Effect of chronic exposure to aspartame on oxidative stress in brain discrete regions of albino rats

This study was aimed at investigating the chronic effect of the artificial sweetener aspartame on oxidative stress in brain regions of Wistar strain albino rats. Many controversial reports are available on the use of aspartame as it releases methanol as one of its metabolite during metabolism. The present study proposed to investigate whether chronic aspartame (75 mg/kg) administration could release methanol and induce oxidative stress in the rat brain. To mimic the human methanol metabolism, methotrexate (MTX)-treated rats were included to study the aspartame effects. Wistar strain male albino rats were administered with aspartame orally and studied along with controls and MTX-treated controls. The blood methanol level was estimated, the animal was sacrificed and the free radical changes were observed in brain discrete regions by assessing the scavenging enzymes, reduced glutathione, lipid peroxidation (LPO) and protein thiol levels. It was observed that there was a significant increase in LPO levels, superoxide dismutase (SOD) activity, GPx levels and CAT activity with a significant decrease in GSH and protein thiol. Moreover, the increases in some of these enzymes were region specific. Chronic exposure of aspartame resulted in detectable methanol in blood. Methanol per se and its metabolites may be responsible for the generation of oxidative stress in brain regions.     

Influence of selenium on heat shock protein 70 expression in heat stressed turkey embryos (Meleagris gallopavo)

Heat shock protein 70 (hsp70) family of proteins, which functions as molecular chaperones, has been associated with tolerance to stressors in avian species. Selenium (Se) is an essential trace mineral incorporated into the seleno-enzymes such as glutathione peroxidase (GSHpx). GSHpx reduces oxidized glutathione (GSSG) to reduced glutathione (GSH) in the GSH/GSSG antioxidant system and protects cells from oxidative damage. This study was conducted to examine if the relationship between dietary supplementation of selenium to turkey (Meleagris gallopavo) hens and the embryonic expression of hsp70 and GSHpx activity in heat stressed embryos. Livers of embryos developing in eggs from turkey hens fed diets with or without supplemental Se were analyzed for hsp70 concentration and GSHpx activity before and after recovery from a heating episode. Before heat stress, hsp70 concentrations were equivalent in each treatment, but GSHpx activity was maximized in the SE treatment group. After recovery from the heating episode, hsp70 concentrations were significantly higher (P<0.05) in the non-Se-supplemented groups, but in the Se-supplemented groups the hsp70 concentrations were not different from pre-stress concentrations. In the pre-stress Se-supplemented group, liver GSHpx activity was significantly higher than GSHpx activity in the non-Se-supplemented embryo livers, and in the livers from embryos recovering from heat stress, GSHpx activity in the non-Se-supplemented group was lower than the pre-stress activity and significantly lower than the GSHpx activity in liver from Se-supplemented embryos recovering from heat distress. Se supplementation to the dams resulted in a significant increase in their embryos and that condition would facilitate a decreased incidence of oxidative damage to cells. A more reduced redox status in embryos from Se-supplemented dams decreased the need for cellular protection attributed to stress induced hsp70 and presumably allows heat distressed embryos to resume normal growth and development than embryos from dams with inadequate selenium nutrition.     

Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: Modulation by reactive oxygen species

We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015–15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg⁹ to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.     

An aqueous extract of Rubus chingii fruits protects primary rat hepatocytes against tert-butyl hydroperoxide induced oxidative stress

Different in vitro free radical generating systems were used to assess the antioxidative activity of aqueous extracts of the five herbal components of Wu-zi-yan-zong-wan, a traditional Chinese medicinal formula with a long history of use for tonic effects. Fructus Rubi [Rubus chingii (Rosaceae) fruits] was found to be the most potent. It was further investigated using the primary rat hepatocyte system. tert-Butyl hydroperoxide (t-BHP) was used to induce oxidative stress. Being a short chain analog of lipid hydroperoxide, t-BHP is metabolized into free radical intermediates by the cytochrome P450 system in hepatocytes, which in turn, initiate lipid peroxidation, glutathione depletion and cell damage. Pre-treatment of hepatocytes with Fructus Rubi extract (50 microg/ml to 200 microg/ml) for 24 h significantly reversed t-BHP-induced cell viability loss, lactate dehydrogenase leakage and the associated glutathione depletion and lipid peroxidation. The amount of reactive oxygen species formed was also decreased as visualized by the fluorescence probe 2',7'-dichlorofluorescin diacetate. These results suggested that Fructus Rubi was useful in protecting against t-BHP-induced oxidative damage and may also be capable of attenuating cytotoxicity of other oxidants.     

