The role of antioxidant systems in the response of <Emphasis Type="Italic">Escherichia coli</Emphasis> to acetamidophenol and some antibiotics

The role of glutathione and other antioxidant systems in the response of Escherichia coli to acetamidophenol (paracetamol), rifampicin, and chloramphenicol was studied. The exposure of aerobically growing E. coli cells to acetamidophenol diminished the intracellular level of glutathione by 40% and the reduced-to-oxidized glutathione ratio in the cells by 50%, while it enhanced the expression of the antioxidant genes soxS and sodA by 2.7 and 1.8 times, respectively. Glutathione-deficient cells were more susceptible to acetamidophenol than were normal cells. All this suggests that acetamidophenol induces a mild oxidative stress in E. coli cells. The oxidative stress induced by rifampicin was still less pronounced, whereas chloramphenicol-treated E. coli cells exhibited no signs of oxidative stress at all.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 149–156.Original Russian Text Copyright © 2005 by Smirnova, Torkhova, Oktyabrskii     

Molecular bases of the treatment of Alzheimer's disease with antioxidants: prevention of oxidative stress

Alzheimer's disease is associated with a systemic oxidative stress situation which can be followed in vivo by determining biomarkers such as plasma lipoperoxides and TBARS levels and the oxidation degree of glutathione in red blood cells. It has been observed that Alzheimer's patients show an increased level of plasma TBARS, which indicates a higher free radical oxidation of plasma unsaturated phospholipids, and an increased oxidation of red blood cells glutathione, which indicates oxidative stress in peripheral cells. This latter, glutathione oxidation, was found to correlate statistically with the cognitive status of the patients. Treatment with vitamin E resulted in an improved cognitive performance only of those patients in which the tocopherol acted as an antioxidant, according to blood indicative markers of oxidative stress. Indeed, the effect of vitamin E on Alzheimer's disease patients showed considerable variations both in its antioxidant function and in its capacity to improve cognitive functions. An important conclusion from the reported results is that epidemiological or clinical studies that aim to test the effect of antioxidant supplementation on given functions should include the determination of the antioxidant status of the patients by the measurement of blood markers of oxidative stress.     

Estrogen administration, postexercise tissue oxidative stress and vitamin C status in male rats

Estrogen can putatively act as an antioxidant and protect tissues from exercise-induced oxidative stress. To test the in vivo efficacy of estrogen, the effects of 2 weeks of daily estrogen (40 microg x kg(-1) body weight beta-estradiol 3-benzoate) injection on indices of immediate postexercise oxidative stress and antioxidant status were determined in adult male rats, with and without 8 weeks of prior dietary vitamin E deprivation. The treadmill running protocol (60 min at 21 m x min(-1), 12% grade) induced significant oxidative stress as indicated by muscle glutathione status. Estrogen administration had little effect on postexercise tissue glutathione status, superoxide dismutase and glutathione peroxidase activity, and vitamin E levels. Estrogen administration induced significant reductions in muscle, liver, and heart vitamin C concentrations following exercise, as well as in unexercised male rats. Tissue vitamin C loss was not directly mediated through liver glycogen or glutathione status. Thus, estrogen administration generally did not appear to influence postexercise tissue indices of oxidative stress or antioxidant status and may have contributed to a decline in overall antioxidant protection by inducing losses in tissue vitamin C content.     

Role of the antioxidant system in response of Escherichia coli bacteria to cold stress

The response of aerobically grown Escherichia coli cells to the cold shock induced by the rapid lowering of growth temperature from 37 to 20 degrees C was found to be basically the same as the oxidative stress response. The enhanced sensitivity of cells deficient in two superoxide dismutases, Mn-SOD and Fe-SOD, and the increased expression of the Mn-SOD gene, sodA, in response to cold stress were interpreted as both oxidative and cold stresses are due to a rise in the intracellular level of superoxide anion. The long-term cultivation of E. coli at 20 degrees C was also accompanied by the typical oxidative stress response reactions--an enhanced expression of the Mn-SOD and catalase HPI genes and a decrease in the intracellular level of reduced glutathione (GSH) and in the GSH/GSSG ratio.     

