RESUMEN
Doxorubicin (DOX) is a potent chemotherapeutic agent used against several cancer types. However, due to its cardiotoxic adverse effects, the use of this drug may be also life-threatening. Although most cancer patients are elderly, they are poorly represented and evaluated in pre-clinical and clinical studies. Considering this, the present work aims to evaluate inflammation and oxidative stress as the main mechanisms of DOX-induced cardiotoxicity, in an innovative approach using an experimental model constituted of elderly animals treated with a clinically relevant human cumulative dose of DOX. Elderly (18-20 months) CD-1 male mice received biweekly DOX administrations, for 3 weeks, to reach a cumulative dose of 9.0 mg/kg. One week (1W) or two months (2 M) after the last DOX administration, the heart was collected to determine both drug's short and longer cardiac adverse effects. The obtained results showed that DOX causes cardiac histological damage and fibrosis at both time points. In the 1W-DOX group, the number of nuclear factor kappa B (NF-κB) p65 immunopositive cells increased and a trend toward increased NF-κB p65 expression was seen. An increase of inducible nitric oxide synthase (iNOS) and interleukin (IL)-33 and a trend toward increased IL-6 and B-cell lymphoma-2-associated X (Bax) expression were seen after DOX. In the same group, a decrease in IL-1ß, p62, and microtubule-associated protein 1A/1B-light chain 3 (LC3)-I, p38 mitogen-activated protein kinase (MAPK) expression was observed. Contrariwise, the animals sacrificed 2 M after DOX showed a significant increase in glutathione peroxidase 1 and Bax expression with persistent cardiac damage and fibrosis, while carbonylated proteins, erythroid-2-related factor 2 (Nrf2), NF-κB p65, myeloperoxidase, LC3-I, and LC3-II expression decreased. In conclusion, our study demonstrated that in an elderly mouse population, DOX induces cardiac inflammation, autophagy, and apoptosis in the heart in the short term. When kept for a longer period, oxidative-stress-linked pathways remained altered, as well as autophagy markers and tissue damage after DOX treatment, emphasizing the need for continuous post-treatment cardiac monitoring.
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Antioxidantes , Neoplasias , Animales , Masculino , Ratones , Antioxidantes/metabolismo , Apoptosis , Proteína X Asociada a bcl-2/metabolismo , Cardiotoxicidad/etiología , Doxorrubicina/farmacología , Fibrosis , Inflamación/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo , Transducción de SeñalRESUMEN
Mitoxantrone (MTX) is a topoisomerase II inhibitor used to treat a wide range of tumors and multiple sclerosis but associated with potential neurotoxic effects mediated by hitherto poorly understood mechanisms. In adult male CD-1 mice, the underlying neurotoxic pathways of a clinically relevant cumulative dose of 6 mg/kg MTX was evaluated after biweekly administration for 3 weeks and sacrifice 1 week after the last administration was undertaken. Oxidative stress, neuronal damage, apoptosis, and autophagy were analyzed in whole brain, while coronal brain sections were used for a closer look in the hippocampal formation (HF) and the prefrontal cortex (PFC), as these areas have been signaled out as the most affected in 'chemobrain'. In the whole brain, MTX-induced redox imbalance shown as increased endothelial nitric oxide synthase and reduced manganese superoxide dismutase expression, as well as a tendency to a decrease in glutathione levels. MTX also caused diminished ATP synthase ß expression, increased autophagic protein LC3 II and tended to decrease p62 expression. Postsynaptic density protein 95 expression decreased in the whole brain, while hyperphosphorylation of Tau was seen in PFC. A reduction in volume was observed in the dentate gyrus (DG) and CA1 region of the HF, while GFAP-ir astrocytes increased in all regions of the HF except in the DG. Apoptotic marker Bax increased in the PFC and in the CA3 region, whereas p53 decreased in all brain areas evaluated. MTX causes damage in the brain of adult CD-1 mice in a clinically relevant cumulative dose in areas involved in memory and cognition.
