Rat lung glutathione release: response to oxidative stress and selenium deficiency

We performed experiments to characterize the glutathione-dependent metabolism occurring during tert-butyl hydroperoxide infusion in isolated perfused rat lungs and to examine the effect of selenium deficiency on this metabolism. Selenium deficiency resulted in decreased lung glutathione peroxidase activity but normal glutathione reductase activity and glutathione content. Infusion of the hydroperoxide into control lungs caused a proportional increase in tissue glutathione disulfide (GSSG) concentration and release of GSSG into the perfusate up to an infusion rate of 250 nmol of tert-butyl hydroperoxide X min-1 X 100 g body wt-1. Infusion rates greater than this resulted in continued rise of tissue GSSG concentrations but GSSG release into the perfusate plateaued. Infusion of tert-butyl hydroperoxide into selenium-deficient rat lungs resulted in much lower concentrations of tissue GSSG and GSSG release into the perfusate; however, release in the selenium-deficient rat lung was also found to be saturable at infusion rates of 450 nmol of tert-butyl hydroperoxide X min-1 X 100 g of body wt-1. Selenium deficiency in the rat decreases the rate of reduction of infused tert-butyl hydroperoxide by glutathione and may predispose the lung to free radical damage.     

Salicylic acid-induced hepatotoxicity triggered by oxidative stress

Salicylic acid is a widely used nonsteroidal anti-inflammatory drug (NSAID). But it is known to cause serious liver damage occasionally. Mitochondrial dysfunction and oxidative stress are predicted to be the major factors of salicylic acid-induced liver injury. We investigated the influence of salicylic acid on ATP contents, oxygen consumption and lipid peroxidation in the presence of the same concentration of salicylic acid. Leakage of lactate dehydrogenase (LDH) was significantly higher in the presence of 5 mM salicylic acid than in its absence. Salicylic acid-induced thiobarbituric acid-reactive substance (TBARS) formation and spontaneous chemiluminescence (CL) in rat hepatocytes, whereas antioxidants, such as promethazine (PMZ) and N,N-diphenylphenylenediamine (DPPD), suppressed both TBARS formation and LDH leakage. TBARS formation in rat liver microsomes was suppressed by diethyldithiocarbamate (a specific inhibitor of cytochrome P450 (CYP)2E1) and diclofenac (a specific inhibitor of CYP2C11). These results suggest that salicylic acid-induced lipid peroxidation was related to oxidative metabolism mediated by CYP2E1 and CYP2C11.On the other hand, 5 mM salicylic acid induced a drastic decrease of ATP contents in rat isolated hepatocytes. Furthermore, mitochondrial respiration control ratio (RC ratio), calculated by State 3/State 4 also decreased with the increase of salicylic acid concentration. These findings suggest that salicylic acid would trigger mitochondrial dysfunction and cause ATP decrease, leading to lethal liver cell injury by lipid peroxidation, although this hypothesis remains to be elucidated in vivo.     

Mycothiol-dependent mycobacterial response to oxidative stress

The effect of exogenous oxidative stress on mycothiol (MSH) levels and redox balance was investigated in mycobacteria. Both the thiol-specific oxidant diamide and hydrogen peroxide induced up to 75% depletion of MSH to form the disulfide form, mycothione (MSSM), in Mycobacterium bovis BCG. In comparison, Mycobacterium smegmatis, a saprophytic mycobacterium, displays a greater tolerance towards these oxidants, reflected by the lack of fluxes in MSH levels and redox ratios upon oxidative stress treatments. The basal ratio of MSH to MSSM was established to be 50:1 in M. bovis BCG and 200:1 in M. smegmatis.     

Telomerase activity in response to mild oxidative stress

We have analysed telomerase activity to determine whether it can be modified when BCL-2 is endogenously overexpressed in response to a mild oxidative stress treatment as part of a survival mechanism, in contrast with an exogenous bcl-2 overexpression due to a retroviral infection. Endogenous bcl-2 overexpression was induced after a low oxidative insult of H2O2 in mice primary lung fibroblasts and L929 cell, whereas bcl-2 exogenous overexpression was performed using a retroviral infection in L929 cells. Telomerase activity was quantified in Bcl-2 overexpressing cells by the TRAP assay. When the cells were treated with different H2O2 concentrations, only those exposed to 50 μM showed increased telomerase activity. This correlates with BCL-2 expression as part of the endogenous response to mild oxidative stress. Oxidative stress generated during the toxic mechanism of chemotherapeutic drugs might induce BCL-2 increment, enhancing telomerase activity and reactivating the oncogenic process. Clinical trials should take into consideration the possibility of telomerase activation following increased BCL-2 expression when treating patients with ROS (reactive oxygen species) generation by anti-cancer drugs.     

