Ebselen prevents mitochondrial ageing due to oxidative stress: in vitro study of fish erythrocytes

Nucleated trout erythrocytes under oxidative stress suffer DNA membrane damage and inactivation of glutathione peroxidase. In addition, oxidative damage increases with the age of the cell. In the present paper, we evaluate the effects of oxidative stress and ageing on mitochondrial functionality by means of transmission electron microscopy and cytofluorimetric determination of mitochondrial membrane potential and intracellular levels of reactive oxygen species. The protective activity of the antioxidant organoselenium compound ebselen, a mimic of glutathione peroxidase, is also evaluated. Ebselen prevents the drastic structural and functional changes in mitochondria in aged RBCs induced by oxidative stress. However, the antioxidant does not prevent swelling of the mitochondria.     

Dehydroepiandrosterone protects tissues of streptozotocin-treated rats against oxidative stress

Chronic hyperglycemia in diabetes determines the overproduction of free radicals, and evidence is increasing that these contribute to the development of diabetic complications. It has recently been reported that dehydroepiandrosterone possesses antioxidant properties; this study evaluates whether, administered daily for three weeks per os, it may provide antioxidant protection in tissues of rats with streptozotocin-induced diabetes. Lipid peroxidation was evaluated on liver, brain and kidney homogenates from diabetic animals, measuring both steady-state concentrations of thiobarbituric acid reactive substances and fluorescent chromolipids. Hyperglycemic rats had higher thiobarbituric acid reactive substances formation and fluorescent chromolipids levels than controls. Dehydroepiandrosterone-treatment (4 mg/day for 3 weeks) protected tissues against lipid peroxidation: liver, kidney and brain homogenates from dehydroepiandrosterone-treated animals showed a significant decrease of both thiobarbituric acid reactive substances and fluorescent chromolipids formation. The effect of dehydroepiandrosterone on the cellular antioxidant defenses was also investigated, as impaired antioxidant enzyme activities were considered proof of oxygen-dependent toxicity. In kidney and liver homogenates, dehydroepiandrosterone treatment restored to near-control values the cytosolic level of reduced glutathione, as well as the enzymatic activities of superoxide-dismutase, glutathione-peroxidase, catalase. In the brain, only an increase of catalase activity was evident (p < .05), which reverted with dehydroepiandrosterone treatment. The results demonstrate that DHEA treatment clearly reduces oxidative stress products in the tissues of streptozotocin-treated rats.     

Ebselen enhances insulin sensitivity and decreases oxidative stress by inhibiting SHIP2 and protects from inflammation in diabetic mice

Ebselen, a multifunctional organoselenium compound, has been recognized as a potential treatment for diabetes-related disorders. However, the underlying mechanisms whereby ebselen regulates metabolic pathways remain elusive. We discovered that ebselen inhibits lipid phosphatase SHIP2 (Src homology 2 domain-containing inositol-5-phosphatase 2), an emerging drug target to ameliorate insulin resistance in diabetes. We found that ebselen directly binds to and inhibits the catalytic activity of the recombinant SHIP2 phosphatase domain and SHIP2 in cultured cells, the skeletal muscle and liver of the diabetic db/db mice, and the liver of the SHIP2 overexpressing (SHIP2-Tg) mice. Ebselen increased insulin-induced Akt phosphorylation in cultured myotubes, enhanced insulin sensitivity and protected liver tissue from lipid peroxidation and inflammation in the db/db mice, and improved glucose tolerance more efficiently than metformin in the SHIP2-Tg mice. SHIP2 overexpression abrogated the ability of ebselen to induce glucose uptake and reduce ROS production in myotubes and blunted the effect of ebselen to inhibit SHIP2 in the skeletal muscle of the SHIP2-Tg mice. Our data reveal ebselen as a potent SHIP2 inhibitor and demonstrate that the ability of ebselen to ameliorate insulin resistance and act as an antioxidant is at least in part mediated by the reduction of SHIP2 activity.     

