Efficacy of antioxidants in the yeast Saccharomyces cerevisiae correlates with their effects on protein thiols

We have found previously that only a limited number of antioxidants are able to protect yeast cells against endogenous and exogenous oxidative stress. In search of factors determining this selectivity of antioxidant action we compared the ability of a set of antioxidants to: (i) protect a thiol-dependent enzyme alcohol dehydrogenase (ADH) against inactivation by superoxide, peroxynitrite and hydrogen peroxide; (ii) prevent H(2)O(2)-induced activation of Yap1 p; and (iii) decrease extracellular redox potential of the medium. The results obtained provide demonstration with respect to yeast that the ability to lower redox potential and to maintain critical thiol groups in the reduced state is an important facet of the action of antioxidants.     

Blood as a reactive species generator and redox status regulator during exercise

The exact origin of reactive species and oxidative damage detected in blood is largely unknown. Blood interacts with all organs and tissues and, consequently, with many possible sources of reactive species. In addition, a multitude of oxidizable substrates are already in blood. A muscle-centric approach is frequently adopted to explain reactive species generation, which obscures the possibility that sources of reactive species and oxidative damage other than skeletal muscle may be also at work during exercise. Plasma and blood cells can autonomously produce significant amounts of reactive species at rest and during exercise. The major reactive species generators located in blood during exercise may be erythrocytes (mainly due to their quantity) and leukocytes (mainly due to their drastic activation during exercise). Therefore, it is plausible to assume that oxidative stress/damage measured frequently in blood after exercise or any other experimental intervention derives, at least in part, from the blood.     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

2-Phenyl-beta-lapachone can affect mitochondrial function by redox cycling mediated oxidation

2-Phenyl-beta-lapachone (3,4-dihydro-2-methyl-2-phenyl-2H-naphtho[1,2b]pyran-5,6-dione) (2PBL) is a o-naphthoquinone synthesized as a possible antitumoral agent. The addition of micromolar concentrations of 2PBL to rat liver mitochondria (in the presence of malate-glutamate or succinate, as respiratory substrates): (1) stimulated O(2) consumption in state 4 and inhibited O(2) consumption in state 3, thus decreasing respiratory control index (RCI); and (2) collapsed the mitochondrial membrane potential. The addition of 2PBL to rat liver submitochondrial particles: (1) stimulated NADH oxidation in the presence of rotenone, antimycin, myxothiazol or cyanide; (2) stimulated (.-)O(2)(-) production in the presence of NADH and antimycin; and (3) led to 2PBL semiquinone radical production. Control studies carried out with two p-naphthoquinones, menadione and atovaquone, did not produced equivalent effects. These findings support the hypothesis that 2PBL, undergoes redox cycling and affects mitochondrial function. The 2PBL effect is complex, involving inhibition of electron transfer, uncoupling of oxidative phosphorylation, collapse of mitochondrial membrane potential and (.-)O(2)(-) production by redox cycling. The mitochondrion could be a target organelle for 2PBL cytotoxicity.     

Antioxidants effectively prevent oxidation-induced protein damage in OLN 93 cells

Oxidative stress is supposed to play an important role in demyelinating diseases. Oligodendrocytes are the myelin-forming cells in the brain and are highly susceptible to oxidative stress due to their low antioxidative defense systems and high metabolic rate. In the present work, we tested the response of the oligodendrocyte cell line OLN 93 to oxidative stress. OLN 93 cell cultures are characterized by a loss of cell viability after oxidation. This loss of cell viability is accompanied by an increase in protein oxidation and consequently an elevated overall proteolysis. To minimize the oxidative damage, we tested the effects of the antioxidants alpha-lipoic acid and coenzyme Q(10). Both compounds were able to elevate cell viability and to decrease intracellular protein turnover and oxidant induced protein oxidation. Therefore, we concluded that the excessive oxidative damage of oligodendrocytes and their protein pool can be prevented by the usage of antioxidants.     

