Ethanolic extracts from three commercial edibe mushrooms: determination of ergosterol contente and evaluation of bioactives properties

Mushrooms are rich in several bioactive metabolites among them are phenolic compounds, terpenoids, polysaccharides, lectins, and steroids including mycosterols, namely ergosterol [1]. Ethanolic extracts prepared by maceration of several mushroom species have been recently described as having antiinflammatory properties [2]. In the present work, ethanolic extracts of Agaricus bisporus L., Lentinus edodes (Berk.) Pegler and Pleurotus ostreatus (Jacq. ex Fr.) P.Kumm., purchased from a local supermarket in the Northeast of Portugal, were obtained by Soxhlet and chemically characterized in terms of ergosterol content by HPLC-UV. The antioxidant properties of these extracts were evaluated through DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity (RSA), reducing power (RP), p. carotene bleaching inhibition (CBI) and lipid peroxidation inhibition in TBARS (thiobarbituric acid reactive substances) assay (LPI); the antioxidant activity of ergosterol was also evaluated by the DPPH assay. The anti-inflammatory activity of the same extracts and ergosterol was evaluated in LPS (lipopolysaccharide) stimulated RAW 264.7 macrophages, through the inhibition of NO production. A. bisporus revealed the highest content in ergosterol (44.8 ± 0.4 mg/ g extract) followed by P. ostreatus (34 ± 3 mg/ g extract) and finally L. edodes (8.9 ± 0.1 mg/ g extract). A. bisporus showed the highest RSA, RP and CBI (EC50 values= 7.0 ± 0.8, 2.3 ± 0.1 and 1.4 ± 0.1 mg/mL, respectively), while L. edodes presented the highest LPI (2.5 ± 0.1 mg/mL ); ergosterol revealed higher RSA (0.46±0. 0 I mg/mL) than the extracts. Concerning the anti-inflammatory potential, the most efficient species was L. edodes (lC50 value = 164 ± 16 J.lg/mL), followed by A. bisporus (185 ± 16 J.lg/mL) and finally P. ostreatus (290 ± 10 J.lg/mL). However, ergosterol presented lower activity (338 ± 23 J.lg/mL) due to its low solubility in the culture medium. The higher antioxidant properties displayed by A. bisporus can be related with its higher ergosterol content, while in the anti-inflammatory activity this relation cannot be established also due to the low solubility of ergosterol in the cells culture medium, decreasing the ergosterol availability. More studies are being conducted regarding the ergosterol solubility. Several compounds have been implicated in the bioactivity of mushrooms and in this study we have found that ergosterol can give an important contribution.

Gamma radiation preserves chemical and bioactive properties of Boletus edulis wild mushrooms

Boletus edulis Bull: Fr. is an edible mushroom quite appreciated for its organoleptic and nutritional properties. However, the seasonality and perishability cause some difficulties in its distribution and marketing in fresh form; losses associated with this type of food during marketing can reach 40% [1]. Irradiation is recognized as a safe and effective method for food preservation, being used worldwide to increase shelf life of fresh and dehydrated products (e.g. fruits, vegetables and spices) [2]. In particular, gamma irradiation has already been applied to cultivated mushrooms (especially Agaricus, Lentinula and Pleurotus Genus) and proved to be an interesting conservation technology [3]. However, the studies with added-value wild species are scarce. In this work, the effects of gamma irradiation on chemical and antioxidant properties of wild B. edulis, were evaluated. Fruiting bodies were obtained in Trás-os-Montes, in the Northeast of Portugal, in November 2012. The irradiation was performed in experimental equipment with 60Co sources at 1 and 2 kGy. All the results were compared with nonirradiated samples (control). Macronutrients and energy value were determined following official procedures of food analysis; fatty acids were analyzed by gas-chromatography coupled to flame ionization detection (GC-FID), while sugars and tocopherols were determined by high performance liquid chromatography (HPLC) coupled to refraction index (RI) and fluorescence detectors, respectively. Antioxidant activity was evaluated in the methanolic extracts by in vitro assays measuring DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity, reducing power, inhibition of β- carotene bleaching and inhibition of lipid peroxidation using thiobarbituric acid reactive substances (TBARS) assay. Total phenolics were also determined by the Folin-Ciocalteu assay. The nutritional profiles were not affected in high extension. Fatty acids and sugars were slightly affected, decreasing with the increasing doses. The performed assays for antioxidant activity, indicate that irradiated samples tended to have lower scavenging activity and reducing power, but higher lipid peroxidation inhibition. Despite the detected differences in individual compounds, the results of nutritional parameters, the most relevant in terms of mushroom acceptability by consumers, were less affected, indicating an interesting potential of gamma-irradiation to be used as an effective conservation technology for the studied mushrooms.

