A simple method to measure sulfonation in man using paracetamol as probe drug

Sulfotransferase enzymes (SULT) catalyse sulfoconjugation of drugs, as well as endogenous mediators, gut microbiota metabolites and environmental xenobiotics. To address the limited evidence on sulfonation activity from clinical research, we developed a clinical metabolic phenotyping method using paracetamol as a probe substrate. Our aim was to estimate sulfonation capability of phenolic compounds and study its intraindividual variability in man. A total of 36 healthy adult volunteers (12 men, 12 women and 12 women on oral contraceptives) received paracetamol in a 1 g-tablet formulation on three separate occasions. Paracetamol and its metabolites were measured in plasma and spot urine samples using liquid chromatography-high resolution mass spectrometry. A metabolic ratio (Paracetamol Sulfonation Index-PSI) was used to estimate phenol SULT activity. PSI showed low intraindividual variability, with a good correlation between values in plasma and spot urine samples. Urinary PSI was independent of factors not related to SULT activity, such as urine pH or eGFR. Gender and oral contraceptive intake had no impact on PSI. Our SULT phenotyping method is a simple non-invasive procedure requiring urine spot samples, using the safe and convenient drug paracetamol as a probe substrate, and with low intraindividual coefficient of variation. Although it will not give us mechanistic information, it will provide us an empirical measure of an individual's sulfonator status. To the best of our knowledge, our method provides the first standardised in vivo empirical measure of an individual's phenol sulfonation capability and of its intraindividual variability. EUDRA-CT 2016-001395-29, NCT03182595 June 9, 2017.publishersversionpublishe

Hydrolysable tannins in aged wine spirits: a fresh perspective using alternative ageing technology and high-resolution mass spectrometry

The authors are very grateful to Victor de Freitas as the Scientific Consultant of the Project POCI-01-0145- FEDER-027819, and to João Pedro Catela, Nádia Santos, Manuela Gomes, Eugénia Gomes and Inês Antunes from Adega Cooperativa da Lourinhã, José Abílio Gonçalves and Sérgio Gonçalves from Tanoaria J. M. Gonçalves, Ana Partidário from INIAV Oeiras, Pedro Rodrigues and Diogo Rodrigues from AZ3Oeno Portugal, Sílvia Lourenço, João Amaral, Deolinda Mota from INIAV Polo de Dois Portos, and A. Pedro Belchior for their technical support.Wine spirits (WSs) are usually aged in wooden barrels, but using wood pieces instead of barrels, with or without micro-oxygenation, is a technological alternative that has been investigated by our team.This research was funded by National Funds through FCT - Foundation for Science and Technology under the Project OXYREBRAND - POCI-01-0145-FEDER-027819 (PTDC/OCE-ETA/27819/2017). The authors thank the research units and Fundação para a Ciência e a Tecnologia, I.P.: CEF (UIDB/00239/2020); CQE (UIDB/00100/2020; UIDP/00100/2020); LEAF (UIDP/04129/2020; UIDB/04129/2020); MED (UIDB/05183/2020) and contracts CEECIND/02725/2018, CEECIND/02001/2017 and DL 57/2016/CP1382/CT0025.info:eu-repo/semantics/publishedVersio

First chemical profile analysis of acacia pods

This study intended to evaluate the potential industrial applications of various Acacia species (Acacia melanoxylon, Acacia longifolia, Acacia cyclops, Acacia retinodes, Acacia pycnantha, Acacia mearnsii, and Acacia dealbata) by examining their chemical composition, antioxidant, and antimicrobial properties. Using high-resolution mass spectrometry, a comprehensive analysis successfully identified targeted compounds, including flavonoids (flavonols/flavones) and phenolic acids, such as 4-hydroxybenzoic acid, p-coumaric acid, and ellagic acid. Additionally, p-coumaric acid was specifically identified and quantified within the hydroxycinnamic aldehydes. This comprehensive characterization provides valuable insights into the chemical profiles of the studied species. Among the studied species, A. pycnantha exhibited a higher concentration of total phenolic compounds, including catechin, myricetin, quercetin, and coniferaldehyde. Furthermore, A. pycnantha displayed notable antibacterial activity against K. pneumoniae, E. coli, S. Typhimurium, and B. cereus. The identified compounds in Acacia pods and their shown antibacterial activities exhibit promising potential for future applications. Moreover, vibrational spectroscopy was a reliable method for distinguishing between species. These significant findings enhance our understanding of Acacia species and their potential for various industrial applications.info:eu-repo/semantics/publishedVersio

