecholamines.
Abstract: 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is a worldwide illegally used amphetamine-derived designer drug known to be hepatotoxic to humans. Jaundice, hepatomegaly, centrilobular necrosis, hepatitis and fibrosis represent some of the adverse effects caused by MDMA in the liver. Although there is irrefutable evidence of MDMA-induced hepatocellular damage, the mechanisms responsible for that toxicity remain to be thoroughly clarified. One well thought-of mechanism imply MDMA metabolism in the liver into reactive metabolites as responsible for the MDMA-elicited hepatotoxicity. However, other factors, including MDMA-induced hyperthermia, the increase in neurotransmitters efflux, the oxidation of biogenic amines, polydrug abuse pattern, and environmental features accompanying illicit MDMA use, may increase the risk for liver complications. Liver damage patterns of MDMA in animals and humans and current research on the mechanisms underlying the hepatotoxic effects of MDMA will be highlighted in this review.
Abstract: Acute renal failure is a common finding in cocaine abusers. While cocaine metabolism may contribute to its nephrotoxic mechanisms, its pharmacokinetics in kidney cells is hitherto to be clarified. Primary cultures of human proximal tubular cells (HPTCs) provide a well-characterized in vitro model, phenotypically representative of HPTCs in vivo. Thus, the present work describes the first sensitive gas chromatography/ion trap-mass spectrometry (GC/IT-MS) method for measurement of cocaine and its metabolites benzoylecgonine (BE) and norcocaine (NCOC) using a primary culture of HPTCs as cellular matrix, following solid phase extraction (SPE) and derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). The application of this methodology also enables the identification of two other cocaine metabolites: ecgonine methyl ester (EME) and anhydroecgonine methyl ester (AEME). The validation of the method was performed through the evaluation of selectivity, linearity, precision and accuracy, limit of detection (LOD), and limit of quantification (LOQ). Its applicability was demonstrated through the quantification of cocaine, BE and NCOC in primary cultured HPTCs after incubation, at physiological conditions, with 1 mM cocaine for 72 h. The developed GC/IT-MS method was found to be linear (r² > 0.99). The intra-day precision varied between 3.6% and 13.5% and the values of accuracy between 92.7% and 111.9%. The LOD values for cocaine, BE and NCOC were 0.97±0.09, 0.40±0.04 and 20.89±1.81 ng/mL, respectively, and 3.24±0.30, 1.34±0.14 and 69.62±6.05 ng/mL as LOQ values.
Abstract: Several studies suggest that regular consumption of nuts, mostly walnuts, may have beneficial effects against oxidative stress mediated diseases such as cardiovascular disease and cancer. Walnuts contain several phenolic compounds which are thought to contribute to their biological properties. The present study reports the total phenolic contents and antioxidant properties of methanolic and petroleum ether extracts obtained from walnut (Juglans regia L.) seed, green husk and leaf. The total phenolic contents were determined by the Folin-Ciocalteu method and the antioxidant activities assessed by the ability to quench the stable free radical 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and to inhibit the 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of human erythrocytes. Methanolic seed extract presented the highest total phenolic content (116 mg GAE/g of extract) and DPPH scavenging activity (EC(50) of 0.143 mg/mL), followed by leaf and green husk. In petroleum ether extracts, antioxidant action was much lower or absent. Under the oxidative action of AAPH, all methanolic extracts significantly protected the erythrocyte membrane from hemolysis in a time- and concentration-dependent manner, although leaf extract inhibitory efficiency was much stronger (IC(50) of 0.060 mg/mL) than that observed for green husks and seeds (IC(50) of 0.127 and 0.121 mg/mL, respectively). Walnut methanolic extracts were also assayed for their antiproliferative effectiveness using human renal cancer cell lines A-498 and 769-P and the colon cancer cell line Caco-2. All extracts showed concentration-dependent growth inhibition toward human kidney and colon cancer cells. Concerning A-498 renal cancer cells, all extracts exhibited similar growth inhibition activity (IC(50) values between 0.226 and 0.291 mg/mL), while for both 769-P renal and Caco-2 colon cancer cells, walnut leaf extract showed a higher antiproliferative efficiency (IC(50) values of 0.352 and 0.229 mg/mL, respectively) than green husk or seed extracts. The results obtained herein strongly indicate that walnut tree constitute an excellent source of effective natural antioxidants and chemopreventive agents.