Chromatographic analysis of antioxidant and related compounds in Polyporus squamosus from different origins

The antioxidant potential of mushrooms is mainly attributed to their composition in polysaccharides, phenolic compounds, tocopherols and some organic acids [1]. Phenolic compounds contribute directly to the antioxidative action and play an important role in stabilizing lipid peroxidation [2]; exhibit a wide range of bioactive properties such as anti-allergenic, anti-inflammatory and antimicrobial, which have been in part related to their antioxidant activity [3]. Tocopherols are important fatsoluble antioxidants, acting in the cellular membrane; due to their role as scavenger of free radicals protecting human cells against degenerative malfunctions [4]. Some organic acids are very common in natural matrices; malic acid contributes to a pleasantly sour taste and is often used as a food additive; citric acid is known due to its antibacterial and antioxidant properties and fumaric acid is important because of its antioxidant, anti-inflammatory, antimicrobial and acidifying properties [5]. The purpose of the present study was to analyze antioxidant and related compounds (phenolic compounds, tocopherols and organic acids) of Polyporus squamosus (Huds.) Fr. samples originated from two different origins (Portugal and Serbia). Specimens of P. squamosus were collected in Bragança (Northeast Portugal) and Jabučki rit (Northern Serbia) during April 2015 and 2012, respectively. Phenolic compounds, organic acids and tocopherols were determined by high performance liquid chromatograph (HPLC) coupled to a diode array detector (DAD), in the two first cases, and a fluorescence detector in the last one. With respect to phenolic and related compounds, p-hydroxybenzoic and cinnamic acids were identified in both samples; the first one predominates in the sample from Portugal, while cinnamic acid was more abundant in the sample from Serbia. Tocopherols (α-, β and γ-isoforms) were found in the sample from Serbia, but in the sample from Portugal, γ-tocopherol was not identified. This sample showed the highest total tocopherols content, and revealed the highest level of β-tocopherol; γ- tocopherol predominated in the sample from Serbia. Among organic acids, it was possible to quantify oxalic, malic and fumaric acids in both samples. Malic acid was found in higher amounts in the sample from Serbia. Overall, the present study shows that mushroom samples from different origins have dissimilar results, but are both rich in bioactive compounds, being a valuable source for the development of natural medicines and nutraceuticals.

Effets préventifs et thérapeutiques des polyphénols dans un modèle in vitro et in vivo de maladie inflammatoire de l’intestin : caractérisation des polyphénols de la pelure de pomme et de la canneberge par spectrométrie de masse