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Deterioro Cognitivo Relacionado con la Quimioterapia , Animales , Autofagia , Masculino , Ratones , Mitoxantrona/toxicidad , Neuronas , Estrés OxidativoRESUMEN
3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") is a recreational hallucinogenic drug of abuse known to elicit neurotoxic properties. Hepatic formation of neurotoxic metabolites is thought to play a major role in MDMA-related neurotoxicity, though the mechanisms involved are still unclear. Here, we studied the neurotoxicity mechanisms and stability of MDMA and 6 of its major human metabolites, namely α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA) and their correspondent glutathione (GSH) and N-acetyl-cysteine (NAC) conjugates, under normothermic (37 °C) or hyperthermic conditions (40 °C), using cultured SH-SY5Y differentiated cells. We showed that MDMA metabolites exhibited toxicity to SH-SY5Y differentiated cells, being the GSH and NAC conjugates more toxic than their catecholic precursors and MDMA. Furthermore, whereas the toxicity of the catechol metabolites was potentiated by hyperthermia, NAC-conjugated metabolites revealed higher toxicity under normothermia and GSH-conjugated metabolites-induced toxicity was temperature-independent. Moreover, a time-dependent decrease in extracellular concentration of MDMA metabolites was observed, which was potentiated by hyperthermia. The antioxidant NAC significantly protected against the neurotoxic effects of MDMA metabolites. MDMA metabolites increased intracellular glutathione levels, though depletion in thiol content was observed in MDMA-exposed cells. Finally, the neurotoxic effects induced by the MDMA metabolite N-Me-α-MeDA involved caspase 3 activation. In conclusion, this study evaluated the stability of MDMA metabolites in vitro, and demonstrated that the catechol MDMA metabolites and their GSH and NAC conjugates, rather than MDMA itself, exhibited neurotoxic actions in SH-SY5Y differentiated cells, which were differently affected by hyperthermia, thus highlighting a major role for reactive metabolites and hyperthermia in MDMA's neurotoxicity.
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Diferenciación Celular/efectos de los fármacos , Fiebre/inducido químicamente , N-Metil-3,4-metilenodioxianfetamina/metabolismo , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Neuronas/efectos de los fármacos , 3,4-Metilenodioxianfetamina/metabolismo , 3,4-Metilenodioxianfetamina/toxicidad , Acetilcisteína/metabolismo , Acetilcisteína/farmacología , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular/efectos de los fármacos , Desoxiepinefrina/análogos & derivados , Desoxiepinefrina/metabolismo , Desoxiepinefrina/toxicidad , Fiebre/metabolismo , Glutatión/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , N-Metil-3,4-metilenodioxianfetamina/farmacocinética , Neuronas/metabolismo , Síndromes de Neurotoxicidad/metabolismo , Síndromes de Neurotoxicidad/patología , TemperaturaRESUMEN
Doxorubicin (DOX) is a topoisomerase II inhibitor used in cancer therapy. Despite its efficacy, DOX causes serious adverse effects, such as short- and long-term cardiotoxicity. This work aimed to assess the short- and long-term cardiotoxicity of DOX and the role of inflammation and antioxidant defenses on that cardiotoxicity in a mice model. Adult CD-1 male mice received a cumulative dose of 9.0 mg/kg of DOX (2 biweekly intraperitoneal injections (ip), for 3 weeks). One week (1W) or 5 months (5M) after the last DOX administration, the heart was collected. One week after DOX, a significant increase in p62, tumor necrosis factor receptor (TNFR) 2, glutathione peroxidase 1, catalase, inducible nitric oxide synthase (iNOS) cardiac expression, and a trend towards an increase in interleukin (IL)-6, TNFR1, and B-cell lymphoma 2 associated X (Bax) expression was observed. Moreover, DOX induced a decrease on nuclear factor erythroid-2 related factor 2 (Nrf2) cardiac expression. In both 1W and 5M, DOX led to a high density of infiltrating M1 macrophages, but only the 1W-DOX group had a significantly higher number of nuclear factor κB (NF-κB) p65 immunopositive cells. As late effects (5M), an increase in Nrf2, myeloperoxidase, IL-33, tumor necrosis factor-α (TNF-α), superoxide dismutase 2 (SOD2) expression, and a trend towards increased catalase expression were observed. Moreover, B-cell lymphoma 2 (Bcl-2), cyclooxygenase-2 (COX-2), and carbonylated proteins expression decreased, and a trend towards decreased p38 mitogen-activated protein kinase (MAPK) expression were seen. Our study demonstrated that DOX induces adverse outcome pathways related to inflammation and oxidative stress, although activating different time-dependent response mechanisms.