Effect of Indigofera tinctoria Linn on liver antioxidant defense system during D-galactosamine/endotoxin-induced acute hepatitis in rodents

Effects of pre-treatment with the alcoholic extract of I. tinctoria (500 mg/kg body wt/day, p.o. for 21 days) on liver antioxidant defense system during acute hepatitis induced by D-galactosamine (D-GalN)/endotoxin (LPS extracted by phenol water method from E. coli serotype 0111.B4; 300 mg and 30 micrograms/kg body wt/day, i.p., 18 hr before the assay) were investigated on the activities of enzymic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase, and levels of total reduced glutathione in the liver of normal and experimental groups of male albino rats. Since lipid peroxidation and associated membrane damage is a key feature of D-galN/LPS-induced liver injury, the levels of lipid peroxides, was estimated and used as an index of oxidative stress. D-GalN/endotoxin-induced hepatic damage was manifested by a significant decrease in the activities of antioxidant enzymes, decreased glutathione levels and increased levels of lipid peroxides. I. tinctoria pre-treated rats showed considerable protection against D-galN/endotoxin, induced oxidative stress as evidenced by a significant increase in the activities of all the antioxidant enzymes studied and significant decrease in the levels of lipid peroxides. Results indicate that pretreatment with I. tinctoria extract in rats is very effective in reducing D-GalN/endotoxin-induced oxidative stress suggesting an antioxidant effect.     

Effects of menadione and hydrogen peroxide on glutathione status in growing Escherichia coli

Menadione (MD) and H2O2 caused distinct effects on glutathione status in growing Escherichia coli. Treatment of E. coli AB1157 with 1-25 mM H2O2 did not result in an appreciable decrease in intracellular total glutathione (reduced glutathione [GSH] + oxidized glutathione [GSSG]). Only when cells were treated with 25 mM H2O2 an increase in GSSG and a decrease in the GSH:GSSG ratio were observed. In cells deficient in catalase HPI, such effect was observed even at 10 mM H2O2. The exposure of E. coli AB1157 to MD caused a dose-dependent decrease in intracellular total glutathione, an increase in GSSG, and a decrease in the ratio of GSH:GSSG. In E. coli deficient in cytosolic superoxide dismutase activity, a decrease in total glutathione after incubation with 0.2 mM MD was not accompanied by an increase in GSSGin, and the ratio of GSHin:GSSGin was three times higher than in the wild-type cells. The changes in the redox status of extracellular glutathione under the action of both oxidants were similar. Although the catalase activity increased several times after exposure to both oxidants, there were little or no changes in the activity of enzymes related to glutathione metabolism. A possible role of changes in redox status of glutathione under oxidative stress is discussed.     

Effects of Glutathione Modulation on Oxidative Stress and Enzymatic Antioxidant Defence in Yeast Pachysolen tannophilus

The aim of this study was to explore the relationship of intracellular glutathione with various oxidative stress markers and the stress protectant marker trehalose. In the first group of yeast cells, diethyl maleate was used for depletion of glutathione. A second group of yeast cells were incubated with amino acids constituting glutathione (GIu, Cys, Gly) to increase glutathione level. Increased level of oxidative stress marker like ROS, protein carbonyl formation and lipid peroxidation and decreased viability in glutathione-depleted cells were observed in the present study. The increased activity of antioxidant enzymes SOD and CAT in the glutathione depleted group suggests the interaction of different antioxidant defence system in Pachysolen tannophilus. Furthermore, the increased levels of trehalose in glutathione-depleted group shows that trehalose acts as a stress reducer in glutathione depleted Pachysolen tannophilus.     