La muqueuse intestinale est exposée à des agents oxydants provenant de l’ingestion d’aliments modifiés, de cellules immuno-inflammatoires et de la flore intestinale. Une diète élevée en fruits et légumes peut diminuer le stress oxydant (SOx) ainsi que l’inflammation via plusieurs mécanismes. Ces effets bénéfiques peuvent être attribuables à leur contenu élevé en polyphénols. La première étude de mon doctorat consistait à tester l’hypothèse que les polyphénols extraits de pelures de pomme (DAPP) pouvaient diminuer le stress oxydant et l'inflammation impliqués dans les maladies inflammatoires de l'intestin (MII). Nous avons caractérisé les polyphénols des DAPP par spectrométrie de masse (LC-MS) et examiné leur potentiel antioxydant et anti-inflammatoire au niveau des cellules intestinales. L’identification des structures chimiques des polyphénols a été effectuée par LC-MS. Le SOx a été induit par l’ajout du complexe fer/ascorbate (Fe/Asc, 200 µM/2 mM) et l’inflammation par la lipopolysaccharide (LPS, 200µg/mL) à des cellules intestinales Caco-2/15 pré-incubées avec les DAPP (250 µg/mL). L’effet du SOx est déterminé par le dosage du malondialdéhyde (MDA), de la composition des acides gras polyinsaturés et de l’activité des enzymes antioxydantes endogènes (SOD et GPx). L’impact des DAPP sur l’inflammation a été testé par l’analyse de l’expression des marqueurs inflammatoires: cyclooxygénase-2 (COX-2), le facteur de nécrose tumorale alpha (TNF-a et l’interleukine-6 (IL-6) et les facteurs de transcription NF-KB, Nrf-2 et PGC1α par immunobuvardage. Nos données ont montré que les flavonols et les flavan-3-ols constituent les composés polyphénoliques majoritaires des DAPP. L’ajout de Fer2+/Asc a provoqué une augmentation de la peroxidation lipidique comparativement aux cellules contrôles, un appauvrissement des acides gras polyinsaturés n-3 et n-6, et une modulation des enzymes antioxydantes, se traduisant par une augmentation de l’activité de la SOD et une diminution de la GPx. En contrepartie, les DAPP ont exhibé leur potentiel à corriger la plupart des perturbations, y compris l’expression protéique anormalement élevée du COX-2 et la production de la prostaglandine E2 (PGE2), ainsi que l’inflammation telle que réflétée par les facteurs NF-κB, TNF-α et IL-6. Par ailleurs, les mécanismes sous-jacents à ces changements bénéfiques des DAPP ont fait intervenir les facteurs de transcription antioxydants (Nrf-2, PGC1α). Vraisemblablement, cette première étude a permis de démontrer la capacité des DAPP à amoindrir le SOx et à réduire l’inflammation, deux processus étroitement impliqués dans les MII. Dans la deuxième étape de mon doctorat, nous avons voulu comparer les résultats de DAPP à ceux des polyphénols dérivant de la canneberge qui est considérée par la communauté scientifique comme le fruit ayant le plus fort potentiel antioxydant. À cette fin, nous avons caractérisé l’effet des composés polyphénoliques de la canneberge (CPC) sur le SOx, la défense antioxydante et l’inflammation au niveau intestinal tout en définissant leur métabolisme intraluminal. Les différents CPC ont été séparés selon leur poids moléculaire par chromatographie et leurs structures chimiques ont été identifiées par LC-MS. Suite à une pré-incubation des cellules Caco-2/15 avec les extraits CPC (250 µg/mL), le Fe/Asc et la LPS ont été administrés comme inducteurs du SOx et de l’inflammation, respectivement. La caractérisation globale des CPC a révélé que les acides phénoliques composaient majoritairement l’extrait de canneberge de petit poids moléculaire (LC) alors que les flavonoïdes et les procyanidines dimériques/trimériques représentaient l’extrait de poids moléculaire moyen (MC) tout en laissant les procyanidines oligo et polymériques à l’extrait de haut poids moléculaire (HC). Les CPC ont permis de restaurer la plupart des perturbations engendrées dans les Caco-2/15 par le Fe/Asc et le LPS. Les CPC exhibaient le potentiel d’abaisser les niveaux de MDA, de corriger la composition des acides gras polyinsaturés n-3 et n-6, d’augmenter l’activité des enzymes antioxydantes (SOD, GPx et CAT) et d’élever l’expression de Nrf2 et PGC1α. En outre, les CPC pouvaient aussi réduire les niveaux élevés des protéines inflammatoires COX-2, TNF-α et IL-6 ainsi que la production des PGE2 par un mécanisme impliquant le NF-κB. Au niveau mitochondrial, les procyanidines oligomériques ont réussi à corriger les dysfonctions reliées à la production d’énergie (ATP), l’apoptose (Bcl-2, Cyt C et AIF) et le statut des facteurs de transcription mitochondriaux (mtTFA, mtTFB1, mtTFB2). Dans le but de bien comprendre les mécanismes d’action des CPC, nous avons défini par LC-MS les composés polyphénoliques qui ont été transportés ou absorbés par l’entérocyte. Nos analyses soulignent le transport (i) des acides cinnamiques et benzoïques (LC); (ii) la quercétine glycosylée et conjuguée et les procyanidines dimériques de type A (MC); et (iii) l’épicatéchine et les procyanidines oligomériques (HC). Les processus de métabolisation (méthylation, glucuronidation et sulfatation) au niveau de l’entérocyte ont probablement permis le transport de ces CPC surtout sous leur forme conjuguée. Les procyanidines oligomériques ayant un degré de polymérisation supérieur à 2 (HC) ont semblé adhérer aux cellules Caco-2/15. L’épicatéchine suivi par les procyanidines dimériques de type A ont été trouvés majoritaires au niveau des mitochondries. Même si nous ignorons encore l’action biologique de chaque composé polyphénolique, nous pouvons suggérer que leurs effets combinatoires exercent des fonctions antioxydantes, anti-inflammatoires et mitochondriales dans le modèle intestinal Caco-2/15. Dans une troisième étape, nous avons procédé à l’évaluation des aspects préventifs et thérapeutique des DAPP tout en sondant les mécanismes sous-jacents dans une étude préclinique. À cette fin, nous avons exploité le modèle de souris avec colite expérimentale provoquée par le Dextran Sulfate de Sodium (DSS). L’induction de l’inflammation intestinale chez la souris C57BL6 a été effectuée par l’administration orale de DSS à 2.5% pendant 10 jours. Des doses physiologiques et supra-physiologiques de DAPP (200 et 400 mg/kg/j, respectivement) ont été administrées par gavage pendant 10 jours pré- et post-DSS. L’inflammation par le DSS a provoqué une perte de poids, un raccourcissement du côlon, le décollement dystrophique de l’épithélium, l’exulcération et les infiltrations de cellules mono et polynucléaires au niveau du côlon. De plus, le DSS a induit une augmentation de la peroxidation lipidique, une régulation à la baisse des enzymes antioxydantes, une expression protéique à la hausse de la myéloperoxidase (MPO), du COX-2 et de la production des PGE2. Par ailleurs, les DAPP ont permis de corriger ou du moins d’alléger la plupart de ces anomalies en situation préventive ou thérapeutique, en plus d’abaisser l’expression protéique de NF-κB et des cytokines inflammatoires (TNF-a et l’IL-6) tout en stimulant les facteurs de transcription antioxydants (Nrf-2, PGC1α). Conséquemment, les polyphénols des DAPP ont exhibé leur puissant pouvoir antioxydant et anti-inflammatoire au niveau intestinal dans un modèle in vivo. Leurs actions sont associées à la régulation des voies de signalisation cellulaire et des changements dans la composition du microbiote. Ces trois projets de recherche permettent d’envisager l’évaluation des effets préventifs et thérapeutiques des DAPP cliniquement chez les patients avec des désordres inflammatoires de l’intestin.

