Severe oxidative damage in multiple sclerosis lesions coincides with enhanced antioxidant enzyme expression

Reactive oxygen species (ROS) and subsequent oxidative damage may contribute to the formation and persistence of multiple sclerosis (MS) lesions by acting on distinct pathological processes. ROS initiate lesion formation by inducing blood–brain barrier disruption, enhance leukocyte migration and myelin phagocytosis, and contribute to lesion persistence by mediating cellular damage to essential biological macromolecules of vulnerable CNS cells. Relatively little is known about which CNS cell types are affected by oxidative injury in MS lesions. Here, we show the presence of extensive oxidative damage to proteins, lipids, and nucleotides occurring in active demyelinating MS lesions, predominantly in reactive astrocytes and myelin-laden macrophages. Oxidative stress can be counteracted by endogenous antioxidant enzymes that confer protection against oxidative damage. Here, we show that antioxidant enzymes, including superoxide dismutase 1 and 2, catalase, and heme oxygenase 1, are markedly upregulated in active demyelinating MS lesions compared to normal-appearing white matter and white matter tissue from nonneurological control brains. Particularly, hypertrophic astrocytes and myelin-laden macrophages expressed an array of antioxidant enzymes. Enhanced antioxidant enzyme production in inflammatory MS lesions may reflect an adaptive defense mechanism to reduce ROS-induced cellular damage.     

Protective effects of peroxiredoxin-1 at the injured blood-brain barrier

Reactive oxygen species (ROS) play a pivotal role in the development of neuroinflammatory disorders, such as multiple sclerosis (MS). Here, we studied the effect of ROS on protein expression in brain endothelial cells (BECs) using proteomic techniques and show that long-term exposure to ROS induces adaptive responses in BECs to counteract an oxidative attack. ROS induce differential protein expression in BECs, among which is peroxiredoxin-1 (Prx1). To further study the role of Prx1 we established a BEC line overexpressing Prx1. Our data indicate that Prx-1 overexpression protects BECs from ROS-induced cell death, reduces adhesion and subsequent transendothelial migration of monocytes by decreasing intercellular adhesion molecule-1 expression, and enhances the integrity of the BEC layer. Interestingly, vascular Prx1 immunoreactivity was markedly upregulated in inflammatory lesions of experimental autoimmune encephalomyelitis (EAE) animals and active demyelinating MS lesions. These findings indicate that enhanced vascular Prx1 expression may reflect the occurrence of vascular oxidative stress in EAE and MS. On the other hand, it may function as an endogenous defense mechanism to inhibit leukocyte infiltration and counteract ROS-induced cellular injury.     

Severe oxidative damage in multiple sclerosis lesions coincides with enhanced antioxidant enzyme expression

Reactive oxygen species (ROS) and subsequent oxidative damage may contribute to the formation and persistence of multiple sclerosis (MS) lesions by acting on distinct pathological processes. ROS initiate lesion formation by inducing blood–brain barrier disruption, enhance leukocyte migration and myelin phagocytosis, and contribute to lesion persistence by mediating cellular damage to essential biological macromolecules of vulnerable CNS cells. Relatively little is known about which CNS cell types are affected by oxidative injury in MS lesions. Here, we show the presence of extensive oxidative damage to proteins, lipids, and nucleotides occurring in active demyelinating MS lesions, predominantly in reactive astrocytes and myelin-laden macrophages. Oxidative stress can be counteracted by endogenous antioxidant enzymes that confer protection against oxidative damage. Here, we show that antioxidant enzymes, including superoxide dismutase 1 and 2, catalase, and heme oxygenase 1, are markedly upregulated in active demyelinating MS lesions compared to normal-appearing white matter and white matter tissue from nonneurological control brains. Particularly, hypertrophic astrocytes and myelin-laden macrophages expressed an array of antioxidant enzymes. Enhanced antioxidant enzyme production in inflammatory MS lesions may reflect an adaptive defense mechanism to reduce ROS-induced cellular damage.     

