The effect of 4-chlororesorcinol on the endogenous levels of IAA, ABA and oxidative enzymes in cuttings

Treatment of bean cuttings with 4-chlororesorcinol (4-CR), known to increase the number of roots and extend their distribution, prevented the accumulation of free indol-3-yl-acetic acid (IAA) in the hypocotyls within 24 h after cutting preparation. In mung bean there was no change in the distribution (upper half vs. 1 ower half of the hypocotyl) of IAA within the hypocotyl as a result of the treatment. In bean cuttings the treatment with 4-CR prevented the accumulation of IAA in the bottom of the cutting. Oxidation of IAA as a measure of IAA oxidase activity in bean was enhanced appreciably by 4-chlororesorcinol. The level of abscisic acid in mung bean, on the other hand, remained 3-4 fold higher than in the control, yet still about 50% lower than the zero time level. In untreated mung bean cuttings the activity of peroxidase increased after cutting preparation. In contrast, the activity of peroxidase in 4-Cr-treated cuttings was consistently lower. In order to relate to the effect of exogenously applied auxin the level of peroxidase was measured also in indol-3-yl-butyric acid-treated cuttings. The overall peroxidase activity in IBA-treated cuttings was not affected. However, when assaying for the different isozymes the drop in peroxidase activity was most evident in the inducible basic isoperoxidases both in 4-CR and IBA treatments. It appears that the exposure to 4-CR exerts an effect that is similar to that of exogenously applied auxin, affecting the activity of basic peroxidases and enhancing the oxidation of endogenous IAA, thus allowing the organization of the primordia.

Thyroid antibody status, subclinical hypothyroidism, and the risk of coronary heart disease: an individual participant data analysis.

CONTEXT: Subclinical hypothyroidism has been associated with increased risk of coronary heart disease (CHD), particularly with thyrotropin levels of 10.0 mIU/L or greater. The measurement of thyroid antibodies helps predict the progression to overt hypothyroidism, but it is unclear whether thyroid autoimmunity independently affects CHD risk. OBJECTIVE: The objective of the study was to compare the CHD risk of subclinical hypothyroidism with and without thyroid peroxidase antibodies (TPOAbs). DATA SOURCES AND STUDY SELECTION: A MEDLINE and EMBASE search from 1950 to 2011 was conducted for prospective cohorts, reporting baseline thyroid function, antibodies, and CHD outcomes. DATA EXTRACTION: Individual data of 38 274 participants from six cohorts for CHD mortality followed up for 460 333 person-years and 33 394 participants from four cohorts for CHD events. DATA SYNTHESIS: Among 38 274 adults (median age 55 y, 63% women), 1691 (4.4%) had subclinical hypothyroidism, of whom 775 (45.8%) had positive TPOAbs. During follow-up, 1436 participants died of CHD and 3285 had CHD events. Compared with euthyroid individuals, age- and gender-adjusted risks of CHD mortality in subclinical hypothyroidism were similar among individuals with and without TPOAbs [hazard ratio (HR) 1.15, 95% confidence interval (CI) 0.87-1.53 vs HR 1.26, CI 1.01-1.58, P for interaction = .62], as were risks of CHD events (HR 1.16, CI 0.87-1.56 vs HR 1.26, CI 1.02-1.56, P for interaction = .65). Risks of CHD mortality and events increased with higher thyrotropin, but within each stratum, risks did not differ by TPOAb status. CONCLUSIONS: CHD risk associated with subclinical hypothyroidism did not differ by TPOAb status, suggesting that biomarkers of thyroid autoimmunity do not add independent prognostic information for CHD outcomes.

Intracellular targeting of GLUT4 in transfected insulinoma cells: evidence for association with constitutively recycling vesicles distinct from synaptophysin and insulin vesicles.

