The Beauty and the Toxic Beast: Use of Comet Assay to Study Antigenotoxicity of Natural Ingredients

The natural cosmetics market has grown since consumers became conscious of natural-based ingredients. A significant number of cosmetics have noxious and chemically potent substances. Thus, the use of natural and organic cosmetics has become increasingly important. An intense investigation into the benefits fruits and plants can bring to our health is required. A healthy lifestyle can reduce these problems, including the consumption or use of substances that protect the genome through various mechanisms that reduce DNA damage. Genotoxicological studies are essential to know the threats to the genome and health, and antigenotoxicological studies are the answer to minimise the instability of the genome. Natural ingredients such as Almond (Prunus dulcis), Elderberry (Sambucus nigra), Olives (Olea europaea), and Grapes (Vitis vinifera) have been shown to possess a variety of biological activities and to hold therapeutic promise. They are the most common ingredients in the Trás-os-Montes region (Portugal). This study aimed to demonstrate, in vivo, the genotoxicological effects of Elderberry, Almonds, Olives, and Grapes in Drosophila melanogaster using the Comet assay

Genotoxic effect of ethanol in drosophila melanogaster neuroblasts

RESUMO - Introdução: O cérebro é um importante alvo do etanol, sendo o consumo excessivo de álcool associado a danos cerebrais. Tem sido demonstrado que os produtos do metabolismo do etanol promovem alterações no DNA. Desta forma, e apesar de se verificar em Portugal uma tendência para a diminuição do consumo de álcool, pensamos ser pertinente a realização deste estudo. Objectivo: Avaliar a toxicidade e o potencial efeito genotóxico do etanol em Drosophila melanogaster in vivo utilizando o ensaio do cometa. Métodos: Realizou-se um estudo de toxicidade para o etanol com as concentrações de 0 %; 0,625 %; 1,25 %; 2,5 % e 5 % (v/v), administradas de forma crónica, seguindo-se a contagem dos descendentes adultos para a avaliação da sobrevivência relativa das drosófilas nas diferentes concentrações de etanol. Seguidamente, utilizaram-se neuroblastos de drosófila para a avaliação do efeito genotóxico pelo ensaio do cometa (ensaio de electroforese de células isoladas). Os danos de DNA são reportados através dos seguintes parâmetros: comprimento da cauda, percentagem de DNA na cauda e momento de cauda. Resultados: O etanol não apresentou toxicidade para a D. melanogaster, verificando-se, pelo contrário, um aumento no número de descendentes em todas as concentrações de etanol testadas, mostrando-se, portanto a presença de etanol benéfica em termos de número de descendentes. A sobrevivência superior ocorreu na concentração de 1,25 %, uma vez que é nesta concentração que se encontraram mais descendentes. No entanto, em termos de genotoxicidade constatou-se nos parâmetros avaliados no ensaio do cometa que, de uma forma genérica, quanto maior a concentração de etanol mais danos são infligidos ao DNA. O etanol evidenciou um efeito genotóxico nas três concentrações mais elevadas em estudo (1,25 %; 2,5 % e 5 %). Os danos foram superiores para a concentração de 5 % de etanol. Por outro lado, a concentração de etanol de 0,625 % não se mostrou genotóxica. Conclusões: A D. melanogaster exposta a diferentes concentrações de etanol manifestou um aumento significativo de danos no DNA, ao nível dos neuroblastos, verificando-se um aumento dose-dependente, nos parâmetros do ensaio do cometa avaliados. Foi, portanto possível demonstrar que o etanol provoca rupturas no DNA, avaliadas pela presença de cometas.ABSTRACT - Introduction: The brain is an important target to ethanol, being the excessive consumption of alcohol related to brain damage. It has been shown that the metabolic products of ethanol induce DNA alterations. Thus, although in Portugal the consumption of alcohol is decreasing, we believe that the present study is pertinent. Purpose: To assess the toxicity and the genotoxicity of ethanol in Drosophila melanogaster in vivo using the comet assay. Methods: The toxicity study of ethanol at 0 %; 0.625 %; 1.25 %; 2.5 % and 5 % (v/v) was conducted; then, we counted the adult descendents to assess the rate of survival of drosophilas in the different concentrations of ethanol. Afterwards, the neuroblasts of drosophila were used to evaluate the genotoxicity of ethanol using the comet assay (single-cell gel electrophoresis). The DNA damage was quantified by determining the following parameters: the percentage of DNA in the tail, tail length, and tail moment. Results: Ethanol did not present toxicity to D. melanogaster, as the number of descendents were higher in all the concentrations tested comparatively to 0 %, hence, the presence of ethanol was beneficial. The higher survival rate was observed in the concentration of 1.25 %, as in this concentration the number of descendents was higher. The parameters assessed by the comet assay showed that in general higher DNA damage is induced by higher concentrations of ethanol. Ethanol showed genotoxicity in the higher concentrations used in the present study (1.25 %, 2.5 %, 5 %). The damages in DNA were superior in the ethanol concentration of 5 %. Conversely, the ethanol concentration of 0.625 % was not genotoxic. Conclusions: The D. melanogaster exposed to different concentrations of ethanol revealed a significant increase in the DNA damage, in neuroblasts, being observed a dose-dependent increase in all the parameters assessed in the comet assay. Thus, the present study demonstrated that ethanol induces DNA ruptures, assessed by the presence of comets.info:eu-repo/semantics/publishedVersio

