Effects of chronic hydrogen peroxide exposure on mitochondrial oxidative stress genes, ROS production and lipid peroxidation in HL60 cells.

Hydrogen peroxide (H2O2) is a reactive species that is also involved in the redox regulation of cells because of it is relative stability. In numerous pathological situations, a chronic increase in the production of reactive species is observed, which is related to oxidative stress and cellular damage. This study aimed to evaluate the effects of long-term exposure to different H2O2 concentrations on oxidative stress biomarkers and mitochondrial dynamics in HL60 cells. HL60 cells were treated with a sustained production (0.1, 1.0 and 10.0 nM/s) of H2O2 for one hour. H2O2 production and malondialdehyde (MDA) levels, as a lipid peroxidation marker, increased progressively in HL60 cells in accordance with higher H2O2 exposure, with significant differences between the 10 nM/s H2O2 group and the control and 0.1 nM/s groups. Similarly, progressive increased expression in genes related to the mitochondrial antioxidant defences and mitochondrial dynamics were also observed. Significantly increased gene expression in the 10 nM/s H2O2 with respect to the control group was observed for manganese superoxide dismutase (MnSOD), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PCG1α), nuclear respiratory factor 2 (Nrf2), mitochondrial transcription factor A (Tfam), mitofusins 1 and 2 (Mfn1 and Mfn2) and uncoupling protein 3 (UCP3), whereas no significant changes were observed in the cytochrome c oxidase subunit IV (COXIV) gene expression. In conclusion, exposure to different sustained production of H2O2 is related to a progressive increase in the gene expression of mitochondrial dynamics and redox processes in HL60 cells, but also to oxidative damage at higher H2O2 production levels.

Breaking the paradigm: Marine sediments hold two-fold microplastics than sea surface waters and are dominated by fibers.

We conducted one of the first studies to integrate the quantification and characterization of microplastics (MPs), including fibers, in different habitats (sea surface, seafloor and beach sediments) of a coastal Mediterranean marine protected area, analyzing their ingestion in several marine species. The objectives of the study were to evaluate the distribution of MPs according to shape and polymer, to assess the contribution of fibers to local plastic pollution and to evaluate their ingestion in fish and invertebrates species that inhabit the study area (Pagrus pagrus, Serranus scriba, Spondyliosoma cantharus, Diplodus vulgaris, Oblada melanura, Holothuria forskalii, Holothuria tubularis, Holothuria polis, Arbacia lixula, Paracentrotus lividus, Modiolus barbatus, Mytilus galloprovincialis and Arca noae). A total of 111 environmental samples were analyzed. The mean abundance of MPs (excluding fibers) quantified in beach sediments (13,418.86 ± 28,787.99 MPs/m2) was two orders of magnitude higher than that found in seafloor sediments (76.92 ± 108.84 MPs/m2), which in turn was two orders of magnitude higher than sea surface samples (0.17 ± 0.39 MPs/m2). The fibers were the most abundant shape of MPs identified in all habitats. Variability in MPs ingestion was detected between species, with ingestion rates ranging from 43 % to 100 % for general MPs and ranging from 7 % to 100 % for fibers. The highest ingestion was observed in Holoturians, representing suitable bioindicators for plastic pollution. The composition of the polymer varies weakly depending on habitats and biota, but the result is strongly correlated with the morphology of the plastic. Fibers were mainly composed of cellulose acetate (29 %), styrofoam of polystyrene (18 %), and filaments, films and fragments of polyethylene and polypropylene. The results highlighted the need to expand integrated approaches to effectively study marine plastic pollution and to undertake efficient actions to limit the input of plastics, particularly fibers, into the marine environment.

Microplastic intake and enzymatic responses in Mytilus galloprovincialis reared at the vicinities of an aquaculture station.

