Space Flight Effects on Antioxidant Molecules in Dry Tardigrades: The TARDIKISS Experiment

The TARDIKISS (Tardigrades in Space) experiment was part of the Biokon in Space (BIOKIS) payload, a set of multidisciplinary experiments performed during the DAMA (Dark Matter) mission organized by Italian Space Agency and Italian Air Force in 2011. This mission supported the execution of experiments in short duration (16 days) taking the advantage of the microgravity environment on board of the Space Shuttle Endeavour (its last mission STS-134) docked to the International Space Station. TARDIKISS was composed of three sample sets: one flight sample and two ground control samples. These samples provided the biological material used to test as space stressors, including microgravity, affected animal survivability, life cycle, DNA integrity, and pathways of molecules working as antioxidants. In this paper we compared the molecular pathways of some antioxidant molecules, thiobarbituric acid reactive substances, and fatty acid composition between flight and control samples in two tardigrade species, namely, Paramacrobiotus richtersi and Ramazzottius oberhaeuseri. In both species, the activities of ROS scavenging enzymes, the total content of glutathione, and the fatty acids composition between flight and control samples showed few significant differences. TARDIKISS experiment, together with a previous space experiment (TARSE), further confirms that both desiccated and hydrated tardigrades represent useful animal tool for space research

Antioxidant Response during the Kinetics of Anhydrobiosis in Two Eutardigrade Species

Anhydrobiosis, a peculiar adaptive strategy existing in nature, is a reversible capability of organisms to tolerate a severe loss of their body water when their surrounding habitat is drying out. In the anhydrobiotic state, an organism lacks all dynamic features of living beings since an ongoing metabolism is absent. The depletion of water in the anhydrobiotic state increases the ionic concentration and the production of reactive oxygen species (ROS). An imbalance between the increased production of ROS and the limited action of antioxidant defences is a source of biomolecular damage and can lead to oxidative stress. The deleterious effects of oxidative stress were demonstrated in anhydrobiotic unicellular and multicellular organisms, which counteract the effects using efficient antioxidant machinery, mainly represented by ROS scavenger enzymes. To gain insights into the dynamics of antioxidant patterns during the kinetics of the anhydrobiosis of two tardigrade species, Paramacrobiotus spatialis and Acutuncus antarcticus, we investigated the activity of enzymatic antioxidants (catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase) and the amount of non-enzymatic antioxidants (glutathione) in the course of rehydration. In P. spatialis, the activity of catalase increases during dehydration and decreases during rehydration, whereas in A. antarcticus, the activity of superoxide dismutase decreases during desiccation and increases during rehydration. Genomic varieties, different habitats and geographical regions, different diets, and diverse evolutionary lineages may have led to the specialization of antioxidant strategies in the two specie

A rapid method for determining arachidonic:eicosapentaenoic acid ratios in whole blood lipids: correlation with erythrocyte membrane ratios and validation in a large Italian population of various ages and pathologies

BACKGROUND: Omega-3 and -6 polyunsaturated fatty acids (LCPUFA), are important for good health conditions. They are present in membrane phospholipids.The ratio of total n-6:n-3 LCPUFA and arachidonic acid:eicosapentaenoic acid (AA and EPA), should not exceed 5:1. Increased intake of n-6 and decreased consumption of n-3 has resulted in much higher, ca 10/15:1 ratio in RBC fatty acids with the possible appearance of a pathological "scenario". The determination of RBC phospholipid LCPUFA contents and ratios is the method of choice for assessing fatty acid status but it is labour intensive and time consuming. AIMS OF THE STUDY: [i] To describe and validate a rapid method, suitable for large scale population studies, for total blood fatty acid assay; [ii] to verify a possible correlation between total n-6:n-3 ratio and AA:EPA ratios in RBC phospholipids and in whole-blood total lipids, [iii] to assess usefulness of these ratio as biomarkers of LCPUFA status. METHODS: 1 Healthy volunteers and patients with various pathologies were recruited.2 Fatty acid analyses by GC of methyl esters from directly derivatized whole blood total lipids and from RBC phospholipids were performed on fasting blood samples from 1432 subjects categorised according to their age, sex and any existing pathologies.AA:EPA ratio and the total n-6:n-3 ratio were determined. RESULTS: AA:EPA ratio is a more sensitive and reliable index for determining changes in total blood fatty acid and it is correlated with the ratio derived from extracted RBC phospholipids. CONCLUSIONS: The described AA:EPA ratio is a simple, rapid and reliable method for determining n-3 fatty acid status