Primary hepatocyte apoptosis is unlikely to relate to caspase-3 activity under sustained endogenous oxidative stress

We previously showed that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in endogenous oxidative stress and apoptosis. For the present study, we determined whether this apoptosis involved activation of caspase-3, which is known to execute apoptosis in many cell types. ATZ and MS increased levels of reactive oxygen species (ROS) from 3-9 h, just before the onset of chromatin condensation (apoptosis) and decreases in protein thiols. Pretreatment with either SKF, a cytochrome P450 inhibitor, or L-ascorbic acid, an antioxidant, completely suppressed the increase in ROS levels and apoptosis, suggesting that the sustained ROS increases may cause the apoptosis. SKF also abolished the decrease in protein thiol content, further supporting the contribution of the P450 system to increased ROS levels. DEVD-CHO, a caspase-3 inhibitor, even at 1 mM had no effect on apoptosis. Caspase-3 activity remained unchanged and pro-caspase-3 processing was not detected during 18 h incubation with ATZ and MS. Moreover, the amount of unoxidized pro-caspase-3 decreased even below the level of untreated hepatocytes. These findings suggest that the sustained oxidative stress is a major cause for the hepatocyte apoptosis, which occurs independently of the caspase-3 related pathway.     

Intracellular oxidative activity and respiratory burst of leukocytes isolated from multiple sclerosis patients

Oxidative damage induced by free radicals and reactive oxygen species (ROS) have been suggested to play an important role in the development of autoimmune diseases such as multiple sclerosis (MS) disease and it has been hypothesised that oxidative injury could mediate demyelination and axonal injury in MS subjects. In our study, we compared intracellular oxidative activity and the respiratory burst activity in MS patients (n=20) and healthy controls (n=15) using leukocytes as cellular model. At this purpose, intracellular ROS levels were evaluated by fluorometric assay using the 2'-7'-dichlorodihydrofluorescin diacetate probe (H(2)DCFDA) in untreated or in leukocytes stimulated with phorbol-12-myristate-13-acetate (PMA). Our results demonstrate that the intracellular spontaneous ROS production in leukocytes from MS patients was higher with respect to cells from control subjects (p<0.001). PMA addition induced a higher formation of ROS both in leukocytes from MS patients and controls (p<0.001). The PMA-induced production of ROS was significantly higher in leukocytes from MS with respect to controls (p<0.001). Significant positive correlations were established between intracellular spontaneous or PMA-induced production of ROS in leukocytes isolated from MS patients and the clinical parameters used to evaluate disease disability such as expanded disability status scale (EDSS), brain lesions evaluated by MRI and visual evoked potential (VEP) (p<0.001). In conclusion, our results demonstrate higher levels of intracellular ROS in untreated or in PMA-treated leukocytes isolated from MS patients with respect to healthy subjects confirming the role of oxidative stress in multiple sclerosis.     

High glucose-induced mitochondrial respiration and reactive oxygen species in mouse cerebral pericytes is reversed by pharmacological inhibition of mitochondrial carbonic anhydrases: Implications for cerebral microvascular disease in diabetes

Hyperglycemia-induced oxidative stress leads to diabetes-associated damage to the microvasculature of the brain. Pericytes in close proximity to endothelial cells in the brain microvessels are vital to the integrity of the blood–brain barrier and are especially susceptible to oxidative stress. According to our recently published results, streptozotocin-diabetic mouse brain exhibits oxidative stress and loose pericytes by twelve weeks of diabetes, and cerebral pericytes cultured in high glucose media suffer intracellular oxidative stress and apoptosis. Oxidative stress in diabetes is hypothesized to be caused by reactive oxygen species (ROS) produced during hyperglycemia-induced enhanced oxidative metabolism of glucose (respiration). To test this hypothesis, we investigated the effect of high glucose on respiration rate and ROS production in mouse cerebral pericytes. Previously, we showed that pharmacological inhibition of mitochondrial carbonic anhydrases protects the brain from oxidative stress and pericyte loss. The high glucose-induced intracellular oxidative stress and apoptosis of pericytes in culture were also reversed by inhibition of mitochondrial carbonic anhydrases. Therefore, we extended our current study to determine the effect of these inhibitors on high glucose-induced increases in pericyte respiration and ROS. We now report that both the respiration and ROS are significantly increased in pericytes challenged with high glucose. Furthermore, inhibition of mitochondrial carbonic anhydrases significantly slowed down both the rate of respiration and ROS production. These data provide new evidence that pharmacological inhibitors of mitochondrial carbonic anhydrases, already in clinical use, may prove beneficial in protecting the brain from oxidative stress caused by ROS produced as a consequence of hyperglycemia-induced enhanced respiration.     

