Perspectives in stabilisation of polyolefins

Polyolefins, which normally undergo high temperature manufacturing and fabrication operations, are susceptible to oxidation during each stage of their lifecycle. Stabilisers and antioxidants are used to inhibit the oxidative damage that is ultimately responsible for loss of physical properties, embrittlement and premature failure. Environmental awareness and health and safety considerations have spurred intense searches for new approaches to procure improved, safe, and more efficient antioxidants and stabiliser systems for polymers. Current activities have concentrated on two approaches: the first advocates the use of biological (naturally occurring) antioxidants, and the second relies on the use of reactive antioxidants that are chemically attached onto the polymer backbone for greater permanence and safety. Stabilisation of polyolefins through the use of vitamin E and some reactive, non-migratory antioxidants is the subject matter of this chapter.

Ultra-endurance exercise:unanswered questions in redox biology and immunology

Ultra-endurance races are extreme exercise events that can take place over large parts of a day, several consecutive days or over weeks and months interspersed by periods of rest and recovery. Since the first ultraendurance races in the late 1970s, around 1000 races are now held worldwide each year, and more than 100000 people take part. Although these athletes appear to be fit and healthy, there have been occasional reports of severe complications following ultra-endurance exercise. Thus there is concern that repeated extreme exercise events could have deleterious effects on health, which might be brought about by the high levels of ROS (reactive oxygen species) produced during exercise. Studies that have examined biomarkers of oxidative damage following ultra-endurance exercise have found measurements to be elevated for several days, which has usually been interpreted to reflect increased ROS production. Levels of the antioxidant molecule GSH (reduced glutathione) are depleted for 1 month or longer following ultra-endurance exercise, suggesting an impaired capacity to copewith ROS. The present paper summarizes studies that have examined the oxidative footprint of ultra-endurance exercise in light of current thinking in redox biology and the possible health implications of such extreme exercise. © The Authors Journal compilation © 2014 Biochemical Society.

Ankyrin is the major oxidised protein in erythrocyte membranes from end-stage renal disease patients on chronic haemodialysis and oxidation is decreased by dialysis and vitamin C supplementation

Chronically haemodialysed end-stage renal disease patients are at high risk of morbidity arising from complications of dialysis, the underlying pathology that has led to renal disease and the complex pathology of chronic kidney disease. Anaemia is commonplace and its origins are multifactorial, involving reduced renal erythropoietin production, accumulation of uremic toxins and an increase in erythrocyte fragility. Oxidative damage is a common risk factor in renal disease and its co-morbidities and is known to cause erythrocyte fragility. Therefore, we have investigated the hypothesis that specific erythrocyte membrane proteins are more oxidised in end-stage renal disease patients and that vitamin C supplementation can ameliorate membrane protein oxidation. Eleven patients and 15 control subjects were recruited to the study. Patients were supplemented with 2 × 500 mg vitamin C per day for 4 weeks. Erythrocyte membrane proteins were prepared pre- and post-vitamin C supplementation for determination of protein oxidation. Total protein carbonyls were reduced by vitamin C supplementation but not by dialysis when investigated by enzyme linked immunosorbent assay. Using a western blot to detect oxidised proteins, one protein band, later identified as containing ankyrin, was found to be oxidised in patients but not controls and was reduced significantly by 60% in all patients after dialysis and by 20% after vitamin C treatment pre-dialysis. Ankyrin oxidation analysis may be useful in a stratified medicines approach as a possible marker to identify requirements for intervention in dialysis patients.

