Browsing by Author "Silva, Diana Dias da"
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- Chemical differences between alternative and traditional tobacco productsPublication . Monteiro, Vânia; Freitas, Inês; Silva, Diana Dias da; Pinho, Paula Guedes de; Pinto, Joana; Dias da Silva, Diana CristinaElectronic cigarettes (E-cigs) and heated tobacco products (HTPs) have gained popularity as alternatives to traditional tobacco products (TTPs), claiming to reduce harm. The carcinogenic proper-ties of chemicals in the smoke of TTPs are widely recognized. However, there is still an incomplete understanding of the different chemicals in E-cigs and HTPs and their toxicity to human cells [1]. Thus, this study aimed at characterizing and comparing the chemical composition of three different brands of E-cigs, HTPs and TTPs. We selected the three top-selling brands of E-cigs, HTPs, and TTPs in Portugal, and each brand (n=9) was analyzed in triplicate. Volatile compounds present in all brands were extracted by headspace solid-phase microextraction (HS-SPME) and solvent extraction (di-chloromethane). The volatile compounds in the headspace and solvent extracts were analysed by gas chromatography-mass spectrometry (GC-MS). Compound annotation was performed by comparing the mass spectrum of each chromatographic peak in the sample with a mass spectral library and standards, where available. A total of 53 compounds were detected in E-cigs, 44 in HTPs and 41 in TTPs by HS-SPME. Solvent extraction revealed 43 compounds in E-cigs, 35 in HTPs and 22 in TTPs. Only 7 compounds were common to E-cigs, HTPs, and TTPs. Overall, the chemical classes included alcohols, aldehydes, ketones, esters, pyridines and others.The composition of HTPs and TTPs was similar (20 compounds in common), particularly in the classes of ketones, alcohols, terpenoids, and pyridines. In contrast, E-cigs contain a larger number of compounds than HTPs and TTPs, including several alcohols, esters, pyranones, and lactones. The volatile composition of HTPs and TPPs showed less variation be-tween different brands, whereas E-cig brands showed greater variability in their composition. HTPs have a volatile chemical composition similar tothat of TTPs in their original form, so their health effects will depend on the impact of the different types of combustion. E-cigs show a distinct chemical profile across all brands, with chemical classes that are potentially relevant for toxicological studies.
- Enantiomeric biodistribution and toxicity of 3-chloromethcathinone (3-CMC) in Wistar rats after acute exposure – preliminary dataPublication . Langa, Ivan; Rocha-Pereira, Carolina; Milhazes, Nuno; Silva, Diana Dias da; Domingues, Susana; Silva, Paula; Barbosa, Joana; Faria, Juliana; Tiritan, Maria Elizabeth; Ribeiro, Cláudia; Dias da Silva, Diana CristinaThere has been a surge in global attention to New Psychoactive Substances (NPS) [1]. Synthetic cathinones stand out as a widely consumed NPS class. Notably, 3-chloromethcathinone (3-CMC) accounted for over 34% of NPS seizures in 2021 [2], which underscores concerns regarding its consumption and health effects. Of note, 3-CMC is chiral and mostly sold as a racemate. As human me-tabolism and pharmacological effects can be enantioselective [3], determination of the impact of enanti-oselectivity in toxicokinetics/toxicodynamics is essential for the assessment of 3-CMC effects. This work aimed to evaluate in vivothe enantioselective biodistribution and toxicity of racemic 3-CMC, after an acute exposure to 3-CMC. Ten-week-old male Wistar rats were administered intraperitoneally with saline or 3-CMC (10 or 20 mg/kg; n=6). Twenty-four hours after, animals were deeply anesthetized and nine organs (brain, liver, kidneys, lungs, heart, spleen, gut, muscle, adipose tis-sue), blood and urine were collected. For evaluation of the enantiomeric biodistribution, a previous in houseestablished indirect method by gas chromatography [3], was adapted and validated. Some biochem-ical analysis was performed using an analyser, whereas TBARS, ATP, glutathione and total protein were determined by spectrophotometry. Organs were also processed for histological analysis. After 24 h, 3-CMC was not found in most organs. Both enantiomers were detected in urine with one dominant enantiomer, suggesting enantioselectivity in metabolism. The histopathological results showed possible central chromatolysis in the brain (20 mg/kg), liver inflammation, renal lesions, lungs’ haemoptysis, and alveolar haemorrhage, in most 3-CMC-exposed animals. No differences were observed inthe heart. Our findings show rapid 3-CMC renal elimination, with enantio selectivity in metabolism. Alt-hough biochemical evaluations are ongoing, the results are expected to give further insights on the 3-CMC toxicity and histological abnormalities found in the brain, kidneys, liver and lungs.