Secreted frizzled‐related protein 5 protects against oxidative stress‐induced apoptosis in human aortic endothelial cells via downregulation of Bax

This study was undertaken to determine the role of secreted frizzled‐related protein 5 (SFRP5) in endothelial oxidative injury. Human aortic endothelial cells (HAECs) were exposed to different oxidative stimuli and examined for SFRP5 expression. The effects of SFRP5 overexpression and knockdown on cell viability, apoptosis, and reactive oxygen species production were measured. HAECs treated with angiotensin (Ang) II (1 μM) or oxidized low‐density lipoprotein (oxLDL) (150 μg/mL) showed a significant increase in SFRP5 expression. Overexpression of SFRP5 significantly attenuated the viability suppression and apoptosis induction by Ang II and oxLDL, whereas the knockdown of SFRP5 exerted opposite effects. Overexpression of SFRP5 prevented ROS formation and β‐catenin activation and reduced Bax expression. Co‐expression of Bax significantly reversed the anti‐apoptotic effect of SFRP5 overexpression, whereas knockdown of Bax restrained Ang II‐ and oxLDL‐induced apoptosis in HAECs. Taken together, SFRP5 confers protection against oxidative stress‐induced apoptosis through inhibition of β‐catenin activation and downregulation of Bax.     

The relationship between the osmotic fragility of human erythrocytes and cell age

Erythrocytes in a normal blood sample are hemolyzed over a range of hypotonic salt concentrations. In order to investigate the relationship between the distribution of osmotic fragilities and the distribution of cellular ages, the osmotic fragility has been compared with three indices of cellular age. The activity of glutamic-oxalacetic transaminase (GOT) and the percentage hemoglobin A_1C were measured in samples hemolyzed in different hypotonic salt concentrations. The osmotic fragility curve was also obtained for cells of different density separated by centrifugation. These experiments indicate that the fragility distribution is not an accurate reflection of the distribution of cellular ages. The mean fragility for older cells is higher than that of younger cells. However, cellular aging does not produce a gradual increase in osmotic fragility. Instead, it seems to produce changes which can both increase and decrease the fragility, resulting in a broader distribution of fragilities with some of the older cells actually less fragile than the younger ones.     

Circadian rhythms in human erythrocytes in vitro not confirmed

Abstract

Human erythrocytes, white and resealed erythrocyte ghosts, and hemolysates were analysed for the occurrence of circadian and ultradian rhythms in vitro. Although the experimental conditions were extensively varied, we did not find any indication for the existence of rhythmicity. These results are at variance with the reports by others.     

Melatonin protects against taurolithocholic‐induced oxidative stress in rat liver

Cholestasis, encountered in a variety of clinical disorders, is characterized by intracellular accumulation of toxic bile acids in the liver. Furthermore, oxidative stress plays an important role in the pathogenesis of bile acids. Taurolithocholic acid (TLC) was revealed in previous studies as the most pro‐oxidative bile acid. Melatonin, a well‐known antioxidant, is a safe and widely used therapeutic agent. Herein, we investigated the hepatoprotective role of melatonin on lipid and protein oxidation induced by TLC alone and in combination with FeCl₃ and ascorbic acid in rat liver homogenates and hepatic membranes. The lipid peroxidation products, malondialdehyde and 4‐hydroxyalkenals (MDA + 4‐HDA), and carbonyl levels were quantified as indices of oxidative damage to hepatic lipids and proteins, respectively. In the current study, the rise in MDA + 4‐HDA levels induced by TLC was inhibited by melatonin in a concentration‐dependent manner in both liver homogenates and in hepatic membranes. Melatonin also had protective effects against structural damage to proteins induced by TLC in membranes. These results suggest that the indoleamine melatonin may potentially act as a protective agent in the therapy of those diseases that involve bile acid toxicity. J. Cell. Biochem. 110: 1219–1225, 2010. Published 2010 Wiley‐Liss, Inc.     