Mass spectrometry-based methods for identifying oxidized proteins in disease:advances and challenges

Many inflammatory diseases have an oxidative aetiology, which leads to oxidative damage to biomolecules, including proteins. It is now increasingly recognized that oxidative post-translational modifications (oxPTMs) of proteins affect cell signalling and behaviour, and can contribute to pathology. Moreover, oxidized proteins have potential as biomarkers for inflammatory diseases. Although many assays for generic protein oxidation and breakdown products of protein oxidation are available, only advanced tandem mass spectrometry approaches have the power to localize specific oxPTMs in identified proteins. While much work has been carried out using untargeted or discovery mass spectrometry approaches, identification of oxPTMs in disease has benefitted from the development of sophisticated targeted or semi-targeted scanning routines, combined with chemical labeling and enrichment approaches. Nevertheless, many potential pitfalls exist which can result in incorrect identifications. This review explains the limitations, advantages and challenges of all of these approaches to detecting oxidatively modified proteins, and provides an update on recent literature in which they have been used to detect and quantify protein oxidation in disease.

Erythrocyte antioxidant protection of rose hips (Rosa spp.)

Rose hips are popular in health promoting products as the fruits contain high content of bioactive compounds. The aim of this study was to investigate whether health benefits are attributable to ascorbic acid, phenols, or other rose-hip-derived compounds. Freeze-dried powder of rose hips was preextracted with metaphosphoric acid and the sample was then sequentially eluted on a C18 column. The degree of amelioration of oxidative damage was determined in an erythrocyte in vitro bioassay by comparing the effects of a reducing agent on erythrocytes alone or on erythrocytes pretreated with berry extracts. The maximum protection against oxidative stress, 59.4 ± 4.0% (mean standard deviation), was achieved when incubating the cells with the first eluted meta-phosphoric extract. Removal of ascorbic acid from this extract increased the protection against oxidative stress to 67.9 ± 1.9% . The protection from the 20% and 100% methanol extracts was 20.8 ± 8.2% and 5.0 ± 3.2% , respectively. Antioxidant uptake was confirmed by measurement of catechin by HPLC-ESI-MS in the 20% methanol extract. The fact that all sequentially eluted extracts studied contributed to protective effects on the erythrocytes indicates that rose hips contain a promising level of clinically relevant antioxidant protection. © Copyright 2012 C. Widén et al.

Protein modification and phospholipid oxidation

Phospholipid oxidation can generate reactive and electrophilic products that are capable of modifying proteins, especially at cysteine, lysine and histidine residues. Such lipoxidation reactions are known to alter protein structure and function, both with gain of function and loss of activity effects. As well as potential importance in the redox regulation of cell behaviour, lipoxidation products in plasma could also be useful biomarkers for stress conditions. Although studies with antibodies suggested the occurrence of lipoxidation adducts on ApoB-100, these products had not previously been characterized at a molecular level. We have developed new mass spectrometry-based approaches to detect and locate adducts of oxidized phospholipids in plasma proteins, as well as direct oxidation modifications of proteins, which avoid some of the problems typically encountered with database search engines leading to erroneous identifications of oxidative PTMs. This approach uses accurate mass extracted ion chromatograms (XICs) of fragment ions from peptides containing oxPTMs, and allows multiple modifications to be examined regardless of the protein that contains them. For example, a reporter ion at 184.074 Da/e corresponding to phosphocholine indicated the presence of oxidized phosphatidylcholine adducts, while 2 reporter ions at 100.078 and 82.025 Da/e were selective for allysine. ApoB-100-oxidized phospholipid adducts were detected even in healthy human samples, as well as LDL from patients with inflammatory disease. Lipidomic studies showed that more than 350 different species of lipid were present in LDL, and were altered in disease conditions. LDL clearly represents a very complex carrier system and one that offers a rich source of information about systemic conditions, with potential as indicators of oxidative damage in ageing or inflammatory diseases.

