Title Participants Abstract "beta-Radiation Stress Responses on Growth and Antioxidative Defense System in Plants: A Study with Strontium-90 in Lemna minor" "Arne Van Hoeck, Nele HOREMANS, May Van Hees, Robin Nauts, Dries Knapen, Hildegarde VANDENHOVE, Ronny Blust" "In the following study, dose dependent effects on growth and oxidative stress induced by -radiation were examined to gain better insights in the mode of action of -radiation induced stress in plant species. Radiostrontium (Sr-90) was used to test for -radiation induced responses in the freshwater macrophyte Lemna minor. The accumulation pattern of Sr-90 was examined for L. minor root and fronds separately over a seven-day time period and was subsequently used in a dynamic dosimetric model to calculate -radiation dose rates. Exposing L. minor plants for seven days to a Sr-90 activity concentration of 25 up to 25,000 kBqL(-1) resulted in a dose rate between 0.084 +/- 0.004 and 97 +/- 8 mGyh(-1). After seven days of exposure, root fresh weight showed a dose dependent decrease starting from a dose rate of 9.4 +/- 0.5 mGyh(-1). Based on these data, an EDR10 value of 1.5 +/- 0.4 mGyh(-1) was estimated for root fresh weight and 52 +/- 17 mGyh(-1) for frond fresh weight. Different antioxidative enzymes and metabolites were further examined to analyze if -radiation induces oxidative stress in L. minor." "$\beta$-radiation stress responses on growth and antioxidative defense system in plants" "Arne Van Hoeck, Nele Horemans, May Van Hees, Robin Nauts, Dries Knapen, Hildegarde Vandenhove, Ronny Blust" "In the following study, dose dependent effects on growth and oxidative stress induced by β-radiation were examined to gain better insights in the mode of action of β-radiation induced stress in plant species. Radiostrontium (90Sr) was used to test for β-radiation induced responses in the freshwater macrophyte Lemna minor. The accumulation pattern of 90Sr was examined for L. minor root and fronds separately over a seven-day time period and was subsequently used in a dynamic dosimetric model to calculate β-radiation dose rates. Exposing L. minor plants for seven days to a 90Sr activity concentration of 25 up to 25,000 kBq·L−1 resulted in a dose rate between 0.084 ± 0.004 and 97 ± 8 mGy·h−1. After seven days of exposure, root fresh weight showed a dose dependent decrease starting from a dose rate of 9.4 ± 0.5 mGy·h−1. Based on these data, an EDR10 value of 1.5 ± 0.4 mGy·h−1 was estimated for root fresh weight and 52 ± 17 mGy·h−1 for frond fresh weight. Different antioxidative enzymes and metabolites were further examined to analyze if β-radiation induces oxidative stress in L. minor." "Anti-oxidative defences are modulated differentially in three freshwater teleosts in response to ammonia-induced oxidative stress" "Amit Kumar Sinha, Hamada Abd Elgawad, Terri Giblen, Gaurav Zinta, Michelle De Rop, Han Asard, Ronny Blust, Gudrun De Boeck" "Oxidative stress and the antioxidant response induced by high environmental ammonia (HEA) were investigated in the liver and gills of three freshwater teleosts differing in their sensitivities to ammonia. The highly ammonia-sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less ammonia sensitive cyprinid Cyprinus carpio (common carp) and the highly ammonia-resistant cyprinid Carassius auratus (goldfish) were exposed to 1 mM ammonia (as NH4HCO3) for 0 h (control), 3 h, 12 h, 24 h, 48 h, 84 h and 180 h. Results show that HEA exposure increased ammonia accumulation significantly in the liver of all the three fish species from 24 h48 h onwards which was associated with an increment in oxidative stress, evidenced by elevation of xanthine oxidase activity and levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA). Unlike in trout, H2O2 and MDA accumulation in carp and goldfish liver was restored to control levels (84 h180 h); which was accompanied by a concomitant increase in superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase activity and reduced ascorbate content. Many of these defence parameters remained unaffected in trout liver, while components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase) enhanced to a greater extent. The present findings suggest that trout rely mainly