Mechanism of vitamin C inhibition of cell death induced by oxidative stress in glutathione-depleted HL-60 cells

Vitamin C is a well known antioxidant whose precise role in protecting cells from oxidative challenge is uncertain. In vitro results have been confounded by pro-oxidant effects of ascorbic acid and an overlapping role of glutathione. We used HL-60 cells as a model to determine the precise and independent role of vitamin C in cellular protection against cell death induced by oxidative stress. HL-60 cells do not depend on glutathione to transport or reduce dehydroascorbic acid. Depletion of glutathione rendered the HL-60 cells highly sensitive to cell death induced by H2O2, an effect that was not mediated by changes in the activities of glutathione reductase, glutathione peroxidase, catalase, or superoxide dismutase. The increased sensitivity to oxidative stress was largely reversed when glutathione-depleted cells were preloaded with ascorbic acid by exposure to dehydroascorbic acid. Resistance to H2O2 treatment in cells loaded with vitamin C was accompanied by intracellular consumption of ascorbic acid, generation of dehydroascorbic acid, and a decrease in the cellular content of reactive oxygen species. Some of the dehydroascorbic acid generated was exported out of the cells via the glucose transporters. Our data indicate that vitamin C is an important independent antioxidant in protecting cells against death from oxidative stress.     

Enhancement of cytotoxicity of hydrogen peroxide by hyperthermia in chinese hamster ovary cells: role of antioxidant defenses

Regional hyperthermia has potential for human cancer treatment, particularly in combination with systemic chemotherapy or radiotherapy. The mechanisms involved in heat-induced cell killing are currently unknown. Hyperthermia may increase oxidative stress in cells, and thus, oxidative stress could have a role in the mechanism of cell death. We use hydrogen peroxide as a model oxidant to improve understanding of interactions between heat and oxidative stress. Heat increased cytotoxicity of hydrogen peroxide in Chinese hamster ovary cells. Altered levels of cellular antioxidants should create an imbalance between prooxidant and antioxidant systems, thus modifying cytotoxic responses to heat and to oxidants. We determine the involvement of the two cellular antioxidant defenses against peroxides, catalase and the glutathione redox cycle, in cellular sensitivity to heat, to hydrogen peroxide, and to heat combined with the oxidant. Defense systems were either inhibited or increased. For inhibition studies, intracellular glutathione was diminished to less than 15% of its initial level by treatment with L-buthionine sulfoximine (1 mM, 24 h). Inhibition of catalase was achieved with 3-amino-1,2,4-triazole (20 mM, 2 h), which caused a 80% decrease in endogenous enzyme activity. To increase antioxidants, cells were pretreated with the thiol-containing reducing agents, N-acetyl-L-cysteine, 2-oxo-4-thiazolidine carboxylate, and 2-mercaptoethane sulfonate. These compounds increased intracellular glutathione levels by 30%. Catalase activity was increased by addition of exogenous enzyme to cells. We show that levels of glutathione and catalase affect cellular cytotoxic responses to heat and hydrogen peroxide, either used separately or in combination. These findings are relevant to mechanisms of cell killing at elevated temperatures and suggest the involvement of oxidative stress.     

The role of thiol redox systems in the peroxide stress response of Escherichia coli

The effect of mutations in the genes encoding glutathione, glutaredoxin, thioredoxin, and thioredoxin reductase on the response of growing Escherichia coli to oxidative stress was studied. The gshA mutants defective in glutathione synthesis had the lowest resistance to high doses of H2O2, whereas the trxB mutants defective in thioredoxin reductase synthesis had the highest resistance to this oxidant, exceeding that of the parent strain. Among the studied mutants, the trxB cells demonstrated the highest basic levels of catalase activity and intracellular glutathione; they were able to rapidly reach the normal GSH level after oxidative stress. At the same time, these bacteria showed high frequency of induced mutations. The expression of the katG and sulA genes suggests that, having different sensitivity to high oxidant concentrations, the studied mutants differ primarily in their ability to induce the antioxidant genes of the OxyR and SOS regulons.     