Prenatal environmental exposure and neurodevelopmentally important gene expression in malformed brain tissue from pediatric intractable epilepsy patients

The primary objective of this proposal was to determine whether mitochondrial oxidative stress and variation in a particular mtDNA lineage contribute to the risk of developing cortical dysplasia and are potential contributing factors in epileptogenesis in children. The occurrence of epilepsy in children is highly associated with malformations of cortical development (MCD). It appears that MCD might arise from developmental errors due to environmental exposures in combination with inherited variation in response to environmental exposures and mitochondrial function. Therefore, it is postulated that variation in a particular mtDNA lineage of children contributes to the effects of mitochondrial DNA damage on MCD phenotype. Quantitative PCR and dot blot were used to examine mitochondrial oxidative damage and single nucleotide polymorphism (SNP) in the mitochondrial genome in brain tissue from 48 pediatric intractable epilepsy patients from Miami Children’s Hospital and 11 control samples from NICHD Brain and Tissue Bank for Developmental Disorders. Epilepsy patients showed higher mtDNA copy number compared to normal health subjects (controls). Oxidative mtDNA damage was lower in non-neoplastic but higher in neoplastic epilepsy patients compared to controls. There was a trend of lower mtDNA oxidative damage in the non-neoplastic (MCD) patients compared to controls, yet, the reverse was observed in neoplastic (MCD and Non-MCD) epilepsy patients. The presence of mtDNA SNP and haplogroups did not show any statistically significant relationships with epilepsy phenotypes. However, SNPs G9804A and G9952A were found in higher frequencies in epilepsy samples. Logistic regression analysis showed no relationship between mtDNA oxidative stress, mtDNA copy number, mitochondrial haplogroups and SNP variations with epilepsy in pediatric patients. The levels of mtDNA copy number and oxidative mtDNA damage and the SNPs G9952A and T10010C predicted neoplastic epilepsy, however, this was not significant due to a small sample size of pediatric subjects. Findings of this study indicate that an increase in mtDNA content may be compensatory mechanisms for defective mitochondria in intractable epilepsy and brain tumor. Further validation of these findings related to mitochondrial genotypes and mitochondrial dysfunction in pediatric epilepsy and MCD may lay the ground for the development of new therapies and prevention strategies during embryogenesis.

Redox Regulation of Ras Proteins in Dictyostelium discoideum

Reactive oxygen species are a normal consequence of life in an aerobic environment. However when they deviate from the narrow permissible range in cells, oxidative damage can occur. Dictyostelium discoideum is a model organism ideal for the study of cell signaling events such as those affected by oxidative stress. It was previously shown that Ras signaling in Dictyostelium is affected by genetic inactivation of the antioxidant enzyme Superoxide dismutase C (SodC) and in vitro data suggests that the NKCD motif of Ras is the redox target of superoxide. The main objective of this project was to determine the mechanism of superoxide mediated Ras regulation in vivo. To accomplish the main objective, we cloned, and in some cases, mutated different Ras proteins and later determined their activity in wild type and sodC- cells. RasC and RasD showed normal activation in sodC- cells, however RasG and RasS displayed high Ras activity. These last two Ras proteins contain the NKC118D motif inside the nucleotide binding region. A mutation of cysteine118 to alanine in RasG rendered the protein less active in sodC- than the wild type RasG protein and a mutation alanine118 to cysteine in RasD conferred redox sensitivity to this small GTPase. Additionally, the propensity of RasG to be targeted by superoxide was evident when the environment of wild type cells was manipulated to induce the internal generation of superoxide through changes in the extracellular ion levels mainly magnesium. Lack of magnesium ions increased the intracellular level of superoxide and severely hampered directional cell migration. Chemotaxis of cells expressing RasG was negatively impacted by the absence of magnesium ions; however rasG- cells did not seem to be affected in their ability to perform chemotaxis. The last experiment implies that RasG is an important mediator of cell signaling during oxidative stress, responsible for preventing cells from continuing their developmental program. Our study suggests that the cysteine residue in the NKCD motif is essential for mediating the redox sensitivity of Ras proteins in Dictyostelium and that RasG is an essential mediator of the response to oxidative stress in this organism.

