Chronic Lipid Hydroperoxide Stress Suppresses Mucosal Proliferation in Rat Intestine: Potentiation of Ornithine Decarboxylase Activity by Epidermal Growth Factor

We recently demonstrated that chronic exposure of rat intestine to sublethal levels of peroxidized lipids suppresses ornithine decarboxylase (ODC) activity, consistent with attenuated intestinal proliferation. The current study was designed to better understand the influence of exogenous epidermal growth factor (EGF) on intestinal proliferation in normal intestine and intestine that was challenged by oxidative stress induced by dietary consumption of peroxidized lipids. Male Sprague-Dawley rats (250-300 g) were fed standard chow (control) or peroxidized lipid chow for 4 weeks. EGF was injected intraperitoneally at a dose of 40 microg/kg. Intestinal proliferation was evaluated by ODC activity in fed or fasted states and at specified times during the circadian phase. Chronic peroxide consumption significantly attenuated ODC activities in association with increased tissue peroxide content. The suppressed ODC activities were restored to control values by EGF in the small intestine; in the colon, EGF increased ODC activity threefold over control rats given EGF. This elevated colonic ODC activity was correlated with decreased tissue GSSG and an increased GSH/GSSG ratio. These results show that EGF administration reverses the suppression of intestinal ODC activities induced by chronic peroxidized lipid intake. In contrast, EGF significantly elevates proliferative activity in the peroxide-stressed colon. This exaggerated proliferation may contribute to a better understanding of colonic susceptibility to oxidant-induced malignant transformation.     

Redox imbalance in patients with coronary artery disease showing progression of atherosclerotic lesions

OBJECTIVES: To clarify the relationship between changes in redox balance and the development of new coronary lesions in patients with coronary artery disease (CAD). METHODS: We studied 82 CAD patients (70 males and 12 females, mean age 61.8 +/- 9.2 years) who underwent repeated coronary angiography within 1 year after percutaneous coronary intervention. Levels of serum lipid peroxide, erythrocyte glutathione peroxidase activity, and the redox state of erythrocyte (ratio of reduced to oxidized glutathione, the GSH/GSSG ratio) were measured at the time of follow-up coronary angiography. According to the development of significant stenotic lesions, we divided the patients into two groups: 57 patients without the development of new stenotic lesions (group A) and 25 patients showing new significant stenotic lesions within 1 year (group B). RESULTS: The serum lipid peroxide level in group B was significantly higher than those of group A (2.61 +/- 0.32 vs 1.74 +/- 0.16 nmol/ml, p < 0.01). Erythrocyte glutathione peroxidase activity did not differ significantly between two groups. The erythrocyte GSH/GSSG ratio in group B was significantly lower than that of group A (83 +/- 9.6 vs 126 +/- 7.3, p < 0.01). The sensitivity and specificity of GSH/GSSG ratio to detect CAD patients with the development of significant coronary stenosis were 80.0% and 61.4%, respectively. CONCLUSIONS: CAD patients who showed development of new coronary lesions within 1 year have increased oxidative stress and imbalanced erythrocyte redox state. The GSH/GSSG ratio, an indicator for redox balance, could be a useful marker to identify high-risk CAD patients.     

Amlodipine and glutathione cycle in hypercholesterolaemia

OBJECTIVE: Effects of amlodipine on lipid peroxidation and alterations in glutathione and related enzymes in blood and aortic tissue were investigated in a cholesterol-induced atherosclerotic rabbit model. METHODS AND RESULTS: New Zealand white male rabbits were fed with regular chow (group I), chow supplemented with I% cholesterol (group II), regular chow plus amlodipine 5 mg/kg/day p.o. (group III) and I% cholesterol diet supplemented with amlodipine (group IV) for 8 weeks. Cholesterol, malondialdehyde (MDA), reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione peroxidase (GSH-PX) and glutathione reductase (GSH-Rd) were determined in blood samples drawn before and after the experimental period. Aortic tissue was examined morphologically for atherosclerotic changes and tissue cholesterol, MDA, GSSG, GSH-PX, GSH-Rd and glutathione-S-transferase (GST) were measured. After 8 weeks, blood cholesterol, MDA, GSSG and GSH-PX were elevated in groups II and IV; GSH was reduced in group IV; MDA levels were higher in group II than in group IV. Aortic tissue investigations revealed higher cholesterol and MDA concentrations in group II than in group IV. Morphological examination of aortic tissues exhibited endothelial disarrangement and lipid deposition in group II. Histopathological alterations related to atherogenesis were less in group IV than in group II. CONCLUSIONS: Amlodipine reduced the increase in oxidative stress by inhibiting excessive MDA production. Accelerated glutathione redox cycle activity of erythrocytes from animals supplemented with amlodipine suggests that this drug may reduce oxidative stress by enhancing the glutathione system. However, this drug does not seem to affect the glutathione redox cycle in the aortic tissue.     