Oxidative stress in plant cell culture: a role in production of beta-thujaplicin by Cupresssus lusitanica suspension culture

Oxidative stress is a common physiological stress that often challenges plants. Reactive oxygen species (ROS) are major factors in oxidative stress that significantly affect plant cell growth and secondary metabolism. Here we used beta-thujaplicin production by Cupressus lusitanica cell culture as an example to demonstrate the common occurrence of oxidative stress in cultivated plant cells and its effect on multiple aspects of cell culture process. C. lusitanica cells cultivated under Fe(2+) stress generate a significant level of ROS, and oxidative stress also occurs at late stages of C. lusitanica cell cultures under normal conditions. ROS production inhibited cell growth, induced lipid peroxidation and cell death, and enhanced ethylene and beta-thujaplicin production. It is demonstrated that Fe(2+) stress enhances ROS production via the Fenton reaction and promotes beta-thujaplicin production via ROS-induced lipid peroxidation that may activate cyclic oxylipin and ethylene pathways. Results further indicate that H(2)O(2) is a positive signal for beta-thujaplicin production, whereas superoxide anion radical (O(2) (- )) negatively affects beta-thujaplicin induction and strongly induces cell death. The study suggests that evaluating the oxidative stress and plant responses in a cell culture process is very necessary and important for understanding biochemical processes and for gaining the maximal productivity of target secondary metabolites.     

Crucial role of antioxidant proteins and hydrolytic enzymes in pathogenicity of Penicillium expansum: analysis based on proteomics approach

Penicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and may lead to the production of mycotoxin that causes harmful effects on human health. In this study, we compared the cellular and extracellular proteomes of P. expansum in the absence and presence of borate, which affects the virulence of the fungal pathogen. The differentially expressed proteins were identified using ESI-Q-TOF-MS/MS. Several proteins related to stress response (glutathione S-transferase, catalase, and heat shock protein 60) and basic metabolism (glyceraldehyde-3-phosphate dehydrogenase, dihydroxy-acid dehydratase, and arginase) were identified in the cellular proteome. Catalase and glutathione S-transferase, the two antioxidant enzymes, exhibited reduced levels of expression upon exposure to borate. Because catalase and glutathione S-transferase are related to oxidative stress response, we further investigated the reactive oxygen species (ROS) levels and oxidative protein carbonylation (damaged proteins) in P. expansum. Higher amounts of ROS and carbonylated proteins were observed after borate treatment, indicating that catalase and glutathione S-transferase are important in scavenging ROS and protecting cellular proteins from oxidative damage. Additionally to find secretory proteins that contribute to the virulence, we studied the extracellular proteome of P. expansum under stress condition with reduced virulence. The expression of three protein spots were repressed in the presence of borate and identified as the same hydrolytic enzyme, polygalacturonase.     

Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria

Although plant cell bioenergetics is strongly affected by abiotic stresses, mitochondrial metabolism under stress is still largely unknown. Interestingly, plant mitochondria may control reactive oxygen species (ROS) generation by means of energy-dissipating systems. Therefore, mitochondria may play a central role in cell adaptation to abiotic stresses, which are known to induce oxidative stress at cellular level. With this in mind, in recent years, studies have been focused on mitochondria from durum wheat, a species well adapted to drought stress. Durum wheat mitochondria possess three energy-dissipating systems: the ATP-sensitive plant mitochondrial potassium channel (PmitoK(ATP)); the plant uncoupling protein (PUCP); and the alternative oxidase (AOX). It has been shown that these systems are able to dampen mitochondrial ROS production; surprisingly, PmitoK(ATP) and PUCP (but not AOX) are activated by ROS. This was found to occur in mitochondria from both control and hyperosmotic-stressed seedlings. Therefore, the hypothesis of a 'feed-back' mechanism operating under hyperosmotic/oxidative stress conditions was validated: stress conditions induce an increase in mitochondrial ROS production; ROS activate PmitoK(ATP) and PUCP that, in turn, dissipate the mitochondrial membrane potential, thus inhibiting further large-scale ROS production. Another important aspect is the chloroplast/cytosol/mitochondrion co-operation in green tissues under stress conditions aimed at modulating cell redox homeostasis. Durum wheat mitochondria may act against chloroplast/cytosol over-reduction: the malate/oxaloacetate antiporter and the rotenone-insensitive external NAD(P)H dehydrogenases allow cytosolic NAD(P)H oxidation; under stress this may occur without high ROS production due to co-operation with AOX, which is activated by intermediates of the photorespiratory cycle.     