The role of protein oxidation in ageing & disease

Ageing is a natural phenomenon of the human lifecycle, yet it is still not understood what causes the deterioration of the human body near the end of the lifespan. One popular theory is the Free Radical Theory of Ageing, which proposes that oxidative damage to biomolecules causes ageing of tissues. The ageing population is affected by many chronic diseases. This study focused on sarcopenia (muscle loss in ageing) and obesity as two models for comparison of oxidative damage in muscle proteins in mice. The aim of the study was to develop advanced mass spectrometry methods to detect specific oxidative modifications to mouse muscle proteins, including oxidation, nitration, chlorination, and carbonyl group formation, but western blotting was also used to provide complementary information on the oxidative state of proteins from aged and obese muscle. Mass spectrometry proved to be a powerful tool, enabling identification of the types of modifications present, the sites at which they were present and percentage of the peptide populations that were modified. Targeted and semi-targeted mass spectrometry methods were optimised for the identification and quantitation of the oxidised residues in muscle proteins. The development of the quantitative methods enabled comparisons of mass spectrometry instruments. Both the Time of Flight and QTRAP systems showed advantages of using the different mass analysers to quantify oxidative modifications. Several oxidised residues were characterised and quantified in both the obese and sarcopenic models, and higher levels of oxidation were found compared to their control counterparts. Residues found to be oxidised were oxidation of proline, tyrosine and tryptophan, dioxidation of methionine, allysine and nitration of tyrosine. However quantification was performed on methionine dioxidation and cysteine trioxidation containing residues in SERCA. The combination of measuring residue susceptibility and functional studies could contribute to understanding the overall role of oxidation in ageing and obesity.

Evaluation of the Genotoxic and Physiological Effects of Decabromodiphenyl Ether (BDE-209) and Dechlorane Plus (DP) Flame Retardants in Marine Mussels (Mytilus galloprovincialis)

Dechlorane Plus (DP) is a proposed alternative to the legacy flame retardant decabromodiphenyl ether (BDE-209), a major component of Deca-BDE formulations. In contrast to BDE-209, toxicity data for DP are scarce and often focused on mice. Validated dietary in vivo exposure of the marine bivalve (Mytilus galloprovincialis) to both flame retardants did not induce effects at the physiological level (algal clearance rate), but induced DNA damage, as determined by the comet assay, at all concentrations tested. Micronuclei formation was induced by both DP and BDE-209 at the highest exposure concentrations (100 and 200 mu g/L, respectively, at 18% above controls). DP caused effects similar to those by BDE-209 but at lower exposure concentrations (5.6, 56, and 100 mu g/L for DP and 56, 100, and 200 mu g/L for BDE-209). Moreover, bioaccumulation of DP was shown to be concentration dependent, in contrast to BDE-209. The results described suggest that DP poses a greater genotoxic potential than BDE-209

Evaluation of the Genotoxic and Physiological Effects of Decabromodiphenyl Ether (BDE-209) and Dechlorane Plus (DP) Flame Retardants in Marine Mussels (Mytilus galloprovincialis)

Dechlorane Plus (DP) is a proposed alternative to the legacy flame retardant decabromodiphenyl ether (BDE-209), a major component of Deca-BDE formulations. In contrast to BDE-209, toxicity data for DP are scarce and often focused on mice. Validated dietary in vivo exposure of the marine bivalve (Mytilus galloprovincialis) to both flame retardants did not induce effects at the physiological level (algal clearance rate), but induced DNA damage, as determined by the comet assay, at all concentrations tested. Micronuclei formation was induced by both DP and BDE-209 at the highest exposure concentrations (100 and 200 mu g/L, respectively, at 18% above controls). DP caused effects similar to those by BDE-209 but at lower exposure concentrations (5.6, 56, and 100 mu g/L for DP and 56, 100, and 200 mu g/L for BDE-209). Moreover, bioaccumulation of DP was shown to be concentration dependent, in contrast to BDE-209. The results described suggest that DP poses a greater genotoxic potential than BDE-209

RECQ5 promotes recombination and mutagenesis at targeted nicks through disruption of RAD51 filaments

Thesis (Ph.D.)--University of Washington, 2016-08

HSP70 abundance and antioxidant capacity in feeding and fasting gray seal pups: suckling is associated with higher levels of key cellular defenses

