Glutathione and alpha-lipoate in diabetic rats: nerve function, blood flow and oxidative state.

BACKGROUND: Increased oxidative stress is considered to be a causal factor in the development of diabetic complications, among which peripheral neuropathy. The pathophysiology of nerve dysfunction in diabetes has been explained both by reduced endoneurial microcirculation and alterations in endoneurial metabolism. It is unclear whether antioxidants primarily improve nerve blood flow or normalise systemic or endoneurial oxidative metabolism. Therefore, we evaluated the effects of the antioxidants glutathione and alpha-lipoic acid on both nerve microcirculation and the antioxidative capacity and lipid peroxidation in experimentally diabetic rats. MATERIALS AND METHODS: Streptozotocin-diabetic rats were treated with different doses of alpha-lipoic acid, reduced glutathione or placebo, and were compared with nondiabetic controls. We measured systemic and endoneurial antioxidants, malondialdehyde and whole blood hydrogen peroxide. Furthermore, we evaluated sciatic and tibial motor and sensory nerve conduction velocity, caudal nerve conduction velocity, and assessed sciatic nerve blood flow and vascular resistance by Laser-Doppler flowmetry. RESULTS: We observed a rise in erythrocyte glutathione by 27 % (P < 0.05), and a trend towards decreased plasma malondialdehyde in alpha-lipoic acid, but not in glutathione-treated animals in comparison with the placebo group. Simultaneously, sciatic nerve blood flow and vascular resistance were improved by daily alpha-lipoic acid administration by 38% (P < 0.05). Peripheral nerve conduction velocity and endoneurial glutathione were not significantly influenced by antioxidant treatment. CONCLUSIONS: Only minor beneficial effects of alpha-lipoic acid on nerve blood flow and oxidative state occur at the given doses; these effects were insufficient to improve nerve conduction deficits.

Nerve conduction and antioxidant levels in experimentally diabetic rats: effects of streptozotocin dose and diabetes duration.

Oxidative stress supposedly plays a role in the pathogenesis of diabetic neuropathy. We have studied whether a variation in the streptozotocin (STZ) dose or diabetes duration affects the outcome of measurements of oxidative damage in relation to nerve conduction. In experiment 1, we induced diabetes in rats using 40 or 60 mg/kg STZ intravenously and assessed sciatic nerve conduction velocity. After 18 weeks, we measured plasma malondialdehyde (MDA) and red blood cell (RBC) and nerve glutathione levels. We observed a dose-dependent effect of STZ on body weight, and to a lesser extent on nerve conduction, but not on RBC or nerve glutathione and plasma MDA. In experiment 2, we administered a fixed dose of STZ (40 mg/kg) and measured antioxidants and MDA in RBCs, plasma, and sciatic nerve after 2, 4, 8, and 18 weeks in diabetic and control rats. RBC glutathione decreased in diabetic animals initially, but did not differ from control values after week 4. Plasma total glutathione increased until week 8. The ratio of total to oxidized glutathione in the sciatic nerve from diabetic animals paralleled the decrease observed in RBCs, and subsequently increased compared with controls. Nerve catalase increased in diabetic animals. Endoneurial MDA remained unchanged, whereas plasma MDA increased and RBC superoxide dismutase (SOD) decreased in the diabetic group. We conclude that differences in antioxidant levels between STZ-diabetic and control rats depend on the duration of hyperglycemia. Furthermore, dose-related effects of STZ on nerve conduction are not reflected in endoneurial lipid peroxidation or glutathione.

Low-dose N-acetylcysteine protects rats against endotoxin-mediated oxidative stress, but high-dose increases mortality.

We evaluated the effect of the antioxidant N-acetylcysteine (NAC) on oxidative stress, lung damage, and mortality induced by an endotoxin (lipopolysaccharide, or LPS) in the rat. Continuous intravenous infusion of 275 mg NAC/kg in 48 h, starting 24 h before LPS challenge, decreased hydrogen peroxide (H2O2) concentrations in whole blood (p < 0.01). This decrease was accompanied by fewer histologic abnormalities of the lung and decreased mortality (p < 0.025), compared with rats receiving LPS alone. N-Acetylserine, which has no sulfhydryl group, did not protect rats against LPS toxicity. Improved survival was not associated with an increase in pulmonary reduced glutathione, nor with inhibition of serum tumor necrosis factor (TNF) activity. In vitro, TNF production and DNA binding of nuclear factor kappa B (NF-kappaB) in human Mono Mac 6 cells was only inhibited at concentrations of NAC above 20 mM. High-dose NAC treatment (550 and 950 mg/kg in 48 h) decreased lung GSH (p < 0.05) and resulted in a significantly smaller number of surviving animals when compared with the low-dose NAC group (p < 0.025). In vitro, NAC increased hydroxyl radical generation in a system with Fe(III)-citrate and H2O2 by reducing ferric iron to its catalytic, active Fe2+ form. We conclude that low-dose NAC protects against LPS toxicity by scavenging H2O2, while higher doses may have the opposite effect.