Reduced Superoxide Dismutase in Lung Cells of Patients with Asthma

Lung cells recovered from symptomatic patients with asthma generate increased amounts of reactive oxygen species (ROS). Animal and in vitro studies indicate that ROS can reproduce many of the features of asthma. The ability of ROS to produce the clinical features of asthma may depend on an individual's lung antioxidant defenses. Patients with asthma are reported to have reduced antioxidant defenses in peripheral blood, but little is known about the antioxidant defenses of their lung cells. To define lung cell antioxidant defenses in asthma, the glutathione concentration and the glutathione reductase, glutathione peroxidase, catalase, and superoxide dismutase (SOD) activities were measured in cells recovered by bronchoalveolar lavage (BAL cells) and by bronchial brushing (bronchial epithelial cells, HBEC) from normal subjects and patients with asthma. Superoxide dismutase activity was reduced 25% in BAL cells (p < .05) and nearly 50% in HBEC (p < .02) from patients with asthma. Alterations in the other antioxidants were not identified. A direct relationship was found between airway reactivity to methacholine, measured as PC₂₀FEV₁, and HBEC SOD activity (r² = 89; p < .005), but not between airway reactivity and the other antioxidants. The finding of reduced SOD activity in lung cells of patients with asthma suggests that diminished SOD activity serves as a marker of the inflammation characterizing asthma. Alternatively, it may play a role in the development or severity of the disease. © 1997 Elsevier Science Inc.     

Impairment of Vα24-Jα18+Vβ11+ natural killer T cells in adult acute lymphoblastic leukemia patients

Type I natural killer T (NKT) cells are attractive candidates for cancer immunotherapy. In this study, we examined the characteristics of type I NKT cells in patients with adult B-cell acute lymphoblastic leukemia (ALL). We first identified type I NKT cells as Vα24-Jα18 and Vβ11 double-positive CD3⁺ lymphocytes. Using this method, we found that the adult B-cell ALL patients presented significantly lower level of type I NKT cells than the age- and sex-matching control subjects. The expression of IL-21 by type I NKT cells was then examined using intracellular flow cytometry, which showed that with α-GalCer stimulation, the adult B-cell ALL patients presented significantly lower level of IL-21⁺ type I NKT cells than control subjects. By both flow cytometry and ELISA, we found that the vast majority of IL-21-expressing type I NKT cells expressed IL-21R, which was also reduced in adult B-cell ALL patients. Using an in vitro co-culture system, we demonstrated that IL-21R⁺, but not IL-21R^-, type I NKT cells could promote the IFN-γ, granzyme B, and perforin expression by CD8 T cells in an IL-21-dependent fashion. This type I NKT cell-mediated stimulatory effect was reduced in adult B-cell ALL patients than in control subjects. In addition, we observed a positive correlation between the frequency of IL-21R⁺ type I NKT cells and the frequencies of IFN-γ-, granzyme B-, and perforin-expressing circulating CD8 T cells in adult B-cell ALL patients directly ex vivo. Overall, this study identified an IL-21-related impairment in type I NKT cells from adult B-cell ALL patients.     

The reductive metabolism of diaziquone (AZQ) in the S9 fraction of MCF-7 cells: Free radical formation and NAD(P)H: Quinone-acceptor oxidoreductase (DT-diaphorase) activity

