Loading...
122 results
Search Results
Now showing 1 - 10 of 122
- Phytochemical profiles and inhibitory effect on free radical-induced human erythrocyte damage of Dracaena draco leaf: a potential novel antioxidant agentPublication . Santos, Rui P.; Mendes, Lídia S.; Silva, Branca M.; Guedes de Pinho, Paula; Valentão, Patrícia; Andrade, Paula B.; Pereira, José A.; Carvalho, MárciaThe present study reports for the first time the metabolite profile and antioxidant activity of aqueous extract obtained from Dracaena draco L. leaf. Volatiles profile was determined by HS-SPME/GC-IT-MS, with 34 compounds being identified, distributed by distinct chemical classes: 2 alcohols, 5 aldehydes, 16 carotenoid derivatives and 8 terpenic compounds. Carotenoid derivative compounds constituted the most abundant class in leaf (representing 45% of total identified compounds). Phenolics profile was determined by HPLC/DAD and 9 constituents were identified: 2 hydroxycinnamic acid derivatives – 5-O-caffeoylquinic and 3,5-O-dicaffeoylquinic acids; 4 hydroxycinnamic acids – caffeic, p-coumaric, ferulic and sinapic acids and 3 flavonol glycosides – quercetin-3-O-rutinoside, kaempferol-3-O-glucoside and kaempferol-3-O-rutinoside. The most abundant phenolic compound is quercetin-3-O-rutinoside (representing 50.2% of total polyphenols). Organic acids composition was also characterised, by HPLC–UV and oxalic, citric, malic and fumaric acids were determined. Oxalic and citric acids were present in higher amounts (representing 47%, each). The antioxidant potential of this material was assessed by the ability to protect against free radical-induced biomembrane damage, using human erythrocyte as in vitro model. Leaf extract strongly protected the erythrocyte membrane from haemolysis (IC50 of 39 ± 11 μg/ml), in a time- and concentration-dependent manner. This is the first report showing that D. draco leaf is a promising antioxidant agent.
- Oxidation process of adrenaline in freshly isolated rat cardiomyocytes: formation of adrenochrome, quinoproteins, and GSH adductPublication . Costa, Vera Marisa; Silva, Renata; Ferreira, Luísa Maria; Branco, Paula Sério; Carvalho, Félix; Bastos, Maria de Lourdes; Carvalho, Rui Albuquerque; Carvalho, Márcia; Remião, FernandoHigh 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.
- Optimisation and validation of a HS-SPME–GC–IT/MS method for analysis of carbonyl volatile compounds as biomarkers in human urine: application in a pilot study to discriminate individuals with smoking habitsPublication . Calejo, Isabel; Moreira, Nathalie; Araújo, Ana Margarida; Carvalho, Márcia; Bastos, Maria de Lourdes; Guedes de Pinho, PaulaA new and simple analytical approach consisting of an automated headspace solid-phase microextraction (HS-SPME) sampler coupled to gas chromatography-ion trap/mass spectrometry detection (GC-IT/MS) with a prior derivatization step with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) was developed to detect volatile carbonyl metabolites with low molecular weights in human urine. A central composite design (CCD) was used to optimise the PFBHA concentration and extraction conditions that affect the efficiency of the SPME procedure. With a sample volume of 1 mL, optimal conditions were achieved by adding 300 mg/L of PFBHA and allowing the sample to equilibrate for 6 min at 62°C and then extracting the samples for 51 min at the same temperature, using a divinylbenzene/polydimethylsiloxane (DVB/PDMS) fibre. The method allowed the simultaneous identification and quantification of 44 carbonyl compounds consisting of aldehydes, dialdehydes, heterocyclic aldehydes and ketones. The method was validated with regards to the linearity, inter- and intra-day precision and accuracy. The detection limits ranged from 0.009 to 0.942 ng/mL, except for 4-hydroxy-2-nonenal (15 ng/mL), and the quantification limits varied from 0.029 to 1.66 ng/mL, except for butanal (2.78 ng/mL), 2-butanone (2.67 ng/mL), 4-heptanone (3.14 ng/mL) and 4-hydroxy-2-nonenal (50.0 ng/mL). The method accuracy was satisfactory, with recoveries ranging from 90 to 107%. The proof of applicability of the methodology was performed in a pilot target analysis of urine samples obtained from 18 healthy smokers and 18 healthy non-smokers (control group). Chemometric supervised analysis was performed using the volatile patterns acquired for these samples and clearly showed the potential of the volatile carbonyl profiles to discriminate urine from smoker and non-smoker subjects. 5-Methyl-2-furfural (p<0.0001), 2-methylpropanal, nonanal and 2-methylbutanal (p<0.05) were identified as potentially useful biomarkers to identify smoking habits.
