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- Microcarrier-based fluorescent yeast estrogen screen assay for fast determination of endocrine disrupting compoundsPublication . Gregório, Bruno J.R.; Ramos, Inês I.; Marques, Sara S.; Barreiros, Luísa; Magalhães, Luís M.; Schneider, Rudolf J.; Segundo, Marcela A.The presence of endocrine-disrupting compounds (EDCs) in water poses a significant threat to human and animal health, as recognized by regulatory agencies throughout the world. The Yeast Estrogen Screen (YES) assay is an excellent method to evaluate the presence of these compounds in water due to its simplicity and capacity to assess the bioaccessible forms/fractions of these compounds. In the presence of a compound with estrogenic activity, Saccharomyces cerevisiae cells, containing a lacZ reporter gene encoding the enzyme β-galactosidase, are induced, the enzyme is synthesised, and released to the extracellular medium. In this work, a YES-based approach encompassing the use of a lacZ reporter gene modified strain of S. cerevisiae, microcarriers as solid support, and a fluorescent substrate, fluorescein di-β-d-galactopyranoside, is proposed, allowing for the assessment of EDCs’ presence after only 2 h of incubation. The proposed method provided an EC50 of 0.17 ± 0.03 nM and an LLOQ of 0.03 nM, expressed as 17β-estradiol. The assessment of different EDCs provided EC50 values between 0.16 and 1.2 × 103 nM. After application to wastewaters, similar results were obtained for EDCs screening, much faster, compared to the conventional 45 h spectrophotometric procedure using a commercial kit, showing potential for onsite high-throughput screening of environmental contamination.
- 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 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.
- 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.
- Preliminary chemical profile and in vitro pharmacological evaluation of the hallucinogenic plant Diplopterys cabreranaPublication . Garcia, Maria Rita; Dutka, Mykhaylo; Guimarães, Sofia; Andrade, Paula B.; Seabra, Vítor; Diana Dias da Silva; Gomes, Nelson G. M.; Dias da Silva, Diana CristinaFor the last few years, Ayahuasca ceremonies have been gaining popularity in recreational settings in Europe and North America [1]. Similar to Psychotria viridis, Diplopterys cabreranais also suggested to contain the psychoactive compound N,N-dimethyltryptamine, and is therefore used in Aya-huasca rituals for its ability to induce hallucinations, euphoria and entheogenic effects [1-3]. However, while information on the toxic profile of D. cabreranaremains very limited, its acquisition is easily ac-complished by consumers. We aimed to characterize the aqueous extracts of D. cabreranaleaves, mimicking those typically consumed, to identify bioactives that underlie the psychoactive or toxic effects, and evaluate their impact on neuronal function, neurotransmission and radical stress. Chemical characterization was attained by HPLC-DAD. Impact upon neuronal viability was assessed by the MTT assay (up to 1000 μg/mL) in SH-SY5Y neuroblastoma cells. Impact on neuromodulation and neuroinflammation was evaluated through acetylcholinesterase and 5-lipoxygenase inhibition, while an-tiradical properties were assessed by evaluating nitric oxide (•NO) and xanthine oxidase (XO) activity. Inhibition of the α-glucosidase enzyme was also evaluated. Statistical comparisons among groups per-formed by one-way ANOVA followed by Dunnett post hoc test. Preliminary characterization results revealed the presence of several catechin derivates, alongside two apigenin derivates and one tryp-tamine derivate. Cytotoxicity was not verified up to the highest concentration tested. Acetylcholinesterase inhibition was recorded starting at 250 μg/mL, and a concentration-dependent inhibition of 5-lipoxygen-ase was found (IC50=79.77 μg/mL). Concentration-dependent scavenging effects upon •NO and XO inhi-bition were verified at concentrations higher than 1.953 μg/mL and 31.25 μg/mL, respectively. At last, inhibition of α-glucosidase occurred with concentration-dependency and an IC50of 4.78 μg/mL. Conclu-sions:Although antiradical, anti-inflammatory and antidiabetic properties were verified, with no in vitrocytotoxicity being detected, further research is needed to elucidate the underlying mechanisms that might be involved in our preliminary results.
- 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.
- 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.
- 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.