Proteolytic response to oxidative stress in mammalian cells

Oxidative stress in mammalian cells is an inevitable consequence of their aerobic metabolism. The production of reactive oxygen and nitric oxide species causes oxidative modifications of proteins often combined with a loss of their biological function. Like most partially denatured proteins, moderately oxidized proteins are more sensitive to proteolytic attack by proteases. The diverse cellular proteolytic systems are an important secondary defense against oxidative stress by degrading oxidized and damaged proteins, thereby preventing their intracellular accumulation. In mammalian cells, a range of proteases exists which are distributed throughout the cell. In this review we summarize the function of the cytosolic (proteasome and calpains), the lysosomal, the mitochondrial and the nuclear proteolytic pathways in response to oxidative stress. Particular emphasis is given to the proteasomal system, since this pathway appears to be the most important proteolytic system involved in the removal of oxidatively modified or damaged proteins.     

Hippocampal damage abolishes the cortisol response to psychosocial stress in humans

The hippocampus (HC) is necessary for learning and memory, but it also plays a role in other behaviors such as those related to stress and anxiety. In support of the latter idea, we show here that bilateral HC damage abolishes the cortisol response to psychosocial stress. We collected salivary cortisol, heart rate, and affective responses to the Trier Social Stress Test (TSST) from 7 participants with bilateral HC lesions, 12 participants with damage outside the HC, and 28 healthy normal comparison participants matched to the HC participants on age and sex. HC participants showed elevated pre-stress cortisol, but no cortisol response to the TSST. Heart rate and affective responses in the HC group were similar to those of the comparison groups. Participants with brain damage outside the HC showed stress responses that were comparable to those of the healthy comparison group. These findings support the idea that the functions of the human HC extend beyond learning and memory, and suggest that the HC is necessary for producing the cortisol response to psychosocial stress.     

Expression of senescence-enhanced genes in response to oxidative stress

Expression of the LSC54 gene, encoding a metallothionein protein, has been shown previously to increase during leaf senescence and cell death. Evidence is presented in this paper to indicate that the extent of LSC54 expression is related to levels of oxidative stress in the tissues. Treatment of Arabidopsis cotyledon and leaf tissues with the catalase inhibitor, 3-amino-1,2,4-triazole, or with silver nitrate result in the enhanced expression of LSC54. Combined treatments with quenchers of reactive oxygen species (ROS), such as ascorbate, tiron and benzoic acid indicated that this induced expression was due to increased levels of ROS. The expression of many other senescence-enhanced genes was also found to be inducible by the increase in ROS. Treatment of plant tissue with 3-amino-1,2,4-triazole, followed by silver nitrate, resulted in protection from the severe damage caused by the silver nitrate treatment and reduced expression of many of the genes examined. However, one gene, encoding a lipid hydroperoxide-dependent glutathione peroxidase, showed increased expression in the protected tissue, which may indicate a role for this enzyme in the protection of plant tissue from oxidative stress. ROS-enhanced expression of at least one of the genes investigated required the presence of the salicylic acid signalling pathway, which was not required for the expression of LSC54.     