- GC-MS-based endometabolome analysis differentiates prostate cancer from normal prostate cellsPublication . Lima, Ana; Araújo, Ana; Pinto, Joana; Jerónimo, Carmen; Henrique, Rui; Bastos, Maria; Carvalho, Márcia; Guedes de Pinho, PaulaProstate cancer (PCa) is an important health problem worldwide. Diagnosis and management of PCa is very complex because the detection of serum prostate specific antigen (PSA) has several drawbacks. Metabolomics brings promise for cancer biomarker discovery and for better understanding PCa biochemistry. In this study, a gas chromatography-mass spectrometry (GC-MS) based metabolomic profiling of PCa cell lines was performed. The cell lines include 22RV1 and LNCaP from PCa with androgen receptor (AR) expression, DU145 and PC3 (which lack AR expression), and one normal prostate cell line (PNT2). Regarding the metastatic potential, PC3 is from an adenocarcinoma grade IV with high metastatic potential, DU145 has a moderate metastatic potential, and LNCaP has a low metastatic potential. Using multivariate analysis, alterations in levels of several intracellular metabolites were detected, disclosing the capability of the endometabolome to discriminate all PCa cell lines from the normal prostate cell line. Discriminant metabolites included amino acids, fatty acids, steroids, and sugars. Six stood out for the separation of all the studied PCa cell lines from the normal prostate cell line: ethanolamine, lactic acid, β-Alanine, L-valine, L-leucine, and L-tyrosine.
- Evaluation of prostate cancer volatilome: an in vitro approachPublication . Lima, A.R.; Araújo, A.M.; Pinto, J.; Jerónimo, C.; Henrique, R.; Bastos, M.D.L.; Carvalho, Márcia; Guedes de Pinho, Paula
- Volatilomics reveals potential biomarkers for identification of renal cell carcinoma: an in vitro approachPublication . Amaro, Filipa; Pinto, Joana; Rocha, Sílvia; Araújo, Ana Margarida; Miranda-Gonçalves, Vera; Jerónimo, Carmen; Henrique, Rui; Bastos, Maria de Lourdes; Carvalho, Márcia; Guedes de Pinho, PaulaThe identification of noninvasive biomarkers able to detect renal cell carcinoma (RCC) at an early stage remains an unmet clinical need. The recognition that altered metabolism is a core hallmark of cancer boosted metabolomic studies focused in the search for cancer biomarkers. The present work aims to evaluate the performance of the volatile metabolites present in the extracellular medium to discriminate RCC cell lines with distinct histological subtypes (clear cell and papillary) and metastatic potential from non-tumorigenic renal cells. Hence, volatile organic compounds (VOCs) and volatile carbonyl compounds (VCCs) were extracted by headspace solid-phase microextraction (HS-SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS). Multivariate and univariate analysis unveiled a panel of metabolites responsible for the separation between groups, mostly belonging to ketones, alcohols, alkanes and aldehydes classes. Some metabolites were found similarly altered for all RCC cell lines compared to non-tumorigenic cells, namely 2-ethylhexanol, tetradecane, formaldehyde, acetone (increased) and cyclohexanone and acetaldehyde (decreased). Furthermore, significantly altered levels of cyclohexanol, decanal, decane, dodecane and 4-methylbenzaldehyde were observed in all metastatic RCC cell lines when compared with the non-metastatic ones. Moreover, some alterations in the volatile composition were also observed between RCC histological subtypes. Overall, our results demonstrate the potential of volatile profiling for identification of noninvasive candidate biomarkers for early RCC diagnosis.
- Metabolic signature of methylone in primary mouse hepatocytes, at subtoxic concentrationsPublication . Araújo, Ana Margarida; Carvalho, Márcia; Bastos, Maria de Lourdes; Carvalho, Félix; Guedes de Pinho, PaulaMethylone (3,4-methylenedioxymethcathinone) is one of the most popular new psychoactive drugs worldwide. Although advertised as a safe drug, its use has been associated to several cases of liver damage. In this work, a metabolomics approach based on gas chromatography-mass spectrometry (GC-MS) combined with chemometric analyses was used to characterize the disturbances occurring in the intra- and extracellular metabolome of primary mouse hepatocytes exposed to two subtoxic concentrations (LC01 and LC10) of methylone to better understand the early hepatotoxic events. Results showed a characteristic metabolic fingerprint for methylone, where aspartate, cysteine, 2-methyl-1-pentanol, 4-methylheptane, dodecane, 2,4-dimethyl-1-heptene, 1,3-di-tert-butylbenzene, acetophenone, formaldehyde and glyoxal levels were significantly changed at both concentrations tested. Furthermore, subtoxic concentrations of methylone caused profound changes in several biochemical pathways, suggesting adaptations in energy production processes (TCA cycle, amino acids metabolism and pyruvate metabolism), cellular antioxidant defenses (glutamate, cysteine and glutathione metabolism) and hepatic enzymes (associated to hydrocarbons, alcohols, aldehydes and ketones metabolism). This metabolic response to the initial methylone challenge most probably reflects the activation of protective mechanisms to restore cellular homeostasis. Overall, this study highlights the potential of untargeted metabolomic analysis to reveal the hepatic metabolic signature of methylone at subtoxic concentrations, and also provides clues to clarify the early mechanisms underlying the toxicity triggered by this new psychoactive substance, opening a new perspective for the study of toxicity mechanisms of new xenobiotics.
- Metabolomics approach in the investigation of hepatic metabolic changes caused by MDMAPublication . Araújo, Ana Margarida; Bastos, Maria de Lourdes; Fernandes, Eduarda; Carvalho, Félix; Carvalho, Márcia; Guedes de Pinho, Paula
- 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.
- Cu2+-induced isoproterenol oxidation into isoprenochrome in adult rat calcium-tolerant cardiomyocytesPublication . Remião, Fernando; Carvalho, Márcia; Carmo, Helena; Carvalho, Félix; Bastos, Maria L.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.