on glutathione dependent defensive mechanism while carp utilize SOD, CAT and ascorbate as anti-oxidative sentinels. Hepatic cells of goldfish appear to utilize each of these protective systems, and showed more effective anti-oxidative compensatory responses towards HEA than carp, while trout were least effective. The present work also indicates that HEA exposure resulted in a relatively mild oxidative stress in the gills of all three species. This probably explains the almost complete lack of anti-oxidative responses in branchial tissue. This research suggests that oxidative stress, as well as the antioxidant potential clearly differ between salmonid and cyprinid species." "Glutathione and mitochondria determine acute defense responses and adaptive processes in cadmium-induced oxidative stress and toxicity of the kidney" "Ambily RAVINDRAN NAIR, Wing-Kee Lee, Karen SMEETS, Quirine SWENNEN, Amparo Sanchez, Frank Thévenod, Ann CUYPERS" "Cadmium (Cd2+) induces oxidative stress that ultimately defines cell fate and pathology. Mitochondria are the main energy-producing organelles in mammalian cells, but they also have a central role in formation of reactive oxygen species, cell injury, and death signaling. As the kidney is the major target in Cd2+ toxicity, the roles of oxidative signature and mitochondrial function and biogenesis in Cd2+-related stress outcomes were investigated in vitro in cultured rat kidney proximal tubule cells (PTCs) (WKPT-0293 Cl.2) for acute Cd2+ toxicity (1–30 µM, 24 h) and in vivo in Fischer 344 rats for sub-chronic Cd2+ toxicity (1 mg/kg CdCl2 subcutaneously, 13 days). Whereas 30 µM Cd2+ caused ~50 % decrease in cell viability, apoptosis peaked at 10 µM Cd2+ in PTCs. A steep, dose-dependent decline in reduced glutathione (GSH) content occurred after acute exposure and an increase of the oxidized glutathione (GSSG)/GSH ratio. Quantitative PCR analyses evidenced increased antioxidative enzymes (Sod1, Gclc, Gclm), proapoptotic Bax, metallothioneins 1A/2A, and decreased antiapoptotic proteins (Bcl-xL, Bcl-w). The positive regulator of mitochondrial biogenesis Pparγ and mitochondrial DNA was increased, and cellular ATP was unaffected with Cd2+ (1–10 µM). In vivo, active caspase-3, and hence apoptosis, was detected by FLIVO injection in the kidney cortex of Cd2+-treated rats together with an increase in Bax mRNA. However, antiapoptotic genes (Bcl-2, Bcl-xL, Bcl-w) were also upregulated. Both GSSG and GSH increased with chronic Cd2+ exposure with no change in GSSG/GSH ratio and augmented expression of antioxidative enzymes (Gpx4, Prdx2). Mitochondrial DNA, mitofusin 2, and Pparα were increased indicating enhanced mitochondrial biogenesis and fusion. Hence, these results demonstrate a clear involvement of higher mitochondria copy numbers or mass and mitochondrial function in acute defense against oxidative stress induced by Cd2+ in renal PTCs as well as in adaptive processes associated with chronic renal Cd2+ toxicity." "Cadmium-induced nephrotoxicity: from defense strategy to acclimation" "Ambily RAVINDRAN NAIR" "Cadmium (Cd) indirectly induces reactive oxygen species (ROS) by (1) a displacement of redox-active metals, (2) depletion of redox scavengers, (3) inhibition of anti-oxidant enzymes and (4) inhibition of the electron transport chain. This ultimately results in mitochondrial damage leading to loss of function or cell death in multiple organs. A disturbance of the redox balance by Cd at the cellular level has been studied repeatedly in different experimental set-ups including differentiated cells at the whole animal level (including humans), tissue level, primary cell cultures and/or cell lines as well as non-differentiated cells. However the outcome of these results as to where the oxidative balance gets compromised at the cellular level and leads to pathologies at the level of the organ depends on a multitude of experimental and environmental conditions. It is important to keep in mind that the comparison of results from different experiments should be done with caution as many studies use high concentrations of Cd that might cause an effect in cell lines, but are irrelevant in terms of environmentally realistic exposures in animals. Several parameters