Lower levels of F2-isoprostanes in serum and livers of long-lived Ames dwarf mice

F₂-isoprostanes (IsoPs), lipid peroxidation products, are markers that quantitatively measure levels of oxidative stress. IsoP levels increase in tissues and serum of aging animals suggesting an increase in oxidative stress. This supports the Free Radical Theory of Aging, which proposes that elevated levels of reactive oxygen species (ROS) cause macromolecular damage, and is a factor in the age-associated decline in tissue function. Numerous studies have shown that the longevity of long-lived mutant mice correlates with their resistance to oxidative stress. However, although the Ames dwarf (DW) mice show resistance to oxidative stress, it has not been shown that these mice have inherently lower levels of ROS. Our results show that the serum and liver IsoP levels in DW mice are lower at all ages suggesting that the lower levels of endogenous ROS production in DW mice may be a factor in their resistance to oxidative stress and longevity.     

Oxytetracycline inactivation by putative reactive oxygen species released to nutrient medium of Helianthus annuus hairy root cultures

When subjected to stress, plants produce reactive oxygen species (ROS) as a part of the defense response. The oxidative response is also used to degrade organic pollutants. Hairy roots of Helianthus annuus (sunflower) are shown to oxidize oxytetracycline (OTC) through the action of the ROS released to the nutrient medium by the hairy root cultures. Methyl jasmonate (MeJA) elicits ROS formation in the hairy root cultures. The activities of the antioxidant enzymes, ascorbate peroxidase (APX), catalase (CAT), and guaiacol peroxidase (GPX), are reported for hairy root cultures treated with increasing concentrations of MeJA. A bioassay using Enterococcus hirae as the test microorganism demonstrates the root-catalyzed oxidation process results in conversion of OTC into product(s) devoid of antibiotic activity. Direct evidence for putative ROS oxidation of OTC is obtained by mass spectrometry (MS) and HPLC/MS showing first quinone formation followed possibly by ring cleavage, which disrupts UV absorption and destroys antibiotic activity.     

Oxidative stress,mitochondrial DNA mutation,and impairment of antioxidant enzymes in aging

Mitochondria do not only produce less ATP, but they also increase the production of reactive oxygen species (ROS) as by-products of aerobic metabolism in the aging tissues of the human and animals. It is now generally accepted that aging-associated respiratory function decline can result in enhanced production of ROS in mitochondria. Moreover, the activities of free radical-scavenging enzymes are altered in the aging process. The concurrent age-related changes of these two systems result in the elevation of oxidative stress in aging tissues. Within a certain concentration range, ROS may induce stress response of the cells by altering expression of respiratory genes to uphold the energy metabolism to rescue the cell. However, beyond the threshold, ROS may cause a wide spectrum of oxidative damage to various cellular components to result in cell death or elicit apoptosis by induction of mitochondrial membrane permeability transition and release of apoptogenic factors such as cytochrome c. Moreover, oxidative damage and large-scale deletion and duplication of mitochondrial DNA (mtDNA) have been found to increase with age in various tissues of the human. Mitochondria act like a biosensor of oxidative stress and they enable cell to undergo changes in aging and age-related diseases. On the other hand, it has recently been demonstrated that impairment in mitochondrial respiration and oxidative phosphorylation elicits an increase in oxidative stress and causes a host of mtDNA rearrangements and deletions. Here, we review work done in the past few years to support our view that oxidative stress and oxidative damage are a result of concurrent accumulation of mtDNA mutations and defective antioxidant enzymes in human aging.     

Anti-Oxidative Effects of Rooibos Tea (Aspalathus linearis) on Immobilization-Induced Oxidative Stress in Rat Brain

