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  • New trends from fungi secondary metabolism in the pharmaceutical industry
    Publication . Calhelha, Ricardo C.; Shiraishi, Carlos S.H.; Ribeiro, Lúcia Marisa Maia; Carocho, Márcio; Abreu, Rui M.V.; Coutinho, Paula; Barros, Lillian; Vaz, Josiana A.; Ferreira, Isabel C.F.R.
    Fungi are eukaryotic organisms that can produce a wide range of secondary metabolites with a significant impact on society. Some metabolites are exploited for their activity as antioxidant, anti-inflammatory, antitumor, and anti-microbial agents, and in the production of cancer vaccines, among other pharmaceutical applications. Since the discovery of penicillin, the pharmaceutical industry has been greatly interested in fungi as sources of natural bioactive compounds, and fungi metabolites have made an indispensable contribution to improving human and animal health throughout the last decades. Starting with the development of antibiotics, the pharmaceutical industry has increasingly turned to these compounds for a variety of applications. The increase in the number of patents registered worldwide is a strong indicator that the market realizes the great potential of fungi secondary metabolites. In general, the pharmaceutical industry trend is centered on adopting different strategies to discover new drugs, and fungi secondary metabolites are viewed as having significant potential. This chapter explores the current pharmaceutical applications of secondary metabolites found in fungi. Initially, the most recent mushroom studies and their commercial pharmaceutical and cosmeceutical applications are explored. An overview of the different classes of fungi secondary metabolites with biologically relevant activities is then presented. Recently marine fungi were found to be a rich source of secondary metabolites. Due to the recent relevancy of marine fungi, an overview of marine fungi secondary metabolites with relevant pharmaceutical-related activities is also presented. Finally, the potential of fungi metabolites as a source of natural pigments and the methodologies used to characterize and explore fungi secondary metabolites are also analyzed.
  • Impact of occupational exposure to wildfire events on systemic inflammatory biomarkers in Portuguese wildland firefighters
    Publication . Esteves, Filipa; Madureira, Joana; Barros, Bela; Alves, Sara; Pires, Joana; Martins, Sandra; Oliveira, Marta; Vaz, Josiana A.; Slezakova, Klara; Pereira, Maria do Carmo; Fernandes, Adília; Morais, Simone; Guimarães, João Tiago; Bonassi, Stefano; Teixeira, João Paulo; Costa, Solange
    While occupational exposure as a firefighter is considered a dangerous occupation, research on the underlying mechanisms remains limited, particularly in wildland firefighters. Inflammation, a key effect of wildfire expo- sure, plays a significant role in the development of various diseases. The current study aims to investigate the impact of wildland firefighting exposure on the levels of pro-inflammatory systemic biomarkers. A pre-post study design investigated 59 wildland firefighters comparing data collected after participation in a wildfire event (Phase II) with data obtained before wildfire season (Phase I). Data on demographics, lifestyle, health and occupational-related factors were assessed. Exposure factors, such as fire combat (e.g., exposure duration), were also registered. Inflammatory biomarkers (i.e. interleukin-6 [IL-6], interleukin-8 [IL-8], tumor necrosis factor α [TNF-α] and high-sensitivity C-reactive protein [hs-CRP]) and hydroxylated polycyclic aromatic hydrocarbons metabolites (1-OHNaph+1-OHAce, 2-OHFlu, 1-OHPhen, 1-OHPyr) were analysed in blood and urine samples, respectively. Serum IL-8 and IL-6 levels were significantly increased after wildland fire combat. IL-8 levels were 2.62 times higher (95 % CI: 1.96–3.50; p < 0.01), whereas IL-6 levels were 1.25 times higher (95 % CI: 1.00–1.57; p = 0.04). Furthermore, IL-8 levels were significantly correlated with urinary 2-hydroxyfluorene levels and fire combat duration (>12 h). In addition, the mean hs-CRP level, in both phases, was above 3.0 mg/L, indicating a potential risk for cardiovascular events. Given the long-term health implications of fire- fighting occupational exposure, biomonitoring and early detection of occupational risks are essential for pro- tecting firefighters’ health. Protective measures must be urgently implemented to enhance occupational health and strengthen preventive strategies in this sector.