Aquaculture is a potential source of microplastics (MPs) that could be strong stressors for marine organisms. In this study, we evaluated the effects of MPs derived from aquaculture in antioxidant defences and oxidative stress markers in gills of Mytilus galloprovincialis. Mussels were distributed in three areas with different impacts: inside aquaculture cages, Control 1 (located inside Andratx harbour) and Control 2 (located in a no-anthropized area). Samples were obtained along three different time periods in May (T0), July (T60) and in September (T120). At each sampling period, mussels' biometric measurements were taken, and tissue samples were kept frozen for biochemical determinations and to determine the intake of MPs. An increase in MPs intake was detected throughout the study, and this increase was significantly higher in samples from the aquaculture cages. Similarly, antioxidant enzyme activities (catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase) were significantly higher in samples from cages at T120. Additionally, a similar tendency was observed in glutathione-s-transferase, with a higher activity in the aquaculture cages at T60 and T120. Malondialdehyde and carbonyl protein derivates as a marker of oxidative damage were also measured and samples from aquaculture cages presented higher oxidative stress markers, mainly in T120. In conclusion, living in environments exposed to aquaculture activities at sea may imply a higher intake of MPs which in turn might cause an antioxidant response in M. galloprovincialis which is not enough to avoid oxidative damage.

Long-term exposure to virgin and seawater exposed microplastic enriched-diet causes liver oxidative stress and inflammation in gilthead seabream Sparus aurata, Linnaeus 1758.

Plastics accumulation in marine ecosystems has notable ecological implications due to their long persistence, potential ecotoxicity, and ability to adsorb other pollutants or act as vectors of pathogens. The present work aimed to evaluate the physiological response of the gilthead seabream (Sparus aurata) fed for 90 days with a diet enriched with virgin and seawater exposed low-density polyethylene microplastics (LDPE-MPs) (size between 100 and 500 µM), followed by 30 days of depuration, applying oxidative stress and inflammatory markers in liver homogenates. No effects of LDPE-MPs treatments on fish growth were observed throughout this study. A progressive increase in antioxidant enzyme activities was observed throughout the study in both treatments, although this increase was higher in the group treated with seawater exposed MPs. This increase was significantly higher in catalase (CAT), glutathione reductase (GRd), and glutathione-s-transferase (GST) in the seawater exposed MPs group, with respect to the virgin group. In contrast, no significant differences were recorded in superoxide dismutase (SOD) and glutathione peroxidase (GPx) between both groups. Exposure to MPs also caused an increase in the oxidative damage markers (malondialdehyde and carbonyls groups). Myeloperoxidase activity significantly increased because of MPs treatments. After 30 days of depuration, antioxidant, inflammatory enzyme activities and oxidative damage markers returned to values similar to those observed in the control group. In conclusion, MPs exposure induced an increase of antioxidant defences in the liver of S. aurata. However, these elevated antioxidant capabilities were not enough to prevent oxidative damage in the liver since, an increased oxidative damage marker was associated with MPs ingestion. The treatment with seawater exposed MPs caused a more significant antioxidant response (CAT, GRs, and GST). Although after a depuration period of 30 days a tendency to recover the initial values of the biomarkers was observed this does not seem to be time enough for a complete normalization.

Enriched environments enhance cognition, exploratory behaviour and brain physiological functions of Sparus aurata.

Environmental enrichment is considered as a recommended tool to guarantee or improve the welfare of captive fish. This study demonstrates for the first time that structural environmental enrichment enhances cognition, exploratory behaviour and brain physiological functions of gilthead seabream (Sparus aurata). Seabream was reared in groups (n = 15) during 60 days under two different treatments: enriched tanks with plant-fibre ropes (EE) or bare/non-enriched tanks (NE). Fish were then exposed to a purpose-built maze for 1 h every second day in four trials. Analysis of video recordings showed that seabream under EE conditions presented higher overall exploratory behaviour, spatial orientation and learning capability compared to seabream from NE conditions. Results from brain monoamines analyses may suggest increased recent dopaminergic activity in telencephalon, known to be involved in learning processes; and increased serotonergic activity in cerebellum, involved in the coordination of balance, movements and orientation. In addition, EE-reared fish showed increased antioxidant activity in whole brain, with no apparent oxidative damage. Structural EE seemed to induce an hormetic response on juvenile seabream, improving their welfare status during captivity. Application of this kind of physical structure might be feasible at fish farms as a passive and non-invasive tool to improve welfare of intensively cultured seabream.

Hypersaline water from desalinization plants causes oxidative damage in Posidonia oceanica meadows.