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Cardiotoxicidad , Factor 2 Relacionado con NF-E2 , Ratones , Masculino , Animales , Cardiotoxicidad/etiología , Cardiotoxicidad/metabolismo , Cardiotoxicidad/patología , Factor 2 Relacionado con NF-E2/metabolismo , Catalasa/metabolismo , Cardiooncología , Doxorrubicina/efectos adversos , Estrés Oxidativo , Interleucina-6/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Inflamación/tratamiento farmacológico , ApoptosisRESUMEN
Paraquat (PQ) is an herbicide implicated in numerous fatalities, mainly caused by voluntary ingestion. Several methods have been used to quantify PQ in plasma and urine samples of intoxicated humans as a predictor of clinical outcome. There is no validated method for the analysis of PQ in postmortem samples. Therefore, the aim of this study was to develop an analytical method, using gas chromatography-ion trap mass spectrometry (GC-IT/MS) after solid-phase extraction, to quantify PQ in postmortem samples, namely in whole blood, urine, liver, lung and kidney, to cover the routes of distribution, accumulation and elimination of PQ. The method proved to be selective as there were no interferences of endogenous compounds with the same retention time as PQ and ethyl paraquat (internal standard). The regression analysis for PQ was linear in the range 0-10 µg/mL. The detection limits ranged from 0.0076 µg/mL for urine to 0.047 µg/mL for whole blood, and the recoveries were suitable for forensic analysis. The proposed GC-IT/MS method provided an accurate and simple assay with adequate precision and recovery for the quantification of PQ in postmortem samples. The proof of applicability was performed in two fatal PQ intoxications. A review of the analytical methods for the determination of quaternary ammonium herbicides is also provided for a better understanding of the presently available techniques.
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Cromatografía de Gases y Espectrometría de Masas/métodos , Paraquat/metabolismo , Cambios Post Mortem , Humanos , Límite de Detección , Paraquat/sangre , Paraquat/orinaRESUMEN
Current cancer therapies are successfully increasing the lifespan of cancer patients. Nevertheless, cardiotoxicity is a serious chemotherapy-induced adverse side effect. Doxorubicin (DOX) and mitoxantrone (MTX) are cardiotoxic anticancer agents, whose toxicological mechanisms are still to be identified. This study focused on DOX and MTX's cardiac mitochondrial damage and their molecular mechanisms. As a hypothesis, we also sought to compare the cardiac modulation caused by 9 mg/kg of DOX or 6 mg/kg of MTX in young adult mice (3 months old) with old control mice (aged control, 18-20 months old) to determine if DOX- and MTX-induced damage had common links with the aging process. Cardiac homogenates and enriched mitochondrial fractions were prepared from treated and control animals and analyzed by immunoblotting and enzymatic assays. Enriched mitochondrial fractions were also characterized by mass spectrometry-based proteomics. Data obtained showed a decrease in mitochondrial density in young adults treated with DOX or MTX and aged control, as assessed by citrate synthase (CS) activity. Furthermore, aged control had increased expression of the peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α) and manganese superoxide dismutase (MnSOD). Regarding the enriched mitochondrial fractions, DOX and MTX led to downregulation of proteins related to oxidative phosphorylation, fatty acid oxidation, amino acid metabolic process, and tricarboxylic acid cycle. MTX had a greater impact on malate dehydrogenase (MDH2) and pyruvate dehydrogenase E1 component subunit α (PDHA1). No significant proteomic changes were observed in the enriched mitochondrial fractions of aged control when compared to young control. To conclude, DOX and MTX promoted changes in several mitochondrial-related proteins in young adult mice, but none resembling the aged phenotype.
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Envejecimiento/efectos de los fármacos , Antibióticos Antineoplásicos/toxicidad , Antineoplásicos/toxicidad , Doxorrubicina/toxicidad , Mitocondrias Cardíacas/efectos de los fármacos , Mitoxantrona/toxicidad , Proteoma/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Citrato (si)-Sintasa/metabolismo , Masculino , Ratones , Mitocondrias Cardíacas/enzimología , Miocitos Cardíacos/patología , Tamaño de los Órganos/efectos de los fármacosRESUMEN
Doxorubicin (DOX) is a topoisomerase II inhibitor commonly used in the treatment of several types of cancer. Despite its efficacy, DOX can potentially cause fatal adverse effects, like cardiotoxicity. This work aimed to assess the role of inflammation in DOX-treated infant and adult mice and its possible link to underlying cardiotoxicity. Two groups of CD-1 male mice of different ages (infants or adults) were subjected to biweekly DOX administrations, to reach a cumulative dose of 18.0 mg/kg, which corresponds approximately in humans to 100.6 mg/m2 for infants and 108.9 mg/m2 for adults a clinically relevant dose in humans. The classic plasmatic markers of cardiotoxicity increased, and that damage was confirmed by histopathological findings in both groups, although it was higher in adults. Moreover, in DOX-treated adults, an increase of cardiac fibrosis was observed, which was accompanied by an increase in specific inflammatory parameters, namely, macrophage M1 and nuclear factor kappa B (NF-κB) p65 subunit, with a trend toward increased levels of the tumor necrosis factor receptor 2 (TNFR2). On the other hand, the levels of myeloperoxidase (MPO) and interleukin (IL)-6 significantly decreased in DOX-treated adult animals. In infants, a significant increase in cardiac protein carbonylation and in the levels of nuclear factor erythroid-2 related factor 2 (Nrf2) was observed. In both groups, no differences were found in the levels of tumor necrosis factor (TNF-α), IL-1ß, p38 mitogen-activated protein kinase (p38 MAPK) or NF-κB p52 subunit. In conclusion, using a clinically relevant dose of DOX, our study demonstrated that cardiac effects are associated not only with the intensity of the inflammatory response but also with redox response. Adult mice seemed to be more prone to DOX-induced cardiotoxicity by mechanisms related to inflammation, while infant mice seem to be protected from the damage caused by DOX, possibly by activating such antioxidant defenses as Nrf2.
