Browsing by Author "Guedes de Pinho, Paula"
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- 3,4-Methylenedioxymethamphetamine hepatotoxicity under the heat stress condition: novel insights from in vitro metabolomic studiesPublication . Araújo, Ana Margarida; Enea, Maria; Fernandes, Eduarda; Carvalho, Félix; de Lourdes Bastos, Maria; Carvalho, Márcia; Guedes de Pinho, PaulaHyperthermia has been extensively reported as a life-threatening consequence of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) abuse. In this work, we used a sensitive untargeted metabolomic approach based on gas chromatography-mass spectrometry to evaluate the impact of hyperthermia on the hepatic metabolic changes caused by MDMA. For this purpose, primary mouse hepatocytes were exposed to subtoxic (LC01 and LC10) and toxic (LC30) concentrations of MDMA for 24 h, at 37 or 40.5 °C (simulating body temperature increase after MDMA consumption), and alterations on both intracellular metabolome and extracellular volatilome were evaluated. Multivariate analysis showed that metabolic patterns clearly discriminate MDMA treated cells from control cells, both in normothermic and hyperthermic conditions. The metabolic signature was found to be largely common to MDMA subtoxic and toxic concentrations, although with evident differences in the magnitude of response, with metabolic changes significantly more pronounced at 40.5 °C. Discriminant metabolites associated with MDMA-induced hepatotoxicity are mostly involved in the amino acid metabolism, aminoacyl tRNA biosynthesis, glutathione metabolism, tricarboxylic acid cycle, and pyruvate metabolism. Moreover, our metabolomic findings were corroborated by classical toxicity parameters, demonstrating the high sensitivity of this omic approach to assess molecular-level effects. Overall, this study indicates that MDMA triggers significant metabolic alterations on hepatic cells, even at low concentrations, that are clearly exacerbated at high temperatures. These findings provide new metabolic pieces to solve the puzzle of MDMA's hepatotoxicity mechanism and emphasize the increased risks of MDMA abuse due to the thermogenic action of the drug.
- 3,4-Methylenedioxypyrovalerone (MDPV): in vitro mechanisms of hepatotoxicity under normothermic and hyperthermic conditionsPublication . Valente, Maria João; Araújo, Ana Margarida; Silva, Renata; Bastos, Maria de Lourdes; Carvalho, Félix; Guedes de Pinho, Paula; Carvalho, MárciaSynthetic cathinones have emerged in recreational drug markets as legal alternatives for classical amphetamines. Though currently banned in several countries, 3,4-methylenedioxypyrovalerone (MDPV) is one of the most commonly abused cathinone derivatives worldwide. We have recently reported the potential of MDPV to induce hepatocellular damage, but the underlying mechanisms responsible for such toxicity remain to be elucidated. Similar to amphetamines, a prominent toxic effect of acute intoxications by MDPV is hyperthermia. Therefore, the present in vitro study aimed to provide insights into cellular mechanisms involved in MDPV-induced hepatotoxicity and also evaluate the contribution of hyperthermia to the observed toxic effects. Primary cultures of rat hepatocytes were exposed to 0.2-1.6 mM MDPV for 48 h, at 37 or 40.5 °C, simulating the rise in body temperature that follows MDPV intake. Cell viability was measured through the MTT reduction and LDH leakage assays. Oxidative stress endpoints and cell death pathways were evaluated, namely the production of reactive oxygen and nitrogen species (ROS and RNS), intracellular levels of reduced (GSH) and oxidized (GSSG) glutathione, adenosine triphosphate (ATP) and free calcium (Ca(2+)), as well as the activities of caspases 3, 8 and 9, and nuclear morphological changes with Hoechst 33342/PI double staining. At 37 °C, MDPV induced a concentration-dependent loss of cell viability that was accompanied by GSH depletion, as one of the first signs of toxicity, observed already at low concentrations of MDPV, with negligible changes on GSSG levels, followed by accumulation of ROS and RNS, depletion of ATP contents and increases in intracellular Ca(2+) concentrations. Additionally, activation of caspases 3, 8, and 9 and apoptotic nuclear morphological changes were found in primary rat hepatocytes exposed to MDPV, indicating that this cathinone derivative activates both intrinsic and extrinsic apoptotic death pathways. The cytotoxic potential of MDPV and all the studied endpoints were markedly aggravated under hyperthermic conditions (40.5 °C). In conclusion, these data suggest that MDPV toxicity in primary rat hepatocytes is mediated by oxidative stress, subsequent to GSH depletion and increased ROS and RNS accumulation, mitochondrial dysfunction, and impairment of Ca(2+) homeostasis. Furthermore, the rise in body temperature subsequent to MDPV abuse greatly exacerbates its hepatotoxic potential.