The <em>w</em>/<em>w</em> ⁺ Somatic Mutation and Recombination Test (SMART) of <em>Drosophila melanogaster</em> for Detecting Antigenotoxic Activity

Genotoxicological studies are emerging as fundamental for knowing the hazards to our genome, to our health. Drosophila melanogaster is one of the preferable organisms for toxicological research considering its metabolic similarities (viz. on dietary input, xenobiotic metabolizing system, antioxidant enzymes and DNA repair systems) to mammals. Accordingly, somatic mutation and recombination tests (SMARTs) of D. melanogaster are fast and low-cost in vivo assays that have shown solid results evaluating genotoxicity. The w/w + SMART uses the white (w) gene as a recessive marker to monitor the presence of mutant ommatidia (eye units), indicating the occurrence of point mutations, deletions, mitotic recombination or/and nondisjunction. Additionally, several studies used SMARTs to assess antigenotoxicity, with some using the w/w + SMART. We reviewed the state of the art of the w/w + SMART used for antigenotoxicity analysis, focusing on published results, aiming to contribute to the conception of a reliable protocol in antigenotoxicity. As such, genotoxic agents with known action mechanisms, as streptonigrin (oxidative stress inducer), were used as a genotoxic insult for proving the antigenotoxic effects of natural substances (e.g. seaweeds), demonstrating the presence of antimutagens in their composition. These antigenotoxicity studies are crucial for promoting preventive measures against environmental genotoxics that affect humans daily

Ginkgo biloba L. Leaf Extract Protects HepG2 Cells Against Paraquat-Induced Oxidative DNA Damage