In adipocytes and muscle cells, the GLUT4 glucose transporter isoform is present in intracellular vesicles which continuously recycle between an intracytoplasmic location and the plasma membrane. It is not clear whether the GLUT4-vesicles represent a specific kind of vesicle or resemble typical secretory granules or synaptic-like microvesicles. To approach this question, we expressed GLUT4 in the beta cell line RINm5F and determined its intracellular localization by subcellular fractionation and by immunofluorescence and immunoelectron microscopy. GLUT4 was not found in insulin granules but was associated with a subpopulation of smooth-surface vesicles present in the trans-Golgi region and in vesicular structures adjacent to the plasma membrane. In the trans-Golgi region, GLUT4 did not colocalize with synaptophysin or TGN38. Incubation of the cells with horseradish peroxidase (HRP) led to colocalization of HRP and GLUT4 in some endosomal structures adjacent to the plasma membrane and in occasional trans-Golgi region vesicles. When cells were incubated in the presence of Bafilomycin A, analysis by confocal microscopy revealed GLUT4 in numerous large spots present throughout the cytoplasm, many of which costained for TGN38 and synaptophysin. By immunoelectron microscopy, numerous endosomes were observed which stained strongly for GLUT4. Together our data demonstrate that ectopic expression of GLUT4 in insulinoma cells reveals the presence of a subset of vesicular structures distinct from synaptic-like vesicles and insulin secretory granules. Furthermore, they indicate that GLUT4 constitutively recycles between the plasma membrane and its intracellular location by an endocytic route also taken by TGN38 and synaptophysin.

Cuticular defects lead to full immunity to a major plant pathogen.

In addition to its role as a barrier, the cuticle is also a source of signals perceived by invading fungi. Cuticular breakdown products have been shown previously to be potent inducers of cutinase or developmental processes in fungal pathogens. Here the question was addressed as to whether plants themselves can perceive modifications of the cuticle. This was studied using Arabidopsis thaliana plants with altered cuticular structure. The expression of a cell wall-targeted fungal cutinase in A. thaliana was found to provide total immunity to Botrytis cinerea. The response observed in such cutinase-expressing plants is independent of signal transduction pathways involving salicylic acid, ethylene or jasmonic acid. It is accompanied by the release of a fungitoxic activity and increased expression of members of the lipid transfer protein, peroxidase and protein inhibitor gene families that provide resistance when overexpressed in wild-type plants. The same experiments were made in the bodyguard (bdg) mutant of A. thaliana. This mutant exhibits cuticular defects and remained free of symptoms after inoculation with B. cinerea. The expression of resistance was accompanied by the release of a fungitoxic activity and increased expression of the same genes as observed in cutinase-expressing plants. Structural defects of the cuticle can thus be converted into an effective multi-factorial defence, and reveal a hitherto hidden aspect of the innate immune response of plants.

The blood-brain barrier: cellular basis.

Perfusion experiments with horseradish peroxidase have established that the morphological substrate of the blood-brain barrier is represented by microvascular endothelial cells. They are characterized by complexly arranged tight junctions and a very low rate of transcytotic vesicular transport. They express transport enzymes, carrier systems and brain endothelial cell-specific molecules of unknown function not expressed by any other endothelial cell population. These blood-brain barrier properties are not intrinsic to these cells but are inducible by the surrounding brain tissue. Type I astrocytes injected into the anterior eye chamber of the rat or onto the chick chorioallantoic membrane are able to induce a host-derived angiogenesis and some blood-brain barrier properties in endothelial cells of non-neural origin. Recently we have shown that this cellular interaction is due to the secretion of a soluble astrocyte derived factor(s). Astrocytes are also implicated in the maintenance, functional regulation and the repair of the blood-brain barrier. Complex interactions between other constituents of the microenvironment surrounding the endothelial cells, such as the basement membrane, pericytes, nerve endings, microglial cells and the extracellular fluid, take place and are required for the proper functioning of the blood-brain barrier, which in addition is regionally different as reflected by endothelial cell heterogeneity.

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis.

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX's cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury.

Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses.

The epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed and surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.