Human polynucleotide phosphorylase protein in response to oxidative stress

8-Oxo-7,8-dihydroguanine (8-oxoGua) is generated in nucleic acids as well as in their precursors due to the actions of oxygen radicals produced through a normal cellular metabolism. Since oxidized guanine can pair with both cytosine and adenine, it causes alterations in the phenotypic expression when it is present in RNA. To prevent such an outcome, organisms must have some mechanism for eliminating such oxidized guanine nucleotides from RNA and its precursors. In mammalian cells, MTH1 and NUDT5 proteins degrade 8-oxoGTP and 8-oxoGDP to 8-oxoGMP, which is an unusable form for RNA synthesis. In a search for proteins functioning at the RNA level, polynucleotide phosphorylase (PNP) protein has been suggested to be a good candidate for such a role. The human PNP protein has an ability to bind specifically to RNA containing 8-oxoGua. When human cells are exposed to agents that induce oxidative stress, such as hydrogen peroxide and menadion, the amounts of PNP protein decrease rapidly while amounts of other proteins in the cells do not change after such treatments. No specific decrease in the PNP protein level is observed when cells are treated with ACNU and cycloheximide at doses sufficient to provide the same degree of growth suppression. These results imply that the PNP protein might thus play a role in excluding oxidized forms of RNA from the translation mechanism.     

Free fatty acid-induced peripheral insulin resistance augments splanchnic glucose uptake in healthy humans

To investigate the effect of elevated plasma free fatty acid (FFA) concentrations on splanchnic glucose uptake (SGU), we measured SGU in nine healthy subjects (age, 44 +/- 4 yr; body mass index, 27.4 +/- 1.2 kg/m(2); fasting plasma glucose, 5.2 +/- 0.1 mmol/l) during an Intralipid-heparin (LIP) infusion and during a saline (Sal) infusion. SGU was estimated by the oral glucose load (OGL)-insulin clamp method: subjects received a 7-h euglycemic insulin (100 mU x m(-2) x min(-1)) clamp, and a 75-g OGL was ingested 3 h after the insulin clamp was started. After glucose ingestion, the steady-state glucose infusion rate (GIR) during the insulin clamp was decreased to maintain euglycemia. SGU was calculated by subtracting the integrated decrease in GIR during the period after glucose ingestion from the ingested glucose load. [3-(3)H]glucose was infused during the initial 3 h of the insulin clamp to determine rates of endogenous glucose production (EGP) and glucose disappearance (R(d)). During the 3-h euglycemic insulin clamp before glucose ingestion, R(d) was decreased (8.8 +/- 0.5 vs. 7.6 +/- 0.5 mg x kg(-1) x min(-1), P < 0.01), and suppression of EGP was impaired (0.2 +/- 0.04 vs. 0.07 +/- 0.03 mg x kg(-1) x min(-1), P < 0.01). During the 4-h period after glucose ingestion, SGU was significantly increased during the LIP vs. Sal infusion study (30 +/- 2 vs. 20 +/- 2%, P < 0.005). In conclusion, an elevation in plasma FFA concentration impairs whole body glucose R(d) and insulin-mediated suppression of EGP in healthy subjects but augments SGU.     

Adaptation to oxidative stress induced by polyunsaturated fatty acids in yeast

To create a conditional system for molecular analysis of effects of polyunsaturated fatty acids (PUFA) on cellular physiology, we have constructed a strain of yeast (Saccharomyces cerevisiae) that functionally expresses, under defined conditions, the Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This strain produces up to 15% PUFA, exclusively under inducing conditions resulting in production of 4-hydroxy-2-nonenal, one of the major end products of n-6 polyunsaturated fatty acid peroxidation. The PUFA-producing yeast was initially more sensitive to oxidative stress than the wild-type strain. However, over extended time of cultivation it became more resistant to hydrogen peroxide indicating adaptation to endogenous oxidative stress caused by the presence of PUFA. Indeed, PUFA-producing strain showed an increased concentration of endogenous ROS, while initially increased hydrogen peroxide sensitivity was followed by an increase in catalase activity and adaptation to oxidative stress. The deletion mutants constructed to be defective in the catalase activity lost the ability to adapt to oxidative stress. These data demonstrate that the cellular synthesis of PUFA induces endogenous oxidative stress which is overcome by cellular adaptation based on the catalase activity.     