- In vitro and in silico evaluation of 5-MeO-DMT, LSD, and mescaline’s interaction with CYP450 enzymesPublication . Brito-da-Costa, Andreia Machado; Carvalho, Mariana; Dinis-Oliveira, Ricardo Jorge; Madureira-Carvalho, Áurea; Sousa, Sérgio F.; Silva, Diana Dias da; Dias da Silva, Diana Cristina5-Methoxy-N,N-dimethyltryptamine(5-MeO-DMT), lysergic acid diethylamide (LSD), and mescaline are classic hallucinogens known for their recreational use, which increased in the last dec-ades. Despite some available data on the metabolism of these drugs [1-3], a scientific gap exists regarding their possible interactions with CYP450 enzymes. Nevertheless, this information is of crucial relevance to predict drug-drug interactions and understand toxicological phenomena, in particular interindividual variability. This study aimed to evaluate in vitroand in silicothe interaction of 5-MeO-DMT, LSD, and mescaline with the enzymes CYP2A6/2B6/2D6/2E1/3A4. The in vitroassessment of CYP450 inhibition was performed using the Vivid®CYP450 screening kits. IC50 was calculated using GraphPad Prism 9.3.0. For in silicoassessment, molecular dynamics were performed using the PMEMD.cuda module in AMBER16. Calculations were made on the last 100 ns of the trajectory (stable zone) to assess the interaction mode/strength between enzyme and ligand, namely MMGBSA, per-residue decomposition energy, and hydrogen bonds. Based on the IC50(μM), LSD (0.35) and 5-MeO-DMT (3.47) present the capacity to be inhibitors of CYP2D6. Based on the MMGBSA (kcal/mol), LSD showed the highest binding affinities for all enzymes, while mescaline showed the lowest. The strong interaction of LSD with CYP2A6 is mediated by a hydrogen bond established with the protein residue Asn297. For interaction with CYP2B6, the residues Thr302 and Lys479 were important in mediating the interaction with 5-MeO-DMT and LSD. Key residues mediating the interaction of 5-MeO-DMT and LSD with CYP2D6 included Phe120, Leu213, and Phe483. For interaction with CYP2E1, residues Phe207, Phe298, and Thr303 are important; and for CYP3A4, an important hydrogen bond between LSD and Ala370 was identified.Conclusions: Both LSD and 5-MeO-DMT are predicted to have strong potential to be CYP2D6 inhibitors. A strong interaction was also identified in silicobetween LSD and CYP2A6.