Changes in glucose-6-phosphate dehydrogenase, copper, zinc-superoxide dismutase and catalase activities, glutathione and its metabolizing enzymes, and lipid peroxidation in rat erythrocytes with age

We have studied the activities of enzymes (glucose-6-phosphate dehydrogenase (G-6-PD), copper, zinc-superoxide dismutase (Cu,Zn-SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GSH-Px) and glutathione-S-transferase (GST)), and the levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and thiobarbituric acid-reactive substances (TBARS) in rat erythrocytes and estimated the ratio of GSH/GSSG and the redox index. Male Wistar rats at ages of 1, 6 and 12 months were used. The activities of G-6-PD and Cu,Zn-SOD, the levels of GSSG and TBARS were increased, while the activity of Se-GSH-Px and the level of GSH were decreased with age. GSH/GSSG ratio was significantly decreased with age. We found a positive correlation between age and G-6-PD (r=0.476, p<0.01), Cu,Zn-SOD (r=0.291, p<0.01), CAT (r=0.254, p<0.01) and GST activities (r=0.250, p<0.05), and GSSG (r=0.708, p<0.05) and TBARS levels (r=0.802, p<0.01), whereas the correlation between age and Se-GSH-Px activity (r=-0.376, p<0.05), GSH level (r=-0.603, p<0.01) and GSH/GSSG ratio (r=-0.685, p<0.05) were negative. We found age-related differences in erythrocyte antioxidant enzyme activities, GSH, GSSG, total GSH and TBARS levels, GSH/GSSG ratio and the redox index.     

Chronic Alcohol Consumption and Cerebral Indices of Oxidative Stress: Is There a Link?

BACKGROUND: It is still difficult to define the biochemical mechanisms that cause alterations in neuronal function and plasticity and neuronal cell loss in the brains of alcohol-dependent patients. METHODS: To evaluate the extent of cerebral alcohol-induced oxidative stress ex vivo, we investigated the levels of glutathione (GSH), its oxidation product glutathione disulfide (GSSG, produced by GSH-peroxidases), and the activities of catalase and superoxide dismutases (SOD). In addition, selected brain regions from up to 22 subjects (versus controls) were studied post mortem to compare the amount of oxidized DNA-base 8-hydroxy-2'-deoxyguanosine (8-OHdG) with levels of deoxyguanosine (dG) in mitochondrial and nuclear DNA. RESULTS: The most prominent findings showed significantly decreased GSH/(GSH+2GSSG) molar redox (oxidation-reduction) ratios in the corpus mamillare and cerebellum, which appeared due to an increase in GSSG caused by chronic alcohol intake. Catalase activity was increased in only the frontal cortex, whereas decreased catalase activity was found in the corpus callosum. In contrast, neither copper-zinc-superoxide dismutase (CuZnSOD) and manganese-superoxide dismutase (MnSOD) activities nor 8-OHdG/dG molar ratios were altered, although a tendency toward higher OHdG/dG ratios in temporal and parietal cortex from alcohol-dependent patients could be detected when mitochondrial DNA was analyzed selectively. CONCLUSIONS: We propose that decreased brain GSH/(GSH+2GSSG) molar redox (oxidation-reduction) ratios in alcohol-dependent patients may reflect neural impairment due to increased peroxide production after chronic alcohol consumption. However, future experiments, investigating the activities of enzymes and cofactors involved in GSH synthesis and metabolism in the human brain, will have to validate the specificity of these results for oxidative stress.     