  • Occupational exposure to wildland firefighting and its effects on systemic DNA damage
    Publication . Esteves, Filipa; Madureira, Joana; Costa, Carla Sofia; Pires, Joana; Barros, Bela; Alves, Sara; Vaz, Josiana A.; Oliveira, Marta; Slezakova, Klara; Fernandes, Adília; Pereira, Maria do Carmo; Morais, Simone; Valdiglesias, Vanessa; Bonassi, Stefano; Teixeira, João Paulo; Costa, Solange
    Portugal is among the European Union countries more devastated by forest fires. Wildland fire- fighters are at the forefront of this battle, facing exposure to a wide range of harmful pollutants. Epidemiological studies have highlighted a potential link between occupational firefighting exposure and several diseases, including cancer. To date, very few studies have explored the biological mechanisms associated with such exposure. The present longitudinal study aims to assess changes in early effect biomarkers following wildland firefighters’ occupational exposure to a real wildfire event. Paired blood samples from 59 healthy Portuguese wildland firefighters were collected at two different time points: before wildfire season and after a fire event during wildfire season. Sociodemographic variables (e. g., age, sex) and work-related factors (e.g., years of service) were assessed via a self-reported questionnaire. Levels of early effect biomarkers, such as primary DNA damage and oxidative DNA damage (oxidised purines) were assessed via comet assay. DNA double-strand breaks (DSBs) were evaluated by phosphorylated H2AX (γH2AX). Moreover, hydroxylated polycyclic aromatic hydrocarbon metabolites (OHPAHs) and metal(loid)s were quantified in urine samples. The influence of urinary OHPAHs, urinary metal(loid)s, and other exposure- related factors (e.g., firefighting duration) on changes (Δ) in early effect biomarkers (post-vs. baseline levels) was investigated. Firefighting activities led to a significant increase in both primary DNA damage and oxidative DNA damage by 22 % (95 % CI: 1.11–1.35; p < 0.05) and 23 % (95 % CI: 1.04–1.45; p < 0.05), respectively. Results from linear regression revealed that per each unit increase of urinary 2-hydroxyfluorene (2-OHFlu) (μmol/mol creatinine), the risk of ⧍ oxidative DNA damage increased by 20 % [FR: 1.20 (1.09–1.32); p < 0.01]. Addi- tionally, each unit increase in urinary cesium (Cs) (μg/L) resulted in a significant 4 % increase in Δ primary DNA damage [FR: 1.04 (1.01–1.06); p < 0.05] and a 3 % increase in Δ oxidative DNA damage [FR: 1.03 (1.01–1.05); p < 0.05]. Post-exposure levels of γH2AX were significantly correlated with urinary 2-OHFlu levels assessed after firefighting (r = 0.30; p < 0.05). Furthermore, exposure duration and reported breathing difficulties during firefighting were significantly associated with increased levels of primary DNA damage. Results obtained provide insights into the potential human health effects of wildland firefighting occupational exposure at the genetic and molecular levels, offering new and important mechanistic data. These findings are crucial for implementing health and safety measures, recommendations, and best practices to mitigate occupational risks and protect the health of wildland firefighters.
  • Clitocybe alexandri extract induces cell cycle arrest and apoptosis in a lung cancer cell line: identification of phenolic acids with cytotoxic potential
    Publication . Vaz, Josiana A.; Almeida, Gabriela M.; Ferreira, Isabel C.F.R.; Martins, Anabela; Vasconcelos, M. Helena
    Mushrooms are a possible rich source of biologically active compounds with potential for drug discovery. The aim of this work was to gain further insight into the citotoxicity mechanism of action of Clitocybe alexandri ethanolic extract against a lung cancer cell line (NCI-H460 cells). The effects on cell cycle profile and levels of apoptosis were evaluated by flow cytometry, and the effect on the expression levels of proteins related to cellular apoptosis was also investigated by Western blot. The extract was characterized regarding its phenolic composition by HPLC-DAD, and the identified compounds were studied regarding their growth inhibitory activity, by sulforhodamine B (SRB) assay. The effect of individual or combined compounds on viable cell number was also evaluated using the Trypan blue exclusion assay. It was observed that the Clitocybe alexandri extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells. In addition, treatment with the GI50 concentration (concentration that was able to cause 50% of cell growth inhibition; 24.8 µg/ml) for 48h caused an increase in the levels of wt p53, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). The main components identified in this extract were protocatechuic, p-hydroxybenzoic and cinnamic acids. Cinnamic acid was found to be the most potent compound regarding cell growth inhibition. Nevertheless, it was verified that the concomitant use of the individual compounds provided the strongest decrease in viable cell number. Overall, we found evidence for alterations in cell cycle and apoptosis, involving p53 and caspase-3. Furthermore, our data suggests that the phenolic acids identified in the extract are at least partially responsible for the cytotoxicity induced by this mushroom extract.