that are inherently connected to the experimental set-up such as the route of Cd administration, the speciation of Cd applied and different cell types to be studied all determine the final outcome of Cd toxicity. In my study, research on kidney cell lines for example, results in direct exposure of Cd on these cells, while an oral or sub-cutaneous administration of Cd to rats in vivo results in Cd absorption by blood and other organs before it reaches the kidney resulting in changes of initial Cd speciation and concentration applied. Nevertheless, in terms of translational toxicology, it is highly interesting to compare the effects of Cd exposure levels in vitro to those in vivo, when Cd concentrations are determined for both experimental set-ups in a comparable manner. Cadmium has a half-life of 15-30 years in the kidney, and considerable damage is mostly confined to the apical domain of the proximal tubular cells (PTCs). These cells possess large amounts of mitochondria, an important source of ROS, but also a highly sensitive organelle to increased ROS levels. As such, mitochondrial alterations are involved in both damaging as well as adaptation responses. This makes the WKPT-0293 cell line derived from the S1 segment of PTCs of the rat a good model to study Cd toxicity, as they also possess transporters and receptors for Cd. The mechanism of Cd-induced ROS production in association with mitochondrial alterations leading to adaptation responses or cell death is not fully elucidated, allthough it is known that Cd can directly damage mitochondria. In general, the aim of this study was to investigate and compare the molecular and cellular responses in renal cells in vitro and in vivo exposed to different doses of Cd in an acute respectively subchronic way. The study specifically focused on the role of oxidative stress and mitochondria, and how they modulate different defense strategies and help cells to acclimate to Cd stress. Gene expression analyses of antioxidant genes and mitochondrial genes formed the core technology of this study and therefore it was very important that the results obtained from qPCR are accurate and reliable. For this purpose, an additional analysis to select appropriate reference genes was performed in both cell lines and kidney tissues of in vitro and in vivo experiments respectively. The choice of reference genes for gene quantification is an important pre-requisite for carrying out new studies. From a set of eight commonly used reference genes, it was found that glyceraldehyde-3-phosphate dehydrogenase (Gapdh), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (Ywhaz) and beta-actin (Actb) were the most stable reference genes in the in vitro experimental set-up, while Gapdh, Ywhaz and peptidylprolyl isomerase A /cyclophilin A (Ppia) were the stable set in vivo. It was shown that the stability and hence selection of reference genes varied between both experimental set-ups even when both the cells/tissues originated from rat. Subsequently all the qPCR results of transcripts analyzed in vitro or in vivo were normalized to their respective set of stable reference genes. The in vitro experiment made use of Cd free medium and of 1, 10 and 30 μM of Cd concentrations that caused low, moderate or high amounts of damage to PTCs. In this case, cells without or with visible alterations were obtained to study the antioxidant signature and role of mitochondria during Cd exposure and/or subsequent adaptation. It was shown that at lower Cd concentrations (1 μM CdCl2), mitochondrial DNA (mtDNA) content and anti-apoptotic gene expression increased with a decrease in reduced glutathione (GSH) content. At higher concentrations (10 and 30 μM CdCl2), there was an increase in hydrogen peroxide (H2O2) and glutathione disulfide (GSSG) content, metallothionein (MT) transcript levels, pro-apoptotic gene induction together with loss of mitochondrial DNA content and function as well as depletion of GSH. From these observations, we noticed that alterations in the balance between the pro- and anti-oxidant status of the cell can lead to cell signalling for survival or damage to cells, called the oxidative challenge. With increasing ROS and thereby cell stress, the mitochondrial