Chromatographic techniques to obtain the biomolecules profile of wild Suillus granulates

In recent years the interest in naturally occurring compounds has been increasing worldwide. Indeed, many of the bioactive compounds currently used as medicines have been synthesized based on the structure of natural compounds [1]. In order to obtain bioactive fractions and subsequently isolated compounds derived from natural matrices, several procedures have been carried out. One of these is to separate and assess the concentration of the active compound(s) present in the samples, a step in which the chromatographic techniques stand out [2]. In the present work the mushroom Sui/Ius granulatus (L.) Roussel was chemically characterized by chromatographic techniques coupled to different detectors, in order to evaluate the presence of nutritional and/or bioactive molecules. Some hydrophilic compounds, namely free sugars, were identified by high performance liquid chromatography coupled to a refraction index detector (HPLC-RI), and organic and phenolic acids were assessed by HPLC coupled to a photodiode array detector (HPLC-PDA). Regarding lipophilic compounds, fatty acids weredetermined by gas chromatography with a flame ionization detector (GC-FID) and tocopherols by HPLC-fluorescence detection. Mannitol and trehalose were the main free sugars detected. Different organic acids were also identified (i.e. oxalic, quinic and fumaric acids), as well as phenolic acids (i.e. gallic and p-hydroxybenzoic acids) and the related compound cinnamic acid. Mono- and polyunsaturated fatty acids were the prevailing fatty acids and a-, ~- and ~-tocopherol were the isoforms of vitamin E detected in the samples. Since this species proved to be a source of biologically active compounds, the antioxidant and antimicrobial properties were evaluated. The antioxidant activity was measured through the reducing power, free radical's scavenging activity and lipid peroxidation inhibition of its methanolic extract, and the antimicrobial activity was also tested in Gram positive and Gram negative bacteria and iri different fungi. S. granulatus presented antioxidant properties in all the performed assays, and proved to inhibit the growth of different bacterial and fungal strains. This study is a first step for classifying S. granulatus as a functional food, highlighting the potential of mushrooms as a source of nutraceutical and biologically active compounds.