Effect of glutathione depletion, hyperthermia, and a 100-mT static magnetic field on an hsp70/luc reporter system

Heat shock proteins, in particular hsp70, are induced under conditions of cellular stress. It has been reported that environmental stimuli such as hyperthermia, oxidative stress, and exposure to magnetic fields increase levels of hsp70. It has also been reported that hyperthermia in combination with magnetic field exposure results in a synergistic increase in hsp70 production. We tested the hypothesis that oxidative stress induced by glutathione (GSH) depletion in combination with static magnetic field (SMF) exposure will produce a similar synergistic increase in hsp70 production. We exposed cells to heat, SMF, and diethylmaleate (DEM), which depletes GSH levels alone and in combination with each other, and measured hsp70 production using an hsp70/luciferase reporter and mRNA levels using PCR. We found that treatment with DEM significantly reduced the rate of luciferase bioluminescence production, particularly in the presence of heat. There was no significant effect of a 100-mT SMF exposure either alone or in combination with heat, DEM, or both on bioluminescence, however there was a significant interaction between SMF and DEM on hsp70 mRNA levels. Therefore, under our exposure conditions, GSH depletion reduced hsp70 levels but a synergistic effect of combining this stress with other external stimuli was only observed at the level of mRNA.     

Induction of heme oxygenase-1 and heat shock protein 70 in rat hepatocytes: The role of calcium signaling

Stress response genes including heat shock proteins are induced under a variety of conditions to confer cellular protection. This study investigated the role of calcium signaling in the induction of two stress response genes, heme oxygenase-1/hsp32 and hsp70, in isolated rat hepatocytes. Both genes were induced by cellular glutathione depletion. This induction could be inhibited by BAPTA-AM. Culturing in a calcium-free medium prevented the induction of hsp70 gene expression after glutathione depletion without affecting heme oxygenase-1 gene expression. Thapsigargin increased the gene expression of heme oxygenase-1 but not that of hsp70. Thapsigargin-induced heme oxygenase-1 induction was completely inhibited by BAPTA-AM. Incubation with the Ca²⁺-ionophore A23187 augmented heme oxygenase-1 (two-fold) and hsp70 (5.2-fold) mRNA levels. Our data suggests a significant role of Ca²⁺-dependent pathways in the induction of the two stress genes. An increase in the cytoplasmic Ca²⁺ activity seems to play a key role in the cascade of signaling leading to the induction of the two genes. However, the source of Ca²⁺ that fluxes into the cytoplasm seems to be different. Our data provides evidence for a compartmentalization of calcium fluxes, i.e. the Ca²⁺ flux from intracellular stores (e.g. the endoplasmic reticulum) plays a major role in the induction of heme oxygenase-1. By contrast, Ca²⁺ flux from the extracellular medium seems to be a mechanism initiating the cellular signaling cascade leading to hsp70 gene induction.     

Antioxidant responses of Chilo suppressalis (Lepidoptera: Pyralidae) larvae exposed to thermal stress

High temperatures cause a variety of physiological stress responses in insects, including increased generation of reactive oxygen species (ROS), which can cause oxidative damage. This study investigated the effects of thermal stress on ROS generation, the expression of heat shock protein 70 (Hsp70) at the mRNA and protein levels, the activity of antioxidant enzymes (SOD, CAT), and apoptosis in hemocytes of Chilo suppressalis larvae. Results indicated that thermal stress significantly elevated the level of ROS and antioxidant enzyme activity in C. suppressalis larvae. Real-time quantitative PCR showed that hsp70 gene expression was induced by heat stress. Flow cytometric results revealed that the expression profile of Hsp70 at the protein level was in agreement with that at the mRNA level. The expression of Hsp70 at both the mRNA and protein levels reached a maximum at 36 °C in larval hemocytes. Exposure to tested temperatures did not cause any significant change in the rate of apoptosis in larval hemocytes. These results suggest that thermal stress leads to oxidative stress and that antioxidant enzymes and the Hsp70 play an important role in reducing oxidative damage in C. suppressalis larvae.     