system can collapse, leading to further damage of mitochondria and ROS production culminating in apoptosis and/or necrosis. Both mitochondrial abundance and a shift from GSH to GSSG as a response to Cd induced oxidative stress are probably involved in the final outcomes towards long-term exposure and were explored in more detail in the in vivo study. In the in vivo study, when rats were treated for 13 days with sub-chronic Cd exposure (1 mg CdCl2/kg bw), the GSSG/GSH ratio remained the same indicating the maintenance of the cellular redox state. Superoxide dismutase 2 and GSH immunohistochemical stainings together with gene expression analyses of antioxidants revealed their up-regulated activities, which points towards the cell’s attempt to defend, and adapt to the Cd-induced oxidative challenge. It is clear that under Cd stress, a new redox equilibrium is installed in the PTCs. The increased expression of genes responsible for regulation and biogenesis of mitochondria indicates a positive regulation for mitochondrial biogenesis. This was also reflected in terms of increased mtDNA content in Cd-treated animals as compared to control animals, which might be the result of an increasing energy demand of the cell necessary for adaptive and or survival mechanisms under Cd stress. Whereas increased caspase activity in Cd-treated animals points towards enhanced apoptosis under Cd stress, the simultaneous up-regulation of both pro- and antiapoptotic genes might represent the varying activities of damaged and undamaged cells present in the heterogeneous kidney tissue. Overall, the in vivo experimental results suggested a clear and obvious involvement of mitochondria in defending against the Cd-induced oxidative stress during long-term subchronic exposure. In conclusion, a comparison of the oxidative stress signature between acute in vitro versus sub-chronic in vivo Cd exposure shows a clear shift in terms of defense and acclimation. It was shown that GSH is a first line of defense in Cd toxicity both in vitro and in vivo due to its chelating and antioxidant properties. There is also a parallel activation of mitochondrial biogenesis in order to meet the increasing energy demand of the cells under stress. Other acclimation strategies i.e. chelation by MTs and antioxidant defense mechanisms come into play during exposure to higher Cd concentrations or prolonged exposure. Once the Cd stress is beyond the withholding limit, these mechanisms fail and the cell enters a point of no return leading to irreversible damage and eventually Cdinduced pathologies." "Stevioside inhibits atherosclerosis by improving insulin signaling and antioxidant defense in obese insulin-resistant mice" "Maarten Hulsmans, Paul Holvoet" "Objective:Stevioside is a non-caloric natural sweetener that does not induce a glycemic response, making it attractive as sweetener to diabetics and others on carbohydrate-controlled diets. Obesity is frequently associated with insulin resistance and increased inflammation and oxidative stress. Therefore, we investigated its effects on insulin resistance, inflammation and oxidative stress related to atherosclerosis in obese insulin-resistant mice.Research design:Twelve-week-old mice were treated with stevioside (10 mg kg(-1), n=14) or placebo (n=20) for 12 weeks.Results:Stevioside had no effect on weight and triglycerides, but lowered glucose and insulin. Stevioside treatment improved adipose tissue maturation, and increased glucose transport, insulin signaling and antioxidant defense in white visceral adipose tissues. Together, these increases were associated with a twofold increase of adiponectin. In addition, stevioside reduced plaque volume in the aortic arch by decreasing the macrophage, lipid and oxidized low-density lipoprotein (ox-LDL) content of the plaque. The higher smooth muscle cell-to-macrophage ratio was indicative for a more stable plaque phenotype. The decrease in ox-LDL in the plaque was likely due to an increase in the antioxidant defense in the vascular wall, as evidenced by increased Sod1, Sod2 and Sod3. Circulating adiponectin was associated with improved insulin signaling and antioxidant defense in both the adipose tissue and the aorta of stevioside-treated