Posidonia oceanica is an endemic marine phanerogam of the Mediterranean Sea for that is very sensitive to the environmental changes, especially those related to human activities. The aim of this study was to evaluate the oxidative stress status of P. oceanica meadows exposed to spillage of hypersaline water from a desalination station by using biomarkers. Leaf samples of P. oceanica were obtained from 4 different points exposed to different levels of salinity water. Samples from the area with the highest salinity conditions were 75% shorter than the samples from the control area. Exposure to high salinity induced an increase in the levels of oxidative stress markers (malondialdehyde (MDA) and protein carbonyls). Interestingly, in the area with the highest salinity the activities of glutathione peroxidase, glutathione reductase and glutathione-s-transferase were significantly induced respect to the other studies areas, while catalase (CAT) and superoxide dismutase (SOD) activities were lower. In addition, salinity induced a progressive increase in non-enzymatic antioxidants (polyphenols and glutathione) and in total antioxidant capacity reaching the highest concentrations in samples directly affected by the saline discharge. In conclusion chronic exposure to high salinity induced an increase in total antioxidant capacity in P. oceanica. However, this increase was not enough to protect the plant against oxidative stress as it is evidenced by the raise in oxidative stress markers. The obtained data suggest that high salinity conditions deactivated CAT and SOD antioxidant enzymes and caused an increase in non-enzymatic antioxidants (polyphenols and glutathione) and in glutathione-related enzymes.

Simultaneous analysis of saturated and unsaturated oxylipins in 'ex vivo' cultured peripheral blood mononuclear cells and neutrophils.

Oxylipins are a family of saturated and unsaturated fatty acids peroxidation products with bioactive properties. We have developed an improved method for the measurement of ex vivo oxylipin production by peripheral blood mononuclear cells (PBMCs) and neutrophils. We aimed to develop an analytical method to determine the production rates of polyunsaturated fatty acids (PUFAs), PUFA-oxylipin, and saturated-oxylipins by stimulated PBMCs and neutrophils based on solid phase extraction and HPLC-MS/MS technology. A UHPLC system coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer was used to identify and quantify oxylipin production. For each oxylipin and PUFA their differential response was calculated with respect to a deuterated internal standard factor (ISF). To calculate oxylipin and PUFAs in the culture samples, the individual ISF was used for each oxylipin and PUFA with respect to the deuterated internal standard. PBMCs and neutrophils showed a different pattern of oxylipin production and fatty acid secretion. Lipopolysaccharide (LPS) did not stimulate oxylipin production or fatty acids secretion in PBMCs, whereas phorbol myristate acetate (PMA) stimulation increased the production rate of 5-HETE, 15-HETE, 15-HEPE, 17-DoHE, PGE2, AA, and DHA. LPS stimulation decreased 16-hydroxyl-palmitatte (16-OHPAL) production and DHA secretion in neutrophils, while PMA stimulation increased the production rate of AA and its derivate oxylipins, 5-HETE, 15-HETE, and PGE2. In conclusion, we have developed a new method to determine oxylipins derived from both saturated and unsaturated fatty acids in culture cell media. This method has enough sensitivity, and accuracy, to determine oxylipin production and fatty acid secretion by PBMCs and neutrophils.

Microencapsulation as a tool to counteract the typical low bioavailability of polyphenols in the management of diabetes.

Polyphenols are secondary metabolites widely distributed in many plant foods, such a tea, coffee, chocolate and fruits. The consumption of these compounds is related to the improvement or amelioration of many diseases, including diabetes. Nevertheless, the great barrier to the therapeutic use of polyphenols is the low bioavailability of these compounds once ingested. For that reason, the encapsulation of polyphenols in different matrices may protect them from digestion and improve their release and subsequent absorption to obtain target-specific health effects. Some studies have reported the beneficial effect of encapsulation to increase both bioavailability and bioaccessibility. However, these works have mostly been carried out in vitro and few studies are specifically addressed at improving diabetes. In the current work, an overview of the knowledge related to nanoparticles and their use in the diabetic condition has been reviewed.

Fatty acids and elemental composition as biomarkers of Octopus vulgaris populations: Does origin matter?

The present study describes the novel use of fatty acids (FAs) and element profiles of Octopus vulgaris inhabiting three coastal areas in the W-Mediterranean Sea. These populations are exposed to different anthropogenic activities, and were compared at different geographical scales. The FA composition in the mantle of O. vulgaris exhibited significant differences in 22:6 n-3 (DHA) and 22:5 n-3 (EPA) among the sampled populations. The essential microelements Fe, Cu, Zn and Ni, and the non-essential microelements As, Sr, Al and Cd were the main contributors of variability among sampled octopus populations, with some notable differences among tissues. The variations in the FAs and elemental composition in octopus tissues were detected with other populations throughout the species distribution range, which might reflect differences in natural habitats and foraging strategies. Therefore, these may be considered biomarkers as a proxy to distinguish the origin of octopus specimens at different scales.