Divergent effects of different oxidants on glutathione homeostasis and protein damage in erythrocytes from diabetic patients: Effects of high glucose

Longterm complications of diabetes mellitus have been ascribed to both the effects of prolonged hyperglycemia and to increased oxidative stress. In an attempt to identify the mechanisms underlying the acute effects of hyperglycemia on oxidative stress, we investigated the hypothesis that high glucose might lead to an insufficiency in reducing equivalents (such as NADPH) and thus to a disruption in the glutathionedependent antioxidant defences and to an incapacity to deal with oxidant attack. For this purpose, erythrocytes from diabetic patients were incubated for 0–90 min in 5.55 or 33.3 mM Dglucose containing tertbutyl hydroperoxide 0.5 and 1 mM, Menadione 100 M, or glucose oxidase. The time course of the changes in nonprotein bound glutathione (reduced and oxidised), lactate and pyruvate, alanine and fluorescent products of oxidative proteolysis, hemolysis and methemoglobin was monitored. The results show that although glucose utilisation was unaffected, all oxidants caused a persistent decrease in total nonproteinbound glutathione suggesting binding to proteins. However, changes in glutathione and redox status differed between the various oxidants and were not directly related to the extent of oxidative cellular damage. In these experimental conditions, with short incubations and using the erythrocyte as the simplest cellular model of glucose metabolism, neither high glucose nor the diabetic condition worsened the susceptibility of erythrocytes to acute in vitro oxidative damage.     

Isopropyl‐phloroglucinol‐DHA protects outer retinal cells against lethal dose of all‐trans‐retinal

All‐trans‐retinal (atRAL) is a highly reactive carbonyl specie, known for its reactivity on cellular phosphatidylethanolamine in photoreceptor. It is generated by photoisomerization of 11‐cis‐retinal chromophore linked to opsin by the Schiff's base reaction. In ABCA4‐associated autosomal recessive Stargardt macular dystrophy, atRAL results in carbonyl and oxidative stress, which leads to bisretinoid A2E, accumulation in the retinal pigment epithelium (RPE). This A2E‐accumulation presents as lipofuscin fluorescent pigment, and its photooxidation causes subsequent damage. Here we describe protection against a lethal dose of atRAL in both photoreceptors and RPE in primary cultures by a lipidic polyphenol derivative, an isopropyl‐phloroglucinol linked to DHA, referred to as IP‐DHA. Next, we addressed the cellular and molecular defence mechanisms in commonly used human ARPE‐19 cells. We determined that both polyunsaturated fatty acid and isopropyl substituents bond to phloroglucinol are essential to confer the highest protection. IP‐DHA responds rapidly against the toxicity of atRAL and its protective effect persists. This healthy effect of IP‐DHA applies to the mitochondrial respiration. IP‐DHA also rescues RPE cells subjected to the toxic effects of A2E after blue light exposure. Together, our findings suggest that the beneficial role of IP‐DHA in retinal cells involves both anti‐carbonyl and anti‐oxidative capacities.     

Ebselen impairs cellular oxidative state and induces endoplasmic reticulum stress and activation of crucial mitogen‐activated protein kinases in pancreatic tumour AR42J cells

Ebselen (2‐phenyl‐1,2‐benzisoselenazol‐3(2H)‐one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti‐inflammatory effects. However, evidence suggests that this compound could exert deleterious actions on cell physiology. In this study, we have analyzed the effect of ebselen on rat pancreatic AR42J cells. Cytosolic free‐Ca²⁺ concentration ([Ca²⁺]_c), cellular oxidative status, setting of endoplasmic reticulum stress, and phosphorylation of major mitogen‐activated protein kinases were analyzed. Our results show that ebselen evoked a concentration‐dependent increase in [Ca²⁺]_c. The compound induced an increase in the generation of reactive oxygen species in the mitochondria. We also observed an increase in global cysteine oxidation in the presence of ebselen. In the presence of ebselen an impairment of cholecystokinin‐evoked amylase release was noted. Moreover, involvement of the unfolded protein response markers, ER chaperone and signaling regulator GRP78/BiP, eukaryotic translation initiation factor 2α and X‐box binding protein 1 was detected. Finally, increases in the phosphorylation of SAPK/JNK, p38 MAPK, and p44/42 MAPK in the presence of ebselen were also observed. Our results provide evidences for an impairment of cellular oxidative state and enzyme secretion, the induction of endoplasmic reticulum stress and the activation of crucial mitogen‐activated protein kinases in the presence of ebselen. As a consequence ebselen exerts a potential toxic effect on AR42J cells.     