mRNA expression of antioxidant enzymes (SOD, CAT and GSH-Px) and lipid peroxidative stress in liver of Atlantic salmon (Salmo salar) exposed to hyperoxic water during smoltification

The mRNA levels of three antioxidant genes, Cu/Zn superoxide dismutase (SOD), catalase (CAT) and phospholipid hydroperoxide glutathione peroxidase (GSH-Px), were quantified with real-time qRT-PCR in liver of Atlantic salmon Salmo salar exposed to 80% (normoxia), 105% and 130% O2 saturation for 54 days. The salmon were then translocated and exposed to 90% and 130% O2 saturation for additional 72 days during smoltification. TBARS and vitamin E levels in liver and the levels of oxidized glutathione (GSSG), total glutathione (GSH) and the resulting oxidative stress index (OSI) in blood were quantified as traditional oxidative stress markers. No significant mean normalized expression (MNE) differences of SOD, CAT or GSH-Px were found in liver after hyperoxia exposure at the two sampling times. Significantly decreased OSI was found in smolt exposed to 130% O2 saturation after 126 days (n = 18, P < 0.0001), indicating hyperoxia-induced oxidative stress. No effects were seen on growth, or on the levels of thiobarbituric reactive substances (TBARS) and vitamin E in liver after the exposure experiment. Overall, the mRNA expression of SOD, CAT and GSH-Px in liver related poorly with the hyperoxic exposure regimes, and more knowledge are needed before the expressed levels of these antioxidant genes can be applied as biomarkers of hyperoxia in Atlantic salmon.     

Effect of chromate stress on Escherichia coli K-12

The nature of the stress experienced by Escherichia coli K-12 exposed to chromate, and mechanisms that may enable cells to withstand this stress, were examined. Cells that had been preadapted by overnight growth in the presence of chromate were less stressed than nonadapted controls. Within 3 h of chromate exposure, the latter ceased growth and exhibited extreme filamentous morphology; by 5 h there was partial recovery with restoration of relatively normal cell morphology. In contrast, preadapted cells were less drastically affected in their morphology and growth. Cellular oxidative stress, as monitored by use of an H2O2-responsive fluorescent dye, was most severe in the nonadapted cells at 3 h postinoculation, lower in the partially recovered cells at 5 h postinoculation, and lower still in the preadapted cells. Chromate exposure depleted cellular levels of reduced glutathione and other free thiols to a greater extent in nonadapted than preadapted cells. In both cell types, the SOS response was activated, and levels of proteins such as SodB and CysK, which can counter oxidative stress, were increased. Some mutants missing antioxidant proteins (SodB, CysK, YieF, or KatE) were more sensitive to chromate. Thus, oxidative stress plays a major role in chromate toxicity in vivo, and cellular defense against this toxicity involves activation of antioxidant mechanisms. As bacterial chromate bioremediation is limited by the toxicity of chromate, minimizing oxidative stress during bacterial chromate reduction and bolstering the capacity of these organisms to deal with this stress will improve their effectiveness in chromate bioremediation.     

Basic Fibroblast Growth Factor Attenuates Bisphosphonate-Induced Oxidative Injury but Decreases Zinc and Copper Levels in Oral Epithelium of Rat