Antioxidant potential of two Apiaceae plant extracts: a comparative study focused on the phenolic composition

The present study aimed to characterize the extracts prepared from Pimpinella anisum L. (anise) and Coriandrum sativum L. (coriander) (Apiaceae plants) seeds in terms of phenolic composition, and to correlate the obtained profiles with the antioxidant activity. Anise gave the highest abundance in phenolic compounds (42.09± 0.11 mg/g extract), mainly flavonoids (28.08±0.17 mg/g extract) and phenolic acids (14.01±0.06 mg/g extract), and also the highest antioxidant potential, measured by the ability to inhibit lipid peroxidation and β-carotene bleaching, the reducing power and the free radical scavenging activity. Apigenin and luteolin derivatives, as also caffeoylquinic acid derivatives seem to be directly related with the higher in vitro antioxidant potential of the anise extract. In contrast, the lower antioxidant potential of coriander seems to be due to its lower abundance in phenolic compounds (2.24±0.01 mg/g extract). Further studies are necessary to evaluate the in vivo antioxidant potential of the tested extracts, but the in vitro experiments already performed highlight them as potential health promoters.

Multibiomarker assessment of cadmium-based quantum dots effects in the marine mussel Mytilus galloprovincialis

Tese de Doutoramento, Ciências do Mar, da Terra e do Ambiente, Ramo: Ciências e Tecnologias do Ambiente, Especialização em Ecotoxicologia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Cuminum cyminum Linn (Apiaceae) extract attenuates MPTP-induced oxidative stress and behavioral impairments in mouse model of Parkinson’s disease

Purpose: To evaluate the protective effects of Cuminum cyminum Linn (Apiaceae, CCY) against 1- methyl-4 phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced oxidative stress and behavioral impairments in mouse model of Parkinson’s disease (PD). Methods: MPTP-intoxicated mice model of PD was used for evaluating the effect of CCY extract on behavioral deficits through rota rod, passive avoidance and open field tasks. The effect of CCY extract on oxidative stress levels were assessed by estimating enzyme status, including superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation(LPO) in brain tissues of MPTP-induced mice. Results: MPTP (25 mg/kg, i.p.)-treated mice resulted in a significant (p < 0.001) behavioral deficit in locomotor behavior (from 56.24 ± 1.21 to 27.64 ± 0.94) and cognitive functions (from 298 ± 3.68 s to 207.28 ± 4.12 s) compared with their respective control groups. Administration of CCY extract (100, 200 and 300 mg/kg, p.o.) for three weeks significantly and dose-dependently improved (p < 0.001 at 300 mg/kg) locomotor and cognitive deficits in MPTP-treated mice. CCY treatment also significantly (p < 0.001 at 300 mg/kg) inhibited MPTP-induced decrease in antioxidant enzyme levels (superoxide dismutase and catalase) and lipid peroxides in mice brain tissues. Conclusion: CCY extract exhibits strong protection against MPTP-induced behavioral deficit through enhancement of antioxidant defense mechanisms. Therefore, CCY may be developed as a therapeutic strategy in the treatment of neurodegeneration seen in PD.

Indigofera tinctoria Linn (Fabaceae) attenuates cognitive and behavioral deficits in scopolamine-induced amnesic mice

Purpose: To investigate the cognition-enhancing effects of aqueous extract of Indigofera tinctoria Linn (ITE, Fabaceae) in experimental amnesic mice. Methods: Scopolamine (2 mg/kg, i.p.) was used to induce amnesia in mice. The cognitive-enhancing activity of the ITE (5, 10 and 20 μg/mL) was studied by passive avoidance response, elevated plus maze and Y-maze behavioral paradigm in normal and scopolamine-induced amnesic mice. Antioxidant activities were also determined based on the ability of ITE to inhibit lipid peroxide, superoxide and hydroxyl radicals. Results: Scopolamine-induced cognitive deficits were significantly reversed by ITE (p < 0.001 at 20 mg/kg) in a dose-dependent fashion in all the behavioral paradigms tested. Furthermore, ITE dosedependently scavenged lipid peroxide, superoxide and hydroxyl free radicals with 50 % inhibition concentration (IC50) of 7.28 ± 0.37, 5.25 ± 0.28 and 7.62 ± 0.43 μg/mL, respectively. Conclusion: ITE possesses cognitive-enhancing properties in amnesic mice due to its potent antioxidant action.