Effects of n-3 PUFAs on breast cancer cells through their incorporation in plasma membrane

<p>Abstract</p> <p>Background</p> <p>PUFAs are important molecules for membrane order and function; they can modify inflammation-inducible cytokines production, eicosanoid production, plasma triacylglycerol synthesis and gene expression. Recent studies suggest that n-3 PUFAs can be cancer chemopreventive, chemosuppressive and auxiliary agents for cancer therapy. N-3 PUFAs could alter cancer growth influencing cell replication, cell cycle, and cell death. The question that remains to be answered is how n-3 PUFAs can affect so many physiological processes. We hypothesize that n-3 PUFAs alter membrane stability, modifying cellular signalling in breast cancer cells.</p> <p>Methods</p> <p>Two lines of human breast cancer cells characterized by different expression of ER and EGFR receptors were treated with AA, EPA or DHA. We have used the MTT viability test and expression of apoptotic markers to evaluate the effect of PUFAs on cancer growth. Phospholipids were analysed by HPLC/GC, to assess n-3 incorporation into the cell membrane.</p> <p>Results</p> <p>We have observed that EPA and DHA induce cell apoptosis, a reduction of cell viability and the expression of Bcl2 and procaspase-8. Moreover, DHA slightly reduces the concentration of EGFR but EPA has no effect. Both EPA and DHA reduce the activation of EGFR.</p> <p>N-3 fatty acids are partially metabolized in both cell lines; AA is integrated without being further metabolized. We have analysed the fatty acid pattern in membrane phospholipids where they are incorporated with different degrees of specificity. N-3 PUFAs influence the n-6 content and vice versa.</p> <p>Conclusions</p> <p>Our results indicate that n-3 PUFA feeding might induce modifications of breast cancer membrane structure that increases the degree of fatty acid unsaturation. This paper underlines the importance of nutritional factors on health maintenance and on disease prevention.</p

Effects of Long-Term Space Flight on Erythrocytes and Oxidative Stress of Rodents

Erythrocyte and hemoglobin losses have been frequently observed in humans during space missions; these observations have been designated as “space anemia”. Erythrocytes exposed to microgravity have a modified rheology and undergo hemolysis to a greater extent. Cell membrane composition plays an important role in determining erythrocyte resistance to mechanical stress and it is well known that membrane composition might be influenced by external events, such as hypothermia, hypoxia or gravitational strength variations. Moreover, an altered cell membrane composition, in particular in fatty acids, can cause a greater sensitivity to peroxidative stress, with increase in membrane fragility. Solar radiation or low wavelength electromagnetic radiations (such as gamma rays) from the Earth or the space environment can split water to generate the hydroxyl radical, very reactive at the site of its formation, which can initiate chain reactions leading to lipid peroxidation. These reactive free radicals can react with the non-radical molecules, leading to oxidative damage of lipids, proteins and DNA, etiologically associated with various diseases and morbidities such as cancer, cell degeneration, and inflammation. Indeed, radiation constitutes on of the most important hazard for humans during long-term space flights. With this background, we participated to the MDS tissue-sharing program performing analyses on mice erythrocytes flown on the ISS from August to November 2009. Our results indicate that space flight induced modifications in cell membrane composition and increase of lipid peroxidation products, in mouse erythrocytes. Moreover, antioxidant defenses in the flight erythrocytes were induced, with a significant increase of glutathione content as compared to both vivarium and ground control erythrocytes. Nonetheless, this induction was not sufficient to prevent damages caused by oxidative stress. Future experiments should provide information helpful to reduce the effects of oxidative stress exposure and space anemia, possibly by integrating appropriate dietary elements and natural compounds that could act as antioxidants

Major artifacts encountered in studying biological samples containing ferric protoporphyrin IX

Heme (ferric protoporphyrin IX, FP) dissolves very rapidly into the lipid phase of membranes, and a large number of studies have focused attention on its possible toxic effect in whole cells or isolated membranes. However, because of its molecular structure and reactivity, different problems can be encountered during the course of studying biological samples containing FP. In this article, we discuss important interferences by FP and artifacts that can affect the experimental values. First, FP interferes with the Lowry's protein determination; therefore, membranes containing FP are overestimated in their protein content determined by this procedure. Second, freezing membranes at -20°C artifactually increases the local concentration of FP, thereby enhancing FP-induced lipid peroxidation. Third, in the presence of thiol compounds such as N-acetyl cysteine, FP is degraded to products that interfere with the thiobarbituric acid assay, one of the most widely used methods to measure the extent of lipoperoxidation