Intrinsic ROS-production capacity of nanoparticles

Engineered nanomaterials (ENMs) exhibit special physicochemical properties and thus are finding their way into an increasing number of industries, enabling products with improved properties. Their increased use brings a greater likelihood of exposure to the nanoparticles (NPs) that could be released during the life cycle of nano-abled products. The field of nanotoxicology has emerged as a consequence of the development of these novel materials, and it has gained ever more attention due to the urgent need to gather information on exposure to them and to understand the potential hazards they engender. However, current studies on nanotoxicity tend to focus on pristine ENMs, and they use these toxicity results to generalize risk assessments on human exposure to NPs. ENMs released into the environment can interact with their surroundings, change characteristics and exhibit toxicity effects distinct from those of pristine ENMs. Furthermore, NPs' large surface areas provide extra-large potential interfaces, thus promoting more significant interactions between NPs and other co-existing species. In such processes, other species can attach to a NP's surface and modify its surface functionality, in addition to the toxicity in normally exhibits. One particular occupational health scenario involves NPs and low-volatile organic compounds (LVOC), a common type of pollutant existing around many potential sources of NPs. LVOC can coat a NP's surface and then dominate its toxicity. One important mechanism in nanotoxicology is the creation of reactive oxygen species (ROS) on a NP's surface; LVOC can modify the production of these ROS. In summary, nanotoxicity research should not be limited to the toxicity of pristine NPs, nor use their toxicity to evaluate the health effects of exposure to environmental NPs. Instead, the interactions which NPs have with other environmental species should also be considered and researched. The potential health effects of exposure to NPs should be derived from these real world NPs with characteristics modified by the environment and their distinct toxicity. Failure to suitably address toxicity results could lead to an inappropriate treatment of nano- release, affect the environment and public health and put a blemish on the development of sustainable nanotechnologies as a whole. The main objective of this thesis is to demonstrate a process for coating NP surfaces with LVOC using a well-controlled laboratory design and, with regard to these NPs' capacity to generate ROS, explore the consequences of changing particle toxicity. The dynamic coating system developed yielded stable and replicable coating performance, simulating an important realistic scenario. Clear changes in the size distribution of airborne NPs were observed using a scanning mobility particle sizer, were confirmed using both liquid nanotracking analyses and transmission electron microscopy (TEM) imaging, and were verified thanks to the LVOC coating. Coating thicknesses corresponded to the amount of coating material used and were controlled using the parameters of the LVOC generator. The capacity of pristine silver NPs (Ag NPs) to generate ROS was reduced when they were given a passive coating of inert paraffin: this coating blocked the reactive zones on the particle surfaces. In contrast, a coating of active reduced-anthraquinone contributed to redox reactions and generated ROS itself, despite the fact that ROS generation due to oxidation by Ag NPs themselves was quenched. Further objectives of this thesis included development of ROS methodology and the analysis of ROS case studies. Since the capacity of NPs to create ROS is an important effect in nanotoxicity, we attempted to refine and standardize the use of 2'7-dichlorodihydrofluorescin (DCFH) as a chemical tailored for the characterization of NPs' capacity for ROS generation. Previous studies had reported a wide variety of results, which were due to a number of insufficiently well controlled factors. We therefore cross-compared chemicals and concentrations, explored ways of dispersing NP samples in liquid solutions, identified sources of contradictions in the literature and investigated ways of reducing artificial results. The most robust results were obtained by sonicating an optimal sample of NPs in a DCFH-HRP solution made of 5,M DCFH and 0.5 unit/ml horseradish peroxidase (HRP). Our findings explained how the major reasons for previously conflicting results were the different experimental approaches used and the potential artifacts appearing when using high sample concentrations. Applying our advanced DCFH protocol with other physicochemical characterizations and biological analyses, we conducted several case studies, characterizing aerosols and NP samples. Exposure to aged brake wear dust engenders a risk of potential deleterious health effects in occupational scenarios. We performed microscopy and elemental analyses, as well as ROS measurements, with acellular and cellular DCFH assays. TEM images revealed samples to be heterogeneous mixtures with few particles in the nano-scale. Metallic and non-metallic elements were identified, primarily iron, carbon and oxygen. Moderate amounts of ROS were detected in the cell-free fluorescent tests; however, exposed cells were not dramatically activated. In addition to their highly aged state due to oxidation, the reason aged brake wear samples caused less oxidative stress than fresh brake wear samples may be because of their larger size and thus smaller relative reactive surface area. Other case studies involving welding fumes and differently charged NPs confirmed the performance of our DCFH assay and found ROS generation linked to varying characteristics, especially the surface functionality of the samples. Les nanomatériaux manufacturés (ENM) présentent des propriétés physico-chimiques particulières et ont donc trouvés des applications dans un nombre croissant de secteurs, permettant de réaliser des produits ayant des propriétés améliorées. Leur utilisation accrue engendre un plus grand risque pour les êtres humains d'être exposés à des nanoparticules (NP) qui sont libérées au long de leur cycle de