Hydrogen peroxide modulation of the superoxide anion production by stimulated neutrophils.

Hydrogen peroxide (H2O2) pretreatment of human neutrophils results in a suppression of the superoxide anion (O2) production in response to surface-acting stimulants such as lipopolysaccharide (LPS) and opsonized zymosan. This effect was not observed when phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP) or tumor necrosis factor alpha (TNF alpha) were used as a stimuli. Since the response to PMA and other stimuli was unimpaired by preincubation with H2O2, we assume that the H2O2 modulated O2 production is probably due to alteration of the LPS receptor conformation rather than effecting directly NADPH-oxidase. The balance of reactive oxygen species (ROS) produced by neutrophils in the state of sepsis may thus be autoregulated by negative feedback phenomena of locally produced H202.

Nerve function and oxidative stress in diabetic and vitamin E-deficient rats.

Nerve dysfunction in diabetes is associated with increased oxidative stress. Vitamin E depletion also leads to enhanced presence of reactive oxygen species (ROS). We compared systemic and endoneurial ROS activity and nerve conduction in vitamin E-depleted control and streptozotocin-diabetic rats (CE- and DE-), and in normally fed control and diabetic animals (CE+ and DE+). Nerve conduction was reduced in both diabetic groups. Vitamin E depletion caused a small further nerve conduction deficit in the diabetic, but not in the control animals. The combination of vitamin E deficiency and streptozotocin-diabetes (group DE-) appeared to be lethal. In the remaining groups, an important rise in sciatic nerve malondialdehyde (MDA) was observed in the vitamin E-depleted control rats. In contrast, plasma MDA levels were elevated in group DE+ only, whereas hydrogen peroxide levels were increased in group CE-. Endoneurial total and oxidized glutathione and catalase were predominantly elevated in group DE+. These data show that nerve lipid peroxidation induced by vitamin E depletion does not lead to reduced nerve conduction or changes in antioxidant concentrations as observed in STZ-diabetes. The marked systemic changes in MDA and antioxidants suggest that nerve dysfunction in experimental hyperglycemia is rather a consequence of systemic than direct nerve damage.

Dimethylthiourea protects rats against gram-negative sepsis and decreases tumor necrosis factor and nuclear factor kappaB activity.

The thiol-containing compound dimethylthiourea (DMTU) is a known protectant in various models of oxidant-mediated tissue damage. Protective effects of DMTU have also been reported in studies on endotoxin-induced (LPS-induced) tissue injury. DMTU may exert this protective effect by reducing oxidative stress. In this study we investigated the effect of DMTU on survival, oxidative stress, and tumor necrosis factor (TNF) activity in two rat models of gram-negative bacterial sepsis. Intraperitoneal injection of 500 mg DMTU/kg protected against the lethal effects of intraperitoneally injected LPS (5 mg/kg) and live Salmonella typhimurium (3.3 x 10(10) CFU/kg). LPS injection resulted in oxidative stress, as indicated by an elevated concentration of hydrogen peroxide (H(2)O(2)) in normal and carbon monoxide-treated deproteinized blood. We also observed increased H(2)O(2) levels in animals injected with live Salmonella typhimurium. Although DMTU improved survival in both models, H(2)O(2) concentrations were not affected by it. This is consistent with our in vitro observation that DMTU is a weak H(2)O(2) scavenger. Serum TNF activity, however, was substantially decreased by DMTU, and this was associated with a reduced activation of nuclear factor kappaB in the peritoneal cells of LPS-treated rats. In addition, LPS-induced TNF production in vitro by rat peritoneal macrophages was inhibited by DMTU (p < 0.05). These results suggest that the protective effect of DMTU in gram-negative bacterial sepsis may be the result of a reduction in TNF activity. DMTU does not exert this effect by H(2)O(2) scavenging but may inactivate toxic H(2)O(2) metabolites.