Stress proteins as biomarkers of oxidative stress: effects of antioxidant supplements

The potential benefits to health of the supply of antioxidants, either through dietary intake or as supplements, is equivocal. There is a need to develop biomarkers that may act as monitors of cellular defense as influenced by antioxidant status. Thirty-two individuals participated in the project and 19 received supplements for 5 weeks in the form of a capsule containing a defined mixture of antioxidants. No change was noted in levels of superoxide dismutase and glutathione peroxidase following antioxidant supplementation. On the other hand, increase in total antioxidant status and decrease in malondialdehyde, protein carbonyl formation, and erythrocyte hemolysis were noted. In lymphocytes isolated from individuals receiving antioxidant supplements and subjected to a heat shock in the presence of the free radical generator 2, 2'-azobis-(2-amidinopropane)-dihydrochloride, enhanced synthesis of heat shock proteins hsp 105, hsp 90, hsp 70, and hsp 40 by contrast with decreased synthesis of heme oxygenase HO-1 (hsp 32) were noted. We conclude that antioxidant status modulates the synthesis of stress proteins.     

Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: modulation by reactive oxygen species

We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015-15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg(9) to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.     

Differences in the expression of heat-shock proteins and antioxidant enzymes between human and rodent pancreatic islets: implications for the pathogenesis of insulin-dependent diabetes mellitus.

BACKGROUND: It has previously been observed that the insulin-producing cells of human pancreatic islets are more resistant to alloxan-, streptozotocin-, nitroprusside-, or cytokine-induced injury than those of mouse and rat islets. MATERIALS AND METHODS: Human pancreatic islets were obtained from heart-beating organ donors. The expression of the stress proteins heat shock protein 70 (hsp70) and heme oxygenase and the anti-apoptosis gene bcl-2 was determined in isolated rat, mouse, and human islets, either cultured in vitro or transplanted under the kidney capsule of nude mice, using immunoblot analysis. Rat and human islet sensitive hydrogen peroxide was assess by glucose oxidation measurements. Isolated islets were also analyzed for their catalase and superoxide dismutase activities, and the islet cell levels of reduced glutathione were determined in response to hydrogen peroxide and nitroprusside. Programmed cell death in human and rat islets in response to streptozotocin was evaluated using TUNEL staining. RESULTS: Cultured human islets expressed higher contents of hsp70 than mouse and rat islets at basal conditions. Also after 4 weeks under the kidney capsule of normoglycemic mice, the hsp70 levels were higher in human islets than in rat islets. The expression of another stress protein, heme oxygenase (HO), was strongly increased in cultured rat islets, but was not affected in human islets. Expression of the bcl-2 gene could not be detected in human islets. In spite of this, 0.5 mM streptozotocin induced apotosis in rat but not in human islet cells. Hydrogen peroxide (0.1 and 0.4 mM) decreased glucose oxidation rates in rat but not in human islets. The levels of reduced glutathione were moderately decreased in human and rat islet cells and sharply decreased in mouse islet cells in response to hydrogen peroxide. Moreover, the activities of catalase and superoxide dismutase (SOD) were markedly lower in mouse islets than in human islets. The activity of catalase was lower in rat islets than in human islets. CONCLUSION: Human islets differ clearly from mouse and rat islets in their increased expression of hsp70, catalase, and SOD, which may explain the increased resistance of human islets to beta cell toxins.     

Generation of free radicals from lipid hydroperoxides by Ni2+ in the presence of oligopeptides.