Effect of difluoromethyl ornithine (DFMO) on small intestine of adult and weanling rats

Oral feeding ofdl-difluoromethyl ornithine (DFMO) (2% in waterad libitum) for 14 days has no detectable effect on the small intestine of adult rats.Similar feeding of DFMO to weanling rat pups caused diarrhea in three to four days accompanied by a decrease in food consumption and body weight compared to age-matched controls. Significant decreases in small intestinal mucosal weight, total protein, DNA, enterokinase, leucine amino peptidase, sucrase, and maltase contents were observed in the DFMO-treated group four days after treatment. Extending the treatment to seven days led to a more severe reduction in these parameters. Villous atrophy of the mucosa was demonstrable by light microscopy and morphometric measurements. The mucosa of the DFMO-treated rat pups showed a reduction in total thickness and villous height but no change in crypt depth. A significant reduction in villus-crypt ratio was also seen.Changes in small intestinal mucosal parameters were not due to a decrease in food intake since pair-fed, agematched rat pups showed no biochemical changes compared to control pups. DFMO-treated weanling rats showed less than 5% of ornithine decarboxylase (ODC) activity when compared to age-matched control animals. The effects observed on the small intestinal mucosa are presumably due to inhibition of ornithine decarboxylase activities by DFMO which prevents the proliferation, regeneration, and maturation of epithelial cells. The relative insensitivity of the adult rat small intestine to DFMO treatment suggests a lesser dependence of its intestinal mucosa to ODC activities.     

Comparison of metabolic rate and oxidative stress between two different strains of mice with varying response to caloric restriction

Metabolic rate and parameters associated with oxidative stress were compared in two strains of mice, one of which, C57BL/6, exhibits an extension of life span in response to caloric restriction while the other, DBA/2, shows no such effect. Metabolic rate was higher in the DBA/2 than in the C57BL/6 mice, when measured at 5-6 months of age as in vivo and in vitro rates of oxygen consumption or body temperature. There were no remarkable inter-strain differences in activities of the antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase or in the rates of mitochondrial superoxide anion radical generation in heart or skeletal muscles. Comparison of glutathione redox state in the heart and skeletal muscles at 3 and 20 months of age indicated that the amount of glutathione (GSH) and the GSH:GSSG (glutathione disulfide) ratio were relatively higher in the young DBA/2 mice, but there were no inter-strain differences in the older mice. The age-related elevation in the level of oxidative stress reflected by GSH:GSSG ratio was greater in the C57BL/6 than DBA/2 mice. The energy balance, indicated by the gain/loss in body weight per unit of food consumed, is higher in C57BL/6 than DBA/2 mice. It is hypothesized that the genotype-specific extension of life span by caloric restriction may involve modulation of oxidative stress produced as a result of an interplay between metabolic rate and energy balance during aging.     

Relationship between gluconeogenesis and glutathione redox state in rabbit kidney-cortex tubules

The intracellular glutathione redox state and the rate of glucose formation were studied in rabbit kidney-cortex tubules. In the presence of substrates effectively utilized for glucose formation, ie, aspartate + glycerol + octanoate, alanine + glycerol + octanoate, malate, or pyruvate, the intracellular reduced glutathione/oxidized glutathione (GSH/GSSG) ratios were significantly higher than those under conditions of negligible glucose production. Changes in the intracellular GSH/GSSG ratio corresponded to those in glucose-6-phosphate content and reduced nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP(+)) ratio obtained from malate/pyruvate measurements. Gluconeogenesis stimulation by extracellular adenosine triphosphate (ATP) or inosine caused an elevation of the intracellular GSH/GSSG and NADPH/NADP(+) ratios, as well as glucose-6-phosphate level. Surprisingly, in the presence of 5 mmol/L glucose, both the intracellular GSH/GSSG and NADPH/NADP(+) ratios and glucose-6-phosphate content were almost as low as under conditions of negligible glucose synthesis. L-buthionine sulfoximine (BSO)-induced decline in both the intracellular glutathione level and redox state resulted in inhibition of gluconeogenesis accompanied by accumulation of phosphotrioses and a decrease in fructose-1,6-bisphosphate content, while cysteine precursors altered neither GSH redox state nor the rate of glucose formation. In view of the data, it seems likely that: (1) intensive gluconeogenesis rather than extracellular glucose is responsible for maintaining a high intracellular GSH/GSSG ratio due to effective glucose-6-phosphate delivery for NADPH generation via the pentose phosphate pathway; (2) a decline in the intracellular glutathione level and/or redox state causes a decrease in glucose synthesis resulting from a diminished flux through aldolase; (3) induced by cysteine precursors, elevation of the intracellular GSH level does not affect the rate of glucose formation, probably due to no changes in the intracellular GSH/GSSG ratio.     