vie. En conséquence, la nanotoxicologie a émergé et gagné de plus en plus d'attention dû à la nécessité de recueillir les renseignements nécessaires sur l'exposition et les risques associés à ces nouveaux matériaux. Cependant, les études actuelles sur la nanotoxicité ont tendance à se concentrer sur les ENM et utiliser ces résultats toxicologiques pour généraliser l'évaluation des risques sur l'exposition humaine aux NP. Les ENM libérés dans l'environnement peuvent interagir avec l'environnement, changeant leurs caractéristiques, et montrer des effets de toxicité distincts par rapport aux ENM originaux. Par ailleurs, la grande surface des NP fournit une grande interface avec l'extérieur, favorisant les interactions entre les NP et les autres espèces présentes. Dans ce processus, d'autres espèces peuvent s'attacher à la surface des NP et modifier leur fonctionnalité de surface ainsi que leur toxicité. Un scénario d'exposition professionnel particulier implique à la fois des NP et des composés organiques peu volatils (LVOC), un type commun de polluant associé à de nombreuses sources de NP. Les LVOC peuvent se déposer sur la surface des NP et donc dominer la toxicité globale de la particule. Un mécanisme important en nanotoxicologie est la création d'espèces réactives d'oxygène (ROS) sur la surface des particules, et les LVOC peuvent modifier cette production de ROS. En résumé, la recherche en nanotoxicité ne devrait pas être limitée à la toxicité des ENM originaux, ni utiliser leur toxicité pour évaluer les effets sur la santé de l'exposition aux NP de l'environnement; mais les interactions que les NP ont avec d'autres espèces environnementales doivent être envisagées et étudiées. Les effets possibles sur la santé de l'exposition aux NP devraient être dérivés de ces NP aux caractéristiques modifiées et à la toxicité distincte. L'utilisation de résultats de toxicité inappropriés peut conduire à une mauvaise prise en charge de l'exposition aux NP, de détériorer l'environnement et la santé publique et d'entraver le développement durable des industries de la nanotechnologie dans leur ensemble. L'objectif principal de cette thèse est de démontrer le processus de déposition des LVOC sur la surface des NP en utilisant un environnement de laboratoire bien contrôlé et d'explorer les conséquences du changement de toxicité des particules sur leur capacité à générer des ROS. Le système de déposition dynamique développé a abouti à des performances de revêtement stables et reproductibles, en simulant des scénarios réalistes importants. Des changements clairs dans la distribution de taille des NP en suspension ont été observés par spectrométrie de mobilité électrique des particules, confirmé à la fois par la méthode dite liquid nanotracking analysis et par microscopie électronique à transmission (MET), et a été vérifié comme provenant du revêtement par LVOC. La correspondance entre l'épaisseur de revêtement et la quantité de matériau de revêtement disponible a été démontré et a pu être contrôlé par les paramètres du générateur de LVOC. La génération de ROS dû aux NP d'argent (Ag NP) a été diminuée par un revêtement passif de paraffine inerte bloquant les zones réactives à la surface des particules. Au contraire, le revêtement actif d'anthraquinone réduit a contribué aux réactions redox et a généré des ROS, même lorsque la production de ROS par oxydation des Ag NP avec l'oxygène a été désactivé. Les objectifs associés comprennent le développement de la méthodologie et des études de cas spécifique aux ROS. Etant donné que la capacité des NP à générer des ROS contribue grandement à la nanotoxicité, nous avons tenté de définir un standard pour l'utilisation de 27- dichlorodihydrofluorescine (DCFH) adapté pour caractériser la génération de ROS par les NP. Des etudes antérieures ont rapporté une grande variété de résultats différents, ce qui était dû à un contrôle insuffisant des plusieurs facteurs. Nous avons donc comparé les produits chimiques et les concentrations utilisés, exploré les moyens de dispersion des échantillons HP en solution liquide, investigué les sources de conflits identifiées dans les littératures et étudié les moyens de réduire les résultats artificiels. De très bon résultats ont été obtenus par sonication d'une quantité optimale d'échantillons de NP en solution dans du DCFH-HRP, fait de 5 nM de DCFH et de 0,5 unité/ml de Peroxydase de raifort (HRP). Notre étude a démontré que les principales raisons causant les conflits entre les études précédemment conduites dans la littérature étaient dues aux différentes approches expérimentales et à des artefacts potentiels dus à des concentrations élevées de NP dans les échantillons. Utilisant notre protocole DCFH avancé avec d'autres caractérisations physico-chimiques et analyses biologiques, nous avons mené plusieurs études de cas, caractérisant les échantillons d'aérosols et les NP. La vielle poussière de frein en particulier présente un risque élevé d'exposition dans les scénarios professionnels, avec des effets potentiels néfastes sur la santé. Nous avons effectué des analyses d'éléments et de microscopie ainsi que la mesure de ROS avec DCFH cellulaire et acellulaire. Les résultats de MET ont révélé que les échantillons se présentent sous la forme de mélanges de particules hétérogènes, desquels une faible proportion se trouve dans l'échelle nano. Des éléments métalliques et non métalliques ont été identifiés, principalement du fer, du carbone et de l'oxygène. Une quantité modérée de ROS a été détectée dans le test fluorescent acellulaire; cependant les cellules exposées n'ont pas été très fortement activées. La raison pour laquelle les échantillons de vielle poussière de frein causent un stress oxydatif inférieur par rapport à la poussière de frein nouvelle peut-être à cause de leur plus grande taille engendrant une surface réactive proportionnellement plus petite, ainsi que leur état d'oxydation avancé diminuant la réactivité. D'autres études de cas sur les fumées de soudage et sur des NP différemment chargées ont confirmé la performance de notre test DCFH et ont trouvé que la génération de ROS est liée à certaines caractéristiques, notamment la fonctionnalité de surface des échantillons.