Phosphoinositide 3-kinase signaling in the cellular response to oxidative stress

Oxidative stress is linked to the pathogenesis and pathobiochemistry of various diseases, including cancer, diabetes and cardiovascular disorders. The non-specific damaging effect of reactive oxygen species (ROS) generated during oxidative stress is involved in the development of diseases, as well as the activation of specific signaling cascades in cells exposed to the higher oxidant load. A cellular signaling cascade that is activated by several types of reactive oxygen species is the phosphoinositide 3'-kinase (PI 3-kinase)/protein kinase B (PKB) pathway, which regulates cellular survival and fuel metabolism, thus establishing a link between oxidative stress and signaling in neoplastic, metabolic or degenerative diseases. Several links of PI 3-kinase/PKB signaling to ROS are discussed in this review, with particular focus on the molecular mechanisms involved in the regulation of PI 3-kinase signaling by oxidative stress and important players such as (i) the glutathione and glutaredoxin system, (ii) the thioredoxin system and (iii) Ser/Thr- and Tyr phosphatases.     

Individual behavioral and autonomic response to emotional stress in humans

Healthy subjects (n = 53) performed a sound version of the proof-reading test under normal conditions and in the state of emotional stress. Stress resistance was evaluated by the overall number of errors. The propensity to active or passive response to stress was evaluated by the number of "false alarms" and signal omissions. The reaction pattern to emotional stress in stress-resistant subjects, irrespective of their behavioral features, consisted in an increase in sympathetic effects on the cardiac rhythm and a decrease in the reaction time to significant signals. In subjects with low stress resistance, no statistically significant changes in the level of sympathetic tone and reaction time were revealed in the state of stress. Subjects with active behavioral response to stress, irrespective of their level of stress resistance, were characterized by aggressiveness, boldness and independence. Subjects with passive response to stress were inclined to conformism, dependence, and passivity.     

Peroxisomal participation in the cellular response to the oxidative stress of endotoxin

Exposure to a sublethal dose of endotoxin offers protection against subsequent oxidative stresses. The cellular mechanisms involved in generating this effect are not well understood. We evaluated the effect of endotoxin on antioxidant enzymes in liver peroxisomes. Peroxisomes have recently been shown to contain superoxide dismutase (SOD) and glutathione peroxidase (GPX) in addition to catalase. Peroxisomes were isolated from liver homogenates by differential and density gradient centrifugations. Endotoxin treatment increased the specific activity of SOD and GPX in peroxisomes to 208% and 175% of control activity, respectively. These findings correlated with increases in peroxisomal SOD and GPX proteins observed by immunoblot. Although the quantity of catalase protein was increased when assessed by immunoblot analysis, the specific activity of catalase was decreased to 68% of control activity. Activation of catalase with ethanol only restored catalase activity to control levels suggesting that catalase had undergone irreversible inactivation. The observed increase in GPX activity may represent a compensatory mechanism triggered by accumulating H₂O₂. The data presented here suggest for the first time that mammalian peroxisomal antioxidant enzymes are altered during the oxidative injury of endotoxin treatment.     

Exercise-induced oxidative stress in humans: cause and consequences

The observation that muscular exercise is associated with oxidative stress in humans was first reported over 30 years ago. Since this initial report, numerous studies have confirmed that prolonged or high-intensity exercise results in oxidative damage to macromolecules in both blood and skeletal muscle. Although the primary tissue(s) responsible for reactive oxygen species (ROS) production during exercise remains a topic of debate, compelling evidence indicates that muscular activity promotes oxidant production in contracting skeletal muscle fibers. Mitochondria, NADPH oxidase, PLA₂-dependent processes, and xanthine oxidase have all been postulated to contribute to contraction-induced ROS production in muscle but the primary site of contraction-induced ROS production in muscle fibers remains unclear. Nonetheless, contraction-induced ROS generation has been shown to play an important physiological function in the regulation of both muscle force production and contraction-induced adaptive responses of muscle fibers to exercise training. Although knowledge in the field of exercise and oxidative stress has grown markedly during the past 30 years, this area continues to expand and there is much more to be learned about the role of ROS as signaling molecules in skeletal muscle.     

Noninvasive measures of oxidative stress status in humans.

Although oxidative stress is thought to be involved in the pathophysiology of several diseases and aging, it is not routinely measured in clinical diagnosis. This is at least partly because accepted and standardized methods for measuring oxidative stress in humans are not yet established. One of the greatest needs in the field of free radical biology is the development of reliable methods for measuring oxidative stress status (OSS) in humans. A listing of some analytical approaches to measuring oxidative stress is provided as well as a listing of some noninvasive techniques that have been used in humans.