The S9 fraction of MCF-7 human breast carcinoma cells has NAD(P)H (quinone-acceptor) oxidoreductase activity as measured by the reduction of dichlorophenol-indophenol (DCPIP). This reduction is dependent on the activators Tween-20 and bovine serum albumin and it is inhibitable by dicumarol. The S9 fraction also has cytochrome c reductase activity which is approximately 29 times less than the two-electron reduction activity of NAD(P)H (quinone-acceptor) oxidoreductase. Diaziquone (AZQ) is a substrate for this NAD(P)H oxidoreductase active S9 fraction as judged by its enzymatic reduction detected spectrophotometrically and by electron spin resonance spectroscopy. Two-electron mediated enzymatic reduction of AZQ was evidenced by the formation of the colorless dihydroquinone (AZQH2) which could be followed at 340 nm. The production of the dihydroquinone was inhibitable by dicumarol implicating NAD(P)H oxidoreductase in its formation. Under aerobic conditions, electron spin resonance spectroscopy showed evidence for the production of AZQ semiquinone (AZQH) and oxygen radicals. Under anaerobic conditions no oxygen radicals were observed, but the semiquinone was stable for hours. These results are also inhibitable by dicumarol and suggest a two-step one-electron oxidation process of the dihydroquinone. The production of semiquinone and oxygen radicals as detected by electron spin resonance spectroscopy was more sensitive to dicumarol when NADPH was used as cofactor (68% inhibition of OH and 65% inhibition of AZQH) than when NADH was used (28% inhibition of OH and 5% inhibition of AZQH). This suggests that NADH flavin reductases play a more important role in the one-electron reduction pathway of AZQ in MCF-7 S9 fraction than NADPH reductases. The reduction of AZQ by NAD(P)H (quinone-acceptor) oxidoreductase may play an important role in the bioreductive alkylating properties of AZQ.     

Collateral sensitivity: ABCG2-overexpressing cells are more vulnerable to oxidative stress

Multidrug resistance (MDR), which is the main obstacle to cancer chemotherapy, is mainly due to overexpression of ATP-binding cassette (ABC) transporters, especially ABCB1 (P-glycoprotein), ABCC1 (MRP1), and ABCG2 (BCRP). A novel idea to overcome MDR is that of collateral sensitivity, i.e., finding a treatment to which cells overexpressing ABC transporters are more sensitive than cells that do not overexpress them. In this study we demonstrate for the first time that MDCKII-BCRP cells, overexpressing ABCG2, are more vulnerable to exogenous oxidative stress induced by several oxidants, viz. paraquat, menadione, hydrogen peroxide, tert-butylperoxide, and 2,2-azobis(2-methylpropionamidine) dihydrochloride. MDCKII-BCRP cells have significantly decreased glutathione level and decreased activities of glutathione S-transferase and glutathione reductase, which may underlie their augmented vulnerability to oxidative stress. These results suggest the possibility of using agents that induce oxidative stress to selectively kill cells overexpressing BCRP.     

Oxidative stress and the development of diabetic retinopathy: Contributory role of matrix metalloproteinase-2

Matrix metalloproteinases (MMPs) degrade extracellular matrix and regulate many functions including cell signaling. Oxidative stress is implicated in the development of diabetic retinopathy, and MMP-2, the most ubiquitous member of the MMP family, is sensitive to oxidative stress. This study aimed to determine the regulation of MMP-2 by oxidative stress in the development of diabetic retinopathy and the role of MMP-2 in the apoptosis of retinal capillary cells. The effects of mitochondrial superoxide scavenger on glucose-induced alterations in MMP-2, and its proenzyme activator MT1-MMP and physiological inhibitor TIMP-2, were determined in retinal endothelial cells, and the regulation of their glucose-induced accelerated apoptosis by the inhibitors of MMP-2 was accessed. To confirm in vitro results, the effects of antioxidant supplementation on MMP-2, MT1-MMP, and TIMP-2 were investigated in the retina of streptozotocin-induced diabetic rats. Glucose-induced activation of retinal capillary cell MMP-2 and MT1-MMP and decrease in TIMP-2 were inhibited by superoxide scavengers, and their accelerated apoptosis was prevented by the inhibitors of MMP-2. Antioxidant therapies, which have been shown to inhibit oxidative stress, capillary cell apoptosis, and retinopathy in diabetic rats, ameliorated alterations in retinal MMP-2 and its regulators. Thus, MMP-2 has a proapoptotic role in the loss of retinal capillary cells in diabetes, and the activation of MMP-2 is under the control of superoxide. This suggests a possible use of MMP-2-targeted therapy to inhibit the development of diabetic retinopathy.     