Exogenous Fatty Acids Modulate ER Lipid Composition and Metabolism in Breast Cancer Cells

(1) Background: Lipid metabolism is a fundamental hallmark of all tumors, especially of breast cancer. Few studies describe the different lipid metabolisms and sensitivities to the microenvironment of breast cancer cell subtypes that influence the proliferation, aggressiveness, and success of therapy. This study describes the impact of lipid microenvironment on endoplasmic reticulum (ER) membrane and metabolic activity in two breast cancer cell lines with Luminal A and triple-negative breast cancer (TNBC) features. (2) Methods: We investigated the peculiar lipid phenotype of a TNBC cell line, MDA-MB-231, and a Luminal A cell line, MCF7, and their different sensitivity to exogenous fatty acids (i.e., palmitic acid (PA) and docosahexaenoic acid (DHA)). Moreover, we verified the impact of exogenous fatty acids on ER lipid composition. (3) Results: The data obtained demonstrate that MDA-MB-231 cells are more sensitive to the lipid microenvironment and that both PA and DHA are able to remodel their ER membranes with consequences on resident enzyme activity. On the contrary, MCF7 cells are less sensitive to PA, whereas they incorporate DHA, although less efficiently than MDA-MB-231 cells. (4) Conclusions: This study sustains the importance of lipid metabolism as an innovative hallmark to discriminate breast cancer subclasses and to develop personalized and innovative pharmacological strategies. The different sensitivities to the lipid environment shown by MCF7 and MDA-MB-231 cells might be related to cell malignancy and chemoresistance onset. In the future, this new approach could lead to a substantial decrease both in deleterious side effects for the patients and in the cost of entire therapeutic treatments coupled with increased therapy efficiency

Exogenous Fatty Acids Modulate ER Lipid Composition and Metabolism in Breast Cancer Cells

(1) Background: Lipid metabolism is a fundamental hallmark of all tumors, especially of breast cancer. Few studies describe the different lipid metabolisms and sensitivities to the microenvironment of breast cancer cell subtypes that influence the proliferation, aggressiveness, and success of therapy. This study describes the impact of lipid microenvironment on endoplasmic reticulum (ER) membrane and metabolic activity in two breast cancer cell lines with Luminal A and triple-negative breast cancer (TNBC) features. (2) Methods: We investigated the peculiar lipid phenotype of a TNBC cell line, MDA-MB-231, and a Luminal A cell line, MCF7, and their different sensitivity to exogenous fatty acids (i.e., palmitic acid (PA) and docosahexaenoic acid (DHA)). Moreover, we verified the impact of exogenous fatty acids on ER lipid composition. (3) Results: The data obtained demonstrate that MDA-MB-231 cells are more sensitive to the lipid microenvironment and that both PA and DHA are able to remodel their ER membranes with consequences on resident enzyme activity. On the contrary, MCF7 cells are less sensitive to PA, whereas they incorporate DHA, although less efficiently than MDA-MB-231 cells. (4) Conclusions: This study sustains the importance of lipid metabolism as an innovative hallmark to discriminate breast cancer subclasses and to develop personalized and innovative pharmacological strategies. The different sensitivities to the lipid environment shown by MCF7 and MDA-MB-231 cells might be related to cell malignancy and chemoresistance onset. In the future, this new approach could lead to a substantial decrease both in deleterious side effects for the patients and in the cost of entire therapeutic treatments coupled with increased therapy efficiency

Antioxidant metabolism in Xenopus laevis embryos is affected by stratospheric balloon flight

To test the effects of low levels of radiation from space on living organisms, we flew Xenopus laevis embryos at different stages of development on a stratospheric balloon (BI.R.BA mission). After recovery, different parameters were analyzed to assess the effects of flight, with particular regard to oxidative stress damage. Because of failed temperature control during flight, the flight shielded embryos (FC) could not be used for biochemical or morphological comparisons. In contrast, the incubation conditions (i.e. temperature, containers, volumes) for the flight embryos (F) were parallel to those for the ground controls. Mortality data show that younger embryos (16 h) flown on the balloon (F) are more sensitive to radiation exposure than older ones (40 h and 6 days). Exposure during flight lowered the antioxidant potential in all embryos, particularly older ones. These preliminary data demonstrate that flight on a stratospheric balloon might affect antioxidant metabolism, though it is not yet possible to correlate these results with low radiation exposure during fligh