Antioxidant pathways in human aged skeletal muscle: relationship with the distribution of type II fibers

Type II fiber loss and reactive oxygen species (ROS)-induced damage are hallmarks of muscle aging. The aim of this study was to analyze whether there exists a relationship between age-dependent changes in cellular antioxidant capacity and type II fiber loss in aged human skeletal muscles. Forty-five male and female subjects ranging in age from 65 to 90 year-old were divided into +40 and -40% type II fiber groups. We measured both total and Mn superoxide dismutase (total and MnSOD), glutathione peroxidase (GSHPx) and catalase (CAT) activities. We also measured the reduced and oxidized forms of glutathione (GSH and GSSG) and lipid peroxide (LPO) levels. Total SOD activity was lower in the -40% type II fiber group than in the +40% group; MnSOD tended to be lower but data are not statistically consistent. Both GSHPx and CAT activities remained unchanged; as did GSH, GSSG and GSH/GSSG ratio. Finally, muscle samples with -40% type II fibers had a significantly higher LPO content compared to those with +40% type II fibers. In summary, a relationship between human skeletal muscle aging, type II fiber loss and ROS reactions seems to exist.     

Insulin decreases intracellular oxidative stress in patients with type 2 diabetes mellitus

Patients affected by diabetes mellitus have oxidative stress with an impaired glutathione (GSH) redox state. The objective of this study was to determine the influence of insulin on oxidative stress, defined as a reduced intracellular GSH/GSH disulfide (GSSG) ratio and lipid peroxidation by plasma thiobarbituric acid reactive substances (TBARSs) in patients with type 2 diabetes. Two experimental interventions were used: (1) measurement of GSH/GSSG ratio after insulin incubation in erythrocytes from 10 type 2 diabetic patients, and (2) measurement of intraerythrocytic GSH/GSSG ratio and plasma TBARS in 14 type 2 diabetic patients during an in vivo hyperinsulinemic condition obtained from a euglycemic hyperinsulinemic clamp study. We confirmed that our patients underwent oxidative stress as shown by the significant difference in intracellular GSH/GSSG ratio in diabetic patients as compared to controls (13.56+/-3.84 vs 27.89+/-8.37, P<.0001). We found a significant elevation in the GSH/GSSG ratio after 2 hours of incubation with insulin in erythrocytes from diabetic patients (11.56+/-1.98 to 15.61+/-2.62, P<.001). During the clamp studies, GSH/GSSG ratio had already increased after 60 minutes and even more after 120 minutes (baseline, 15.04+/-4.19; at 60 minutes, 19.74+/-6.33; at 120 minutes, 25.33+/-11.15; P<.0001). On the contrary, no significant changes were observed in plasma TBARS (3.59+/-0.77 to 3.56+/-0.83, NS). We conclude that insulin in patients with type 2 diabetes mellitus can reduce intracellular oxidative stress through increased GSH/GSSG ratio.     

氧化型谷胱甘肽和辅酶Ⅱ与颈动脉粥样硬化的关系研究

目的 探讨氧化还原态失衡与动脉粥样硬化的关系。方法 应用高分辨超声技术检测颈动脉内膜增厚度,将167例住院患者分为：斑块形成组（A组）79例,内膜中层增厚组（B组）52例,内膜中层正常组（C组）36例。取静脉血,测血浆还原型谷胱甘肽（GSH）与氧化型谷胱甘肽（GSSG）,还原型辅酶Ⅱ（NADPH）与氧化型辅酶Ⅱ（NADP^＋）,氧化型低密度脂蛋白（OX—LDL）和丙二醛（MDA）浓度。计算GSH／GSSG,NADP^＋／NADPH的氧化还原电位,该电位与OX—LDL行相关性分析。结果随内膜增厚（从C组到A组）,GSH、GSH／GSSG渐次减低,GSH／GSSG氧化还原电位渐次升高（P〈0．05）;NADPH、NADPH／NADP^＋氧化还原态亦显示出与GSH、GSH／GSSG类似,但较弱的变化,氧化应激损伤产物ox-LDL、MDA随内膜增厚明显增加（P〈0．05）;GSH／GSSG氧化还原电位与OX-LDL明显正相关（P〈0．05）。随着动脉内膜增厚、斑块形成,血浆GSH／GSSG氧化还原态向氧化方向明显偏移。结论 氧化还原态失衡、向氧化方向偏移与脂质的氧化损伤、内膜增厚相关,与动脉粥样硬化的发生发展可能存在某种紧密的内在联系。     