Abstract: The present study reports the phenolic profile and antiproliferative properties of quince (Cydonia oblonga Miller) leaf and fruit (pulp, peel, and seed) against human kidney and colon cancer cells. The phenolic profiles of quince methanolic extracts were determined by high-performance liquid chromatography (HPLC)/diode array detector (DAD). 5-O-Caffeoylquinic acid was always one of the two major phenolic compounds present in all extracts, except for seed. Our results revealed that quince leaf and fruit extracts exhibited distinctive antiproliferative activities. The extracts from quince leaf showed concentration-dependent growth inhibitory activity toward human colon cancer cells (IC(50) = 239.7 +/- 43.2 microg/mL), while no effect was observed in renal adenocarcinoma cells. Concerning the fruit, seed extracts exhibited no effect on colon cancer cell growth, whereas strong antiproliferative efficiency against renal cancer cells was observed for the highest concentration assayed (500 microg/mL). The antiproliferative activity of pulp and peel extracts was low or absent in the selected range of extract concentrations. This is the first report showing that C. oblonga may be useful as a cancer chemopreventive and/or chemotherapeutic agent.
Abstract: In recent years, the scientific community, food industry, consumers and media have revealed a growing interest regarding the potential benefits of tea consumption for human health. Several studies indicate that phenolic compounds, especially flavan-3-ols (catechins), are the main components responsible for the medicinal effects of this beverage due to their recognized antioxidant properties. Antioxidants are ascribed to reduce cells and biomolecules oxidative damage caused by reactive oxygen/nitrogen species (ROS/RNS). In fact, ROS and RNS have been implicated in the oxidative deterioration of food products, as well as in the pathogenesis of several chronic and/or ageing diseases such as atherosclerosis, diabetes mellitus, chronic inflammation, neurodegenerative disorders, including Alzheimer's disease, and certain types of cancer. (-)-Epigallocatechin-3-gallate, the main component of white and green teas, for which chemical synthesis is complex, not only possesses strong antiradicalar activity but also can inhibit nitration reactions, modulate carcinogen-metabolizing enzymes, trap ultimate carcinogens and inhibit cancer cell proliferation. The recent patents on the tea manufacture, extracts preparation and health-promoting properties are reviewed in this manuscript, especially in what concerns the chemopreventive and chemotherapeutic activities of tea polyphenols.
Abstract: In several pathologic conditions, like cardiac ischemia/reperfusion, the sustained elevation of plasma and interstitial catecholamine levels, namely adrenaline (ADR), and the generation of reactive oxygen species (ROS) are hallmarks. The present work aimed to investigate in cardiomyocytes which intracellular signalling pathways are altered by ADR redox ability. To mimic pathologic conditions, freshly isolated calcium tolerant cardiomyocytes from adult rat were incubated with ADR alone or in the presence of a system capable of generating ROS [(xanthine with xanthine oxidase) (X/XO)]. ADR elicited a pro-oxidant signal with generation of reactive species, which was largely magnified by the ROS generating system. However, no change in cardiomyocytes viability was observed. The pro-oxidant signal promoted the translocation to the nucleus of the transcription factors, Heat shock factor-1 (HSF-1) and Nuclear factor-kappaB (NF-kappaB). In addition, proteasome activity was compromised in the experimental groups where the generation of reactive species occurred. The decrease in the proteasome activity of the ADR group resulted from its redox sensitivity, since the activity was recovered by adding the ROS scavenger, tiron. Proteasome inhibition seemed to elicit an increase in HSP70 levels. Furthermore, retention of mitochondrial cytochrome c and inhibition of caspase 3 activity were observed by X/XO incubation in presence or absence of ADR. In conclusion, in spite of all the insults inflicted to the cardiomyocytes, they were capable to activate intracellular responses that enabled their survival. These mechanisms, namely the pathways altered by catecholamine proteasome inhibition, should be further characterized, as they could be of relevance in the ischemia preconditioning and the reperfusion injury.
Abstract: The aim of this study was to determine the phenolic content and evaluate the antioxidant activity of quince (Cydonia oblonga) fruit. For this purpose, fruits were separated into pulps, peels and seeds and methanolic extracts were prepared. The phenolic profiles were determined by HPLC/UV and antioxidant properties were studied for their ability to quench the stable free radical 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and to inhibit the 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of human erythrocytes. The main phenolic compounds were 5-O-caffeoylquinic acid for pulp and peel (57% and 29%, respectively) and stellarin-2 for seed (18%). Total phenolics content was 2.5, 6.3 and 0.4g/kg of methanolic extract for pulp, peel and seed, respectively. Pulp and peel extracts showed similar DPPH free radical scavenging activities (EC(50) of 0.6 and 0.8 mg/ml, respectively), while seed extract presented much lower antioxidant potential (EC(50) of 12.2mg/ml). Under the oxidative action of AAPH, pulp and peel extracts showed significant protection of the erythrocyte membrane from hemolysis, in a time- and concentration-dependent manner. Seed extracts by themselves induced extensive hemolysis. These results indicate higher antioxidant activity for certain parts of quince fruit, namely pulp and peel, that may therefore represent accessible sources of natural antioxidants with potential application in nutritional/pharmaceutical fields, as preventive or therapeutic agents in diseases in which free radicals are implicated.