Sodium chloride added to transport water and physiological responses of Matrinxã Brycon amazonicus (Teleost: Characidae)

A adição de sal à água tem sido utilizada para a mitigação de estresse e aumento da taxa de sobrevivência em peixes. O presente estudo avaliou o efeito do cloreto de sódio (0,0; 1,0; 3,0 e 6.0 g/l) nas concentrações de cortisol plasmático, glicemia, triglicerídios, proteínas total plasmática, hematócrito, hemoglobina, número de eritrócitos, glicogênio e lipídio hepáticos, e lipídio muscular em matrinxã Brycon amazonicum adultos após quatro horas de transporte e durante período de recuperação de 96 h. Amostras foram coletadas antes e depois do transporte, bem como 24 e 96 h após a chegada. O nível de cortisol plasmático estava mais elevado logo após o transporte quando comparado à condição inicial (pré-transporte), exceto para os peixes transportados com sal nas concentrações 3,0 e 6,0 g/l. Comportamento semelhante foi observado para a glicemia, porém os peixes dos tratamentos 0,0, 1,0 e 3,0 g/l necessitaram de período superior a 24 h para recuperar a condição inicial. Foram registrados níveis mais baixos de glicogênio hepático em peixes do tratamento controle (0,0 g/l). Os parâmetros hemoglobina, número de eritrócitos, proteína plasmática total e lipídio hepático não apresentaram alterações durante o período experimental. Os valores de hematócrito diminuíram logo após o transporte em todos os tratamentos, retornando aos níveis iniciais após 24 h. Todos os tratamentos apresentaram redução nos níveis de lipídio muscular e triglicerídios durante o período de recuperação. Os resultados sugerem que a adição de 6,0 g/l de sal na água de transporte reduz as alterações fisiológicas de estresse e que é necessário período de 96 h após o transporte para a recuperação da condição inicial de matrinxãs transportados sem a adição de sal.

A systematic review of observational studies on oxidative/nitrosative stress involvement in dengue pathogenesis

Objective: Our objective was to systematically review the published observational research related to the role of oxidative-nitrosative stress in pathogenesis of dengue. Methods: We searched electronic databases (PubMed, EMBASE, The COCHRANE library, ScienceDirect, Scopus, SciELO, LILACS via Virtual Health Library, Google Scholar) using the term: dengue, dengue virus, severe dengue, oxidative stress, nitrosative stress, antioxidants, oxidants, free radicals, oxidized lipid products, lipid peroxides, nitric oxide, and nitric oxide synthase. Articles were selected for review by title and abstract excluding letter, review, in vivo and in vitro studies, and duplicates studies. Selected articles were reviewed for study design, original purposes, sample size, main outcomes, methods, and oxidative-nitrosative stress markers values. Results: In total, 4,331 non-duplicates articles were identified from electronic databases searches, of which 16 were eligible for full text searching. Data from the observational studies originate from Asian countries (50%; 8/16), South American countries (31.2%; 5/16), and Central America and the Caribbean countries (18.8%; 3/16). Casecontrol study was the type of design most common in researches reviewed. The 1997 World Health Organization (WHO) dengue case classification criteria were used in all studies included in this review. Conclusions: Based on published data found in peer-reviewed literature, oxidative and nitrosative stress are demonstrated by changes in plasma levels of nitric oxide, antioxidants, lipid peroxidation and protein oxidation markers in patients with dengue infection. Additionally, elevated serum protein carbonyls and malondialdehyde levels appear to be associated with dengue disease severity.