Role of N-acetylcysteine and GSH redox system on total and active MMP-2 in intestinal myofibroblasts of Crohn's disease patients

Purpose

Intestinal subepithelial myofibroblasts (ISEMFs)¹ are the predominant source of matrix metalloproteinase-2 (MMP-2) in gut, and a decrease in glutathione/oxidized glutathione (GSH/GSSG) ratio, intracellular redox state index, occurs in the ISEMFs of patients with Crohn’s disease (CD). The aim of this study is to demonstrate a relationship between MMP-2 secretion and activation and changes of GSH/GSSG ratio in ISEMFs stimulated or not with tumor necrosis factor alpha (TNFα).

Methods

ISEMFs were isolated from ill and healthy colon mucosa of patients with active CD. Buthionine sulfoximine, GSH synthesis inhibitor, and N-acetylcysteine (NAC), precursor of GSH synthesis, were used to modulate GSH/GSSG ratio. GSH and GSSG were measured by HPLC and MMP-2 by ELISA Kit.

Results

In cells, stimulated or not with TNFα, a significant increase in MMP-2 secretion and activation, related to increased oxidative stress, due to low GSH/GSSG ratio, was detected. NAC treatment, increasing this ratio, reduced MMP-2 secretion and exhibited a direct effect on the secreted MMP-2 activity. In NAC-treated and TNFα-stimulated ISEMFs of CD patients’ MMP-2 activity were restored to physiological value. The involvement of c-Jun N-terminal kinase pathway on redox regulation of MMP-2 secretion has been demonstrated.

Conclusion

For the first time, in CD patient ISEMFs, a redox regulation of MMP-2 secretion and activation related to GSH/GSSG ratio and inflammatory state have been demonstrated. This study suggests that compounds able to maintain GSH/GSSG ratio to physiological values can be useful to restore normal MMP-2 levels reducing in CD patient intestine the dysfunction of epithelial barrier.     

Maternal Alcohol Use During Pregnancy Causes Systemic Oxidation of the Glutathione Redox System

Background: Increased systemic oxidant stress contributes to a variety of maternal complications of pregnancy. Although the antioxidant glutathione (GSH) and its oxidized component glutathione disulfide (GSSG) have been demonstrated to be significantly altered in the adult alcoholic, the effects of maternal alcohol use during pregnancy on oxidant stress in the postpartum female remain under investigation. We hypothesized that maternal alcohol use would increase systemic oxidant stress in the pregnant female, evidenced by an oxidized systemic GSH redox potential. Methods: As a subset analysis of a larger maternal language study, we evaluated the effects of alcohol consumption during pregnancy on the systemic GSH redox status of the postpartum female. Using an extensive maternal questionnaire, postpartum women where queried regarding their alcohol consumption during pregnancy. Any drinking, the occurrence of drinking >3 drinks/occasion, and heavy drinking of >5 drinks/occasion during pregnancy were noted. Using HPLC, maternal plasma samples were analyzed for GSH, oxidized GSSG and the redox potential of the GSH/GSSG antioxidant pair calculated. Results: Maternal alcohol use occurred in 25% (83/321) of our study sample. Two in ten women reported consuming >3 drinks/occasion during pregnancy, while 1 in 10 women reported consuming alcohol at >5 drinks/occasion. Any alcohol use during pregnancy significantly decreased plasma GSH (p < 0.05), while alcohol at >3 drinks/occasion or >5 drinks/occasion significantly decreased plasma GSH concentration (p < 0.05), increased the percent of oxidized GSSG (p < 0.05), and substantially oxidized the plasma GSH redox potential (p < 0.05). Conclusions: Alcohol use during pregnancy, particularly at levels >3 drinks/occasion, caused significant oxidation of the systemic GSH system in the postpartum women. The clinical ramifications of the observed alcohol‐induced oxidation of the GSH redox system on high risk pregnancies or on the exposed offspring require more accurate identification and further investigation.     