Ginkgo biloba L. leaf extracts and herbal infusions are used worldwide due to the health benefits that are attributed to its use, including anti-neoplastic, anti-aging, neuro-protection, antioxidant and others. The aim of this study was to evaluate the effect of an aqueous Ginkgo biloba extract on HepG2 cell viability, genotoxicity and DNA protection against paraquat-induced oxidative damage. Exposure to paraquat (PQ), over 24 h incubation at 1.0 and 1.5 µM, did not significantly reduce cell viability but induced concentration and time-dependent oxidative DNA damage. Ginkgo biloba leaf extract produced dose-dependent cytotoxicity (IC50 = 540.8 ± 40.5 µg/mL at 24 h exposure), and short incubations (1 h) produced basal and oxidative DNA damage (>750 and 1500 µg/mL, respectively). However, lower concentrations (e.g., 75 µg/mL) of Ginkgo biloba leaf extract were not cytotoxic and reduced basal DNA damage, indicating a protective effect at incubations up to 4 h. On the other hand, longer incubations (24 h) induced oxidative DNA damage. Co-incubation of HepG2 cells for 4 h, with G. biloba leaf extract (75 µg/mL) and PQ (1.0 or 1.5 µM) significantly reduced PQ-induced oxidative DNA damage. In conclusion, the consumption of Ginkgo biloba leaf extract for long periods at high doses/concentrations is potentially toxic; however, low doses protect the cells against basal oxidative damage and against environmentally derived toxicants that induce oxidative DNA damage.Financial support was received from the INTERACT project—“Integrative Research in Environment, Agro-Chains and Technology”, no. NORTE-01-0145-FEDER-000017, in its line of research entitled ISAC, co-financed by the European Regional Development Fund (ERDF) through NORTE 2020 (North Regional Operational Program 2014/2020). C.M.-G. was supported by a grant from INTERACT (BIM/UTAD/30/2018). The Portuguese Science and Technology Foundation (FCT) is also acknowledged under the projects UID/AGR/04033/2019 (CITAB) and UID/CVT/00772/2019 (CECAV).info:eu-repo/semantics/publishedVersio

Platanus hybrida’s phenolic profile, antioxidant power, and antibacterial activity against methicillin-resistant staphylococcus aureus (MRSA)

Methicillin-resistant S. aureus (MRSA) are a threat to public health as they frequently reveal a multidrug-resistant pattern. Researchers all over the world are on an urgent hunt for new treatments to help fight infections before antibiotics become obsolete, and some natural alternatives, such as polyphenols, have already exhibited therapeutic properties. Therefore, this study aimed to determine the phenolic profile, antioxidant capacity, and antimicrobial activity against MRSA of the leaf, fruit, and stem bark extracts of Platanus hybrida. The polyphenols were extracted with a water/ethanol (20:80) mixture and the methodology included HPLC-DAD, DPPH, FRAP, and CuPRAC. To address this issue from a One Health perspective, the Kirby–Bauer disc diffusion method was performed against nine MRSA strains from three different sources (livestock, wild animals, and humans). Fourteen phenolics were identified and the leaf extract showed the highest phenolic content, followed by the fruit extract. The leaf extract also showed the highest antioxidant capacity while the fruit extract had the lowest antioxidant capacity. Both leaf and fruit extracts inhibited the growth of strains from all sources, while the stem bark extract did not inhibit the growth of human strains. This work highlights the complex chemical composition and the antioxidative and antimicrobial potential of extracts derived from P. hydrida.This work was supported by the projects UIDB/CVT/00772/2020 and LA/P/0059/2020 funded by the Portuguese Foundation for Science and Technology (FCT). This work was also supported by the Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020) funded by FCT. The authors acknowledge the financial support of FCT by national funds under the project UIDB/04033/2020. Vanessa Silva is grateful to FCT for financial support through PhD grant SFRH/BD/137947/2018. To FCT, Portugal for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020) and the contract of L. Barros through the institutional scientific employment program-contract. This work was supported.info:eu-repo/semantics/publishedVersio

Oxidative stress function in women over 40 years of age, considering their lifestyle

Aging is dependent on biological processes that determine the aging of the organism at the cellular level. The Oxidative Stress Theory of Aging might explain some of the age-related changes in cell macromolecules. Moreover, exposome and lifestyle may also induce changes in cell damage induced by oxidative stress. The aim of the present study was to analyze the related redox changes in lymphocyte function of healthy women over 40 years old. Three groups: younger (YG: 40–49 years), middle aged (MAG: 50–59 years), and older (OG: ≥60 years) were evaluated on anthropometric variables, blood pressure, cardiovascular fitness, lifestyle habits, perceived stress, DNA damage, malondialdehyde, catalase activity, and total antioxidant capacity. Physical activity and cardiovascular fitness were significantly higher in YG and MAG as compared to the OG. Systolic blood pressure increased significantly with group age. Frequency and total amount of alcohol intake were lower in the OG and higher in the MAG. No significant differences were observed between the three groups in oxidative stress parameters. Only alcohol consumption was associated with the higher DNA FPG-sensitive sites, and only in the YG (p < 0.05). Healthy lifestyle is critical to avoiding major ailments associated with aging. This may be inferred from the lack of significant differences in the various oxidative stress parameters measured in the healthy women over the age of 40 who took part in the study. Conscious lifestyle behaviors (decrease in alcohol and smoking habits) could have impaired the expected age-related oxidative stress increase.info:eu-repo/semantics/publishedVersio

