Inhibiting catalase activity sensitizes 36B10 rat glioma cells to oxidative stress

Gliomas are extremely resistant to anticancer therapies resulting in poor patient survival, due, in part, to altered expression of antioxidant enzymes. The primary antioxidant enzyme, catalase, is elevated constitutively in gliomas compared to normal astrocytes. We hypothesized that downregulating catalase in glioma cells would sensitize these cells to oxidative stress. To test this hypothesis, we implemented two approaches. The first, a pharmacological approach, used 3-amino-1,2,4-triazole, an irreversible inhibitor that reduced catalase enzymatic activity by 75%. Pharmacological inhibition of catalase was not associated with a reduction in rat 36B10 glioma cell viability until the cells were challenged with additional oxidative stress, i.e., ionizing radiation or hydrogen peroxide (H(2)O(2)). In the second molecular approach, we generated 36B10 glioma cells stably expressing catalase shRNA; a stable cell line displayed a 75% reduction in catalase immunoreactive protein and enzymatic activity. This was accompanied by an increase in intracellular reactive oxygen species and extracellular H(2)O(2). These cells exhibited increased sensitivity to radiation and H(2)O(2), which was rescued by the antioxidant, N-acetylcysteine. These results support the hypothesis that catalase is a major participant in the defense of 36B10 glioma cells against oxidative stress mediated by anticancer agents capable of increasing steady-state levels of H(2)O(2).     

Cloning, characterization and tissue expression of disk abalone (Haliotis discus discus) catalase

Catalase is an antioxidant enzyme that plays a significant role in protection against oxidative stress by detoxification of hydrogen peroxide (H2O2). A gene coding for a putative catalase was isolated from the disk abalone (Haliotis discus discus) cDNA library and denoted as Ab-catalase. The full-length (2864 bp) Ab-catalase cDNA contained 1,503 bp open reading frame (ORF), encoding 501 amino acid residues with 56 kDa predicted molecular weight. The deduced amino acid sequence of Ab-catalase has characteristic features of catalase family such as catalytic site motif (61FNRERIPERVVHAKGAG77), heme-ligand signature motif (351RLYSYSDT358), NADPH and heme binding residues. Phylogenetic and pairwise identity results indicated that Ab-catalase is more similar to scallop (Chlamys farreri) catalase with 80% amino acid identity except for other reported disk abalone catalase sequences. Constitutive Ab-catalase expression was detected in gill, mantle, gonad, hemocytes, abductor muscle and digestive tract in tissue specific manner. Ab-catalase mRNA was up-regulated in gill and digestive tract tissues for the first 3h post injection of H2O2, showing the inducible ability of abalone catalase against oxidative stress generated by H2O2. The purified recombinant catalase showed 30,000 U/mg enzymatic activity against H2O2 and biochemical properties of higher thermal stability and broad spectrum of pH. Our results suggest that abalone catalase may play an important role in regulating oxidative stress by scavenging H2O2.     

Seasonal variation in the activity of selected antioxidant enzymes and malondialdehyde level in worker honey bees

The recent decline in managed honey bee populations, Apis mellifera L. (Hymenoptera: Apidae), has caused scientific, ecological, and economic concern. Research into the formation of reactive oxygen species (ROS), antioxidative defense mechanisms, and oxidative stress can contribute to our understanding of bee survival and conservation of this species. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S‐transferase (GST) enzymes together with levels of malondialdehyde (MDA) were measured in summer and winter honey bees sampled from three colonies. One colony was stationary (C1), entering the winter period having accumulated Robinia pseudoacacia L. (Fabaceae) honey, and two were migratory (C2 and C3), entering the winter period with mainly Tilia (Malvaceae) and Brassica (Brassicaceae) honey, respectively. Compared to summer workers, winter worker bees had decreased SOD and GST activity, and MDA level, whereas CAT activity increased in all three colonies. We also demonstrated that seasonality is the main factor responsible for changes in antioxidant enzymes and MDA levels in worker honey bees. Overall, our results indicate a difference between summer and winter worker bees, pointing at a reduced level of antioxidant enzyme defenses during overwintering which may be due to a decrease in production of ROS. The decreased levels of MDA measured in winter honey bees confirm this. As ROS are actively used by insects as a defense mechanism to fight pathogens, we suggest that reduced production of ROS contributes to higher susceptibility of winter honey bees to infections and reduced overwinter survival.     

Differential expression of antioxidant system genes in honey bee (Apis mellifera L.) caste development mitigates ROS-mediated oxidative damage in queen larvae

The expression of morphological differences between the castes of social bees is triggered by dietary regimes that differentially activate nutrient-sensing pathways and the endocrine system, resulting in differential gene expression during larval development. In the honey bee, Apis mellifera, mitochondrial activity in the larval fat body has been postulated as a link that integrates nutrient-sensing via hypoxia signaling. To understand regulatory mechanisms in this link, we measured reactive oxygen species (ROS) levels, oxidative damage to proteins, the cellular redox environment, and the expression of genes encoding antioxidant factors in the fat body of queen and worker larvae. Despite higher mean H₂O₂ levels in queens, there were no differences in ROS-mediated protein carboxylation levels between the two castes. This can be explained by their higher expression of antioxidant genes (MnSOD, CuZnSOD, catalase, and Gst1) and the lower ratio between reduced and oxidized glutathione (GSH/GSSG). In worker larvae, the GSG/GSSH ratio is elevated and antioxidant gene expression is delayed. Hence, the higher ROS production resulting from the higher respiratory metabolism in queen larvae is effectively counterbalanced by the up-regulation of antioxidant genes, avoiding oxidative damage. In contrast, the delay in antioxidant gene expression in worker larvae may explain their endogenous hypoxia response.     