Serum levels of lycopene, beta-carotene, and retinol and their correlation with sperm DNA damage in normospermic and infertile men

Background: Oxidative stress in reproductive system leads to sperm DNA damage and sperm membrane lipid peroxidation and may play an important role in the pathogenesis of male infertility, especially in idiopathic cases. Antioxidants such as carotenoids function against free radical damages. Objective: The aim of this study was to determine the levels of lycopene, beta-carotene and retinol in serum and their relationship with sperm DNA damage and lipid peroxidation in infertile and normospermic males. Materials and Methods: Sixty two infertile men and 71 normospermic men participated in this study. Blood and semen samples were collected from all subjects. Sperm DNA damage was measured using TUNEL method. Carotenoids, retinol, and malonedildehyde in serum were also determined. Results: DNA fragmentation was higher in infertile group comparing to control group. Serum levels of lycopene, beta-carotene and, vitamin A in infertile men were significantly lower than normospermic men (p< 0.001, =0.005, and =0.003 respectively). While serum MDA was not significantly different between two groups, MDA in seminal plasma of infertile men was significantly higher than control group (p< 0.001). Conclusion: We concluded that lycopene, beta-carotene, and retinol can reduce sperm DNA fragmentation and lipid peroxidation through their antioxidant effect. Therefore the DNA fragmentation assay and determination of antioxidants factors such as lycopene, beta-carotene and retinol, along with sperm analysis can be useful in diagnosis and treatment of men with idiopathic infertility.

Chronic intermittent hypoxia increases rat sternohyoid muscle NADPH oxidase expression with attendant modest oxidative stress

Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling.

Development of an ecotoxicological protocol for the deep-sea fauna using the hydrothermal vent shrimp Rimicaris exoculata

In light of deep-sea mining industry development, particularly interested in massive-sulphide deposits enriched in metals with high commercial value, efforts are increasing to better understand potential environmental impacts to local fauna. The aim of this study was to assess the natural background levels of biomarkers in the hydrothermal vent shrimp Rimicaris exoculata and their responses to copper exposure at in situ pressure (30MPa) as well as the effects of depressurization and pressurization of the high-pressure aquarium IPOCAMP. R. exoculata were collected from the chimney walls of the hydrothermal vent site TAG (Mid Atlantic Ridge) at 3630m depth during the BICOSE cruise in 2014. Tissue metal accumulation was quantified in different tissues (gills, hepatopancreas and muscle) and a battery of biomarkers was measured: metal exposure (metallothioneins), oxidative stress (catalase, superoxide dismutase, glutathione-S-transferase and glutathione peroxidase) and oxidative damage (lipid peroxidation). Data show a higher concentration of Cu in the hepatopancreas and a slight increase in the gills after incubations (for both exposed groups). Significant induction of metallothioneins was observed in the gills of shrimps exposed to 4μM of Cu compared to the control group. Moreover, activities of enzymes were detected for the in situ group, showing a background protection against metal toxicity. Results suggest that the proposed method, including a physiologically critical step of pressurizing and depressurizing the test chamber to enable the seawater exchange during exposure to contaminants, is not affecting metal accumulation and biomarkers response and may prove a useful method to assess toxicity of contaminants in deep-sea species.

Función endotelial y lipemia postprandial en adultos normolipídicos: Efecto del estado nutricional

El objetivo del presente estudio fue cuantificar la contribución del sobrepeso en la magnitud de la lipemia posprandial en sujetos normolipídicos. Se incluyeron 33 adultos normolipídicos en dos grupos (n=20, sobrepeso y (n=13 eutróficos, 66% hombres, edad media 31,2±7,6 años). Se midió la vasodilatación mediada por flujo (VMF), la velocidad de onda de pulso (VOP), el perfil lipídico, el cociente Log TG/c-HDL, la glucosa y presión arterial tras una ingesta estándar con alto contenido de grasa (79% Kcal/grasa). Se calculó, el Z-score de riesgo cardiovascular a partir de la suma de los residuos tipificados (Z) de las variables de riesgo cardiovascular. El estado de lipemia posprandial se midió en ayuno (0 min) y a los (60, 120, 180, y 240 min) posprandiales. El valor basal de la VMF y la VOP fue de 6,9±5,9% y 7.0±0.8 m/s, respectivamente. Se identificó que la lipemia posprandial reducía la WMF en 19,2% a los 60 min (5,9±1,5%) y a los 240 min (3,7±1,2%) (p<0,04), respectivamente. Este hallazgo se acompañó con un aumento en la VOP (p<0,05). Al dividir los sujetos en dos grupos según el IMC, los participantes en sobrepeso muestran cifras más elevadas en el Z-score de riesgo cardiovascular, la VOP, el Log TG/c-HDL y el Δ-VOP, (p<0,001). En conclusión los sujetos clasificados en sobrepeso muestran un perfil cardiometabolico asociado con un mayor riesgo cardiovascular.