Metabolic stability and metabolite profiling of emerging synthetic cathinones

We thank Fundação para a Ciência e a Tecnologia (FCT), Portugal, for financial support through projects UIDB/QUI/00100/2020 and UIDP/00100/2020 (to CQE), LA/P/0056/2020 (to IMS), UIDB/04046/2020 and UIDP/04046/2020 (to BioISIBiosystems & Integrative Sciences Institute), UIDB/04292/2020 and UIDP/04292/2020 (to MARE-Marine and Environmental Sciences Centre). FCT is also acknowledged for the PhD grant 2022.04738. PTDC to RPL. Joint funding from FCT and the COMPETE Program through grant RNEMLISBOA-01-0145-FEDER-022125 funding are also gratefully acknowledged.Synthetic cathinones constitute the second largest groups of new psychoactive substances (NPS), which are especially popular among adolescents and young adults. Due to their potential toxicity, the recreational use of these NPS constitute a serious worldwide public health problem. However, their fast appearance in the market renders the continuous updating of NPS information highly challenging for forensic authorities. The unavailability of pharmacokinetic data for emerging NPS is critical for forensic and clinical verifications. With the ultimate goal of having a proactive approach towards the NPS issue, high resolution mass spectrometry was used in the current work to assess preliminary pharmacokinetic data for 8 selected cathinones: 4 reported substances (4-CIC, 3-CMC, 4-CMC and 4-MEAP) and 4 previously unreported ones (3-CIC, 4-MDMB, 4-MNEB and 4-MDMP) for which the emergence on the NSP market is expected to be eminent, were also included in this study. Based on the calculation of pharmacokinetic parameters, half-life and intrinsic clearance, 4-CMC and 4-MDMB are low and high clearance compounds, respectively, and all the remaining cathinones included in this study are intermediate clearance compounds. This fact anticipates the key role of metabolites as suitable biomarkers to extend detection windows beyond those provided by the parent cathinones. Reduction of the keto group and hydroxylation on the alkyl chains were the common metabolic pathways identified for all cathinones. However, the relative importance of these metabolic transformations is dependent on the cathinone substituents. The glucuronic acid conjugation to metabolites stemming for keto group reduction constituted the sole Phase II transformation identified. To our knowledge, this study constitutes the first metabolite profiling of the already reported synthetic cathinones 4-CIC, 3-CMC and 4-CMC. Noteworthy is the fact that 3-CMC accounts for almost a quarter of the quantity of powders seized during 2020. The analytical methods developed, and the metabolites characterized, are now available to be included in routine screening methods to attest the consumption of the 8 cathinones studied.info:eu-repo/semantics/publishedVersio

An integrated in vitro approach unveils the biocompetence and glutathiolomic profile of a human hepatocyte-like cell 3d model

Funding: This work was supported by FCT (Portugal) through the research grant PTDC/MED-TOX/29183/2017. Acknowledgments: The authors thank ECBio S.A. for providing the hnMSCs and F.A. Beland (NCTR, Jefferson, AR, USA) for the kind donation of nevirapine. FCT (UID/DTP/04138/2019, UID/QUI/00100/2019, RECI/QEQ-MED/0330/2012, SFRH/BD/144130/2019 to J.S.R., SFRH/BD/110945/2015 to P.F.P. and CEECIND/02001/2017 to A.M.M.A) are also acknowledged.The need for competent in vitro liver models for toxicological assessment persists. The differentiation of stem cells into hepatocyte-like cells (HLC) has been adopted due to its human origin and availability. Our aim was to study the usefulness of an in vitro 3D model of mesenchymal stem cell-derived HLCs. 3D spheroids (3D-HLC) or monolayer (2D-HLC) cultures of HLCs were treated with the hepatotoxic drug nevirapine (NVP) for 3 and 10 days followed by analyses of Phase I and II metabolites, biotransformation enzymes and drug transporters involved in NVP disposition. To ascertain the toxic effects of NVP and its major metabolites, the changes in the glutathione net flux were also investigated. Phase I enzymes were induced in both systems yielding all known correspondent NVP metabolites. However, 3D-HLCs showed higher biocompetence in producing Phase II NVP metabolites and upregulating Phase II enzymes and MRP7. Accordingly, NVP-exposure led to decreased glutathione availability and alterations in the intracellular dynamics disfavoring free reduced glutathione and glutathionylated protein pools. Overall, these results demonstrate the adequacy of the 3D-HLC model for studying the bioactivation/metabolism of NVP representing a further step to unveil toxicity mechanisms associated with glutathione net flux changes.publishersversionpublishe

Cysteine as a Multifaceted Player in Kidney, the Cysteine-Related Thiolome and Its Implications for Precision Medicine