Effects of glucose, Trolox-C, and glutathione disulphide on lipid peroxidation and cell death induced by oxidant stress in rat heart.

OBJECTIVE: The aim was to find effective protection of myocytes against peroxide induced damage in terms of preservation of contractile activity, protection against lipid peroxidation, and protection against cell death. METHODS: The components of the glutathione redox cycle, the production of malondialdehyde, cell contractions, and enzyme release from myocytes were measured in cultured neonatal rat heart cells before and after administration of cumene hydroperoxide, 80 mumol.litre-1. The protective action was tested of (1) glucose (10 mmol.litre-1) which stimulates the production of NADPH; (2) Trolox-C (0.16 mmol.litre-1) which is a water soluble analogue of alpha tocopherol and a scavenger of free radicals; and (3) GSSG (0.6 mmol.litre-1) which increases the intracellular concentrations of GSH and GSSG. RESULTS: Although the three substances tested were equally effective in reducing the formation of malondialdehyde, exogenous GSSG afforded only slight protection against cumene hydroperoxide induced cell death, whereas glucose and Trolox-C were highly effective protectors. The depressant effect of cumene hydroperoxide on beating frequency was not influenced by preincubation with GSSG, nor by coadministration of glucose, but Trolox-C was able to diminish the negative chronotropic action of cumene hydroperoxide. CONCLUSIONS: Effective protection against cumene hydroperoxide induced lipid peroxidation is not associated per se with effective protection against cumene hydroperoxide induced loss of beating frequency and cell death.     

Non-invasive evaluation of pulmonary glutathione in the exhaled breath condensate of otherwise healthy alcoholics

BACKGROUND: Chronic alcoholism is associated with an elevated risk for pulmonary infection and a 3-fold chance for incidence and mortality of acute respiratory distress syndrome with critical injury. Limited sampling of the alveolar lining fluid has restricted clinical studies of the role of glutathione (GSH) redox balance in pulmonary function and diseased states. Non-invasive sampling in the exhaled breath condensate (EBC) to monitor alveolar GSH would facilitate research in pulmonary oxidative stress. METHODS: EBC was collected from otherwise healthy subjects with and without a history of alcohol abuse. Reduced and oxidized EBC glutathione (GSH and GSSG, respectively), pH, and hydrogen peroxide were measured. RESULTS: GSH was statistically decreased in alcohol abusers only when normalized to protein (4.7nmol/mg protein [0.75, 11.4] vs. 13.4 [7.8, 26.4], p=0.03). In contrast, GSSG was significantly elevated in the EBC from alcohol abusers when compared to controls, 5.62 [0.45, 8.94] vs. 0.50nM [0.38, 0.80], p=0.03. Thus, a greater percentage was in the oxidized GSSG form when subjects abused alcohol (35.3% [11.8, 58.1] vs. 5.2 [3.6, 6.1], p<0.001). These concentrations represented a 40mV shift in GSH redox state towards a more oxidized state. CONCLUSIONS: Proper sample preparation was essential to prevent GSH loss and artificial oxidation. The shift in redox potential or %GSSG, which were not affected by dilution, may serve as better markers of pulmonary oxidative stress. Furthermore, these data suggested that the oxidant stress observed in the lavage fluid of otherwise healthy alcoholics could be measured non-invasively in the EBC.     