Abstract: This study aimed to determine the phenolic profile and to investigate the antioxidant potential of quince (Cydonia oblonga) leaf, comparing it with green tea (Camellia sinensis). For these purposes, methanolic extracts were prepared and phenolics content of quince leaf was determined by HPLC/UV. The antioxidant properties were assessed by Folin-Ciocalteu reducing capacity assay and by the ability to quench the stable free radical 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and to inhibit the 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of human erythrocytes.5-O-Caffeoylquinic acid was found to be the major phenolic compound in quince leaf extract. Quince leaf exhibited a significantly higher reducing power than green tea (mean value of 227.8 +/- 34.9 and 112.5 +/- 1.5 g/kg dry leaf, respectively). Quince leaf extracts showed similar DPPH radical-scavenging activities (EC50 mean value of 21.6 +/- 3.5 microg/ml) but significantly lower than that presented by green tea extract (EC50 mean value of 12.7 +/- 0.1 microg/ml). Under the oxidative action of AAPH, quince leaf methanolic extract significantly protected the erythrocyte membrane from hemolysis in a similar manner to that found for green tea (IC50 mean value of 30.7 +/- 6.7 and 24.3 +/- 9.6 microg/ml, respectively, P > 0.05). These results point that quince leaf may have application as preventive or therapeutic agent in diseases in which free radicals are involved.
Abstract: Isolated heart cells are highly susceptible to the toxicity of catecholamine oxidation products, namely, to catecholamine-glutathione adducts. Although cellular uptake and/or efflux of these products may constitute a crucial step, the knowledge about the involvement of transporters is still very scarce. This work aimed to contribute to the characterization of membrane transport mechanisms, namely, extraneuronal monoamine transporter (EMT), the multidrug resistant protein 1 (MRP1), and P-glycoprotein (P-gp) in freshly isolated cardiomyocytes from adult rats. These transporters may be accountable for uptake and/or efflux of adrenaline and an adrenaline oxidation product, 5-(glutathion-S-yl)adrenaline, in cardiomyocyte suspensions. Our results showed that 5-(glutathion-S-yl)adrenaline efflux was mediated by MRP1. Additionally, we demonstrated that the adduct formation occurs within the cardiomyocytes, since EMT inhibition reduced the intracellular adduct levels. The classical uptake2 transport in rat myocardial cells was inhibited by the typical EMT inhibitor, corticosterone, and surprisingly was also inhibited by low concentrations of another drug, a well-known P-gp inhibitor, GF120918. The P-gp activity was absent in the cells since P-gp-mediated efflux of quinidine was not blocked by GF120918. In conclusion, this work showed that freshly isolated cardiomyocytes from adult rats constitute a good model for the study of catecholamines and catecholamines metabolites membrane transport. The cardiomyocytes maintain EMT and MRP1 fully active, and these transporters contribute to the formation and efflux of 5-(glutathion-S-yl)adrenaline. In the present experimental conditions, P-gp activity is absent in the isolated cardiomyocytes.
Abstract: The sustained elevation of plasma and interstitial catecholamine levels, namely adrenaline (ADR), and the generation of reactive oxygen species (ROS) are well recognized hallmarks of several cardiopathologic conditions, like cardiac ischemia/reperfusion (I/R) and heart failure (HF). The present work aimed to investigate the proteomics and energetic metabolism of cardiomyocytes incubated with ADR and/or ROS. To mimic pathologic conditions, freshly isolated calcium-tolerant cardiomyocytes from adult rat were incubated with ADR alone or in the presence of a system capable of generating ROS [(xanthine with xanthine oxidase) (XXO)]. Two-dimensional electrophoresis with matrix-assisted laser desorption/ionization and time-of-flight mass spectrometer analysis were used to define protein spot alterations in the cardiomyocytes incubated with ADR and/or ROS. Moreover, the energetic metabolism and the activity of mitochondrial complexes were evaluated by nuclear magnetic resonance and spectrophotometric determinations, respectively. The protein extract was mainly constituted by cardiac mitochondrial proteins and the alterations found were included in five functional classes: (i) structural proteins, notably myosin light chain-2; (ii) redox regulation proteins, in particular superoxide dismutase (SOD); (iii) energetic metabolism proteins, encompassing ATP synthase alpha chain and dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex; (iv) stress response proteins, like the heat shock proteins; and (v) regulatory proteins, like cytochrome c and voltage-dependent anion channel 1. The XXO system elicited alterations in cardiac contractile proteins, as they showed high levels of cleavage, and also altered energetic metabolism, through increased lactate and alanine levels. The cardiomyocytes incubation with ADR resulted in an accentuated increase in mitochondrial complexes activity and the decrease in alanine/lactate ratio, thus reflecting a high cytosolic NADH/NAD(+) ratio. Furthermore, an increase in manganese SOD expression and total SOD activity occurred in the ADR group, as the increase in the mitochondrial complexes presumably led to higher 'electron leakage'. The modifications in proteins, enzymes activity, and energetic metabolism were indicative that different pathways are activated by catecholamines and ROS. These alterations altogether determine the I/R and HF specific features and contribute for the initiation or aggravation of those cardiopathologic conditions.