Recent studies have reported oxidative damage due to bisphosphonate (BP) in various cancer tissues and neurons, although basic fibroblast growth factor (bFGF) induced antioxidant effects in the cells. The bFGF may modulate the BP-induced oxidative stress in oral epithelium of rats. This study was undertaken to explore possible beneficial antioxidant effects of bFGF on oxidative stress induced by BP in oral epithelium of rats. Twenty-eight rats were equally divided into four groups. The first group was used as control. The second, third and fourth groups intraperitoneally received BP (zoledronic acid), bFGF and BP + bFGF. At the end of 10 weeks, the rats were sacrificed, and oral epithelium samples were taken for analyses. In BP group, the lipid peroxidation levels were increased in the oral epithelium, while the activities of glutathione peroxidase (GSH-Px) and the concentrations of total antioxidant status (TAS) were decreased. In rats treated with bFGF, lipid peroxidation levels decreased, and the activities of GSH-Px and concentrations of TAS improved in the oral epithelium. However, zinc and copper levels were decreased in the oral epithelium by BP and bFGF treatments. Concentrations of vitamin E and reduced glutathione in the samples did not change in the groups. In conclusion, treatment with bFGF modulated the antioxidant redox system and reduced the oral epithelium oxidative stress induced by BP. However, zinc and copper levels were decreased by BP and bFGF treatments.     

Effect of bacoside A on brain antioxidant status in cigarette smoke exposed rats

Free radicals mediated oxidative stress has been implicated in the pathogenesis of smoking-related diseases and antioxidant nutrients are reported to prevent the oxidative damage induced by smoking. Therefore, the present study was conducted to evaluate the antioxidant role of bacoside A (triterpenoid saponin isolated from Bacopa monniera) against chronic cigarette smoking induced oxidative damage in rat brain. Adult male albino rats were exposed to cigarette smoke for a period of 12 weeks and simultaneously administered with bacoside A (10 mg/kg b.w./day, p.o.). Antioxidant status of the brain was assessed from the levels of reduced glutathione, vitamin C, vitamin E, and vitamin A and the activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. The levels of copper, iron, zinc and selenium in brain and serum ceruloplasmin activity were also measured. Oxidative stress was evident from the diminished levels of both enzymatic and non-enzymatic antioxidants. Alterations in the levels of trace elements with accumulation of copper and iron, and depletion of zinc and selenium were also observed. Bacoside A administration improved the antioxidant status and maintained the levels of trace elements. These results suggest that chronic cigarette smoke exposure enhances oxidative stress, thereby disturbing the tissue defense system and bacoside A protects the brain from the oxidative damage through its antioxidant potential.     

Increased lymphocyte antioxidant defences in response to exhaustive exercise do not prevent oxidative damage

It has been reported that exercise induces oxidative stress and causes adaptations in antioxidant defences. The aim of this study was to determine the adaptations of lymphocytes to the oxidative stress induced by an exhaustive exercise. Nine voluntary male subjects participated in the study. The exercise was a cycling mountain stage (171.8 km), and the cyclists took a mean of 283 min to complete it. Blood samples were taken the morning of the cycling stage day, after overnight fasting, and 3 h after finishing the stage. We determined the blood glutathione redox status (GSSG/GSH), lymphocyte antioxidant enzyme activities and superoxide dismutase (SOD) levels; the plasma and lymphocyte vitamin E levels; the serum lactate dehydrogenase (LDH) and creatine kinase (CK) activities and urate levels; the plasma carotene and malonaldehyde (MDA) levels; and the lymphocyte carbonyl index. The cycling stage induced significant increases in blood-oxidized (glutathione/GSSG), plasma MDA and serum urate levels. The exercise also produced increases in CK and LDH serum activities. The mountain cycling stage induced significant increases in lymphocyte vitamin E levels, glutathione peroxidase and glutathione reductase activities as well as increased SOD activity and protein levels. The protein carbonyl levels increased significantly in lymphocytes after the stage. In conclusion, in spite of increasing antioxidant defences in response to the oxidative stress induced by the exhaustive exercise, lymphocyte oxidative damage was produced after the stage as demonstrated by the increased carbonyl index even in very well trained athletes.     