Effects of an oral glucose challenge on free radicals/antioxidants balance in an older population with type II diabetes

BACKGROUND: Our goal was to assess the effect of chronic vs acute hyperglycemia on free radicals (FR)/antioxidants balance in serum and leukocytes in an older (>65 years old) population with type II diabetes mellitus (DM). METHODS: Case-control study comparing older male patients with type II DM with normal controls. FRs and antioxidants were measured at baseline and 120 min after an oral 100-g glucose load. RESULTS: Baseline measurements showed an increased level of oxidized glutathione (GSSG) (p = .01) in the serum of diabetic subjects. Similar findings were observed at the intracellular level in the same group for GSSG (p = .0004), total glutathione (GSH + GSSG) (p = .0001) (GSH is reduced glutathione), decreased GSH/GSSG ratio (p = .0001), and ascorbic acid (p = .008). Monocytes from diabetic subjects produced larger amounts of nitric oxide (NO) in vitro (p = .03). After the oral glucose challenge, between-group comparisons demonstrated similar findings at the intracellular level for increased oxidized glutathione (p = .0004), GSH + GSSG (p = .0001), decreased GSH/GSSG (p = .0001), ascorbic acid (p = .02), and increased NO (p = .02) for the diabetic subjects. Within-group comparisons showed a significant drop of ascorbic acid in the control and the diabetic groups (p < .01), an increased level of GSSG in the diabetic group (p < .01), a decreased GSH/GSSG ratio (p < .05), and decreased production of NO by monocytes after in vitro stimulation in the control group (p < .05). CONCLUSIONS: Our results suggest that type II diabetes in an older population is associated with increased basal oxidative stress. Hyperglycemic challenge is associated with an accentuation of this phenomenon as measured in the leukocytes.     

Modulation of endometrial redox balance during the menstrual cycle: relation with sex hormones

This study aimed to evaluate the effects of changes in sex hormones occurring during the menstrual cycle on the redox balance and lipid peroxidation in normal human endometrial cells. Forty women, ages 21-41 yr, who were admitted to the Department of Gynecology and Obstetrics of the University of Bari for routine checkups or were treated for benign uterine disease, underwent endometrial biopsy and venipuncture. On the basis of histological examination, patients were allocated as follows: 10 in the early proliferative phase, 12 in the late proliferative phase, 8 in the early secretory phase, and 10 in the late secretory phase. LH, FSH (immunoradiometric essay), estradiol (E2), and progesterone (P(4)) (RIA) were determined in plasma samples. On the endometrial specimens, total glutathione (GSH), oxidized GSH (GSSG), malondialdehyde, and GSH peroxidase activity (GSH-Px) were determined. Significant cycle-dependent changes in endometrial GSH-Px (P < 0.0001), GSH (P < 0.001), and GSSG as a percentage of GSH (P < 0.0001) were observed. Malondialdehyde did not show significant differences. A linear regression model correlating sex hormone changes with redox indexes was performed. A significant positive correlation was observed between E2 and GSH-Px (r = 0.74; P = 0.0001), E2 and GSSG, as percentage of total (r = 0.84; P < 0.0001); a negative correlation was found between E2 and GSH (r = -0.57; P = 0.0001). No significant correlation was found between P(4) or FSH and oxidative balance. LH was found to be correlated with GSH-Px (r = 0.66; P = 0.0001) and GSSG as percentage of GSH (r = 0.5; P < 0.001). We conclude that the hormonal pattern is involved in maintaining the optimal redox balance in endometrium, mainly through modulation of GSH level and metabolism.     

Protein and non-protein sulfhydryls and disulfides in gastric mucosa and liver after gastrotoxic chemicals and sucralfate： Possible new targets of pharmacologic agents

AIM： To investigate the role of major non-protein and protein sulfhydryls and disulfides in chemically induced gastric hemorrhagic mucosal lesions （HML） and the mechanism of gastroprotective effect of sucralfate.
METHODS： Rats were given 1 mL of 75% ethanol, 25%NaCl, 0.6 mol/L HCI, 0.2 mol/L NaOH or 1% ammonia solutions intragastrically （i.g.） and sacrificed 1, 3, 6 or 12 min later. Total （reduced and oxidized） glutathione （GSH ＋ GSSG）, glutathione disulfide （GSSG）, protein free sulfhydryls （PSH）, protein-glutathione mixed disulfides （PSSG） and protein cystine disulfides （PSSP） were measured in gastric mucosa and liver.
RESULTS： Reduced glutathione （GSH） was depleted in the gastric mucosa after ethanol, HCI or NaCl exposure,while oxidized glutathione （GSSG） concentrations increased, except by HCI and NaOH exposure. Decreased levels of PSH after exposure to ethanol were observed,NaCl or NaOH while the total protein disulfides were increased. Ratios of reduced to oxidized glutathione or sulfhydrils to disulfides were decreased by all chemicals.No changes in thiol homeostasis were detected in the liver after i.g. abbreviation should be spelled out the first time here administration of ethanol. Sucralfate increased the concentrations of GSH and PSH and prevented the ethanol-induced changes in gastric mucosal thiol concentrations.
CONCLUSION： Our modified methods are now suitable for direct measurements of major protein and nonprotein thiols/disulfides in the gastric mucosa or liver.A common element in the pathogenesis of chemically induced HML and in the mechanism of gastroprotective drugs seems to be the decreased ratios of reduced and oxidized glutathione as well as protein sulfhydryls and disulfides.     