- Advances and perspectives in prostate cancer biomarker discovery in the last 5 years through tissue and urine metabolomicsPublication . Lima, Ana Rita; Pinto, Joana; Amaro, Filipa; Bastos, Maria de Lourdes; Carvalho, Márcia; Guedes de Pinho, PaulaProstate cancer (PCa) is the second most diagnosed cancer in men worldwide. For its screening, serum prostate specific antigen (PSA) test has been largely performed over the past decade, despite its lack of accuracy and inability to distinguish indolent from aggressive disease. Metabolomics has been widely applied in cancer biomarker discovery due to the well-known metabolic reprogramming characteristic of cancer cells. Most of the metabolomic studies have reported alterations in urine of PCa patients due its noninvasive collection, but the analysis of prostate tissue metabolome is an ideal approach to disclose specific modifications in PCa development. This review aims to summarize and discuss the most recent findings from tissue and urine metabolomic studies applied to PCa biomarker discovery. Eighteen metabolites were found consistently altered in PCa tissue among different studies, including alanine, arginine, uracil, glutamate, fumarate, and citrate. Urine metabolomic studies also showed consistency in the dysregulation of 15 metabolites and, interestingly, alterations in the levels of valine, taurine, leucine and citrate were found in common between urine and tissue studies. These findings unveil that the impact of PCa development in human metabolome may offer a promising strategy to find novel biomarkers for PCa diagnosis.
- Analysis of extracellular metabolome by HS-SPME/GC–MS: optimization and application in a pilot study to evaluate galactosamine-induced hepatotoxicityPublication . Araújo, Ana Margarida; Moreira, Nathalie; Lima, Ana Rita; Bastos, Maria de Lourdes; Carvalho, Félix; Carvalho, Márcia; Guedes de Pinho, PaulaTwo methods based on headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS) were developed to study in vitro the volatile exometabolome, which were then further tested in a pilot study to evaluate galactosamine-induced hepatotoxicity. The analysis of volatile organic compounds (VOCs) was carried out directly in the headspace of the cell culture medium, while some other volatile organic compounds such as volatile carbonyl compounds (VCCs) (aldehydes and ketones) were determined in the headspace of the cell culture medium after a previous derivatization step with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA). Fiber selection was performed using a univariate mode, whereas a central composite design (CCD) was used in the optimization of several other parameters that affect the extraction conditions. VOCs showed optimal extraction results using a DVB/CAR/PDMS fiber, by adding 0.43 g of NaCl to a sample volume of 2 mL and allowing the sample to equilibrate for 10 min at 45 °C with a subsequent extraction for 39 min at the same temperature. For VCCs, the best extraction response was achieved after in-solution (2 mL) derivatization with 0.94 g L-1 of PFBHA (final concentration), followed by an incubation period of 6 min and an extraction time of 37 min at 53 °C, using a PDMS/DVB fiber. The applicability of both optimized methods was then tested, through a untargeted study, on cell culture medium samples obtained from primary mouse hepatocytes (PMH) exposed to three low concentrations (LC01, LC10 and LC30) of the well-known hepatotoxic agent galactosamine (GalN). The results obtained by both methods showed that volatile compounds from GalN exposed cells are separated from controls in a concentration-dependent manner. Several volatile compounds, namely aldehydes, ketones and alcohols, suffered significant alterations, suggesting that GalN induces marked metabolic alterations in cells even at low, non-toxic concentrations. Although preliminary, this metabolomics approach proved its potential to be used in future studies to evaluate toxicity of different xenobiotics.
- Analysis of volatile human urinary metabolome by solid-phase microextraction in combination with gas chromatography–mass spectrometry for biomarker discovery: application in a pilot study to discriminate patients with renal cell carcinomaPublication . Monteiro, Márcia; Carvalho, Márcia; Henrique, Rui; Jerónimo, Carmen; Moreira, Nathalie; Bastos, Maria de Lourdes; Guedes de Pinho, PaulaA new and simple analytical approach consisting of headspace-solid phase microextraction (HS-SPME) sampling coupled with gas chromatography-ion trap/mass spectrometry (GC-IT/MS) was developed to study the volatile human urinary metabolome. A central composite design (CCD) was used in the optimisation of extraction conditions. Fibre selection and evaluation of pH influence were performed using an univariate mode and the influence of other parameters, such as the time and temperature of extraction, time of incubation and salt addition, that affect the efficiency of the SPME sampling, was carried out using a CCD. With a sample volume of 2 mL, the optimal conditions in terms of total response values and reproducibility were achieved by performing analyses with a divinylbenzene/polydimethylsiloxane (DVB/PDMS) fibre, in an acidic pH (pH 2) with the addition of 0.59 g of NaCl, allowing the sample to equilibrate for 9 min and extracting at 68 °C for 24 min. The applicability of the optimised method was then tested in a pilot non-target analysis of urine samples obtained from patients with renal cell carcinoma (RCC) and healthy individuals. Chemometric unsupervised analyses performed on the volatile pattern acquired for these samples clearly showed the potential of volatile urinary metabolome to discriminate between RCC and control patients.