The status and the role of glutathione under disturbed ionic balance and pH homeostasis in Escherichia coli

The study of glutathione status in aerobically grown Escherichia coli cultures showed that the total intracellular glutathione (GSHin + GSSGin) level falls by 63% in response to a rapid downshift in the extracellular pH from 6.5 to 5.5. The incubation of E. coli cells in the presence of 50 mM acetate or 10 micrograms/ml gramicidin S decreased the total intracellular glutathione level by 50 and 25%, respectively. The fall in the total intracellular glutathione level was accompanied by a significant decrease in the (GSHin:GSSGin) ratio. The most profound effect on the extracellular glutathione level was exerted by gramicidin S, which augmented the total glutathione level by 1.8 times and the (GSHout:GSSGout) ratio by 2.1 times. The gramicidin S treatment and acetate stress inhibited the growth of mutant E. coli cells defective in glutathione synthesis 5 and 2 times more severely than the growth of the parent cells. The pH downshift and the exposure of E. coli cells to gramicidin S and 50 mM acetate enhanced the expression of the sodA gene coding for superoxide dismutase SodA.     

Evaluation of parameters of oxidative stress after in vitro exposure to FMCW- and CDMA-modulated radiofrequency radiation fields

The goal of this study was to determine whether radiofrequency (RF) radiation is capable of inducing oxidative stress or affecting the response to oxidative stress in cultured mammalian cells. The two types of RF radiation investigated were frequency-modulated continuous-wave with a carrier frequency of 835.62 MHz (FMCW) and code division multiple access centered on 847.74 MHz (CDMA). To evaluate the effect of RF radiation on oxidative stress, J774.16 mouse macrophage cells were stimulated with gamma-interferon (IFN) and bacterial lipopolysaccharide (LPS) prior to exposure. Cell cultures were exposed for 20-22 h to a specific absorption rate of 0.8 W/kg at a temperature of 37.0 +/- 0.3 degrees C. Oxidative stress was evaluated by measuring oxidant levels, antioxidant levels, oxidative damage and nitric oxide production. Oxidation of thiols was measured by monitoring the accumulation of glutathione disulfide (GSSG). Cellular antioxidant defenses were evaluated by measuring superoxide dismutase activity (CuZnSOD and MnSOD) as well as catalase and glutathione peroxidase activity. The trypan blue dye exclusion assay was used to measure any changes in viability. The results of these studies indicated that FMCW- and CDMA-modulated RF radiation did not alter parameters indicative of oxidative stress in J774.16 cells. FMCW- and CDMA-modulated fields did not alter the level of intracellular oxidants, accumulation of GSSG or induction of antioxidant defenses in IFN/LPS-stimulated cells. Consistent with the lack of an effect on oxidative stress parameters, no change in toxicity was observed in J774.16 cells after either optimal (with or without inhibitors of nitric oxide synthase) or suboptimal stimulation.     

Attenuation of TCDD-induced oxidative stress by 670 nm photobiomodulation in developmental chicken kidney

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent developmental teratogen inducing oxidative stress and sublethal changes in multiple organs, provokes developmental renal injuries. In this study, we investigated TCDD-induced biochemical changes and the therapeutic efficacy of photobiomodulation (670 nm; 4 J/cm(2)) on oxidative stress in chicken kidneys during development. Eggs were injected once prior to incubation with TCDD (2 pg/g or 200 pg/g) or sunflower oil vehicle control. Half of the eggs in each dose group were then treated with red light once per day through embryonic day 20 (E20). Upon hatching at E21, the kidneys were collected and assayed for glutathione peroxidase, glutathione reductase, catalase, superoxide dimutase, and glutathione-S-transferase activities, as well as reduced glutathione and ATP levels, and lipid peroxidation. TCDD exposure alone suppressed the activity of the antioxidant enzymes, increased lipid peroxidation, and depleted available ATP. The biochemical indicators of oxidative and energy stress in the kidney were reversed by daily phototherapy, restoring ATP and glutathione contents and increasing antioxidant enzyme activities to control levels. Photobiomodulation also normalized the level of lipid peroxidation increased by TCDD exposure. The results of this study suggest that 670 nm photobiomodulation may be useful as a noninvasive treatment for renal injury resulting from chemically induced cellular oxidative and energy stress.