The generation of free radicals from lipid hydroperoxides by Ni2+ in the presence of several oligopeptides was investigated by electron spin resonance (ESR) utilizing 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. Incubation of Ni2+ with cumene hydroperoxide or t-butyl hydroperoxide did not generate any detectable free radical. In the presence of glycylglycylhistidine (GlyGlyHis), however, Ni2+ generated cumene peroxyl (ROO.) radical from cumene hydroperoxide, with the free radical generation reaching its saturation level within about 3 min. The reaction was first order with respect to both cumene hydroperoxide and Ni2+. Similar results were obtained using t-butyl hydroperoxide, but the yield of t-butyl peroxyl radical generation was about 7-fold lower. Other histidine-containing oligopeptides such as beta-alanyl-L-histidine (carnosine), gamma-aminobutyryl-L-histidine (homocarnosine), and beta-alanyl-3-methyl-L-histidine (anserine) caused the generation of both cumene alkyl (R.) and cumene alkoxyl (RO.) radicals in the reaction of Ni2+ with cumene hydroperoxide. Similar results were obtained using t-butyl hydroperoxide. Glutathione also caused generation of R. and RO. radicals in the reaction of Ni2+ with cumene hydroperoxide but the yield was approximately 25-fold greater than that produced by the histidine-containing peptides, except GlyGlyHis. The ratio of DMPO/R. and DMPO/RO. produced with glutathione and cumene hydroperoxide was approximately 3:1. Essentially the same results were obtained using t-butyl hydroperoxide except that the ratio of DMPO/R. to DMPO/RO. was approximately 1:1. The free radical generation from cumene hydroperoxide reached its saturation level almost instantaneously while in the case of t-butyl hydroperoxide, the saturation level was reached in about 3 min. In the presence of oxidized glutathione, the Ni2+/cumene hydroperoxide system caused DMPO/.OH generation from DMPO without forming free hydroxyl radical. Since glutathione, carnosine, homocarnosine, and anserine are considered to be cellular antioxidants, the present work suggests that instead of protecting against oxidative damage, these oligopeptides may facilitate the Ni(2+)-mediated free radical generation and thus may participate in the mechanism(s) of Ni2+ toxicity and carcinogenicity.     

Role of glutathione and hsp 70 in the acquisition of thermotolerance in postimplantation rat embryos

Studies were initiated to determine the extent to which reduced glutathione (GSH) may be involved in the capacity of cultured rat embryos to develop heat-induced tolerance to the deleterious effects of exposure to high temperatures (heat shock). Investigations of the modulation of dysmorphogenic responses of embryos to heat shock (43 degrees C, 30 min) as well as to the expression of the hsp70 gene and subsequent formation of hsps indicated that the acquisition of thermotolerance by rat embryos could be significantly influenced by the inhibition of GSH synthesis. Treatment of conceptuses with L-buthionine-S,R-sulfoximine (BSO) reduced intracellular GSH concentrations and compromised the capacity of embryos to mount a thermotolerance response as assessed by alterations in indices of growth and development. Embryonic thermotolerance elicited by preexposure to 42 degrees C for 30 min was accompanied by increases in GSH to levels greater than those measured in control embryos at 37 degrees C just prior to the subsequent 43 degrees C heat exposure. Expression of hsp70 mRNA was detectable soon after elevation of the temperature to 42 degrees C and reached its highest level of accumulation 1.5 hr after the 43 degrees C heat shock. BSO treatment had little if any effect on hsp70 message levels or on the synthesis of hsp70. The fact that BSO-treatment attenuated the thermotolerance response but did not produce a decrease in hsp70 RNA or the synthesis of hsp70 suggests that hsp70 alone is not sufficient to confer thermotolerance upon cultured rat embryos.     

Influence of organic selenium on hsp70 response of heat-stressed and enteropathogenic Escherichia coli-challenged broiler chickens (Gallus gallus)