Triheptanoin protects against status epilepticus‐induced hippocampal mitochondrial dysfunctions,oxidative stress and neuronal degeneration

Triheptanoin, the triglyceride of heptanoate, is anaplerotic (refills deficient tricarboxylic acid cycle intermediates) via the propionyl‐CoA carboxylase pathway. It has been shown to be neuroprotective and anticonvulsant in several models of neurological disorders. Here, we investigated the effects of triheptanoin against changes of hippocampal mitochondrial functions, oxidative stress and cell death induced by pilocarpine‐induced status epilepticus (SE ) in mice. Ten days of triheptanoin pre‐treatment did not protect against SE , but it preserved hippocampal mitochondrial functions including state 2, state 3 ADP , state 3 uncoupled respiration, respiration linked to ATP synthesis along with the activities of pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex 24 h post‐SE . Triheptanoin prevented the SE ‐induced reductions of hippocampal mitochondrial superoxide dismutase activity and plasma antioxidant status as well as lipid peroxidation. It also reduced neuronal degeneration in hippocampal CA 1 and CA 3 regions 3 days after SE . In addition, heptanoate significantly reduced hydrogen peroxide‐induced cell death in cultured neurons. In situ hybridization localized the enzymes of the propionyl‐CoA carboxylase pathway, specifically Pcc α, Pcc β and methylmalonyl‐CoA mutase to adult mouse hippocampal pyramidal neurons and dentate granule cells, indicating that anaplerosis may occur in neurons. In conclusion, triheptanoin appears to have anaplerotic and antioxidant effects which contribute to its neuroprotective properties.

     

CTRP3 protects against uric acid-induced endothelial injury by inhibiting inflammation and oxidase stress in rats

Hyperuricemia, which contributes to vascular endothelial damage, plays a key role in multiple cardiovascular diseases. This study was designed to investigate whether C1q/tumor necrosis factor (TNF)-related protein 3 (CTRP3) has a protective effect on endothelial damage induced by uric acid and its underlying mechanisms. Animal models of hyperuricemia were established in Sprague-Dawley (SD) rats through the consumption of 10% fructose water for 12 weeks. Then, the rats were given a single injection of Ad-CTRP3 or Ad-GFP. The animal experiments were ended two weeks later. In vitro, human umbilical vein endothelial cells (HUVECs) were first infected with Ad-CTRP3 or Ad-GFP. Then, the cells were stimulated with 10 mg/dL uric acid for 48 h after pretreatment with or without a Toll-like receptor 4 (TLR4)-specific inhibitor. Hyperuricemic rats showed disorganized intimal structures, increased endothelial apoptosis rates, increased inflammatory responses and oxidative stress, which were accompanied by reduced CTRP3 and elevated TLR4 protein levels in the thoracic aorta. In contrast, CTRP3 overexpression decreased TLR4 protein levels and ameliorated inflammatory responses and oxidative stress, thereby improving the morphology and apoptosis of the aortic endothelium in rats with hyperuricemia. Similarly, CTRP3 overexpression decreased TLR4-mediated inflammation, reduced oxidative stress, and rescued endothelial damage induced by uric acid in HUVECs. In conclusion, CTRP3 ameliorates uric acid-induced inflammation and oxidative stress, which in turn protects against endothelial injury, possibly by inhibiting TLR4-mediated inflammation and downregulating oxidative stress.     

Distribution patterns of trace metals and of lipid peroxidation in plasma and erythrocytes of rat exposed to aluminum

Significant decreases of the hematocrit, hemoglobin, and plasma iron levels were observed in rats receiving daily intraperitoneal injections of aluminum at a dose of 27 mg Al/kg body wt for 3 wk, as compared to untreated controls. The activity of alkaline phosphatase was also significantly lower in the treated animals as a result of the accumulation of aluminum in the liver (p<0.05). Following aluminum administration, the plasma concentrations of aluminum and copper were also significantly increased, whereas the plasma zinc levels and oxidative stress measured through thiobarbituric acid reaction products showed nonsignificant differences between the two groups (p>0.05). The erythrocyte concentrations of aluminum, copper, zinc, and iron and of superoxide dismutase activity were found to be significantly higher in the study group as compared to controls. The treated animals also showed evidence of higher oxidative stress in comparison to controls. These results suggest that erythrocyte aluminum accumulation could result in abnormal trace element homeostasis and increasing oxidative stress, which might be a mechanism of aluminum-induced anemia.