Abstract: High concentrations of circulating biogenic catecholamines often exist during the course of several cardiovascular disorders. Additionally, coronary dysfunctions are prominent and frequently related to the ischemic and reperfusion phenomenon (I/R) in the heart, which leads to the release of large amounts of catecholamines, namely adrenaline, and to a sustained generation of reactive oxygen species (ROS). Thus, this work aimed to study the toxicity of adrenaline either alone or in the presence of a system capable of generating ROS [xanthine with xanthine oxidase (X/XO)], in freshly isolated, calcium tolerant cardiomyocytes from adult rats. Studies were performed for 3 h, and cardiomyocyte viability, ATP level, lipid peroxidation, protein carbonylation content, and glutathione status were evaluated, in addition to the formation of adrenaline's oxidation products and quinoproteins. Intracellular GSH levels were time-dependently depleted with no GSSG formation when cardiomyocytes were exposed to adrenaline or to adrenaline with X/XO. Meanwhile, a time-dependent increase in the rate of formation of adrenochrome and quinoproteins was observed. Additionally, as a new outcome, 5-(glutathion- S-yl)adrenaline, an adrenaline adduct of glutathione, was identified and quantified. Noteworthy is the fact that the exposure to adrenaline alone promotes a higher rate of formation of quinoproteins and glutathione adduct, while adrenochrome formation is favored where ROS production is stimulated. This study shows that the redox status of the surrounding environment greatly influences adrenaline's oxidation pathway, which may trigger cellular changes responsible for cardiotoxicity.
Abstract: The sustained high release of catecholamines to circulation is a deleterious condition that may induce toxicity, which seems to be partially related to the products formed by oxidation of catecholamines that can be further conjugated with glutathione (GSH). The aim of the present study was to develop a method for the determination of GSH adducts of adrenaline in biological samples. Two position isomers of the glutathion-S-yl-adrenaline were synthesized and characterized by HPLC using diode array, coulometric and mass detectors. A method for the extraction of these adducts from human plasma was also developed, based on adsorption to activated alumina, which showed adequate recoveries and proved to be crucial in removing interferences from plasma. The selectivity, precision and linearity of the method were all within the accepted values for these parameters. Furthermore, the sensitivity of this method allows the detection of adduct amounts that are within the range of the expected concentrations for these adducts under certain pathophysiological conditions and/or drug treatments. In conclusion, the development of this method allows the direct analysis of GSH adducts of adrenaline in human plasma, providing a valuable tool for the study of the catecholamine oxidation process and its related toxicity.
Abstract: The objective of the present work was to evaluate the toxic effects of sodium arsenite, As(III), in mice and the protective effect of 2 antioxidants, hesperidin and lipoic acid, against the observed As(III)-induced toxicity. In each study, mice were assigned to 1 of 4 groups: control, antioxidant, antioxidant + arsenite, and arsenite. Animals were first injected with the vehicle or 25 mg antioxidant/kg BW. After 30 minutes they received an injection of 10 mg arsenite/kg BW or 0.9% NaCl. Two hours after the first injection, the liver, kidney, and testis were collected for histological evaluation. Liver samples were also taken for quantification of arsenic. In mice exposed only to As(III), various histopathological effects were observed in the liver, kidneys, and testes. In mice pretreated with either hesperidin or lipoic acid, a reduction of histopathologic effects on the liver and kidneys was observed. No protective effects were observed in the testes for either of the 2 studied antioxidants. In conclusion, hesperidin and lipoic acid provided protective effects against As(III)-induced acute toxicity in the liver and kidneys of mice. These compounds may potentially play an important role in the protection of populations chronically exposed to arsenic.