Actions of Neurotoxic β-Amyloid on Calcium Homeostasis and Viability of PC12 Cells Are Blocked by Antioxidants but Not by Calcium Channel Antagonists

Abstract: The fragment of β-amyloid comprised of amino acids 25–35 induces a rapid, concentration-dependent increase in cytosolic free calcium levels in suspensions of PC12 neuronal cells. This action of β-amyloid 25–35 is not altered by pretreatment with the calcium channel blockers nifedipine or cobalt, with the depleter of intracellular calcium stores cyclopiazonic acid, or with the phospholipase C inhibitor neomycin. However, the effects of β-amyloid 25–35 on cytosolic free calcium are absent in calcium-free buffer and are blocked by the antioxidant lazaroid U-83836E and by vitamin E. β-Amyloid 25–35 is also neurotoxic and produces a concentration-dependent reduction in the viability of PC12 cells in culture. The neurotoxic action of β-amyloid is blocked by U-83836E and vitamin E but not by nifedipine or cobalt. These data indicate that both the disruption of calcium homeostasis and the reduction of cell viability produced by β-amyloid in PC12 cells are mediated by free radical-based processes.     

Are antioxidants useful for treating skeletal muscle atrophy?

Changes in the skeletal muscle protein mass frequently occur in both physiological and pathological states. Muscle hypotrophy, in particular, is commonly observed during aging and is characteristic of several pathological conditions such as neurological diseases, cancer, diabetes, and sepsis. The skeletal muscle protein content depends on the relative rates of synthesis and degradation, which must be coordinately regulated to maintain the equilibrium. Pathological muscle depletion is characterized by a negative nitrogen balance, which results from disruption of this equilibrium due to reduced synthesis, increased breakdown, or both. The current view, mainly based on experimental data, considers hypercatabolism as the major cause of muscle protein depletion. Several signaling pathways that probably contribute to muscle atrophy have been identified, and there is increasing evidence that oxidative stress, due to reactive oxygen species production overwhelming the intracellular antioxidant systems, plays a role in causing muscle depletion both during aging and in chronic pathological states. In particular, oxidative stress has been proposed to enhance protein breakdown, directly or by interacting with other factors. This review focuses on the possibility of using antioxidant treatments to target molecular pathways involved in the pathogenesis of skeletal muscle wasting.     

Oxidant stress-induced loss of IRS-1 and IRS-2 proteins in rat skeletal muscle: Role of p38 MAPK

Oxidative stress is characterized as an imbalance between the cellular production of oxidants and the cellular antioxidant defenses and contributes to the development of numerous cardiovascular and metabolic disorders, including hypertension and insulin resistance. The effects of prolonged oxidant stress in vitro on the insulin-dependent glucose transport system in mammalian skeletal muscle are not well understood. This study examined the in vitro effects of low-level oxidant stress (60–90 μM, H₂O₂) for 4 h on insulin-stimulated (5 mU/ml) glucose transport activity (2-deoxyglucose uptake) and on protein expression of critical insulin signaling factors (insulin receptor (IR), IR substrates IRS-1 and IRS-2, phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3 (GSK-3)) in isolated soleus muscle of lean Zucker rats. This oxidant stress exposure caused significant (50%, p < 0.05) decreases in insulin-stimulated glucose transport activity that were associated with selective loss of IRS-1 (59%) and IRS-2 (33%) proteins, increased (64%) relative IRS-1 Ser³⁰⁷ phosphorylation, and decreased phosphorylation of Akt Ser⁴⁷³ (50%) and GSK-3β Ser⁹ (43%). Moreover, enhanced (37%) phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was observed. Selective inhibition of p38 MAPK (10 μM A304000) prevented a significant portion (29%) of the oxidant stress-induced loss of IRS-1 (but not IRS-2) protein and allowed partial recovery of the impaired insulin-stimulated glucose transport activity. These results indicate that in vitro oxidative stress in mammalian skeletal muscle leads to substantial insulin resistance of distal insulin signaling and glucose transport activity, associated with a selective loss of IRS-1 protein, in part due to a p38 MAPK-dependent mechanism.