mice.Conclusion:Stevioside treatment was associated with improved insulin signaling and antioxidant defense in both the adipose tissue and the vascular wall, leading to inhibition of atherosclerotic plaque development and inducing plaque stabilization.International Journal of Obesity advance online publication, 15 December 2009; doi:10.1038/ijo.2009.261." "Antioxidant defense in quiescent cells determines selectivity of electron transport chain inhibition-induced cell death" "Jan Blecha, Silvia Magalhaes Novais, Katerina Rohlenova, Eliska Novotna, Sandra Lettlova, Sabine Schmitt, Hans Zischka, Jiri Neuzil, Jakub Rohlena" "Mitochondrial electron transport chain (ETC) targeting shows a great promise in cancer therapy. It is particularly effective in tumors with high ETC activity where ETC-derived reactive oxygen species (ROS) are efficiently induced. Why modern ETC-targeted compounds are tolerated on the organismal level remains unclear. As most somatic cells are in non-proliferative state, the features associated with the ETC in quiescence could account for some of the specificity observed. Here we report that quiescent cells, despite increased utilization of the ETC and enhanced supercomplex assembly, are less susceptible to cell death induced by ETC disruption when glucose is not limiting. Mechanistically, this is mediated by the increased detoxification of ETC-derived ROS by mitochondrial antioxidant defense, principally by the superoxide dismutase 2 - thioredoxin axis. In contrast, under conditions of glucose limitation, cell death is induced preferentially in quiescent cells and is correlated with intracellular ATP depletion but not with ROS. This is related to the inability of quiescent cells to compensate for the lost mitochondrial ATP production by the upregulation of glucose uptake. Hence, elevated ROS, not the loss of mitochondrially-generated ATP, are responsible for cell death induction by ETC disruption in ample nutrients condition, e.g. in well perfused healthy tissues, where antioxidant defense imparts specificity. However, in conditions of limited glucose, e.g. in poorly perfused tumors, ETC disruption causes rapid depletion of cellular ATP, optimizing impact towards tumor-associated dormant cells. In summary, we propose that antioxidant defense in quiescent cells is aided by local glucose limitations to ensure selectivity of ETC inhibition-induced cell death." "Metabolism and antioxidant defense in the larval chironomid **Tanytarsus minutipalpus**" "Lucas F. Bianchini, Chris M. Wood, Harold L. Bergman, Ora E. Johannsson, Pierre Laurent, Claudine Chevalier, Mosiany L. Kisipan, Geraldine D. Kavembe, Michael B. Papah, Kevin V. Brix, Gudrun De Boeck, John N. Maina, Rodi O. Ojoo, Adalto Bianchini" "Insect larvae are reported to be a major component of the simple but highly productive trophic web found in Lake Magadi (Kenya, Africa), which is considered to be one of the most extreme aquatic environments on Earth. Previous studies show that fish must display biochemical and physiological adjustments to thrive under the extreme conditions of the lake. However, information for invertebrates is lacking. In the present study, the occurrence of the larval chironomid Tanytarsus minutipalpus is reported in Lake Magadi for the first time. Additionally, changes in larval metabolism and antioxidant defense correlated with diel variations in the extremely hostile environmental conditions of the lake are described. Wide variations in water temperature (20.2-29.3°C) and dissolved oxygen content (3.2-18.6 mg O2 l−1) were observed at different times of day, without significant change in water pH (10.0±0.03). Temperature and dissolved oxygen were higher at 13:00 h (29.3±0.4°C and 18.6±1.0 mg O2 l−1) and 19:00 h (29.3±0.8°C and 16.2±1.6 mg O2 l−1) and lower at 01:00 h (21.1±0.1°C and 10.7±0.03 mg O2 l−1) and 07:00 h (20.2±0.4°C and 3.2±0.7 mg O2 l−1). Significant and parallel increases in parameters related to metabolism (cholinesterase, glucose, cholesterol, urea, creatinine and hemoglobin) and the antioxidant system (SOD, GPx, GR, GSH and GSSG) were observed in larvae collected at 13:00 h. In contrast, no significant changes were observed in pro-oxidants (ROS and NO), TOSC and oxidative