Abstract: Cardiovascular complications associated with 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) abuse have increasingly been reported. The indirect effect of MDMA mediated by a sustained high level of circulating biogenic amines may contribute to the cardiotoxic effects, but other factors, like the direct toxic effects of MDMA and its metabolites in cardiac cells, remain to be investigated. Thus, the objective of the present in vitro study was to evaluate the potential cardiotoxic effects of MDMA and its major metabolites 3,4-methylenedioxyamphetamine (MDA), N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA), and alpha-methyldopamine (alpha-MeDA) using freshly isolated adult rat cardiomyocytes. The cell suspensions were incubated with these compounds in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 4 h. alpha-MeDA, N-Me-alpha-MeDA, and their respective aminochromes (oxidation products) were quantified in cell suspensions by HPLC-DAD. The toxic effects were evaluated at hourly intervals for 4 h by measuring the percentage of cells with normal morphology, glutathione (GSH), and glutathione disulfide (GSSG); intracellular Ca(2+), ATP, and ADP; and the cellular activities of glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. No toxic effects were found after exposure of rat cardiomyocytes to MDMA or MDA at any of the tested concentrations for 4 h. In contrast, their catechol metabolites N-Me-alpha-MeDA and alpha-MeDA induced significant toxicity in rat cardiomyocytes. The toxic effects were characterized by a loss of normal cell morphology, which was preceded by a loss of GSH homeostasis due to conjugation of GSH with N-Me-alpha-MeDA and alpha-MeDA, sustained increase of intracellular Ca(2+) levels, ATP depletion, and decreases in the antioxidant enzyme activities. The oxidation of N-Me-alpha-MeDA and alpha-MeDA into the toxic compounds N-methyl-alpha-methyldopaminochrome and alpha-methyldopaminochrome, respectively, was also verified in cell suspensions incubated with these MDMA metabolites. The results obtained in this study provide evidence that the metabolism of MDMA into N-Me-alpha-MeDA and alpha-MeDA is required for the expression of MDMA-induced cardiotoxicity in vitro, being N-Me-alpha-MeDA the most toxic of the studied metabolites.
Abstract: Hypericum androsaemum L. (Guttiferae) is a medicinal plant with antioxidant activity. Increasing evidence regarding free radical generating agents suggests that hepatotoxic-related disorders may involve reactive oxygen species (ROS). The purpose of this study was to investigate the protective effect of Hypericum androsaemum infusion on isolated rat hepatocytes oxidative injury induced by tert-butyl hydroperoxide (t-BHP). The results showed that pretreatment of the cells with this infusion (16, 62 and 250 microg/ml) prevented the leakage of lactate dehydrogenase (LDH) and lipid peroxidation caused by a 30-min treatment with t-BHP (1mM). However, infusion-induced alterations on glutathione homeostasis were noticed, as it was observed by the increase in glutathione oxidised form (GSSG) and depletion in total glutathione levels, which indicates that plant-derived antioxidant extracts may not be considered a generalised way of treating pro-oxidant-related diseases.
Abstract: The amphetamine designer drugs 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy") and its N-demethylated analogue 3,4-methylenedioxyamphetamine (MDA or "love") have been extensively used as recreational drugs of abuse. MDA itself is a main MDMA metabolite. MDMA abuse in humans has been associated with numerous reports of hepatocellular damage. Although MDMA undergoes extensive hepatic metabolism, the role of metabolites in MDMA-induced hepatotoxicity remains unclear. Thus, the aim of the present study was to evaluate the effects of MDA and alpha-methyldopamine (alpha-MeDA), a major metabolite of MDA, in freshly isolated rat hepatocyte suspensions. The cells were incubated with MDA or alpha-MeDA at final concentrations of 0.1, 0.2, 0.4, 0.8, or 1.6 mM for 3 h. The toxic effects induced following incubation of hepatocyte suspensions with these metabolites were evaluated by measuring cell viability, the extent of lipid peroxidation, levels of glutathione (GSH) and glutathione disulfide (GSSG), the formation of GSH conjugates, and the activities of GSSG reductase (GR), GSH peroxidase (GPX), and GSH S-transferase (GST). MDA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on lipid peroxidation, cell viability, or on the activities of GR, GPX, and GST. In contrast, alpha-MeDA (1.6 mM, 3 h) induced a marked depletion of GSH accompanied by a loss on cell viability, and decreases in GR, GPX and GST activities, although no significant effect on lipid peroxidation was found. For both metabolites, GSH depletion was not accompanied by increases in GSSG levels; rather, 2-(glutathion- S-yl)-alpha-MeDA and 5-(glutathion- S-yl)-alpha-MeDA were identified by HPLC-DAD/EC within cells incubated with MDA or alpha-MeDA. The results provide evidence that one of the early consequences of MDMA metabolism is a disruption of thiol homeostasis, which may result in loss of protein function and the initiation of a cascade of events leading to cellular damage.