- Aplicação do volatiloma urinário no diagnóstico não invasivo do carcinoma de células renaisPublication . Pinto, Joana; Amaro, Filipa; Lima, Ana Rita; Carvalho-Maia, Carina; Jerónimo, Carmen; Henrique, Rui; Bastos, Maria de Lourdes; Carvalho, Márcia; Guedes de Pinho, PaulaO carcinoma de células renais do subtipo células claras (ccCCR) representa o tipo mais comum (~70%) de cancro renal. Investigações recentes sugerem que o desenvolvimento deste tipo de cancro está relacionado com alterações metabólicas induzidas por mutações que ocorrem em genes que controlam o metabolismo celular (p.ex., von Hippel–Lindau e fumarato hidratase). Deste modo, o estudo de potenciais biomarcadores de ccCCR, através de uma abordagem metabolómica, torna-se bastante pertinente e atual. Em particular, a fração volátil do metaboloma urinário tem revelado resultados muito promissores na identificação de painéis de biomarcadores com elevada sensibilidade para a deteção de cancros urológicos. Devido aos avanços consideráveis na área dos sensores bioelectrónicos, espera-se que num futuro próximo este tipo de cancro possa ser diagnosticado de uma forma simples, rápida e não invasiva em contexto clínico. Objetivos: Este trabalho teve como objetivo principal a identificação de um painel de biomarcadores para deteção não invasiva de ccCCR através da análise do perfil volátil da urina de doentes com ccCCR (n=75) e de indivíduos controlo (sem cancro, n=75). Material e Métodos: Os compostos orgânicos voláteis (COVs), e mais especificamente os compostos carbonílicos voláteis (CCVs), presentes na urina foram analisados por microextração em fase sólida por headspace e cromatografia gasosa acoplada à espectrometria de massa (HS-SPME-GC-MS). Os dados obtidos foram pré-processados e analisados com recurso a ferramentas bioinformáticas. Resultados: Os modelos de classificação obtidos para as análises de COVs e CCVs mostraram uma boa separação entre as urinas de doentes com ccCCR e controlos, com áreas sob a curva (AUC) de 0,846 e 0,818, respetivamente. No total, 22 compostos revelaram alterações estatisticamente significativas entre os dois grupos, incluindo vários aldeídos, cetonas, hidrocarbonetos aromáticos e terpenos. Foi ainda encontrado um conjunto de seis potenciais biomarcadores de ccCCR que revelou uma AUC de 0.869, sensibilidade de 80%, especificidade de 82% e exatidão de 81%. Deste painel fazem parte o octanal, 3-metilbutanal, benzaldeído, 2-furaldeído, 4-heptanona e p-cresol. As desregulações observadas nos níveis destes compostos sugerem alterações no metabolismo energético e uma maior expressão das enzimas ligadas a processos de carcinogénese. Conclusões: Estes resultados confirmam o potencial da assinatura volátil da urina para a deteção não invasiva de ccCCR e revelam um conjunto de biomarcadores moleculares que podem ser utilizados no desenvolvimento de novos materiais que permitam o seu reconhecimento por sensores bioelectrónicos, comummente chamados de “E-noses”.