- In vitro and in silico evaluation of psilocybin and psilocin’s interaction with CYP450 enzymesPublication . Brito-da-Costa, Andreia Machado; Carvalho, Mariana; Dinis-Oliveira, Ricardo Jorge; Madureira-Carvalho, Áurea; Sousa, Sérgio F.; Silva, Diana Dias da; Dias da Silva, Diana CristinaPsilocybin is a hallucinogen produced by “magic mushrooms”, being rapidly metabolized in the organism into psilocin [1, 2]. A scientific gap exists regarding the possible interactions between psilocybin/psilocin and CYP450 enzymes. Given their biological importance, and since the binding of drugs to CYP450 enzymes can interfere with the metabolism of other substrates leading to drug-drug interactions, this research topic is of utmost importance. This study aimed to evaluate in silicoand in vitrothe interaction of psilocybin and psilocin with the enzymes CYP2A6/2B6/2D6/2E1/3A4. The in vitroassessment of inhibition was performed using the Vivid®CYP450 screening kits. IC50 was calculated using GraphPad Prism 9.3.0. For in silico assessment, molecular dynamics were per-formed using the PMEMD.cuda module in AMBER16. Calculations were made on the last 100 ns of the trajectory (stable zone) to assess the interaction between enzyme and ligand, namely MMGBSA, per-residue decomposition energy, and hydrogen bonds. Psilocin showed the capacity to be an in-hibitor of CYP2A6/2B6/2D6/2E1/3A4, based on the respective IC50 values (μM) of 2.06, 6.17, 11.89, 6.37, and 2.36. Considering the MMGBSA, higher values were obtained for psilocin, corroborating the stronger binding affinity of this compound. The interaction of psilocybin/psilocin with CYP2A6 is medi-ated by a hydrogen bond established with the protein residue Asn297. Other important residues include Phe107 and Ile366. For CYP2B6, the strong binding of psilocin is mediated by interactions with Ile114, Thr302 (hydrogen bond), and Leu363. For interaction with CYP2D6, the most important residue seems to be Ser304, with which it forms a hydrogen bond; for CYP2E1, key residues include Phe207, Thr303, and Phe478. A strong hydrogen bond is formed between psilocin and CYP3A4 residue Phe304, contrib-uting to the high binding affinity. The results suggest a potential for psilocin to inhibit all enzymes, especially CYP2A6 and CYP3A4.
- Influence of puncture devices on the accuracy of cyclophosphamide dosing for chemotherapy administrationPublication . Carvalho, Susana; Cardoso, Andreia; Ferreira, Débora; Silva, Diana Dias da; Moreira, Fernando; Moreira, Fernando; Dias da Silva, Diana CristinaCyclophosphamide is one of the most commonly used cytotoxic drugs in chemotherapy protocols. Its preparation in the hospital setting involves handling concentrated solutions, which pose occupational exposure risks and potential variations in the final dose administered. The aim of this study was to evaluate the effect of aspiration devices on the concentration of cyclophosphamide in reconstituted solutions. An analytical method was validated using high-performance liquid chromatography coupled to a diode-array detector (HPLC-DAD) for quality control. Cyclophosphamide solutions were prepared and aspirated using either a conventional needle or spike device with or without a filtration system. The validated method demonstrated linearity (R2 = 0.9999), high precision (0.22–4.59%) and accuracy (88.9–99.4%), with a limit of quantification of 4.03 µg/mL. Significant differences (p < 0.001) were observed between samples aspirated with a needle and those aspirated with a spike fitted with a 5 µm filter, with the latter showing lower cyclophosphamide concentrations, suggesting partial retention of the drug. No significant differences were found between the needle and filterless spike preparations. These results suggest that the choice of aspiration device influences the final drug concentration, potentially affecting therapeutic efficacy. Standardisation of preparation techniques and an awareness of device limitations are essential to ensure accurate chemotherapy dosing and patient safety.
- Mexican calea (Calea zacatechichi Schltdl.) interferes with cholinergic and dopaminergic pathways and causes neuroglial toxicityPublication . Garcia, Maria Rita; Gil-Izquierdo, Ángel; Ferreres, Federico; Andrade, Paula B.; Mineiro, Tiago; Seabra, Vítor; Videira, Romeu A.; Silva, Diana Dias da; Gomes, Nelson G.M.; Dias da Silva, Diana CristinaThe use of “Mexican calea” (Calea zacatechichi Schltdl.) in ritualistic ceremonies, due to its dream-inducing effects, was until recently limited to indigenous communities in Mexico. However, the plant has recently gained popularity in Western societies being commonly used in recreational settings. Despite the traditional and recreational uses, mechanisms underlying its reported oneirogenic effects remain unknown, with no data available on its neurotoxic profile. The scarcity of toxicological data and the unknown role of major neurotransmitter systems in the dream-inducing properties of the plant prompted us to investigate which neurotransmitters might be affected upon its consumption, as well as the potential cytotoxic effects on neurons and microglial cells. Furthermore, we aimed to explore a relationship between