Abstract: Amphetamine derivatives are a class of compounds increasingly abused as recreational drugs in various regions of the world. Although d-amphetamine (AMPH) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are among the most commonly used, the abuse of other designer drugs such as 4-bromo-2,5-dimethoxyphenethylamine (2C-B) and 4-methylthioamphetamine (4-MTA) and their involvement in acute intoxications has been increasingly reported. There is evidence that abusers ingest these compounds either alone or in combination and the respective monitoring is important for both legal and health care purposes in hospital emergency. In the present study a simple and clean solid-phase extraction procedure from urine of AMPH and MDMA, and their major metabolites p-hydroxyamphetamine (OH-AMPH) and methylenedioxyamphetamine (MDA) and 2C-B and 4-MTA was developed. Analysis was performed by HPLC-UV and the precision of the technique was between 2.9 and 5.3% for all compounds. For the overall procedure, the precision values were between 3.3 and 5.9%. Recoveries obtained from spiked urines at three concentration levels were better than 84 +/- 4% for the six compounds. The limit of detection of the method for the compounds (between 5.3 and 84.0 ng) enables their identification in urine after ingestion of fatal and non-fatal doses. The main advantages of the present method lie in its simple, clean and reliable SPE extraction method of the six amphetamine derivatives from urine followed by their simultaneous detection and quantification by liquid chromatography with UV detection.
Abstract: Increasing evidence regarding free radical generating agents indicates that the sustained production of high levels of reactive oxygen species (ROS) can cause hepatotoxicity. Being a short chain analog of lipid peroxide, tert-butyl hydroperoxide (t-BHP) is metabolized into free radical intermediates by cytochrome P450 in hepatocytes, which initiate lipid peroxidation, glutathione depletion and cell damage. The aim of the present study was to evaluate the putative protective effect of Hypericum androsaemum lyophilised infusion against t-BHP-induced mice hepatotoxicity in vivo, which has already been shown to be antioxidant in vitro. However, the results showed that the oral pretreatment with Hypericum androsaemum infusion (4, 20 and 100 mg/kg) for 4 days before a single intraperitoneal dose of t-BHP (1.8 mmol/kg) potentiated the t-BHP-induced hepatotoxicity. In fact, it was observed a potentiation in the depletion of total glutathione and reduced glutathione (GSH) contents and increase in oxidised glutathione (GSSG) level. Also the histopathological evaluation of the mice livers revealed that the infusion raised the incidence of liver lesions induced by t-BHP. These data do not corroborate any effect of Hypericum androsaemum infusion as hepatoprotector, but rather as a potentiator of hepatotoxicity in the present experimental conditions.
Abstract: In the past decade, clinical evidence has increasingly shown that the liver is a target organ for 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") toxicity. The aims of the present in vitro study were: (1) to evaluate and compare the hepatotoxic effects of MDMA and one of its main metabolites, N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and (2) to investigate the ability of antioxidants, namely ascorbic acid and N-acetyl-L-cysteine (NAC), to prevent N-Me-alpha-MeDA-induced toxic injury, using freshly isolated rat hepatocytes. Cell suspensions were incubated with MDMA or N-Me-alpha-MeDA in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 3 h. To evaluate the potential protective effects of antioxidants, cells were preincubated with ascorbic acid in the final concentrations of 0.1 and 0.5 mM, or NAC in the final concentrations of 0.1 and 1 mM for 15 min before treatment with 1.6 mM N-Me-alpha-MeDA for 3 h (throughout this incubation period the cells were exposed to both compounds). The toxic effects were evaluated by measuring the cell viability, glutathione (GSH) and glutathione disulfide (GSSG), ATP, and the cellular activities of GSH peroxidase (GPX), GSSG reductase (GR), and GSH S-transferase (GST). MDMA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on cell viability, ATP levels, or on the activities of GR, GPX, and GST. In contrast, N-Me-alpha-MeDA was shown to induce not only a concentration- and time-dependent depletion of GSH, but also a depletion of ATP levels accompanied by a loss in cell viability, and decreases in the antioxidant enzyme activities. For both compounds, GSH depletion was not accompanied by increases in GSSG levels, which seems to indicate GSH depletion by adduct formation. Importantly, the presence of ascorbic acid (0.5 mM) or NAC (1 mM) prevented cell death and GSH depletion induced by N-Me-alpha-MeDA. The results provide evidence that MDMA and its metabolite N-Me-alpha-MeDA induce toxicity to freshly isolated rat hepatocytes. Oxidative stress may play a major role in N-Me-alpha-MeDA-induced hepatic toxicity since antioxidant defense systems are impaired and administration of antioxidants prevented N-Me-alpha-MeDA toxicity.