Dietary supplementation with chestnut (Castanea sativa) reduces abdominal adiposity in FVB/n mice: a preliminary study

The production of chestnut (Castanea sativa Miller) is mostly concentrated in Europe. Chestnut is recognized by its high content of antioxidants and phytosterols. This work aimed to evaluate the e ects of dietary chestnut consumption over physiological variables of FVB/n mice. Eighteen FVB/n male 7-month-old mice were randomly divided into three experimental groups (n = 6): 1 (control group) fed a standard diet; 2 fed a diet supplemented with 0.55% (w/w) chestnut; and 3 supplemented with 1.1% (w/w) chestnut. Body weight, water, and food intake were recorded weekly. Following 35 days of supplementation, the mice were sacrificed for the collection of biological samples. Chestnut supplementation at 1.1% reduced abdominal adipose tissue. Lower serum cholesterol was also observed in animals supplemented with chestnut. There were no significant di erences concerning the incidence of histological lesions nor in biochemical markers of hepatic damage and oxidative stress. These results suggest that chestnut supplementation may contribute to regulate adipose tissue deposition.This work is supported by National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020, CIMO (UIDB/00690/2020) and UIDB/CVT/00772/2020 Interreg Program for the financial support of the Project IBERPHENOL, Project Number 0377_IBERPHENOL_6_E; co-financed by European Regional Development Fund (ERDF) through POCTEP 2014-2020. This work was also supported by VALORIZEBYPRODUCTS Project, reference n.º 029152, funded by Portuguese Foundation for Science and Technology (FCT) and co-financed by the European Regional Development Fund (FEDER) through COMPETE 2020 - Operational Competitiveness and Internationalization Programme (POCI). This work was also financially supported by Project UID/EQU/00511/2019 - Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE funded by national funds through FCT/MCTES (PIDDAC) and Project “LEPABE-2-ECO-INNOVATION” – NORTE-01-0145-FEDER-000005, funded by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), by the Research Centre of the Portuguese Institute of Oncology of Porto (CI-IPOP 37-2016) and by the Interact R&D project, operation number NORTE-01-0145-FEDER-000017, in its ISAC research line, co-financed by the ERDF through NORTE 2020. This work was also supported by PhD fellowship SFRH/BD/136747/2018.info:eu-repo/semantics/publishedVersio

Toxicological and anti-tumor effects of a linden extract (Tilia platyphyllos Scop.) in a HPV16-transgenic mouse model