the recorded effects and specific constituents. Effects on cholinergic and monoaminergic pathways were investigated using enzymatic assays, with the latter also being conducted in neuronal SH-SY5Y cells along with the impact on glutamate-induced excitotoxicity. Investigation of the neurotoxic profile was approached in neuronal SH-SY5Y and microglial BV-2 cells, evaluating effects on metabolic performance and membrane integrity using MTT and LDH leakage assays, respectively. Potential interference with oxidative stress was monitored by assessing free radical's levels, as well as 5-lipoxygenase mediated lipid peroxidation. Phenolic constituents were identified through HPLC-DAD-ESI(Ion Trap)MSn analysis. Based on the significant inhibition upon acetylcholinesterase (p < 0.05) and tyrosinase (IC50 = 60.87 ± 7.3 μg/mL; p < 0.05), the aqueous extract obtained from the aerial parts of C. zacatechichi interferes with the cholinergic and dopaminergic systems, but has no impact against monoamine oxidase A. Additionally, a notable cytotoxic effect was observed in SH-SY5Y and BV-2 cells at concentrations as low as 125 and 500 μg/mL (p < 0.05), respectively, LDH leakage suggesting apoptosis may occur at these concentrations, with necroptosis observed at higher ones. Despite the neurocytotoxic profile, these effects appear to be independent of radical stress, as the C. zacatechichi extract scavenged nitric oxide and superoxide radicals at concentrations as low as 62.5 μg/mL, significantly inhibiting also 5-lipoxygenase (IC50 = 72.60 ± 7.3 μg/mL; p < 0.05). Qualitative and quantitative analysis using HPLC-DAD-ESI(Ion Trap)MSn enabled the identification of 28 constituents, with 24 of them being previously unreported in this species. These include a series of dicaffeoylquinic, caffeoylpentoside, and feruloylquinic acids, along with 8 flavonols not previously known to occur in the species, mainly 3-O-monoglycosylated derivatives of quercetin, kaempferol, and isorhamnetin. Our findings regarding the neuroglial toxicity elicited by C. zacatechichi emphasize the necessity for a thorough elucidation of the plant's toxicity profile. Additionally, evidence is provided that the aerial parts of the plant inhibit both acetylcholinesterase and tyrosinase, potentially linking its psychopharmacological effects to the cholinergic and dopaminergic systems, with an apparent contribution from specific phenolic constituents previously unknown to occur in the species. Collectively, our results lay the groundwork for a regulatory framework on the consumption of C. zacatechichi in recreational settings and contribute to elucidating previous contradictory findings regarding the mechanisms underlying the dream-inducing effects of the plant.
- Optimization of the derivatization procedure for the separation of the stereoisomers of 1,3-dimethylamylamine (1,3-DMAA) by gas chromatography - preliminary dataPublication . Almeida, Maria Mexia de; Silva, Diana Dias da; Oliveira, Ricardo Jorge Dinis; Ribeiro, Cláudia; Dias da Silva, Diana Cristina1,3-Dimethylamylamine (1,3-DMAA),also known as methylhexanamine, is a central nervous system stimulant with structural similarities with amphetamines and therefore presenting over-lapping biological and detrimental effects [1]. Despite being banned, the presence of 1,3-DMAA in dop-ing controls and dietary supplements continues to be of significant concern. This molecule has two stere-ogenic centres and thus four stereoisomers [2]. It is widely recognized that enantiomers may exhibit dif-ferent biological activity, including pharmacokinetics, pharmacodynamics, and toxicity. Consequently, the development of analytical methods for enantioselective separation of 1,3-DMAA is crucial for an accurate determination of the risks associated with each of these stereoisomers. To develop an indirect method by gas chromatography coupled to mass spectrometry (GC-MS) for the separation and identification of the stereoisomers of the 1,3-DMAA. 1,3-DMAA was regenerated with sodium hydroxide, extracted with 0.1% triethylamine in hexane and then derivatized using the enantiomeric pure reagent (R)-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride ((R)-MTPA-Cl). Subsequently, the sample was evaporated, reconstituted in anhydrous ethyl acetate, and analyzed by GC-MS. The chroma-tographic conditions were established using a capillary column containing 5% diphenyl-95% dime-thylpolysiloxane (30 m × 0.25 mm × 0.25 μm), an injector temperature set to 280 ºC, with a temperature ramp starting at 140 ºC and increasing up to 215 ºC at a flow rate of 1 mL/min to a total run of 12.32 min. Results: As preliminary data indicate, the derivatization procedure allowed the formation of 4 diastere-omers of 1,3-DMAA. The chromatographic conditions were optimised, allowing for the separation of the four diastereomers within 12 min. Derivatization and chromatographic conditions were established for enantioselective separation of 1,3-DMAA by GC-MS. Further validation of the method will be crucial for understanding the diastereomers' differential pharmacokinetics and pharmacodynamics, and consequently, the perils associated with their presence in food supplement samples.