Asunto(s)
Cardiotoxicidad , Animales , Doxorrubicina , Ratones , FN-kappa B , Estrés OxidativoRESUMEN
Mitoxantrone (MTX) is a pharmaceutical drug used in the treatment of several cancers and refractory multiple sclerosis (MS). Despite its therapeutic value, adverse effects may be severe, namely the frequently reported cardiotoxicity, whose mechanisms need further research. This work aimed to assess if inflammation or oxidative stress-related pathways participate in the cardiotoxicity of MTX, using the mouse as an animal model, at two different age periods (infant or adult mice) using two therapeutic relevant cumulative doses. Histopathology findings showed that MTX caused higher cardiac toxicity in adults. In MTX-treated adults, at the highest dose, noradrenaline cardiac levels decreased, whereas at the lowest cumulative dose, protein carbonylation increased and the expression of nuclear factor kappa B (NF-κB) p65 subunit and of M1 macrophage marker increased. Moreover, MTX-treated adult mice had enhanced expression of NF-κB p52 and tumour necrosis factor (TNF-α), while decreasing interleukin-6 (IL-6). Moreover, while catalase expression significantly increased in both adult and infant mice treated with the lowest MTX cumulative dose, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glutathione peroxidase only significantly increased in infant animals. Nevertheless, the ratio of GAPDH to ATP synthase subunit beta decreased in adult animals. In conclusion, clinically relevant doses of MTX caused dissimilar responses in adult and infant mice, being that inflammation may be an important trigger to MTX-induced cardiotoxicity.
RESUMEN
"Ecstasy" [(+/-)-3,4-methylenedioxymethamphetamine, MDMA, XTC, X, E] is a psychoactive recreational hallucinogenic substance and a major worldwide drug of abuse. Several reports raised the concern that MDMA has the ability to induce neurotoxic effects both in laboratory animals and humans. Despite more than two decades of research, the mechanisms by which MDMA is neurotoxic are still to be fully elucidated. MDMA induces serotonergic terminal loss in rats and also in some mice strains, but also a broader neuronal degeneration throughout several brain areas such as the cortex, hippocampus, and striatum. Meanwhile, in human "ecstasy" abusers, there are evidences for deficits in seronergic biochemical markers, which correlate with long-term impairments in memory and learning. There are several factors that contribute to MDMA-induced neurotoxicity, namely, hyperthermia, monoamine oxidase metabolism of dopamine and serotonin, dopamine oxidation, the serotonin transporter action, nitric oxide, and the formation of peroxinitrite, glutamate excitotoxicity, serotonin 2A receptor agonism, and, importantly, the formation of MDMA neurotoxic metabolites. The present review covered the following topics: history and epidemiology, pharmacological mechanisms, metabolic pathways and the influence of isoenzyme genetic polymorphisms, as well as the acute effects of MDMA in laboratory animals and humans, with a special focus on MDMA-induced neurotoxic effects at the cellular and molecular level. The main aim of this review was to contribute to the understanding of the cellular and molecular mechanisms involved in MDMA neurotoxicity, which can help in the development of therapeutic approaches to prevent or treat the long-term neuropsychiatric complications of MDMA abuse in humans.