Acclimation,Hydrogen Peroxide,and Abscisic Acid Protect Mitochondria against Irreversible Chilling Injury in Maize Seedlings

Our previous results indicated that 3-d-old dark-grown chilling-sensitive maize (Zea mays L.) seedlings did not survive 7 d of 4[deg]C chilling stress, but 69% of them survived similar stress when the seedlings were either preexposed to 14[deg]C for 3 d or pretreated with 0.1 mM H2O2 for 4 h at 27[deg]C (T.K. Prasad, M.D. Anderson, B.A. Martin, C.R. Stewart [1994] Plant Cell 6: 65-74) or 1 mM abscisic acid (ABA) for 24 h at 27[deg]C (M.D. Anderson, T.K. Prasad, B.A. Martin, C.R. Stewart [1994] Plant Physiol 105: 331-339). We discovered that chilling imposed oxidative stress on the seedlings. Since H2O2 accumulated during the periods of both acclimation and nonacclimation, we concluded that H2O2 had dual effects at low temperature: (a) During acclimation, its early transient accumulation signals the induction of antioxidant enzymes such as catalase 3 and peroxidase to scavenge H2O2; and (b) at 4[deg]C in nonacclimated seedlings, it accumulates to damaging levels in the tissues because of low levels of these and perhaps other antioxidant enzymes. Three-day-old seedlings pretreated with H2O2 (a mild oxidative stress) or ABA showed induced chilling tolerance. In the present study, we investigated whether mitochondria are a target for chilling-induced oxidative stress and, if so, what differences do acclimation, H2O2, or ABA make to protect mitochondria from irreversible chilling injury. The results indicated that chilling, in general, impairs respiratory activity, the cytochrome pathway of electron transport, and ATPase activity regardless of the treatment. In pretreated seedlings, the activities of catalase 3 and peroxidase in the mitochondria increased severalfold compared with control and nonacclimated seedlings. The increases in these antioxidant enzymes imply that mitochondria are under oxidative stress and such increases could initiate a protective mechanism in the mitochondria. Mitochondrial respiration is partially cyanide resistant during chilling stress and also after the 1st d of recovery. Upon further recovery over 3 d, in contrast to nonacclimated seedlings, the mitochondria of acclimation-, H2O2-, and ABA-treated seedlings showed the following recovery features. (a) The mitochondrial respiration changed from a cyanide-resistant to a cyanide-sensitive cytochrome pathway, (b) cytochrome oxidase activity recovered to control levels, (c) the ability of mitochondria to generate ATP was regained, and (d) the antioxidant enzyme activities remained at or above control levels. Based on these results, we conclude that chilling impairs mitochondrial function and that chilling-induced oxidative stress seems to be a factor, at least in part, for causing possible irreversible damage to the mitochondrial membrance components. Acclimation, H2O2, and ABA provide a protective mechanism by inducing antioxidant enzymes to protect mitochondria from irreversible oxidative damage that is absent in nonacclimated seedlings. Therefore, we conclude that the ability of the seedlings to recover from chilling injury is, at least in part, due to the ability of the mitochondria to resume normal function.     

Penicillin-induced oxidative stress: effects on antioxidative response of midgut tissues in instars of Galleria mellonella

Penicillin and other antibiotics are routinely incorporated in insect culture media. Although culturing insects in the presence of antibiotics is a decades-old practice, antibiotics can exert deleterious influences on insects. In this article, we test the hypothesis that one of the effects of dietary penicillin is to increase oxidative stress on insects. The effects of penicillin on midgut concentrations of the oxidative stress indicator malondialdehyde (MDA) and on midgut antioxidant enzyme (superoxide dismutase [SOD], catalase [CAT], glutathione S-transferase [GST], and glutathione peroxidase [GPx]) and transaminases (alanine aminotransferase and aspartate aminotransferase) activities in greater wax moth, Galleria mellonella (L.), were investigated. The insects were reared from first instars on artificial diets containing 0.001, 0.01, 0.1, or 1.0 g penicillin per 100 g of diets. MDA content was significantly increased in the midgut tissues of each larval instar reared in the presence of high penicillin concentrations. Activities of antioxidant and transaminase enzymes did not show a consistent pattern with respect to penicillin concentrations in diet or age of larvae. Despite the increased penicillin-induced oxidative stress in gut tissue, antioxidant and transaminase enzymes did not correlate with oxidative stress level or between each other in larvae of other age stages except for the seventh instar. We found a significant negative correlation of MDA content with SOD and GST activities in seventh instars. SOD activity was also negatively correlated with CAT activity in seventh instars. These results suggest that exposure to dietary penicillin resulted in impaired enzymatic antioxidant defense capacity and metabolic functions in wax moth larval midgut tissues and that the resulting oxidative stress impacts midgut digestive physiology.     