Biomarkers of physiological responses of Octopus vulgaris to different coastal environments in the western Mediterranean Sea.

The increase of pollutants in coastal seawater could produce several harmful biological effects on marine organisms related to the production of reactive oxygen species (ROS) causing cellular and tissue damages through oxidative stress mechanisms. Common octopuses (Octopus vulgaris) inhabiting coastal areas under high anthropogenic activity of Mallorca (W-Mediterranean Sea) have the ability to control oxidative damage by triggering antioxidant enzyme responses. Analyzing the digestive glands, octopuses from human-altered coastal areas showed higher activity of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) compared to octopuses from non-influenced coastal waters (i.e. marine reserve area). Higher metallothionein (MT) concentrations and lack of malondialdehyde (MDA) variations also reflect adaptations of O. vulgaris to polluted areas. This is the first study assessing the levels of the oxidative stress biomarkers on O. vulgaris in the Mediterranean Sea, revealing their usefulness to assess diverse environmental pollution effects on this relevant ecological and commercial species.

Microplastic ingestion by Mullus surmuletus Linnaeus, 1758 fish and its potential for causing oxidative stress.

A total of 417 striped red mullet, Mullus surmuletus, were analyzed to study microplastic ingestion and livers of fish were assessed to study effects of microplastics. Nearly one third (27.30%) of the individuals were quantified to ingest microplastics although there was no evidence of oxidative stress or cellular damage in the liver of fish which had ingested microplastics. A small increase in the activity of glutathione S-transferase (GST) of M. surmuletus was detected which could be suggesting an induction of the detoxification systems but these findings should be tested in laboratory conditions under a controlled diet and known concentration of microplastics. Fish from trammel fisheries, operating closer to land and targeting larger individuals, showed higher mean ingestion values than fish from trawling fisheries, and were related to body size, as microplastics ingested increased with total fish length. Consequently, ingestion values of microplastics were not related to sampling distance from land giving further evidence of the ubiquity of microplastics in the marine environment. Finally, Fourier Transform Infrared Spectroscopy (FTIR) analysis showed that the vast majority of microplastics were filament type and polyethylene terephthalate (PET) was the main identified component.

Effects of dietary almond- and olive oil-based docosahexaenoic acid- and vitamin E-enriched beverage supplementation on athletic performance and oxidative stress markers.

Functional beverages based on almonds and olive oil and enriched with α-tocopherol and docosahexaenoic acid (DHA) could be useful in modulating oxidative stress and enhancing physical performance in sportsmen. The aim of this work was to evaluate the effects of supplementation with functional beverages on physical performance, plasma and erythrocyte fatty acids' and polyphenol handling, oxidative and nitrative damage, and antioxidant and mitochondrial gene expression in young and senior athletes. Athletes performed maximal exercise tests before and after one month of dietary supplementation and blood samples were taken immediately before and one hour after each test. The beverages did not alter performance parameters during maximal exercise. Supplementation increased polyunsaturated and reduced saturated plasma fatty acids while increasing the DHA erythrocyte content; it maintained basal plasma and blood polyphenol levels, but increased the blood cell polyphenol concentration in senior athletes. Supplementation protects against oxidative damage although it enhances nitrative damage in young athletes. The beverages enhance the gene expression of antioxidant enzymes in peripheral blood mononuclear cells after exercise in young athletes.

Effects of dietary Docosahexaenoic, training and acute exercise on lipid mediators.