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N-Metil-3,4-metilenodioxianfetamina/farmacología , Síndromes de Neurotoxicidad/metabolismo , Síndromes de Neurotoxicidad/patología , Animales , Humanos , N-Metil-3,4-metilenodioxianfetamina/química , N-Metil-3,4-metilenodioxianfetamina/farmacocinética , Polimorfismo Genético , Factores de TiempoRESUMEN
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.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Desoxiepinefrina/análogos & derivados , Epinefrina/metabolismo , Glutatión/análogos & derivados , Miocitos Cardíacos/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Acridinas/farmacología , Animales , Catecolaminas/metabolismo , Catecolaminas/toxicidad , Corticosterona/farmacología , Desoxiepinefrina/metabolismo , Glutatión/metabolismo , Masculino , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Tetrahidroisoquinolinas/farmacologíaRESUMEN
5,7-Dihydroxytryptamine (5,7-DHT), is an experimentally widely used selective serotonergic neurotoxin, though the mechanisms of toxicity remain to be fully elucidated. In the present study, we evaluated 5,7-dihydroxitryptamine (5,7-DHT) induced serotonergic neurotoxicity in foetal raphe serum free cultures from the rat. For this purpose, a model of foetal raphe serum free neuronal cultures from the rat was established, containing about 16% serotonergic neurons and studied up to 3 months. Two weeks old raphe cultures were exposed to the serotonergic neurotoxin 5,7-DHT (concentration range 10-100 microM) for 72 h, after which the medium was replaced and neurotoxicity was evaluated by immunocitochemistry 1 week later. Lactate dehydrogenase release into the medium, 72 h after exposure to 5,7-DHT, showed a concentration-dependent neurotoxicity. To access morphologically the serotonergic toxicity tryptophan hydroxylase (TPH) was used as a specific marker of these neurons. Immunocitochemistry using TPH antisera showed a concentration-dependent serotonergic neurotoxicity induced by 5,7-DHT. Serotonergic neurons showed the typical pattern of "pruning" accompanied by axon terminals and dendrites loss, which were either partial or total. The axotomy induced by the neurotoxin was morphologically characteristic of retrograde axonal degeneration. Fluoxetine (0.1 microM) pre-treatment reduced 5,7-DHT-induced serotonergic neurotoxicity. These results indicate that the mechanism by which 5,7-DHT-induces serotonergic neurotoxicity is, at least partially, dependent on the toxin uptake by the serotonin transporter. Finally, we have established a robust model of primary raphe neuronal culture to evaluate serotonergic neurons development and the mechanisms of toxicity involving this neuronal population.
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5,7-Dihidroxitriptamina/toxicidad , Núcleos del Rafe/efectos de los fármacos , Animales , Células Cultivadas , Medio de Cultivo Libre de Suero , Relación Dosis-Respuesta a Droga , Fluoxetina/farmacología , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Degeneración Nerviosa , Fibras Nerviosas/efectos de los fármacos , Núcleos del Rafe/patología , RatasRESUMEN
Ethanol is frequently consumed along with 3,4-methylenedioxymethamphetamine (MDMA; ecstasy). Since both compounds are hepatotoxic and are metabolized in the liver, an increased deleterious interaction resulting from the concomitant use of these two drugs seems plausible. Another important feature of MDMA-induced toxicity is hyperthermia, an effect known to be potentiated after continuous exposure to ethanol. Considering the potential deleterious interaction, the aim of the present study was to evaluate the hepatotoxic effects of ethanol and MDMA mixtures to primary cultured rat hepatocytes and to elucidate the mechanism(s) underlying this interaction. For this purpose, the toxicity induced by MDMA to primary cultured rat hepatocytes in absence or in presence of ethanol was evaluated, under normothermic (36.5 degrees C) and hyperthermic (40.5 degrees C) conditions. While MDMA and ethanol, by themselves, had discrete effects on the analysed parameters, which were slightly aggravated under hyperthermia, the simultaneous incubation of MDMA and ethanol for 24h, resulted in high cell death ratios accompanied by a significant disturbance of cellular redox status and decreased energy levels. Evaluation of apoptotic/necrotic features provided clear evidences that the cell death occurs preferentially through a necrotic pathway. All the evaluated parameters were dramatically aggravated when cells were incubated under hyperthermia. In conclusion, co-exposure of hepatocytes to ethanol and MDMA definitely results in a synergism of the hepatotoxic effects, through a disruption of the cellular redox status and enhanced cell death by a necrotic pathway in a temperature-dependent extent.