Lipid mobilization and gluconeogenesis in plants: Do glyoxylate cycle enzyme activities constitute a real cycle? A hypothesis

Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves. They are the site of beta-oxidation and of the glyoxylate cycle. These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids. In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors. The necessity of NAD(+) regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles. Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system. Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD(+) regeneration). Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.

FemHab: The effects of bed rest and hypoxia on oxidative stress in healthy women.

Independently, both inactivity and hypoxia augment oxidative stress. This study, part of the FemHab project, investigated the combined effects of bed rest-induced unloading and hypoxic exposure on oxidative stress and antioxidant status. Healthy, eumenorrheic women were randomly assigned to the following three 10-day experimental interventions: normoxic bed rest (NBR;n= 11; PiO2 = 133 mmHg), normobaric hypoxic bed rest (HBR;n= 12; PiO2 = 90 mmHg), and ambulatory hypoxic confinement (HAMB;n= 8: PiO2 = 90 mmHg). Plasma samples, obtained before (Pre), during (D2, D6), immediately after (Post) and 24 h after (Post+1) each intervention, were analyzed for oxidative stress markers [advanced oxidation protein products (AOPP), malondialdehyde (MDA), and nitrotyrosine], antioxidant status [superoxide dismutase (SOD), catalase, ferric-reducing antioxidant power (FRAP), glutathione peroxidase (GPX), and uric acid (UA)], NO metabolism end-products (NOx), and nitrites. Compared with baseline, AOPP increased in NBR and HBR on D2 (+14%; +12%;P< 0.05), D6 (+19%; +15%;P< 0.05), and Post (+22%; +21%;P< 0.05), respectively. MDA increased at Post+1 in NBR (+116%;P< 0.01) and D2 in HBR (+114%;P< 0.01) and HAMB (+95%;P< 0.05). Nitrotyrosine decreased (-45%;P< 0.05) and nitrites increased (+46%;P< 0.05) at Post+1 in HAMB only. Whereas SOD was higher at D6 (+82%) and Post+1 (+67%) in HAMB only, the catalase activity increased on D6 (128%) and Post (146%) in HBR and HAMB, respectively (P< 0.05). GPX was only reduced on D6 (-20%;P< 0.01) and Post (-18%;P< 0.05) in HBR. No differences were observed in FRAP and NOx. UA was higher at Post in HBR compared with HAMB (P< 0.05). These data indicate that exposure to combined inactivity and hypoxia impairs prooxidant/antioxidant balance in healthy women. Moreover, habitual activity levels, as opposed to inactivity, seem to blunt hypoxia-related oxidative stress via antioxidant system upregulation.