Tilia platyphyllos Scop. is a popular broad-leaved tree, native to Central and Southern Europe. Hydroethanolic extracts rich in phenolic compounds obtained from T. platyphyllos Scop. have shown in vitro antioxidant, anti-inflammatory and antitumor properties. The aim of this work was to evaluate the therapeutic properties of a hydroethanolic extract obtained from T. platyphyllos in HPV16-transgenic mice. The animals were divided into eight groups according to their sex and phenotype. Four groups of female: HPV+ exposed to linden (HPV linden; n = 6), HPV+ (HPV water; n = 4), HPV- exposed to linden (WT linden; n = 5) and HPV- (WT water; n = 4) and four groups of male: HPV+ exposed to linden (HPV linden; n = 5), HPV+ (HPV water; n = 5), HPV- exposed to linden (WT linden; n = 5) and HPV- (WT water; n = 7). The linden (Tilia platyphyllos Scop.) extract was orally administered at a dose of 4.5 mg/10 mL per animal (dissolved in water) and changed daily for 33 days. The hydroethanolic extract of T. platyphyllos consisted of protocatechuic acid and (-)-epicatechin as the most abundant phenolic acid and flavonoid, respectively, and was found to be stable during the studied period. In two male groups a significant positive weight gain was observed but without association with the linden extract. Histological, biochemical, and oxidative stress analyses for the evaluation of kidney and liver damage support the hypothesis that the linden extract is safe and well-tolerated under the present experimental conditions. Skin histopathology does not demonstrate the chemopreventive effect of the linden extract against HPV16-induced lesions. The linden extract has revealed a favourable toxicological profile; however, additional studies are required to determine the chemopreventive potential of the linden extract. This journal isThis work was supported by the project IBERPHENOL, project number 0377_IBERPHENOL_6_E; Interact R&D project, operation number NORTE-01-0145-FEDER-000017, National Funds by FCT – Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020 (CITAB), and project UIDB/ CVT/00772/2020 (CECAV) and the post-graduation grant SFRH/ BD/136747/2018 and 2020.04789.BD; the authors are also grateful to FCT, Portugal and FEDER under programme PT2020 for financial support to CIMO (UIDB/00690/2020) and L. Barros acknowledges the national funding by FCT, P. I., through the institutional scientific employment program-contract. The authors would like to thank Cantinho das Aromáticas organic farmers from Vila Nova de Gaia (Portugal) for providing the samples. This work was financially supported by: Base Funding - UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE - funded by national funds through the FCT/MCTES (PIDDAC).info:eu-repo/semantics/publishedVersio

Pulegone and Eugenol Oral Supplementation in Laboratory Animals: Results from Acute and Chronic Studies

Essential oils are natural compounds used by humans for scientific purposes due to their wide range of properties. Eugenol is mostly present in clove oil, while pulegone is the main constituent of pennyroyal oil. To guarantee the safe use of eugenol and pulegone for both humans and animals, this study addressed, for the first time, the effects of these compounds, at low doses (chronic toxicity) and high doses (acute toxicity), in laboratory animals. Thirty-five FVB/n female mice were randomly assigned to seven groups (n = 5): group I (control, non-additive diet); group II (2.6 mg of eugenol + 2.6 mg of pulegone); group III (5.2 mg of eugenol + 5.2 mg of pulegone); group IV (7.8 mg of eugenol + 7.8 mg of pulegone); group V (7.8 mg of eugenol); group VI (7.8 mg of pulegone); and group VII (1000 mg of eugenol + 1000 mg of pulegone). The compounds were administered in the food. Groups I to VI were integrated into the chronic toxicity study, lasting 28 days, and group VII was used in the acute toxicity study, lasting 7 days. Animals were monitored to assess their general welfare. Water and food intake, as well as body weight, were recorded. On the 29th day, all animals were euthanized by an overdose of ketamine and xylazine, and a complete necropsy was performed. Blood samples were collected directly from the heart for microhematocrit and serum analysis, as well as for comet assay. Organs were collected, weighed, and fixed in formaldehyde for further histological analysis and enzymatic assay. Eugenol and pulegone induced behavioral changes in the animals, namely in the posture, hair appearance and grooming, and in mental status. These compounds also caused a decrease in the animals' body weight, as well as in the food and water consumption. A mortality rate of 20% was registered in the acute toxicity group. Both compounds modulated the serum levels of triglycerides and alanine aminotransferase. Eugenol and pulegone induced genetic damage in all animals. Eugenol increased the activity of the CAT enzyme. Both compounds increased the GR enzyme at the highest dose. Moreover, pulegone administered as a single compound increased the activity of the GST enzyme. Histopathological analysis revealed inflammatory infiltrates in the lungs of groups II, III, and IV. The results suggest that eugenol and pulegone may exert beneficial or harmful effects, depending on the dose, and if applied alone or in combination