Abstract: 2-Styrylchromones are a novel class of chromones, vinylogues of flavones (2-phenylchromones), which have recently been found in nature. The best described property of almost every group of flavones and other flavonoids, especially the hydroxylated derivatives, is their capacity to act as antioxidants. Indeed there is a widely accepted view that the positive health effects of flavones are due to their antioxidant activity. As oxidative stress is a main cause of liver toxicity induced by several hepatotoxicants, agents with the ability to protect the liver against reactive pro-oxidant species may be therapeutically useful. The present study evaluated the possible protective activity of six new synthetic polyhydroxylated 2-styrylchromone derivatives against the pro-oxidant hepatotoxicity exerted by tert-butylhydroperoxide ( t-BHP) in freshly isolated rat hepatocytes. The cells were preincubated with the 2-styrylchromones in the final concentrations of 3.125, 12.5, 25, 50, 100, and 200 microM for 5 min before treatment with 1.0 mM t-BHP for 30 min (throughout this incubation period the cells were exposed to both compounds). The well-known antioxidant 3-hydroxyflavone (quercetin) was used as positive control. At the end of the 30-min incubation period, aliquots of cells suspensions were taken for measurement of lactate dehydrogenase leakage, thiobarbituric acid reactive substances, reduced glutathione, and oxidized glutathione contents. The tested compounds exhibited in vitro protective activity against t-BHP induced hepatotoxicity (1.0 mM, 30 min). Three of the tested compounds, at the concentrations of 3.125, 12.5, 25, and 50 microM, prevented the t-BHP induced glutathione depletion, lipid peroxidation, and cell death in freshly isolated rat hepatocytes to a comparable potency with that of quercetin.
Abstract: The consumption of 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) is known to cause severe hyperthermia and liver damage in humans. The thermogenic response induced by MDMA is complex and partially determined by the prevailing ambient temperature (AT). This is of extreme importance since ecstasy is often consumed at "rave" parties, where dancing takes place in a warm environment, which may exacerbate the effect of MDMA on thermoregulation. In view of the fact that hyperthermia is a well-known pro-oxidant aggressive condition, its potential role in ecstasy-induced hepatocellular toxicity should be further studied. Thus, the present study was performed in order to evaluate the influence of AT on the effects of single administration of MDMA on body temperature and liver toxicity in Charles River mice. Animals were given an acute intraperitoneal dose of MDMA (5, 10 or 20 mg/kg) and placed in AT of 20+/-2 degrees C or 30+/-2 degrees C for 24 h. Body temperature was measured during the study using implanted transponders and a temperature probe reading device. Plasma and liver samples were used for biochemical analysis. Liver sections were also taken for histological examination. The parameters evaluated were (1) plasma levels of transaminases and alkaline phosphatase, (2) hepatic glutathione (GSH), (3) hepatic lipid peroxidation, (4) activity of hepatic antioxidant enzymes (catalase, glutathione peroxidase, glutathione reductase, glutathione- S-transferase, copper/zinc superoxide dismutase and manganese superoxide dismutase), and (5) liver histology. The hyperthermic response elicited by MDMA was clearly dose-related and potentiated by high AT. Administration of MDMA produced some evidence of oxidative stress, expressed as GSH depletion at both ATs studied, as well as by lipid peroxidation and decreased catalase activity at high AT. High AT, by itself, decreased glutathione peroxidase activity. Histological examination of the liver revealed abnormalities of a dose- and AT-dependent nature. These changes included vacuolation of the hepatocytes, presence of blood clots and loss of typical hepatic cord organisation. The results obtained in the present study suggest that oxidative stress plays a part in the first stage of MDMA-induced liver damage and that liver antioxidant status is aggravated by increased AT. Thus, these findings are in accordance with the hypothesis that high AT may potentiate ecstasy-induced hepatotoxicity by increasing body hyperthermia.
Abstract: Sustained high levels of circulating catecholamines may induce cardiotoxicity. There is increasing evidence that this could result from catecholamine oxidation into aminochromes, which is catalyzed by transition metals. In fact, it has already been shown that copper-induced oxidation of the beta-agonist isoproterenol decreases the viability of isolated cardiomyocytes. Thus, the aim of this work was to contribute for the clarification of the mechanisms underlying the toxic effects of isoproterenol, Cu2+ and their concomitant effect in isolated rat cardiomyocytes. Freshly isolated calcium-tolerant cardiomyocytes from adult rat were incubated with 1 mM isoproterenol, 20 microM Cu2+ or with both during 4 h. Isoproterenol and its aminochrome (isoprenochrome), and reduced and oxidized glutathione were measured at each hour in the incubation medium and in the cells. The intracellular activities of the selenium-dependent glutathione peroxidase, glutathione reductase, and glutathione-S-transferase were determined after 4 h of incubation. Isoprenochrome was found in both cells and incubation medium in samples incubated with isoproterenol alone. However, in the isoproterenol plus Cu2+ samples, a greater depletion of isoproterenol accompanied by a proportional increase of isoprenochrome was observed. This higher ISO oxidation resulted in the depletion of intracellular glutathione and in the release of oxidized glutathione to the incubation medium. The content of total glutathione (intra- and extracellular) and the intracellular activity of the selenium-dependent glutathione peroxidase, glutathione reductase, and glutathione-S-transferase were also decreased in the isoproterenol plus Cu2+ samples. These results seem to indicate that the oxidative stress resulting from catecholamine/transition metal association may contribute to catecholamine cardiotoxicity.