Heat shock proteins (HSPs) and antioxidants are key cellular defenses against stress. Seals routinely undergo protracted fasting, which is normally associated with physiological stress in other animals. We tested the hypotheses that (1) relative HSP70 protein abundance is higher in liver and blubber of fasting relative to suckling wild gray seal pups; (2) differences in HSP70 are mirrored in tissue superoxide dismutase (SOD) and catalase activity, as well as glutathione levels; (3) extracellular HSP70 correlates with hepatic and blubber HSP70 abundance; and (4) protein carbonylation, an index of oxidative damage, is lower in tissues with higher levels of these cellular stress markers. In contrast to our expectation, suckling pups had higher relative HSP70 abundance and glutathione levels in liver and blubber and higher hepatic catalase activity. Plasma HSP70 did not correlate with liver or blubber abundance of the protein. Suckling pups did not experience greater protein carbonylation, suggesting that cellular protective mechanisms prevent protein damage despite an apparent increase in cellular stress. SOD activity was not affected by nutritional state, but in blubber tissue, it was positively correlated with blubber thickness. Greater requirements for antioxidants and HSPs in suckling pups or in animals with thicker blubber could arise from rapid protein synthesis, high metabolic fuel availability, and/or exposure to lipophilic toxins. Developmental and nutritional changes in cellular defenses have important implications for gray seals’ susceptibility to additional stress exposure.

Effets du stress oxydatif induit par les rayons UVA et endogène sur la cornée humaine : étude des délétions de l’ADN mitochondrial, des modifications dans la matrice extracellulaire cornéenne et de la dystrophie cornéenne endothéliale de Fuchs

Le stress oxydatif peut provenir de sources exogènes comme les UVA ou de sources endogènes comme la chaîne respiratoire (OXPHOS). L’oxydation des composants cellulaires a été associée avec la dégénération, des phénotypes de vieillissement et des pertes de fonctionnalités des tissus. Les UVA sont les plus efficaces des rayons UV à induire de l’oxydation, tel que démontré par la formation de dommages oxydatifs à l’ADN et par l’apparition de délétions mitochondriales qui en résultent. La délétion mitochondriale de 4977 pb (ADNmtCD4977), la plus commune, et celle de 3895 pb (ADNmt3895) sont deux délétions reliées au photovieillissement cutané et à l’exposition au stress oxydant. Le phénomène de vieillissement dans la peau est bien documenté et se traduit par une dégradation de la matrice extracellulaire, une perte d’élasticité et la formation de rides. Toutefois, peu d’études portent sur la cornée humaine alors qu’elle est un tissu exposé directement aux rayonnements UV au même titre que la peau. Nous avons donc tenté mieux comprendre l’effet de l’oxydation exogène et endogène sur cette structure. L’analyse de la localisation des délétions ADNmtCD4977 et ADNmtCD4977 dans l’oeil humain a permis de révéler qu’elles se concentrent principalement dans le stroma cornéen et s’accumule avec l’âge. Le stroma cornéen est la couche cellulaire qui confère la transparence et la rigidité à la cornée humaine. Ces résultats nous ont suggéré une implication des UVA dans le photovieillissement de la cornée. Nous avons donc entrepris de vérifier les changements liés à l’exposition aux UVA dans le stroma cornéen puisque les UVA sont connus pour causer des altérations à la matrice extracellulaire (ECM) au niveau cutané. Nous avons donc créé un modèle de photovieillisement par une exposition chronique aux UVA sur des kératocytes avec lesquels nous avons fait sécréter une ECM. Nos résultats nous ont démontré qu’une exposition chronique aux UVA cause des altérations à l’ECM cornéen semblable à des phénotypes de photvieillissement. En effet, nous avons dénoté des changements transcriptomiques et protéomiques pour certains collagènes et protéoglycans. Une atteinte aux collagènes par le vieillissement cornéen se traduit entre autres par une rigidification, une opacification et un changement dans son pouvoir réfractif qui mène à une perte de la vision. Par ailleurs, notre avons également investigué l’implication du stress oxydatif dans la dystrophie cornéenne endothéliale de Fuchs (FECD), une maladie dégénérative de l’endothélium cornéen, qui mène à une perte de vision et est une cause principale de greffe cornéenne. L’étiologie de la maladie est encore inconnue, mais le stress oxydatif est soupçonné de jouer un rôle important dans la pathogenèse. Nos résultats ont amené de nouvelles évidences de l’implication de l’oxydation dans la maladie par l’augmentation de la quantité d’ADN mitochondrial et un raccourcissement des télomères dans des explants de cornées pathologiques. Nos résultats nous ont également démontré que la mise en culture de cellules FECD permettait la sélection de cellules fonctionnelles et comparables à des cellules saines en termes de quantité d’ADN mitochondrial et de son intégrité, de sensibilité à l’oxydation et de longueur télomérique. Les résultats obtenus soutiennent ainsi la possibilité d’employer les cellules FECD fonctionnelles sélectionnées pour utilisation en génie tissulaire afin de créer des cornées autologues pour pallier aux manques de greffes cornéennes. Enfin, nos résultats apportent de nouvelles évidences quant à l’implication du stress oxydatif dans le photovieillissement cornéen et dans l’étiologie de la FECD.