  • Antimicrobial activity and bioactive compounds of portuguese wild edible mushrooms methanolic extracts
    Publication . Barros, Lillian; Calhelha, Ricardo C.; Vaz, Josiana A.; Ferreira, Isabel C.F.R.; Baptista, Paula; Estevinho, Leticia M.
    The antimicrobial properties of phenolic extracts of Portuguese wild edible mushroom species (Lactarius deliciosus, Sarcodon imbricatus and Tricholoma portentosum) against pathogens were investigated. The minimal inhibitory concentrations (MICs) were evaluated for the entire mushroom, the cap and the stipe, separately; the portion of the mushroom used proved to be influenced in the results obtained, which are directly correlated with the content of total phenols and flavonoids in the extracts. The growth of Grampositive bacteria (Bacillus cereus, B. subtilis,) was well inhibited by these mushrooms, while Escherichia coli (Gramnegative bacteria) was resistant. The study on the antifungal effect of these mushrooms revealed that Candida albicans and Cryptococcus neoformans were differently inhibited for the mushrooms used.
  • Occupational exposure of firefighters in non-fire settings
    Publication . Slezakova, Klara; Esteves, Filipa; Vaz, Josiana A.; Alves, Maria José; Madureira, Joana; Costa, Solange; Fernandes, Adília; Teixeira, João Paulo Fernandes; Morais, Simone; Pereira, Maria do Carmo
    This work assessed firefighters’ exposure to particulate matter (PM) in non-fire work settings during pre-fire season, as a baseline for the respective occupational exposure characterization. Indoor and outdoor air sampling was conducted for two weeks in pre-fire season of 2021 in seven fire corporations (FC1-FC7) in north of Portugal. PM fractions (PM2.5, PM10) were continuously monitored concurrently in indoors (living rooms, rest areas, truck bays) and outdoors. The results showed low levels of pollution. Indoor PM10 was between 2 and 205 µg m–3 (mean 10 µg m–3); PM2.5 were 2—115 µg m–3 (8.5 µg m–3). Both indoor PM fractions were highly and significantly correlated (rs = 0.959–0.997). PM2.5 accounted for 85% of indoor PM; indoor to outdoor ratios (I/O) of PM2.5 ranged between 1.4 and 3.0, thus emphasizing the contribution of indoor emission sources for fine fraction. Outdoor PM10 were 2–6 times higher than indoors (6–894 µg m–3; mean 21 µg m–3); and 2–9 times higher for PM2.5 (5 – 169 µg m–3; 5 µg m–3).
  • Bioactive activities of the phenolic extract from sterile bracts of Araucaria angustifolia
    Publication . Fischer, Thais E.; Marcondes, Amanda; Zardo, Danianni M.; Nogueira, Alessandro; Calhelha, Ricardo C.; Vaz, Josiana A.; Barros, Lillian; Zielinski, Acacio A.F.; Alberti, Aline
  • Wild mushrooms Clitocybe alexandri and Lepista inversa: In vitro antioxidant activity and growth inhibition of human tumour cell lines
    Publication . Vaz, Josiana A.; Heleno, Sandrina A.; Martins, Anabela; Almeida, Gabriela M.; Vasconcelos, M. Helena; Ferreira, Isabel C.F.R.
    The in vitro antioxidant and growth inhibitory activity of extracts obtained from two Portuguese wild mushrooms (Clitocybe alexandri and Lepista inversa) was studied in human tumour cell lines. The extracts were phenolic (methanolic and ethanolic) and polysaccharidic (boiling water). The antioxidant activity assays included evaluation of radical-scavenging capacity, reducing power and inhibition of lipid peroxidation measured in liposome solutions. Extract-induced cell growth inhibition was measured in four different tumour cell lines (lung, breast, colon and gastric cancer) using the SRB assay. The polysaccharidic extract oft. inversa was the most potent as antioxidant (EC50 < 1.8 +/- 0.1 mg/ml), while the phenolic ethanolic extract of C alexandri was the most potent as inhibitor of growth of the studied cancer cell lines (Gl(50) <26.0 +/- 1.3 mu g/ml). Together, these activities indicate that these mushrooms are promising sources of bioactive compounds. (C) 2010 Elsevier Ltd. All rights reserved.