Abstract: The metabolism of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has recently been implicated in the mechanisms underlying ecstasy-induced neurotoxicity and hepatotoxicity. However, its potential role in ecstasy-induced kidney toxicity has yet to be investigated. Thus, primary cultures of rat and human renal proximal tubular cells (PTCs) were used to investigate the cytotoxicity induced by MDMA and its metabolites methylenedioxyamphetamine (MDA), alpha-methyldopamine (alpha-MeDA), and the glutathione (GSH) conjugates 5-(glutathion- S-yl)-alpha-MeDA and 2,5- bis(glutathion- S-yl)-alpha-MeDA. Cell viability was evaluated using the mitochondrial MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. MDMA and MDA were not found to be toxic to either rat or human PTCs at any concentration tested (100-800 micro M). In contrast, 800 micro M alpha-MeDA caused 60% and 40% cell death in rat and human PTCs, respectively. Conjugation of alpha-MeDA with GSH resulted in the formation of even more potent nephrotoxicants. Thus, exposure of rat and human PTC monolayers to 400 micro M 5-(glutathion- S-yl)-alpha-MeDA caused approximately 80% and 70% cell death, respectively. 5-(Glutathion- S-yl)-alpha-MeDA (400 micro M) was more toxic than 2,5- bis(glutathion- S-yl)-alpha-MeDA to rat renal PTCs but equally potent in human renal PTCs. Pre-incubation of rat PTCs with either acivicin, an inhibitor of gamma-glutamyl transpeptidase (gamma-GT), or bestatin, an inhibitor of aminopeptidase M, resulted in increased toxicity of 5-(glutathion- S-yl)-alpha-MeDA but had no effect on 2,5- bis(glutathion- S-yl)-alpha-MeDA-mediated cytotoxicity. The present data provide evidence that metabolism is required for the expression of MDMA-induced renal toxicity in vitro. In addition, metabolism of 5-(glutathion- S-yl)-alpha-MeDA by gamma-GT and aminopeptidase M to the corresponding cystein- S-yl-glycine and/or cystein- S-yl conjugates is likely to be associated with detoxication of this compound. Thus, it appears that toxicity induced by thioether metabolites of ecstasy at the apical membrane of renal proximal tubular cells is the result of extracellular events, presumably redox cycling.
Abstract: The consumption of 3,4-methylenedioxymethamphetamine (ecstasy; MDMA) may cause hepatocellular damage in humans, a toxic effect that has been increasing in frequency in the last few years, although the underlying mechanisms are still unknown. The metabolism of MDMA involves the production of reactive metabolites which form adducts with intracellular nucleophilic sites, as is the case with glutathione (GSH). Also, MDMA administration elicits hyperthermia, a potentially deleterious condition that may aggravate its direct toxic effects. Thus, the objective of this study was to evaluate the extent of MDMA-induced depletion of GSH, induction of lipid peroxidation and loss of cell viability in freshly isolated mouse hepatocytes under normothermic conditions (37 degrees C) and to compare the results with the effects obtained under hyperthermic conditions (41 degrees C). By itself, hyperthermia was an important cause of cell toxicity. A rise in incubation temperature from 37 degrees C to 41 degrees C caused oxidative stress in freshly isolated mouse hepatocytes, reflected as a time-dependent induction of lipid peroxidation and consequent loss of cell viability (up to 40-45%), although the variations in GSH and GSSG levels were similar to those under normothermic conditions. MDMA (100, 200, 400, 800 and 1600 microM) induced a concentration- and time-dependent GSH depletion at 37 degrees C but had a negligible effect on lipid peroxidation and cell viability at this temperature. It is particularly noteworthy that hyperthermia (41 degrees C) potentiated MDMA-induced depletion of GSH, production of lipid peroxidation and loss of cell viability (up to 90-100%). It is therefore concluded that hyperthermia potentiates MDMA-induced toxicity in freshly isolated mouse hepatocytes.