Melatonin attenuates diabetes-induced oxidative stress in rabbits

Oxidative stress is considered to be the main cause of diabetic complications. As the role of antioxidants in diabetes therapy is still underestimated, the aim of the present investigation was to study the antioxidative action of melatonin in comparison with N-acetylcysteine (NAC) under diabetic conditions. Alloxan-diabetic rabbits were treated daily with either melatonin (1 mg/kg, i.p.), NAC (10 mg/kg, i.p.) or saline. Blood glutathione redox state and serum hydroxyl free radicals (HFR), creatinine and urea levels were monitored. After 3 wk of treatment animals were killed and HFR content, reduced glutathione/oxidized glutathione (GSH/GSSG) ratio as well as the activities of glutathione reductase, glutathione peroxidase and gamma-glutamylcysteine synthetase were estimated in both liver and kidney cortex. Diabetes evoked a several-fold increase in HFR levels accompanied by a significant decline in GSH/GSSG ratio in serum and the examined organs. In contrast to NAC, melatonin (at 1/10 the dose of NAC) attenuated diabetes-induced alterations in glutathione redox state and HFR levels, normalized creatinine concentration and diminished urea content in serum. Moreover, the indole resulted in an increase in glutathione reductase activity in both studied organs and in a rise in glutathione peroxidase and gamma-glutamylcysteine synthetase activities in the liver. In contrast to NAC, melatonin seems to be beneficial for diabetes therapy because of its potent antioxidative and nephroprotective action. The indole-induced increase in the activities of the enzymes of glutathione metabolism might be of importance for antioxidative action of melatonin under diabetic conditions.     

Protection of myocytes against free radical-induced damage by accelerated turnover of the glutathione redox cycle

The primary defence mechanism of myocytes against peroxidesand peroxide-derived peroxyl and alkoxyl radicals is the glutathioneredox cycle. The purpose of the present study was to increasethe turnover rate of this cycle by stimulating the glutathioneperoxidase catalysed reaction (2GSHGSSG), the glutathione reductasecatalysed reaction (GSSG2GSH), or both, Neonatal rat heart cellcultures were subjected to a standardized protocol of oxidativestress using 80 µmol. l^–1 cumene hydroperoxide (CHPO)for 0–90 min. The consequences of this protocol were describedin terms of cellular concentrations of GSH, GSSG, NADPH andATP, formation of malondialdehyde (MDA), release of GSSG andof ATP catabolites, depression of contraction frequency, cellularcalcium overload, and enzyme release. Trolox-C, an analogue of vitamin E, accelerated the glutathioneperoxidase reaction leading to lowering of GSH concentrationand the GSH/GSSG ratio, less MDA formation, diminished negativechronotropy, delayed calcium overload, and less enzyme release.Glucose was used to accelerate the glutathione reductase reactionby supplying NADPH, leading to higher GSH concentration anda higher GSH/GSSG ratio, less MDA formation, diminished negativechronotropy, unchanged development of calcium overload, andless enzyme release. As a full turn of the glutathione redoxcycle involves both the peroxidase and the reductase reactions,the combination of Trolox-C and glucose was superior to eitherof the two alone: 90 min following addition of CHPO togetherwith Trolox-C and glucose, the GSH concentration and the GSH/GSSGratio were almost normal, MDA formation was extremely low, calciumoverload was markedly delayed, and enzyme release hardly occurredat all. Cells remained beating in the observation period of30 min. We conclude that the capacity of the glutathione redoxcycle to withstand oxidative stress can be increased by stimulationof either the peroxidase reaction or the reductase reaction,and that optimal redox cycling is achieved by stimulation ofboth reactions.