Cannabinoids impact on pregnancy: effects in trophoblast cells

Cannabinoids (CBs) can be classified as: phytocannabinoids, the constituents of the Cannabis sativa plant; synthetic cannabinoids lab-synthesized and the endocannabinoids that are endogenous lipid mediators. Cannabinoid compounds activate cannabinoid receptors – CB1 and CB2. The most prevalent psychoactive phytocannabinoid is Δ9tetrahydrocannabinol (THC), but more than 60 different CBs were already identified in the plant. The best characterized endocannabinoids (eCBs) are anandamide (AEA) and 2arachidonoylglycerol (2-AG), that are involved in several physiological processes including synaptic plasticity, pain modulation, energy homeostasis and reproduction. On the other hand, some synthetic cannabinoids that were initially designed for medical research, are now used as drugs of abuse. During the period of placental development, highly dynamic processes of remodeling occur, involving proliferation, apoptosis, differentiation and invasion of trophoblasts. It is known that a tight control of eCBs levels is required for normal pregnancy progression and that eCBs are involved in trophoblast cells turnover. Therefore, by sharing activation of the same receptors, exposure to exocannabinoids either by recreational or medicinal use may lead to alterations in the eCBs levels and in the endocannabinoid system homeostasis In this work, it was studied the impact of CBs in BeWo trophoblastic cells and in primary cultures of human cytotrophoblasts. Cells were treated for 24 hours with different concentrations of THC, the synthetic cannabinoid WIN‐55,212 (WIN) and 2-AG. Treatment with THC did not affect BeWo cells viability while WIN and 2-AG caused a dose-dependent viability loss. Morphological studies together with biochemical markers indicate that 2-AG is able to induce apoptosis in cytotrophoblasts. On the other hand, morphological studies after acridine orange staining suggest that autophagy may take part in WIN-induced loss of cell viability. All cannabinoids caused a decrease in mitochondrial membrane potential (Δψm) but only 2-AG led to ROS/RNS generation, though no changes in glutathione levels were observed. In addition, ER-stress may be involved in the 2-AG induced-oxidative stress, as preliminary results point to an increase in CCAAT-enhancer-binding protein homologous protein (CHOP) expression. Besides the decrease in cell viability, alterations in cell cycle progression were observed. WIN treatment induced a cell cycle arrest in G0/G1 phase, whereas 2-AG induced a cell cycle arrest in G2/M phase. Here it is reinforced the relevance of cannabinoid signaling in fundamental processes of cell proliferation and cell death in trophoblast cells. Since cannabis-based drugs are the most consumed illicit drugs worldwide and some of the most consumed recreational drugs by pregnant women, this study may contribute to the understanding of the impact of such substances in human reproduction.