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Etanol/toxicidad , Hepatocitos/efectos de los fármacos , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Adenosina Trifosfato/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Sinergismo Farmacológico , Calor , Masculino , Estrés Oxidativo , Ratas , Ratas WistarRESUMEN
3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) is an amphetamine derivative drug with entactogenic, empathogenic and hallucinogenic properties, commonly consumed at rave parties in a polydrug abuse pattern, especially with cannabis, tobacco and ethanol. Since both MDMA and ethanol may cause deleterious effects to the liver, the evaluation of their putative hepatotoxic interaction is of great interest, especially considering that most of the MDMA users are regular ethanol consumers. Thus, the aim of the present study was to evaluate, in vivo, the acute hepatotoxic effects of MDMA (10mg/kg i.p.) in CD-1 mice previously exposed to 12% ethanol as drinking fluid (for 8 weeks). Body temperature was continuously measured for 12h after MDMA administration and, after 24h, hepatic damage was evaluated. The administration of MDMA to non pre-treated mice resulted in sustained hyperthermia, which was significantly increased in ethanol pre-exposed mice. A correspondent higher increase of hepatic heat shock transcription factor (HSF-1) activation was also observed in the latter group. Furthermore, MDMA administration resulted in liver damage as confirmed by histological analysis, slight decrease in liver weight and increased plasma transaminases levels. These hepatotoxic effects were also exacerbated when mice were pre-treated with ethanol. The activities of some antioxidant enzymes (such as SOD, GPx and Catalase) were modified by ethanol, MDMA and their joint action. The hepatotoxicity resulting from the simultaneous exposure to MDMA and ethanol was associated with a higher activation of NF-kappaB, indicating a pro-inflammatory effect in this organ. In conclusion, the obtained results strongly suggest that the consumption of ethanol increases the hyperthermic and hepatotoxic effects associated with MDMA abuse.
Asunto(s)
Depresores del Sistema Nervioso Central/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Etanol/toxicidad , Fiebre/inducido químicamente , Alucinógenos/toxicidad , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Alanina Transaminasa/sangre , Animales , Antioxidantes/metabolismo , Aspartato Aminotransferasas/sangre , Temperatura Corporal/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Sinergismo Farmacológico , Ensayo de Cambio de Movilidad Electroforética , Fiebre/fisiopatología , Glutatión/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Masculino , Ratones , Microscopía Electrónica de Transmisión , FN-kappa B/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Carbonilación Proteica/efectos de los fármacosRESUMEN
3,4-Methylenedioxymethamphetamine (MDMA) is frequently consumed at "rave" parties by polydrug users that usually take this drug in association with ethanol. In addition, many young people are repeatedly exposed to ethanol, which likely leads to tolerance phenomena. Both compounds are metabolized in the liver, with formation of hepatotoxic metabolites, which gives high relevance to the evaluation of their putative toxicological interaction. Therefore, the aim of this study was to evaluate the toxicity induced by 0.8 and 1.6 mM MDMA to freshly isolated hepatocytes obtained from ethanol-treated mice whose tap drinking water was replaced by a 5% ethanol solution for 1 week and, afterwards, by a 12% ethanol solution for 8 weeks (ethanol group) comparatively to non-treated animals (non-ethanol group). The hepatocytes were incubated under normothermic and hyperthermic conditions in order to simulate in vitro the hyperthermic response induced in vivo by MDMA, a condition that has been recognized as a life-threatening effect associated with MDMA exposure and implicated in its hepatotoxicity. Six mice treated under the same protocol as the ethanol group were used for histological analysis, and compared to non-ethanol-treated animals. The pre-treatment of mice with ethanol caused a significant decrease in the hepatocytes yield in the isolation procedure comparatively to the non-ethanol group, which can be explained by an increase in collagen deposition along the hepatic parenchyma as observed in the histological analysis. The initial cell viability of hepatocytes suspensions was similar between ethanol and non-ethanol groups. However, the ethanol group showed a higher GSH oxidation rate, which was enhanced under hyperthermia. Additionally, a concentration-dependent MDMA-induced loss of cell viability and ATP depletion was observed for both groups, at 41 degrees C. In conclusion, the repeated treatment with ethanol seems to increase the vulnerability of freshly isolated mice hepatocytes towards pro-oxidant conditions, as ascertained by the increase in collagen deposition, lower hepatocyte yield and decreased glutathione levels. However, MDMA toxicity to the isolated hepatocytes was independent of ethanol pre-treatment, while significantly dependent on incubation temperature.
