Impact of transplacental exposure to tobacco smoke in the DNA of newborn. Evaluation of genetic damage and epigenetic changes. Evolution throughout childhood.

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Protective effect of Crataegus monogyna Jacq. ethanolic extracts in oxidant-induced DNA damage evaluated through comet assay with human peripheral lymphocytes

Publication . Barreira, João C.M.; Costa, Carla Sofia; Teixeira, João Paulo Fernandes; Ferreira, Isabel C.F.R.; Oliveira, Beatriz

Much attention of preventive medicine research is focused on natural antioxidants. This interest refers not only to isolation and identification of new biologically active molecules for the pharmaceutical industry, but also because of the emergent public interest in using crude plant extracts, such as infusions for self-medication (Krishnaiah et al., 2011). The use of antioxidants, such as the well-known polyphenolic compounds, to prevent genetic damage induced by physical or chemical agents is of considerable interest. This bioactivity might be related to their anticlastogenic effect, due to the presence of specific functional groups. Other antioxidant compounds, such as vitamins C and D, were reported for their DNA-damage decreasing effect, suggesting that reactive oxygen species may be involved in this activity (Benavente-García et al., 2004). Evaluating the antioxidant activity of natural matrices represents one of our primary research challenges (Ferreira et al., 2009). Among hundreds of studied species, Crataegus monogyna Jacq. stood out as being one of the most promising plants due to its high bioactivity. Besides the antioxidant activity, C. monogyna was also studied for the human tumour cells growth inhibitory capacity of its phenolic extracts; furthermore, individual phenolic compounds were fully characterized by high performance liquid chromatography-photo diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI/MS), revealing high levels of flavonols, flavones, hydroxycinnamic acid derivatives and anthocyanins (Rodrigues et al., 2012). However, there is a high limitation in examining if the detected bioactivity is actually transferred from in vitro to in vivo systems (Carocho and Ferreira, 2013). Nevertheless, there have been major advances to accurately measure end products of oxidative damage to proteins, lipids, and DNA. Short-term in vitro tests are commonly used to identify genotoxicants and antigenotoxicants. Comet assay, in particular, has been considered to be a useful biomarker to this purpose as it can easily establish a link between natural extracts and DNA repair damage (Cemeli et al., 2009). Although this assay might be carried out virtually with any cell type, lymphocytes are often used as these are in contact with xenobiotics after absorption and have proved to be good surrogate cells (Cemeli et al., 2009). Herein, the potential genotoxic and antigenotoxic effect of ethanolic extracts from different botanical parts of C. monogyna was evaluated by the alkaline comet assay. Extracts were assayed to match the previously determined effective concentrations (EC) corresponding to 25% (EC25), 37.5% (EC37.5), 50% (EC50), 62.5% (EC62.5) and 75% (EC75) of antioxidant activity. Blood samples were obtained from healthy non-smoking donors in EDTA tubes and lymphocytes were isolated by Ficoll density gradient. A pool of lymphocytes was exposed to extract in different concentrations in addition to a positive and negative control for 1h to test for extracts’ genotoxicity. To test for extracts antigenotoxicity, two concentrations were selected and cells were exposed to a mutagenic compound (MMS) and extract concentrations simultaneously for 1 h. In one experiment, comet assay was immediately performed after the exposure period while in the second experiment, cell media were renewed and the comet assay was performed after a recovery-period of 1 h. All experiments were carried out with and without metabolic activation by liver fraction (S9). The results showed significant differences within the assessed botanical parts and also among the assayed concentrations. The performed investigation might be considered as representing a step further in the evaluation of the in vivo bioactive potential of this highly promising species. Furthermore, it has established some practical bases for the evaluation of additional natural matrices with high scoring in bioactivity screening studies.

2015Conference object

Open access

Evaluation of the cytotoxicity (HepG2) and chemical composition of polar extracts from the ruderal species Coleostephus myconis (L.) Rchb.f.

Publication . Bessada, Sílvia M.F.; Barreira, João C.M.; Santos, J.V. Araújo; Costa, Carla Sofia; Pimentel, Filipa B.; Bessa, Maria João; Teixeira, João Paulo Fernandes; Oliveira, Beatriz

Coleostephus myconis (L.) Rchb.f. (Asteraceae) is a highly disseminated plant species with ruderal and persistent growth. Owing to its advantageous agronomic properties, C. myconis might have industrial applications. However, this species needs to be comprehensively characterized before any potential use. In a previous study, the phenolic composition and antioxidant activity of different C. myconis tissues were characterized. This investigation was extended to examine the cytotoxic potential of selected plant tissues (flowers and green parts) using a HepG2 cell line by utilizing the lysosomal neutral red uptake assay or mitochondrial (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. In addition, the macronutrients content, lipophilic compounds (fatty acids, tocopherols), and amino acids were also determined. C. myconis flowers were used in the senescence stage, which was previously identified as the stage that presented maximal phenolic content and highest antioxidant activity. In contrast, stems and leaves were employed due to their high biomass proportion. Regarding cytotoxicity, mitochondrial and lysosomal damage was only significant when HepG2 cells were exposed to the highest extract concentrations (stems and leaves, 0.9 mg/ml; senescent flowers, 0.3 mg/ml). Chemically, the senescent flowers were mostly characterized by their high levels of fat, amino acids (especially threonine), oleic acid, ß-, and ?-tocopherol, while stems and leaves contained high concentrations of carbohydrates, linolenic acid, and a-tocopherol. In general, these results provide information regarding the threshold concentrations of C. myconis extracts that might be used in different applications without toxicity hazards.