The effect of dietary selenium yeast, a source of organic selenium, on heat shock protein 70 (hsp70) responses, redox status, growth and feed utilization were evaluated either in enteropathogenic Escherichia coli-challenged (EPEC) or in heat-stressed (HS) male broiler chickens grown to 42 days of age. One day-old chicks in experiment 1 were challenged orally with EPEC (10(6) cfu/chicken on day 1 and boosted by water application on days 2, 3, and 4) and fed diets with or without selenium yeast. Body weight (BW), feed conversion ratio (FCR), and total mortality were determined at 42 days of age, and this was followed by collection of ileal tissue for the quantification of total glutathione (TGSH), reduced glutathione (GSH), oxidized glutathione (GSSG), and hsp70 in randomly selected chickens from each treatment. In experiment 2, male broiler chickens were fed diets with or without selenium yeast under a thermoneutral rearing condition. At four weeks of age, blood and hepatic tissue were collected from chickens maintained in the thermoneutral environment and from chickens subjected to HS (40 degrees C for 1 h) and analyzed for TGSH, GSH, GSSG, and hsp70. Selenium yeast improved BW, FCR, and decreased mortality in both control and EPEC-challenged chicks. Selenium yeast significantly attenuated hsp70 expression in EPEC-challenged chickens and in those subjected to HS. The EPEC challenge increased TGSH and GSSG levels and decreased GSH/GSSG ratio. However, GSSG level accumulated in chickens fed diets without selenium supplementation resulting in a lower GSH/GSSG ratio in the selenium yeast-fed group. Heat stress increased GSSG level and decreased GSH/GSSG ratio. Selenium yeast-fed groups maintained higher levels of GSSG before and after HS with a resultant lower GSH/GSSG ratio. The hsp70 response was significantly less in those chickens fed selenium yeast and challenged with either EPEC or HS than in those chickens given no supplemental selenium. The results of this study suggest that selenium yeast supplementation had imparted resistance to oxidative stress associated with enteric bacteria infection and to high temperature exposure. It is believed that the resistance to the stressors was due to an improved redox status of the selenium yeast-fed chickens.     

In-vivo glutathione elevation protects against hydroxyl free radical-induced protein oxidation in rat brain

Glutathione deficiency has been associated with a number of neurodegenerative diseases including Lou Gehrig's disease, Parkinson's disease, and HIV. A crucial role for glutathione is as a free radical scavenger. Alzheimer's disease (AD) brain is characterized by oxidative stress, manifested by protein oxidation, lipid oxidation, oxidized glutathione, and decreased activity of glutathione S-transferase, among others. Reasoning that elevated levels of endogenous glutathione would offer protection against free radical-induced oxidative stress, rodents were given in vivo injections of N-acetylcysteine (NAC), a known precursor of glutathione, to study the vulnerability of isolated synaptosomal membranes treated with Fe2+/H2O2, a known hydroxyl free radical producer. Protein carbonyls, a marker of protein oxidation, were measured. NAC significantly increased endogenous glutathione levels in cortical synaptosome cytosol (P < 0.01). As reported previously, protein carbonyl levels of the Fe2+/H2O2-treated synaptosomes were significantly higher compared to that of non-treated controls (P < 0.01), consistent with increased oxidative stress. In contrast, protein carbonyl levels in Fe2+/H2O2-treated synaptosomes isolated from NAC-injected animals were not significantly different from saline-injected non-treated controls, demonstrating protection against hydroxyl radical induced oxidative stress. These results are consistent with the notion that methods to increase endogenous glutathione levels in neurodegenerative diseases associated with oxidative stress, including AD, may be promising.     

Melatonin prevents oxidant damage in various tissues of rats with hyperthyroidism

Impairment of thyroid functions brings about pathological changes in different organs of body. Findings of in vivo and in vitro studies indicate that thyroid hormones have a considerable impact on oxidative stress. Melatonin reduces oxidative damage through its free radical eliminating and direct anti-oxidant effects. The present study was undertaken to determine how a 3-week period of intraperitoneal melatonin administration affected oxidative damage caused in experimental hyperthyroidism in rat. The experimental animals were divided into 3 groups (control, hyperthyroidism, hyperthyroidism+melatonin). Malondialdehyde (MDA) and glutathione (GSH) levels were determined in different tissues. MDA levels in cerebral, liver and cardiac tissues in hyperthyroidism group were significantly higher than those in control and hyperthyroidism+melatonin supplemented groups (p<0.001). The highest GSH levels were observed in the group that was administered melatonin in addition to having hyperthyroidism (p<0.001). These results show that hyperthyroidism increased oxidative damage in cerebral, hepatic and cardiac tissues of rat. Melatonin supplementation may also suppress oxidative damage.     

