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In vitro neurotoxicity evaluation of piperazine designer drugs in differentiated human neuroblastoma SH-SY5Y cells

Publication . Arbo, M.D.; Silva, R.; Barbosa, D.J.; da Silva, D. Dias; Silva, S.P.; Teixeira, João Paulo; Bastos, M.L.; Carmo, H.

Abuse of synthetic drugs is widespread worldwide. Studies indicate that piperazine designer drugs act as substrates at dopaminergic and serotonergic receptors and/or transporters in the brain. This work aimed to investigate the cytotoxicity of N-benzylpiperazine, 1-(3-trifluoromethylphenyl)piperazine, 1-(4-methoxyphenyl)piperazine and 1-(3,4-methylenedioxybenzyl)piperazine in the differentiated human neuroblastoma SH-SY5Y cell line. Cytotoxicity was evaluated after 24 h incubations through the MTT reduction and neutral red uptake assays. Oxidative stress (reactive oxygen and nitrogen species production and glutathione content) and energetic (ATP content) parameters, as well as intracellular Ca(2+) , mitochondrial membrane potential, DNA damage (comet assay) and cell death mode were also evaluated. Complete cytotoxicity curves were obtained after 24 h incubations with each drug. A significant decrease in intracellular total glutathione content was noted for all the tested drugs. All drugs caused a significant increase of intracellular free Ca(2+) levels, accompanied by mitochondrial hyperpolarization. However, ATP levels remained unchanged. The investigation of cell death mode revealed a predominance of early apoptotic cells. No genotoxicity was found in the comet assay. Among the tested drugs, 1-(3-trifluoromethylphenyl)piperazine was the most cytotoxic. Overall, piperazine designer drugs are potentially neurotoxic, supporting concerns on risks associated with the abuse of these drugs. Copyright © 2015 John Wiley & Sons, Ltd.

2015-04-20Journal article

Restricted access

Protective effect of Crataegus monogyna Jacq. ethanolic extracts in oxidant-induced DNA damage evaluated through comet assay with human peripheral lymphocytes

Publication . Barreira, Joao; Costa, Carla; Teixeira, João Paulo; Ferreira, Isabel; Oliveira, Beatriz

Much attention of preventive medicine research is focused on natural antioxidants. This interest refers not only to isolation and identification of new biologically active molecules for the pharmaceutical industry, but also because of the emergent public interest in using crude plant extracts, such as infusions for self-medication (Krishnaiah et al., 2011). The use of antioxidants, such as the well-known polyphenolic compounds, to prevent genetic damage induced by physical or chemical agents is of considerable interest. This bioactivity might be related to their anticlastogenic effect, due to the presence of specific functional groups. Other antioxidant compounds, such as vitamins C and D, were reported for their DNA-damage decreasing effect, suggesting that reactive oxygen species may be involved in this activity (Benavente-García et al., 2004). Evaluating the antioxidant activity of natural matrices represents one of our primary research challenges (Ferreira et al., 2009). Among hundreds of studied species, Crataegus monogyna Jacq. stood out as being one of the most promising plants due to its high bioactivity. Besides the antioxidant activity, C. monogyna was also studied for the human tumour cells growth inhibitory capacity of its phenolic extracts; furthermore, individual phenolic compounds were fully characterized by high performance liquid chromatography-photo diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI/ MS), revealing high levels of flavonols, flavones, hydroxycinnamic acid derivatives and anthocyanins (Rodrigues et al., 2012). However, there is a high limitation in examining if the detected bioactivity is actually transferred from in vitro to in vivo systems (Carocho and Ferreira, 2013).

2015-09Conference object

Open access

Chemical characterization and in vitro cyto- and genotoxicity of ‘legal high’ products containing Kratom (Mitragyna speciosa)

Publication . Oliveira, Ana Sofia; Fraga, Sónia; Carvalho, Félix; Araújo, Ana Margarida; Pereira, Cristiana Costa; Teixeira, João Paulo; de Lourdes Bastos, Maria; de Pinho, Paula Guedes

Kratom is a popular ‘legal high’ mainly constituted by alkaloids extracted from the Mitragyna speciosa plant with mitragynine (MG) as the dominant active substance. The increasing use of Kratom for recreational purposes has alerted risk assessment bodies of the lack of information on the real composition and its potential health risks. The present study aimed to determine and compare the MG composition of 13 commercial products of Kratom sold online and in “smartshops”, by gas chromatography–mass spectrometry. For the first time, the cytotoxicity induced by pure MG and Kratom, extracts was evaluated in in vitro models of human intestinal (Caco-2) and neuronal (SH-SY5Y) cells after 6 and 24 h. Genotoxicity was also evaluated in intestinal Caco-2 cells following 24 h of exposure to subtoxic concentrations using the comet assay. The obtained results revealed an inconsistency between the information (‘power’) provided in labels and the MG content. Cytotoxicity tests revealed a concentration-dependent decrease in cell viability in both cellular models, with the SH-SY5Y cells being more sensitive to the Kratom extracts. The resin and the ‘powered extracts’ were the most cytotoxic samples, with IC50 values significantly lower than the leaf extracts and pure MG (P < 0.0001 vs. leaf extracts and MG). In addition, significant DNA damage was observed in Caco-2 cells exposed to these extracts but not to pure MG, which suggests that other substances present in the extracts or interactions involving Kratom components might be responsible for the observed effects.

2016-07Journal article

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Resistência de Clostridium difficile do ribotipo 017 ao imipenemo: contributo da sequenciação do genoma completo

Publication . Isidro, Joana; Santos, Andrea; Nunes, Alexandra; Borges, Vítor; Silva, Catarina; Vieira, Luís; Mendes, Aristides L; Serrano, Mónica; Henriques, Adriano O; Gomes, João Paulo; Oleastro, Mónica

A infeção por Clostridium difficile é a principal causa de diarreia infeciosa associada aos cuidados de saúde. Neste estudo, caracterizámos um conjunto de estirpes clínicas de Clostridium difficile, provenientes de diversos hospitais portugueses, com o objetivo de estudar a resistência aos carbapenemos neste agente patogénico. Um total de 191 estirpes clínicas, isoladas entre 2012 e 2015 de 15 hospitais em Portugal, foram incluídas no estudo; a suscetibilidade ao imipenemo foi determinada por um método de gradiente de difusão em agar. Foram selecionadas estirpes sensíveis e resistentes ao imipenemo, para estudos fenotípicos adicionais e para contributo da sequenciação do genoma completo. A resistência ao imipenemo foi detetada em 24 (12,6%) das estirpes, 22 das quais pertencentes ao ribotipo (RT) 017 (apenas toxina B positivo), todas provenientes do mesmo hospital, durante o período em estudo, e com perfil de multiresistência. Pela análise dos dados de sequenciação dos genomas, foram identificadas duas substituições de aminoácidos (Ala555Thr e Tyr721Ser) nos domínios funcionais de duas enzimas envolvidas na síntese do peptidoglicano (penicillin-binding proteins - PBP). Uma PBP adicional foi também identificada nas estirpes RT017. Este estudo descreve pela primeira vez alterações em PBPs como base genética provável da resistência ao imipenemo em C. difficile.

2018-12Journal article

Open access