Funding Information: This research was supported by Fundação para a Ciência e Tecnologia (PTDC/MED-TOX/30418/2017) and iNOVA4Health (UID/Multi/04462/2013). M.J.C., D.G.F.F. and J.M. were supported by FCT (PhD grant SFRH/BD/131331/2017, PhD grant PD/BD/135484/2018 and postdoctoral contract PTDC/MED-TOX/30418/2017, respectively).In this review encouraged by original data, we first provided in vivo evidence that the kidney, comparative to the liver or brain, is an organ particularly rich in cysteine. In the kidney, the total availability of cysteine was higher in cortex tissue than in the medulla and distributed in free reduced, free oxidized and protein-bound fractions (in descending order). Next, we provided a comprehensive integrated review on the evidence that supports the reliance on cysteine of the kidney beyond cysteine antioxidant properties, highlighting the relevance of cysteine and its renal metabolism in the control of cysteine excess in the body as a pivotal source of metabolites to kidney biomass and bioenergetics and a promoter of adaptive responses to stressors. This view might translate into novel perspectives on the mechanisms of kidney function and blood pressure regulation and on clinical implications of the cysteine-related thiolome as a tool in precision medicine.publishersversionpublishe

Targeting Glutathione and Cystathionine β-Synthase in Ovarian Cancer Treatment by Selenium-Chrysin Polyurea Dendrimer Nanoformulation

The research was funded by iNOVA4Health UID/Multi/04462, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência (FCT-MCTES), through national funds, and co-funded by FEDER under the PT2020 Partnership Agreement. We also acknowledge funding from FCT-MCTES through project DREAM PTDC/MEC-ONC/29327/2017.Ovarian cancer is the main cause of death from gynecological cancer, with its poor prognosis mainly related to late diagnosis and chemoresistance (acquired or intrinsic) to conventional alkylating and reactive oxygen species (ROS)-generating drugs. We and others reported that the availability of cysteine and glutathione (GSH) impacts the mechanisms of resistance to carboplatin in ovarian cancer. Different players in cysteine metabolism can be crucial in chemoresistance, such as the cystine/glutamate antiporter system Xc (xCT) and the H2S-synthesizing enzyme cystathionine β-synthase (CBS) in the pathway of cysteine catabolism. We hypothesized that, by disrupting cysteine metabolic flux, chemoresistance would be reverted. Since the xCT transporter is also able to take up selenium, we used selenium-containing chrysin (SeChry) as a plausible competitive inhibitor of xCT. For that, we tested the effects of SeChry on three different ovarian cancer cell lines (ES2, OVCAR3, and OVCAR8) and in two non-malignant cell lines (HaCaT and HK2). Results showed that, in addition to being highly cytotoxic, SeChry does not affect the uptake of cysteine, although it increases GSH depletion, indicating that SeChry might induce oxidative stress. However, enzymatic assays revealed an inhibitory effect of SeChry toward CBS, thus preventing production of the antioxidant H2S. Notably, our data showed that SeChry and folate-targeted polyurea dendrimer generation four (SeChry@PUREG4-FA) nanoparticles increased the specificity for SeChry delivery to ovarian cancer cells, reducing significantly the toxicity against non-malignant cells. Collectively, our data support SeChry@PUREG4-FA nanoparticles as a targeted strategy to improve ovarian cancer treatment, where GSH depletion and CBS inhibition underlie SeChry cytotoxicity.publishersversionpublishe

Elucidating the environmental causes of disease"

Adductomics studies represent effective tools for better understand how exposure to exogenous and endogenous reactive chemical agents underlie mechanism of diseases. This special issue is focused not only on summarizing the analytical methodologies used for DNA, protein, and mercapturic acid adductomics tools but also on highlighting the opportunities and challenges for the application of this type of studies in biomedical research.publishersversionpublishe

Casting light on the chemical characterization of Acacia pods

Invasive species impose a strain on natural ecosystems by contributing to the loss of certain native species. Acacia species are amongst the most aggressive invasive species in Portugal. In this work, Acacia retinodes, A. longifolia, A. melanoxylon, A. pycnantha and A. dealbata pods were studied concerning the extraction of compounds for potential industrial application.This research was funded by project PCIF/GVB/0145/2018 (Acacia4fireprev). This work is also funded by National Funds through the FCT Foundation for Science and Technology under the Projects UIDB/00239/2020 (CEF), (UI0204): UIDB/00313/2020 and UIDB/00690/2020 (CIMO),CICS-UBI (UIDB/00709/2020 and UIDP/00709/2020), CQE (UIDB/00100/2020 and UIDP/00100/2020).info:eu-repo/semantics/publishedVersio