Hsp70 level in progeny of aging grasshoppers from variously polluted habitats and additionally exposed to zinc during diapause

The hsp70 level in the bodies of 1st instars of grasshoppers Chorthippus brunneus from unpolluted (Pilica) and polluted (Olkusz, Szopienice) sites and additionally exposed to various doses of zinc during diapause and embryonic development prior to hatching were measured by Western blotting. The main aim of our work was to assess the relationship between the age of female grasshoppers originating from variously polluted habitat and the hsp70 level in their progeny. Possible reasons for population variation in hsp70 levels were discussed. The hsp70 level in the offspring's body depended on the place of origin. The strongest expression of hsp70 was found in the bodies of larvae hatching from the eggs laid by young females from Pilica (reference site). In contrast, a low initial level of hsp70 in larvae from polluted sites, especially in young females’ progeny, was observed. The application of zinc during diapause influenced the hsp70 level in grasshopper larvae; however, the direction of the changes depended on the insects’ place of origin. In larvae from the reference site, and also (but to a lesser degree) from Olkusz, the increase in the hsp70 level after zinc treatment was most pronounced. Whereas in grasshoppers from Szopienice, zinc (in 100 μg g⁻¹ dry weight of sand) did not change the hsp70 level, or (in 500 μg g⁻¹ dry weight of sand) caused a reduction in hsp70. The differences may result from maternal effects; however, possible adaptation also cannot be excluded. To confirm this statement further studies are needed.     

Human hsp27, Drosophila hsp27 and human alphaB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFalpha-induced cell death.

Expression of small stress proteins (shsp) enhances the survival of mammalian cells exposed to heat or oxidative injuries. Recently, we have shown that the expression of shsp from different species, such as human hsp27, Drosophila hsp27 or human alphaB-crystallin protected murine L929 cells against cell death induced by tumor necrosis factor (TNFalpha), hydrogen peroxide or menadione. Here, we report that, in growing L929 cell lines, the presence of these shsp decreased the intracellular level of reactive oxygen species (ROS). shsp expression also abolished the burst of intracellular ROS induced by TNFalpha. Several downstream effects resulting from the TNFalpha-mediated ROS increment, such as NF-kappaB activation, lipid peroxidation and protein oxidation, were inhibited by shsp expression. We also report that the expression of these different shsp raised the total glutathione level in both L929 cell lines and transiently transfected NIH 3T3-ras cells. This phenomenon was essential for the shsp-mediated decrease in ROS and resistance against TNFalpha. Our results therefore suggest that the protective activity shared by human hsp27, Drosophila hsp27 and human alphaB-crystallin against TNFalpha-mediated cell death and probably other types of oxidative stress results from their conserved ability to raise the intracellular concentration of glutathione.     

Effect of cholesterol and 7beta-hydroxycholesterol on glutathione status and expression of Hsp70 in cultured murine peritoneal macrophages

Using cultured murine peritoneal macrophages, the change in redox ratio (oxidized/reduced glutathione) was studied at different incubation intervals (6, 12, 18 and 24 hr) with different concentrations (2.5, 5 and 7.5 microg/ml) of cholesterol and 7beta-hydroxycholesterol (7beta-OH), using fluorimeter. The changes in the levels of heat shock protein, hsp70 was determined using ELISA. Both cholesterol/7beta-OH caused a decrease in hsp70 protein levels at all the incubation intervals in dose dependent manner but the decrease was significantly higher with 7beta-OH. Treatment with 7beta-OH also resulted in significantly increased levels of oxidized glutathione (GSSG) and decreased reduced glutathione (GSH) while cholesterol showed no effect on GSSG levels. Moreover, GSH levels were lowered only at the highest concentration (7.5 microg/ml) at longer incubation intervals (18 and 24 hr) with cholesterol exposure. This altered the redox status in both cholesterol/7beta-OH treated macrophages. These results suggest that cholesterol and more likely 7beta-OH may exert their pro-atherogenic effects by lowering hsp70 protein production and inhibiting glutathione synthesis by macrophages present in the arterial wall.