Exposure to chronic psychological stress may be related to increased reactive oxygen species (ROS) or free radicals, and thus, long-term exposure to high levels of oxidative stress may cause the accumulation of oxidative damage and eventually lead to many neurodegenerative diseases. Compared with other organs, the brain appears especially susceptible to excessive oxidative stress due to its high demand for oxygen. In the case of excessive ROS production, endogenous defense mechanisms against ROS may not be sufficient to suppress ROS-associated oxidative damage. Dietary antioxidants have been shown to protect neurons against a variety of experimental neurodegenerative conditions. In particular, Rooibos tea might be a good source of antioxidants due to its larger proportion of polyphenolic compounds. An optimal animal model for stress should show the features of a stress response and should be able to mimic natural stress progression. However, most animal models of stress, such as cold-restraint, electric foot shock, and burn shock, usually involve physical abuse in addition to the psychological aspects of stress. Animals subjected to chronic restraint or immobilization are widely believed to be a convenient and reliable model to mimic psychological stress. Therefore, in the present study, we propose that immobilization-induced oxidative stress was significantly attenuated by treatment with Rooibos tea. This conclusion is demonstrated by Rooibos tea’s ability to (i) reverse the increase in stress-related metabolites (5-HIAA and FFA), (ii) prevent lipid peroxidation (LPO), (iii) restore stress-induced protein degradation (PD), (iv) regulate glutathione metabolism (GSH and GSH/GSSG ratio), and (v) modulate changes in the activities of antioxidant enzymes (SOD and CAT).     

Drug resistance to 5-FU linked to reactive oxygen species modulator 1

While acute oxidative stress triggers cell apoptosis or necrosis, persistent oxidative stress induces genomic instability and has been implicated in tumor progression and drug resistance. In a previous report, we demonstrated that reactive oxygen species modulator 1 (Romo1) expression was up-regulated in most cancer cell lines and suggested that increased Romo1 expression might confer chronic oxidative stress to tumor cells. In this study, we show that enforced Romo1 expression induces reactive oxygen species (ROS) production in the mitochondria leading to massive cell death. However, tumor cells that adapt to oxidative stress by increasing manganese superoxide dismutase (MnSOD), Prx I, and Bcl-2 showed drug resistance to 5-FU. To elucidate the relationship between 5-FU-induced ROS production and Romo1 expression, Romo1 siRNA was used to inhibit 5-FU-triggered Romo1 induction. Romo1 siRNA treatment efficiently blocked 5-FU-induced ROS generation, demonstrating that 5-FU treatment stimulated ROS production through Romo1 induction. Based on these results we suggest that cellular adaptive response to Romo1-induced ROS is another mechanism of drug resistance to 5-FU and Romo1 expression may provide a new clinical implication in drug resistance of cancer chemotherapy.     

Oxidative stress tolerance in plants: Novel interplay between auxin and reactive oxygen species signaling

Biotic and abiotic stress conditions produce reactive oxygen species (ROS) in plants causing oxidative stress damage. At the same time, ROS have additional signaling roles in plant adaptation to the stress. It is not known how the two seemingly contrasting functional roles of ROS between oxidative damage to the cell and signaling for stress protection are balanced. Research suggests that the plant growth regulator auxin may be the connecting link regulating the level of ROS and directing its role in oxidative damage or signaling in plants under stress. The objective of this review is to highlight some of the recent research on how auxin’s role is intertwined to that of ROS, more specifically H₂O₂, in plant adaptation to oxidative stress conditions.     

Deregulated Cdk5 promotes oxidative stress and mitochondrial dysfunction

Oxidative stress is one of the earliest events in Alzheimer's disease (AD). A chemical genetic screen revealed that deregulated cyclin-dependent kinase 5 (Cdk5) may cause oxidative stress by compromising the cellular anti-oxidant defense system. Using novel Cdk5 modulators, we show the mechanism by which Cdk5 can induce oxidative stress in the disease's early stage and cell death in the late stage. Cdk5 dysregulation upon neurotoxic insults results in reactive oxygen species (ROS) accumulation in neuronal cells because of the inactivation of peroxiredoxin I and II. Sole temporal activation of Cdk5 also increases ROS, suggesting its major role in this process. Cdk5 inhibition rescues mitochondrial damage upon neurotoxic insults, thereby revealing Cdk5 as an upstream regulator of mitochondrial dysfunction. As mitochondrial damage results in elevated ROS and Ca(2+) levels, both of which activate Cdk5, we propose that a feedback loop occurs in late stage of AD and leads to cell death (active Cdk5 --> ROS --> excess ROS --> mitochondrial damage --> ROS --> hyperactive Cdk5 --> severe oxidative stress and cell injury --> cell death). Cdk5 inhibition upon neurotoxic insult prevents cell death significantly, supporting this hypothesis. As oxidative stress and mitochondrial dysfunction play pivotal roles in promoting neurodegeneration, Cdk5 could be a viable therapeutic target for AD.