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Consumo de Bebidas Alcohólicas/efectos adversos , Etanol/toxicidad , Hepatocitos/efectos de los fármacos , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Adenosina Trifosfato/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Fiebre/metabolismo , Glutatión/efectos de los fármacos , Glutatión/metabolismo , Hepatocitos/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Ratones , N-Metil-3,4-metilenodioxianfetamina/administración & dosificación , Oxidación-Reducción/efectos de los fármacos , TemperaturaRESUMEN
Pixantrone (PIX) is an anticancer drug approved for the treatment of multiple relapsed or refractory aggressive B-cell non-Hodgkin's lymphoma. It is an aza-anthracenedione synthesized to have the same anticancer activity as its predecessors, anthracyclines (e.g. doxorubicin) and anthracenediones (e.g. mitoxantrone), with lower cardiotoxicity. However, published data regarding its possible cardiotoxicity are scarce. Therefore, this work aimed to assess the potential cytotoxicity of PIX, at clinically relevant concentrations (0.1; 1; and 10 µM) in both non-differentiated and 7-day differentiated H9c2 cells. Cells were exposed to PIX for 48 h and cytotoxicity was evaluated through phase contrast microscopy, Hoescht staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and neutral red (NR) uptake assays. Cytotoxicity was observed in differentiated and non-differentiated H9c2 cells, with detached cells and round cells evidenced by phase contrast microscopy, mainly at the highest concentration tested (10 µM). In the Hoechst staining, PIX 10 µM showed a marked decrease in the number of cells when compared to control but with no signs of nuclear condensation. Furthermore, significant concentration-dependent mitochondrial dysfunction was observed through the MTT reduction assay. The NR assay showed similar results to those obtained in the MTT reduction assay in both differentiated and non-differentiated H9c2 cells. The differentiation state of the cells was not crucial to PIX effects, although PIX toxicity was slightly higher in differentiated H9c2 cells. To the best of our knowledge, this was the first in vitro study performed with PIX in H9c2 cells and it discloses worrying cytotoxicity at clinically relevant concentrations.
RESUMEN
Abstract Prolonged overexposure to catecholamines causes toxicity, usually credited to continuous adrenoceptor stimulation, autoxidation, and the formation of reactive pro-oxidant species. Non-differentiated SH-SY5Y cells were used to study the possible contribution of oxidative stress in adrenaline (ADR)-induced neurotoxicity, as a model to predict the toxicity of this catecholamine to peripheral nerves. Cells were exposed to several concentrations of ADR (0.1, 0.25, 0.5 and 1mM) and two cytotoxicity assays [lactate dehydrogenase (LDH) release and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction] were performed at several time-points (24, 48, and 96h). The cytotoxicity of ADR was concentration- and time-dependent in both assays, since the lowest concentration tested (0.1mM) also caused significant cytotoxicity at 96h. N-acetyl-cysteine (1mM), a precursor of glutathione synthesis, prevented ADR-induced toxicity elicited by 0.5mM and 0.25mM ADR following a 96-h exposure, while the antioxidant Tiron (100µM) was non-protective. In conclusion, ADR led to mitochondrial distress and ultimately cell death in non-differentiated SH-SY5Y cells, possibly because of ADR oxidation products. The involvement of such processes in the catecholamine-induced peripheral neuropathy requires further analysis.
Asunto(s)
Epinefrina/agonistas , Enfermedades del Sistema Nervioso Periférico/clasificación , Toxicidad , Neuronas/clasificación , Nervios Periféricos/anomalías , Bromuros/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacos , Antioxidantes/farmacologíaRESUMEN
3,4-Methylenedioxymethamphetamine (MDMA or "Ecstasy") is a psychoactive and hallucinogenic drug of abuse. MDMA has been shown to produce neurotoxicity both in animals and humans. MDMA and other amphetamines induce serotonergic and dopaminergic terminal neurotoxicity and also neurodegeneration in areas including the cortex, hippocampus, striatum and thalamus. Herein, we investigated the mechanisms involved in MDMA-induced neurotoxicity to neuronal serum free cultures from rat cortex. The hyperthermic effect produced by MDMA has been shown to be a clinically relevant aspect for the neurotoxic events. Thus, MDMA-induced toxicity to cortical neurons was evaluated both under normothermic (36.5 degrees C) and hyperthermic (40 degrees C) conditions. Our findings showed that MDMA produced neuronal apoptosis, accompanied by activation of caspase 3, in a concentration dependent manner. MDMA neurotoxicity was completely prevented by pre-treatment with a 5-HT(2A)-receptor antibody, which acted as an "irreversible non-competitive antagonist" of this receptor. Furthermore, MDMA depleted intracellular glutathione (GSH) levels in a concentration dependent manner, an effect that was attenuated by Ketanserin, a competitive 5-HT(2A)-receptor antagonist. Accordingly, N-acetylcysteine, an antioxidant and GSH precursor, also reduced MDMA-induced toxicity. Specific inhibitors of the inducible and neuronal nitric oxide synthase (NOS) partially prevented MDMA neurotoxicity, ascertaining the involvement of reactive nitrogen species, in the toxic effect. In conclusion, direct MDMA 5-HT(2A)-receptor stimulation produces intracellular oxidative stress that leads to neuronal apoptosis accompanied by caspase 3 activation.