Role of glutathione S-transferases in protection against lipid peroxidation. Overexpression of hGSTA2-2 in K562 cells protects against hydrogen peroxide-induced apoptosis and inhibits JNK and caspase 3 activation

The physiological significance of the selenium-independent glutathione peroxidase (GPx) activity of glutathione S-transferases (GSTs), associated with the major Alpha class isoenzymes hGSTA1-1 and hGSTA2-2, is not known. In the present studies we demonstrate that these isoenzymes show high GPx activity toward phospholipid hydroperoxides (PL-OOH) and they can catalyze GSH-dependent reduction of PL-OOH in situ in biological membranes. A major portion of GPx activity of human liver and testis toward phosphatidylcholine hydroperoxide (PC-OOH) is contributed by the Alpha class GSTs. Overexpression of hGSTA2-2 in K562 cells attenuates lipid peroxidation under normal conditions as well as during the oxidative stress and confers about 1.5-fold resistance to these cells from H(2)O(2) cytotoxicity. Treatment with 30 microm H(2)O(2) for 48 h or 40 microm PC-OOH for 8 h causes apoptosis in control cells, whereas hGSTA2-2-overexpressing cells are protected from apoptosis under these conditions. In control cells, H(2)O(2) treatment causes an early (within 2 h), robust, and persistent (at least 24 h) activation of JNK, whereas in hGSTA2-2-overexpressing cells, only a slight activation of JNK activity is observed at 6 h which declines to basal levels within 24 h. Caspase 3-mediated poly(ADP-ribose) polymerase cleavage is also inhibited in cells overexpressing hGSTA2-2. hGSTA2 transfection does not affect the function of antioxidant enzymes including GPx activity toward H(2)O(2) suggesting that the Alpha class GSTs play an important role in regulation of the intracellular concentrations of the lipid peroxidation products that may be involved in the signaling mechanisms of apoptosis.     

氯化钠胁迫对嫁接茄子生长、抗氧化酶活性和活性氧代谢的影响

以日本引进的茄子设施栽培专用耐盐品种‘Torvum Vigor’为砧木,栽培品种‘苏崎茄’为接穗,在营养液栽培条件下,对80 mmol·L^-1 NaCl胁迫下茄子嫁接苗和自根苗的光合特性、叶片抗氧化酶活性、活性氧代谢进行了比较.结果表明：NaCl胁迫下,茄子嫁接苗的干质量、叶绿素含量和光合速率分别比苏崎茄自根苗高67。8％、8。8％和31。1％,均有显著差异;抗氧化酶活性均显著高于苏崎茄自根苗,而O₂^-_·生成速率、H₂O₂和MDA含量则显著低于苏崎茄自根苗.NaCl胁迫明显降低了叶片叶绿素含量和光合速率,显著增加了O₂^-_·生成速率、H₂O₂和MDA含量,但嫁接苗受NaCl胁迫的影响小于苏崎茄自根苗,嫁接苗较强的耐盐性与NaCl胁迫下较高的抗氧化酶活性和较低的氧化损伤有关.     

Reduction in molecular synthesis or enzyme activity of superoxide dismutases and catalase contributes to oxidative stress and neurogenic hypertension in spontaneously hypertensive rats

A balance between production and elimination of reactive oxygen species such as superoxide anion (O2*-) and hydrogen peroxide (H2O2) tightly regulates the homeostasis of cellular oxidative stress, which contributes to a variety of cardiovascular diseases, including hypertension. The present study assessed the hypothesis that O2*- or H2O2 levels augmented by the reduced molecular synthesis or enzyme activity of superoxide dismutase (SOD), catalase (CAT), or glutathione peroxidase (GPx) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons that generate tonic vasomotor tone are located, contribute to the pathogenesis of hypertension. We found that copper/zinc SOD (SOD1), manganese SOD (SOD2), or CAT, but not GPx, mRNA or protein expression and enzyme activity in the RVLM of spontaneously hypertensive rats (SHR) were significantly lower than those in normotensive Wistar-Kyoto (WKY) rats, along with a significantly higher level of O2*- or H2O2. A causative relationship between these biochemical correlates of oxidative stress and neurogenic hypertension was established when gene transfer by microinjection of adenovirus encoding SOD1, SOD2, or CAT into the bilateral RVLM promoted a long-lasting reduction in arterial pressure in SHR, but not WKY rats, accompanied by an enhanced SOD1, SOD2, or CAT protein expression or enzyme activity and reduced O2*- or H2O2 level in the RVLM. These results together suggest that downregulation of gene expression and enzyme activity of the antioxidant SOD1, SOD2, or CAT may underlie the augmented levels of O2*- and H2O2 in the RVLM, leading to oxidative stress and hypertension in SHR.