- Sequencing technologies in forensic microbiology: current trends and advancementsPublication . Oliveira, Manuela; Dias da Silva, Diana Cristina; Marszałek, Kamila; Kowalski, Michał; Frolova, Alina; Łabaj, Paweł P.; Branicki, Wojciech; Carvalho, Áurea Madureira; Silva, Diana Dias da; Oliveira, Ricardo Jorge DinisForensic microbiology is a subject of interest and research development across the forensic community. Studies of pathogen outbreaks, biocrime or bioterrorism attacks, and analyses of crime scenes fall within this field. Significant progress has been made in evolving DNA sequencing technologies in recent decades. These newly emerged transformative tools have become available to both biomedicine and forensics. Based on the published literature, this review provides an overview of the current trends and developments of NGS (next-generation sequencing) technologies applied to forensic microbiology. These new methods present numerous advantages over traditional sequencing technology and are useful in several practical applications within this forensic field. This article then examines the main challenges and limitations of this technology in forensics, providing a comprehensive review of NGS technology capabilities in expanding the precision and effectiveness of microbial forensic investigations—with the aim of inspiring scientists, forensic experts, lawyers, public health professionals, and policymakers alike to approach this newly powerful sequencing tool appropriately.
- The effect of synthetic cannabinoid ADB-FUBI-NACA on primary neuronal cultures ß-galacto-sidase activity: preliminary findingsPublication . Roque-Bravo, Rita; Carmo, Helena; Silva, João Pedro; Carvalho, Félix; Silva, Diana Dias da; Dias da Silva, Diana CristinaADB-FUBINACA (ADB-FUB) is a synthetic cannabinoid (SC) that has gained popularity among users as a new psychoactive substance. This stems from SC's pharmacological similarity to the active principle of cannabis, D9-tetrahydrocannabinol (THC). However, unlike THC, SCs demonstrate full agonism of cannabinoid receptors 1 and 2 [1]. Recent scientific developments have shown that can-nabis use may aggravate ageing-related parameters [2,3]. Moreover, a study using human fibroblasts re-vealed that 1 μM THC (2h-longexposure, for 15 days) can increase ß-galactosidase activity [3], which serves as a first-line marker for cellular senescence. This study was designed to investigate whether these biologically-relevant concentrations could accelerate neuronal ageing. PHC were isolated from Wistar rat day 18-19 embryos and cultured for up to 21 days-in-vitro (DIV). Exposure to 1 pM, 1 nM and 1 μM ADB-FUB (concentrations previously shown to be non-cytotoxic to PHC) started either on DIV3 or DIV7 and was maintained up to 21 DIV. At that final timepoint, ß-galactosidase activity was evaluated. DMSO at 0.02% was employed as solvent control. Under these experi-mental conditions, PHC exposed to 1 nM and 1 μM ADB-FUB in the DIV3-21 protocol had lower ß-galactosidase activity when compared to control conditions (p<0.05, 1 nM; p<0.001, 1 μM). No statisti-cally significant results were registered for PHC under the DIV7-21 exposure protocol. These findings are, to the best of our knowledge, the first evidence of a potential “anti-ageing” effect of ADB-FUB. Evaluation of other senescence-related endpoints will follow. Moreover, experiments using another in vitroneuronal model (human neuroblastoma cell line SH-SY5Y) are underway to compare the effects of the same drug in different models and further substantiate conclusions on ADB-FUB’s effect.