  • Cogumelos silvestres portugueses: valorização como alimentos funcionais e fonte de nutracêuticos
    Publication . Ferreira, Isabel C.F.R.; Vaz, Josiana A.; Barros, Lillian; Almeida, Gabriela M.; Martins, Anabela; Vasconcelos, M. Helena
    Os cogumelos silvestres possuem grande diversidade de biomoléculas com valor nutricional e/ou propriedades medicinais. Têm sido reconhecidos como alimentos funcionais e como fonte de compostos para o desenvolvimento de fármacos e nutracêuticos, incluindo compostos com propriedades anti-tumorais. Exemplos disso são os β-glucanos “Lentinan” (do cogumelo Lentinus edodes) e “Schizophyllan” (do meio de cultura de Schizophyllum commune) ou os glucopéptidos PSP e “Krestin” (PSK) (do micélio de Coriolus versicolor). Para além dos cogumelos mencionados, existem muitos outros conhecidos como sendo medicinais e existe um potencial enorme nas espécies ainda não exploradas do Nordeste Português. Por esse motivo, o nosso grupo analisou algumas destas espécies ainda não estudadas, tais como Clitocybe alexandri, Lepista inversa e Suillus collinitus. Os seus extratos fenólicos foram caracterizados e mostraram potenciais efeitos anti-tumorais em linhas celulares tumorais humanas, que se traduzem especificamente na indução de paragem do ciclo de divisão celular e na indução de morte celular programada (apoptose) em linhas celulares modelo, derivadas de diferentes tipos de tumores humanos. Verificámos que extratos de Clitocybe alexandri induzem paragem no ciclo celular e apoptose numa linha celular de cancro do pulmão, constituindo-se assim como fonte de potenciais novos agentes citotóxicos. O nosso grupo de investigação descobriu também que o extrato metanólico de Suillus collinitus aumenta a expressão da proteína supressora de tumores p53, causa paragem no ciclo de divisão celular e aumenta a apoptose numa linha celular modelo de cancro da mama. Tendo em conta o potencial anti-tumoral evidenciado em condições in vitro pelas espécies mencionadas, os extratos/compostos fenólicos prefiguram-se como potenciais agentes citotóxicos e poderão encontrar, caso estudos futuros o comprovem, potencial utilização no tratamento do cancro, a segunda causa de morte nos países desenvolvidos. Em específico, podem potencialmente servir para acrescentar e diversificar os “pipelines” de desenvolvimento de fármacos da indústria farmacêutica ou de alimentos funcionais.
  • HPLC-DAD-ESI/MS phenolic characterization and biological activity of Equisetum giganteum L.
    Publication . Jabeur, Inès; Martins, Natália; Barros, Lillian; Calhelha, Ricardo C.; Vaz, Josiana A.; Achour, Lotfi; Santos-Buelga, Celestino; Ferreira, Isabel C.F.R.
    Naturally-occurring phytochemicals have received a pivotal attention in the last years, due to the increasing evidences of biological activities. Equisetum giganteum L., commonly known as “giant horsetail”, is a native plant from Central and South America, being largely used in dietary supplements as diuretic, hemostatic, antiinflammatory and anti-rheumatic agents [1,2]. The aim of the present study was to evaluate the antioxidant (scavenging effects on 2,2-diphenyl-1-picrylhydrazyl radicals- RSA, reducing power- RP, β-carotene bleaching inhibition- CBI and lipid peroxidation inhibition- LPI), anti-inflammatory (inhibition of NO production in lipopolysaccharidestimulated RAW 264.7 macrophages) and cytotoxic (in a panel of four human tumor cell lines: MCF-7- breast adenocarcinoma, NCI-H460- non-small cell lung cancer, HeLa- cervical carcinoma and HepG2- hepatocellular carcinoma; and in non-tumor porcine liver primary cells- PLP2) properties of E. giganteum, providing a phytochemical characterization of its extract (ethanol/water, 80:20, v/v), by using highperformance liquid chromatography coupled to diode array detection and electrospray ionisation mass spectrometry (HPLC-DAD–ESI/MS). E. giganteum presented fourteen phenolic compounds, two phenolic acids and twelve flavonol glycoside derivatives, mainly kaempferol derivatives, accounting to 81% of the total phenolic content, being kaempferol-O-glucoside-O-rutinoside, the most abundant molecule (7.6 mg/g extract). The extract exhibited antioxidant (EC50 values = 123, 136, 202 and 57.4 μg/mL for RSA, RP, CBI and LPI, respectively), anti-inflammatory (EC50 value = 239 μg/mL) and cytotoxic (GI50 values = 250, 258, 268 and 239 μg/mL for MCF-7, NCI-H460, HeLa and HepG2, respectively) properties, which were positively correlated with its concentration in phenolic compounds. Furthermore, up to 400 μg/mL, it did not revealed toxicity in non-tumor liver cells. Thus, this study highlights the potential of E. giganteum extracts as rich sources of phenolic compounds that can be used in the food, pharmaceutical and cosmetic fields.