- Biomarker discovery in human prostate cancer: an update in metabolomics studiesPublication . Lima, Ana Rita; Bastos, Maria de Lourdes; Carvalho, Márcia; Guedes de Pinho, PaulaProstate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer death among men in Western countries. Current screening techniques are based on the measurement of serum prostate specific antigen (PSA) levels and digital rectal examination. A decisive diagnosis of PCa is based on prostate biopsies; however, this approach can lead to false-positive and false-negative results. Therefore, it is important to discover new biomarkers for the diagnosis of PCa, preferably noninvasive ones. Metabolomics is an approach that allows the analysis of the entire metabolic profile of a biological system. As neoplastic cells have a unique metabolic phenotype related to cancer development and progression, the identification of dysfunctional metabolic pathways using metabolomics can be used to discover cancer biomarkers and therapeutic targets. In this study, we review several metabolomics studies performed in prostatic fluid, blood plasma/serum, urine, tissues and immortalized cultured cell lines with the objective of discovering alterations in the metabolic phenotype of PCa and thus discovering new biomarkers for the diagnosis of PCa. Encouraging results using metabolomics have been reported for PCa, with sarcosine being one of the most promising biomarkers identified to date. However, the use of sarcosine as a PCa biomarker in the clinic remains a controversial issue within the scientific community. Beyond sarcosine, other metabolites are considered to be biomarkers for PCa, but they still need clinical validation. Despite the lack of metabolomics biomarkers reaching clinical practice, metabolomics proved to be a powerful tool in the discovery of new biomarkers for PCa detection.
- Biomarkers in bladder cancer: a metabolomic approach using in vitro and ex vivo model systemsPublication . Rodrigues, Daniela; Jerónimo, Carmen; Henrique, Rui; Belo, Luís; Bastos, Maria de Lourdes; Guedes de Pinho, Paula; Carvalho, MárciaMetabolomics has recently proved to be useful in the area of biomarker discovery for cancers in which early diagnostic and prognostic biomarkers are urgently needed, as is the case of bladder cancer (BC). This article presents a comprehensive review of the literature on the metabolomic studies on BC, highlighting metabolic pathways perturbed in this disease and the altered metabolites as potential biomarkers for BC detection. Current disease model systems used in the study of BC metabolome include in vitro-cultured cancer cells, ex vivo neoplastic bladder tissues and biological fluids, mainly urine but also blood serum/plasma, from BC patients. The major advantages and drawbacks of each model system are discussed. Based on available data, it seems that BC metabolic signature is mainly characterized by alterations in metabolites related to energy metabolic pathways, particularly glycolysis, amino acid and fatty acid metabolism, known to be crucial for cell proliferation, as well as glutathione metabolism, known to be determinant in maintaining cellular redox balance. In addition, purine and pyrimidine metabolism as well as carnitine species were found to be altered in BC. Finally, it is emphasized that, despite the progress made in respect to novel biomarkers for BC diagnosis, there are still some challenges and limitations that should be addressed in future metabolomic studies to ensure their translatability to clinical practice.
- Biomarkers in renal cell carcinoma: a metabolomics approachPublication . Monteiro, Márcia Sá; Carvalho, Márcia; Bastos, Maria de Lourdes; Guedes de Pinho, PaulaSince Otto Warburg, many studies have explored the unique metabolic phenotype of cancer cells highlighting the value and applicability of metabolomics in the oncology field, particularly in the development of cancer biomarkers. With respect to renal cell carcinoma (RCC), a metabolomics approach would own a great potential since urinary system is intimately connected with urine and, this biofluid, offers some advantages allowing the development of an assay suitable for use in clinical practice. Moreover, the assessment of metabolic derangements characteristics of RCC might provide a complete health assessment of this pathology, enabling the development of novel targeted therapies and even the stratification of responsive patients to specific therapeutic options improving the effectiveness of therapy. Metabolomic studies performed so far showed that the RCC metabolic signature is characterized by alterations in metabolites related to energy metabolic pathways, particularly glycolysis, amino acid and fatty acid catabolism, known to be crucial to cell proliferation. Despite some of those alterations are common to carcinogenesis, the potential role of acylcarnitines, gentisate, α-ketoglutarate and quinolinate in RCC pathophysiology has been proposed recently. The ability of metabolomics to discriminate between RCC and normal samples shows convincing evidence of its applicability in RCC management. Furthermore, the studies already carried out have not only tried to unveil the metabolic profile of RCC but also to evaluate the impact of some factors, namely technical, in RCC-metabolomics research. This type of study is pivotal in the design of metabolomics studies, helping to increase the reliability of the results. The present review updates the current knowledge on the metabolic alterations recognized in the RCC, and provides insight to the characteristics, strengths, limitations, and recent advances in RCC-metabolomics studies, always keeping in mind its potential application in clinical/health areas as a biomarker discovery tool.
- Caracterização da assinatura metabólica do cancro da próstata em urinaPublication . Lima, Ana Rita; Pinto, Joana; Barros‑Silva, Daniela; Jerónimo, Carmen; Henrique, Rui; Bastos, Maria de Lourdes; Carvalho, Márcia; Guedes de Pinho, Paula