BACKGROUND: Eicosanoids mediate initiation and resolution of inflammation. Our aim was evaluating the effects of training, exercise and docosahexaenoic (DHA) supplementation on plasma eicosanoids levels and peripheral blood mononuclear cells (PBMCs) eicosanoids production. METHODS: Fifteen male footballers were distributed to placebo and experimental groups. Experimental group consumed DHA-enriched beverage (1.16 g DHA/day) for 8 weeks, placebo group consumed a placebo beverage. Blood samples were taken before and after the nutritional intervention in basal conditions and 2 h after acute exercise. RESULTS: Training increased basal Prostaglandin E1 (PGE1) plasma levels and PBMCs cyclooxygenase 2 (COX-2) protein levels in both groups, but COX-1 protein levels only in the experimental group. Acute exercise increased plasma PGE2 and PBMCs active NFκß levels. Lipopolysaccharide (LPS)-stimulated PBMCs increases eicosanoids production (PGE1, PGE2, RvD1) in both groups and increased LPS-stimulated PBMCs active NFκß. DHA supplementation increased COX-2 levels but decreased LPS-stimulated PBMCs PGE1 and PGE2 production. Neither DHA supplementation nor acute exercise altered the expression of NFκß, COX-2, 15-LOX2, 5-LOX, or IL-1ß genes in PBMCs. CONCLUSIONS: The increase of PGE1 plasma levels after training promoted systemic anti-inflammatory and vasodilator environment. Exercise and DHA supplementation acted synergistically by increasing plasma PGE2 with anti-inflammatory effects. Exercise primed PBMCs to enhance PGE1, PGE2 and RvD1 production in response to LPS. TRIAL REGISTRATION: The project was registered at ClinicalTrial.gov (NCT02177383).

Docosahexaenoic acid supplementation promotes erythrocyte antioxidant defense and reduces protein nitrosative damage in male athletes.

The aim of this study was to determine the influence of long-term docosahexaenoic acid (DHA) dietary supplementation on the erythrocyte fatty acid profile and oxidative balance in soccer players after training and acute exercise. Fifteen volunteer male athletes (age 20.0 ± 0.5 years) were randomly assigned to a placebo group that consumed an almond-based beverage (n = 6), or to an experimental group that consumed the same beverage enriched with DHA (n = 9) for 8 weeks. Blood samples were taken in resting conditions at the beginning and after 8 weeks of nutritional intervention and training in resting and in post-exercise conditions. Oxidative damage markers (malonyldialdehyde, carbonyl and nitrotyrosine indexes) and the activity and protein level of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase and peroxidase) were assessed. The results showed that training increased antioxidant enzyme activities in erythrocytes. The experimental beverage increased DHA from 34.0 ± 3.6 to 43.0 ± 3.6 nmol/10(9) erythrocytes. DHA supplementation increased the catalytic activity of superoxide dismutase from 1.48 ± 0.40 to 10.5 ± 0.35 pkat/10(9) erythrocytes, and brought about a reduction in peroxidative damage induced by training or exercise. In conclusion, dietary supplementation with DHA changed the erythrocyte membrane composition, provided antioxidant defense and reduced protein peroxidative damage in the red blood cells of professional athletes after an 8-week training season and acute exercise.

Docosahexanoic acid diet supplementation attenuates the peripheral mononuclear cell inflammatory response to exercise following LPS activation.

Exercise induces changes in circulating pro- and anti-inflammatory cytokines. The aim was to investigate the effect of docosahexaenoic acid (DHA) diet supplementation on the plasma cytokine levels and on the peripheral mononuclear (PBMCs) cells cytokine production after a training season or an acute bout of exercise. Fifteen male soccer players were randomly assigned to a placebo or an experimental group. The experimental group consumed an almond-based beverage enriched with DHA, whereas the placebo group consumed the same beverage without DHA. Three blood samples were taken: in basal conditions at the beginning of the nutritional intervention and after eight weeks of training season in basal and post-exercise conditions. The DHA content increased in erythrocytes after 8weeks of training and supplementation. Neither diet supplementation with DHA nor training season altered the basal plasma cytokines and growth factors. Only acute exercise significantly increased plasma IL6 in experimental and placebo groups. Lipopolysaccharide (LPS) activation induced the inflammatory response in PBMCs, with a significant production rate of TNFα, IL6 and IL8 mainly after acute exercise. DHA supplementation significantly reduced the rate of TNFα and IL6 production by stimulated PBMCs. Acute exercise increased the Toll-like receptor 4 (TLR4) protein levels in PBMCs, although the increase was only statistically significant in the placebo group. In conclusion, a training season does not induce significant changes in the circulating cytokine profile in well-trained soccer players. Exercise increases the PBMCs cell capabilities to produce cytokines after TLR4 stimulation with LPS and this rate of cytokine production is attenuated by diet DHA supplementation.