Asunto(s)
Apoptosis/efectos de los fármacos , Corteza Cerebral/citología , N-Metil-3,4-metilenodioxianfetamina/farmacología , Neuronas/efectos de los fármacos , Receptor de Serotonina 5-HT2A/fisiología , Serotoninérgicos/farmacología , Animales , Anticuerpos/farmacología , Caspasa 3/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Estrés Oxidativo/efectos de los fármacos , Ratas , Receptor de Serotonina 5-HT2A/inmunologíaRESUMEN
An electrothermal atomization atomic absorption spectrometry method was validated to quantify residues of copper, aluminum, cadmium, chromium, iron, lead, and nickel in olive fruit. The linearity ranges under the optimized conditions were 0.19-20.0, 1.11-50.0, 0.02-2.0, 0.15-20.0, 0.80-20.0, 0.35-50.0, and 0.60-50.0 microg/L, respectively. The limits of quantification were, expressed in nanograms per gram of dry weight, 12.6, 74.0, 1.34, 10.0, 53.4, 23.4, and 40.0, respectively. For all of the metals the precision of the instrumental method was <6.3% and that of the analytical method was always <10%, except for aluminum, for which the precision was 12%. The accuracy of the method was evaluated according to the standard additions method, the recoveries being >90% for all of the added concentrations. An interference study was also carried out in a simulated matrix, and it was verified that the deviations of the expected values were <6% for all of the metals. The method was applied to the monitoring of the residues of the referred metals in olive fruits collected from trees pulverized with three different copper formulations available on the market to control fungal diseases.
Asunto(s)
Cobre/análisis , Frutas/química , Metales/análisis , Olea/crecimiento & desarrollo , Espectrofotometría Atómica/métodos , Cobre/administración & dosificación , Fungicidas Industriales/administración & dosificación , Olea/químicaRESUMEN
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.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Desoxiepinefrina/análogos & derivados , Desoxiepinefrina/antagonistas & inhibidores , Desoxiepinefrina/toxicidad , Depuradores de Radicales Libres/farmacología , Hepatocitos/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Separación Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Glutatión Transferasa/metabolismo , Alucinógenos/antagonistas & inhibidores , Alucinógenos/toxicidad , Hepatocitos/enzimología , Técnicas In Vitro , Masculino , N-Metil-3,4-metilenodioxianfetamina/antagonistas & inhibidores , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Ratas , Espectrofotometría UltravioletaRESUMEN
"Ecstasy" (3,4-methylenedioxymethamphetamine or MDMA) is a widely abused recreational drug, reported to produce neurotoxic effects, both in laboratory animals and in humans. MDMA metabolites can be major contributors for MDMA neurotoxicity. This work studied the neurotoxicity of MDMA and its catechol metabolites, α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA) in human dopaminergic SH-SY5Y cells differentiated with retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate. Differentiation led to SH-SY5Y neurons with higher ability to accumulate dopamine and higher resistance towards dopamine neurotoxicity. MDMA catechol metabolites were neurotoxic to SH-SY5Y neurons, leading to caspase 3-independent cell death in a concentration- and time-dependent manner. MDMA did not show a concentration- and time-dependent death. Pre-treatment with the antioxidant and glutathione precursor, N-acetylcysteine (NAC), resulted in strong protection against the MDMA metabolites' neurotoxicity. Neither the superoxide radical scavenger, tiron, nor the inhibitor of the dopamine (DA) transporter, GBR 12909, prevented the metabolites' toxicity. Cells exposed to α-MeDA showed an increase in intracellular glutathione (GSH) levels, which, at the 48 h time-point, was not dependent in the activity increase of γ-glutamylcysteine synthetase (γ-GCS), revealing a possible transient effect. Importantly, pre-treatment with buthionine sulfoximine (BSO), an inhibitor of γ-GCS, prevented α-MeDA induced increase in GSH levels, but did not augment this metabolite cytotoxicity. Even so, BSO pre-treatment abolished NAC protective effects against α-MeDA neurotoxicity, which were, at least partially, due to GSH de novo synthesis. Inversely, pre-treatment of cells with BSO augmented N-Me-α-MeDA-induced neurotoxicity, but only slightly affected NAC neuroprotection. In conclusion, MDMA catechol metabolites promote differential toxic effects to differentiated dopaminergic human SH-SY5Y cells.