damage parameters (LPO and DNA damage). Therefore, the observed increases in temperature and dissolved O2 content in Lake Magadi were associated with changes in the antioxidant system of T. minutipalpus larvae. Adjustments performed by the chironomid larvae were efficient in maintaining body homeostasis, as well as protecting biomolecules against oxidative damage, so that oxidative stress did not occur. GSH-GSSG and GPx-GR systems appeared to play an essential role in the adjustments displayed by the chironomid larvae during the diel changes in the extreme conditions of Lake Magadi." "Mycorrhized wheat and bean plants tolerate bismuth contaminated soil via improved metal detoxification and antioxidant defense systems" "Afrah E. Mohammed, Elke Pawelzik, Mudawi M. Nour, Modhi O. Alotaibi, Hamada Abd Elgawad, Ahmed M. Saleh" "Contamination of agricultural fields with bismuth (Bi) reduces crop yield and quality. Arbuscular mycorrhizal fungi (AMF) are known to enhance plant growth and crop production, even under stressful conditions such as soil contamination with heavy metals. The objective of this study was to investigate the effect of AMF on the mitigation of Bi-phytotoxicity in wheat (Triticum aestivum) and beans (Phaseolus vulgaris) and to provide a comprehensive evaluation of the physiological and biochemical basis for the growth and development of AMF-induced plants under Bi stress conditions. Wheat and bean were treated by Bi and AMF individually and in combination. Then the physiological and biochemical responses in the shoot and roots of the two crop species were studied. Evident retardations in plant growth and key photosynthesis-related parameters and accumulation of MDA, H2O2, as markers of oxidative stress, were observed in plants subjected to Bi. AMF colonization reduced the uptake and translocation of Bi in the plant organs by enhancing the exudation of polyphenols and organic acids into the rhizospheric soil. Mycorrhized wheat and bean plants were able to attenuate the effects of Bi by improving metal detoxification (phytochelatins, metallothionein, total glutathione, and glutathione-S-transferase activity) and antioxidant defense systems (both enzymatic and non-enzymatic) and maintaining C assimilation and nutrient status. The current results suggest the manipulation of AMF as a powerful approach to alleviate the phytotoxicity of Bi in legumes and grasses." "Constitutive defense mechanisms have a major role in the resistance of woodland strawberry leaves against Botrytis cinerea" "Yijie Zhao, Liese Vlasselaer, Wim Van den Ende, Maarten Hertog, Bart Nicolai, Barbara De Coninck" "The necrotrophic fungus Botrytis cinerea is a major threat to strawberry cultivation worldwide. By screening different Fragaria vesca genotypes for susceptibility to B. cinerea, we identified two genotypes with different resistance levels, a susceptible genotype F. vesca ssp. vesca Tenno 3 (T3) and a moderately resistant genotype F. vesca ssp. vesca Kreuzkogel 1 (K1). These two genotypes were used to identify the molecular basis for the increased resistance of K1 compared to T3. Fungal DNA quantification and microscopic observation of fungal growth in woodland strawberry leaves confirmed that the growth of B. cinerea was restricted during early stages of infection in K1 compared to T3. Gene expression analysis in both genotypes upon B. cinerea inoculation suggested that the restricted growth of B. cinerea was rather due to the constitutive resistance mechanisms of K1 instead of the induction of defense responses. Furthermore, we observed that the amount of total phenolics, total flavonoids, glucose, galactose, citric acid and ascorbic acid correlated positively with higher resistance, while H2O2 and sucrose correlated negatively. Therefore, we propose that K1 leaves are more resistant against B. cinerea compared to T3 leaves, prior to B. cinerea inoculation, due to a lower amount of innate H2O2, which is attributed to a higher level of antioxidants and antioxidant enzymes in K1. To conclude, this study provides important insights into the resistance mechanisms against B. cinerea, which highly depend on the innate antioxidative profile and specialized metabolites of woodland strawberry leaves."