2017Journal article

Open access

Antioxidant activity and cytotoxic effects of polar extracts from saffron (Crocus sativus L) flowers

Publication . Barreira, João C.M.; Rebelo, José Miguel; Costa, Carla Sofia; Costa, Anabela S.G.; Ferreira, Isabel C.F.R.; Teixeira, João Paulo Fernandes; Oliveira, Beatriz

Saffron (Crocus sativus L.) flower is composed of six purple tepals, three yellow stamens and a white filiform style ending in a stigma with three threads, which only represents less than 10% (w/w) of the flower weight. Nevertheless, saffron is cultivated for the stigma of its flowers which, after being dried, is the most valued spice [1]. For each kg of this spice, about 63 kg of floral bio-residues are produced, which so far are not exploited, being usually thrown away. However, the floral bio-residues were reported as having high phenolic content and bioactive properties, such as antioxidant, antityrosinase, antidepressant, antinociceptive, anti-inflammatory, antifungal and arterial pressure reducer activities [2]. In view of the reported bioactivity, it is important to study the cytotoxic effects of saffron [3]. Furthermore, it is a wellknown fact that the effectiveness of bioactive compounds extraction from plants, as well as their corresponding activity, is highly dependent on factors such as different types of solvent, solvent-to-solid ratios and specially the solvent polarity [4]. In the present work, the antioxidant activity of different polar extracts (ethanol, ethanol:water 1:1 v/v, and water) of saffron was evaluated using different in vitro assays (2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity, ferric reducing reducing power and inhibition of β-carotene bleaching assay). In addition, the cytotoxicity of the extracts was also evaluated in Caco-2 (ATCC HTB-37TM) cultures by using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay. All samples proved to have antioxidant activity, despite the higher effectiveness of the hydroalcoholic extract. Up to the assayed concentrations, none of the extracts showed cytotoxicity against Caco-2 cell lines. Accordingly, saffron flowers might be used in different applications such as the development of food supplements or pharmaceutic related products.

2014Conference object

Open access

Wood smoke exposure of Portuguese wildland firefighters: DNA and oxidative damage evaluation

Publication . Abreu, Ana M.; Costa, Carla Sofia; Silva, Susana Pinho; Morais, Simone; Pereira, Maria do Carmo; Fernandes, Adília; Andrade, Vanessa Moraes; Teixeira, João Paulo Fernandes; Costa, Solange

Portugal is among the European Union countries most devastated by forest fires each year. In the last three decades, more than 3.8 million hectares of forest were burned. Wildland firefighters are exposed to a variety of hazards, including many toxic combustion products that may lead to deleterious health effects. Epidemiological studies showed a positive association between firefighting and several chronic diseases, including cancer. Results from biomonitoring studies in firefighters, particularly concerning genotoxicity evaluation, constitute a valuable tool for investigating important occupational hazards. Thus, the aim of this study was to assess genotoxicity in a group of wildland firefighters using the comet assay for DNA damage and oxidative stress. Both parameters were increased in firefighters compared to controls, but significance was only found for basal DNA damage. No significant influence was found regarding major confounding variables on the genotoxic endpoints studied, with the exception of age. Data obtained provide preliminary information on human health effects of wildland firefighting exposure at genetic and molecular levels. These findings may also provide new important data to serve as public awareness to the potential adverse health risks involving wildland firefighting. Implementation of security and hygiene measures in this sector as well as good practices campaigns may be crucial to decrease risk.

2017Journal article

Open access

Antioxidant activity and cytotoxic effects of polar extracts from saffron (Crocus sativus L.) flowers

Publication . Barreira, João C.M.; Rebelo, José Miguel; Costa, Carla Sofia; Costa, Anabela S.G.; Ferreira, Isabel C.F.R.; Teixeira, João Paulo Fernandes; Oliveira, Beatriz

Saffron is cultivated exclusively to obtain the stigma of its flowers, which represents less than 10% of the flower weight. In fact, to achieve a single kg of dried stigma, more than 60 kg of floral bio-residues are produced. These bio-residues were reported as having high bioactivity and important contents in bioactive compounds. Before considering the use of these bioresidues in different applications, it is important to assess its cytotoxicity. Herein, the antioxidant activity and cytotoxicity of saffron flower polar extracts were evaluated. All samples showed antioxidant activity, despite the higher effectiveness of the hydroalcoholic extract. Up to the concentrations corresponding to the EC50 values obtained in the antioxidant activity assays, none of the extracts showed high cytotoxicity against Caco-2 cell lines. Accordingly, saffron flowers might be used in different applications such as the development of food supplements or pharmaceutical related products.

2014Conference object

Open access