Effects of insulin treatment on endoneurial and systemic oxidative stress in relation to nerve conduction in streptozotocin-diabetic rats.

As increased oxidative stress is probably a pathogenetic factor in the development of diabetic complications, we studied nerve function and endogenous antioxidants in plasma, erythrocytes and sciatic nerve of untreated and insulin-treated streptozotocin-diabetic rats. After 18 weeks, the diabetes-induced sciatic nerve conduction velocity deficits were approximately 65% improved by insulin (P < 0.001). Plasma superoxide dismutase was significantly reduced in diabetes (P < 0.01); smaller decreases in plasma catalase and glutathione levels were observed. These changes were corrected by insulin treatment. In erythrocytes, decreased superoxide dismutase (P < 0.05) and increased total glutathione levels (P < 0.05) were found. All effects of diabetes, including a rise in plasma malonyldialdehyde (P < 0.05), were partially reversed by insulin treatment. In nervous tissue, diabetes caused increased catalase activity, uninfluenced by insulin (P < 0.05). Nerve superoxide dismutase and glutathione did not change. The data suggest that in diabetes, changes in systemic rather than endoneurial oxidative stress lead to nerve dysfunction.

Effect of iron chelators on paraquat toxicity in rats and alveolar type II cells.

There is general agreement that the lung damage seen in paraquat poisoning is due to the generation of free radicals in alveolar epithelial cells. We have recently shown that the iron chelator and antioxidant deferoxamine (DF) reduces the mortality caused by paraquat in vitamin-E-deficient rats. In the present study we investigated the effect of DF and the lipid soluble iron chelator compound 51 (CP51) of the hydroxypyridin-4-one family on paraquat poisoning in rats with a normal vitamin E status and on isolated alveolar type II cells (ATTC). Adult rats were intravenously injected with a lethal dose of paraquat (40 mg/kg) while concurrent treatment with a continuous intravenous infusion of DF or CP51 was started. Survival of rats receiving DF at 25 and 50 mg/kg/24 h was not significantly increased compared with PBS-treated control animals. CP51, however, significantly (p less than 0.01) reduced the mortality caused by paraquat. When rats were treated with 25 mg/kg/24 h, eight of 15 rats survived the study period of 35 days compared with three in the PBS-treated control group (n = 27). In ancillary in vitro studies radiolabeled [51Cr]ATTC were incubated in a medium containing 100 microM paraquat in the absence or presence of DF and CP51. Paraquat-induced ATTC lysis increased to approximately 25% after 7 h of incubation. At the highest tested concentration (500 microM) of chelator, injury decreased markedly (80%), whereas at the lowest tested concentration (50 microM) cytotoxicity was not prevented.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of protection of alveolar type II cells against paraquat-induced cytotoxicity by deferoxamine.

Paraquat toxicity has been associated with the generation of free radicals in alveolar epithelial cells in which paraquat specifically accumulates via a polyamine uptake system. In the present study we investigated whether deferoxamine (DF), an iron chelator that has antioxidant capacity and that also has a polyamine-like structure, could protect alveolar type II cells (ATTC) against injury by paraquat. Radiolabeled [3H]adenine ATTC were incubated in a medium containing 75 microM paraquat in the absence or presence of DF (500 microM). After 3 hr of incubation paraquat-mediated cytotoxicity of ATTC, as measured by [3H]adenine release, was significantly (P less than 0.005) decreased by addition of DF (26.6 +/- 2.6% vs 7.4 +/- 1.7%). Accumulation of radiolabeled [14C]paraquat at a concentration of 75 microM was also decreased (70%) by 500 microM DF from 94.8 +/- 2.1 to 28.9 +/- 6.7 nmoles paraquat/2.5 x 10(5) ATTC. This effect of DF was dose dependent and comparable with the protective effect of equimolar concentrations of putrescine. However, per cent uptake of paraquat at a concentration of 500 microM was not significantly inhibited by DF (1 mM), whereas paraquat-induced injury was still markedly reduced (36.2 +/- 2.5% vs 2.6 +/- 4.2%). This indicated that the protective effect of DF could not be explained by its competition with paraquat on uptake alone. In the same series of experiments using another iron chelator, pyridoxal benzoyl hydrazone (PBH), which has antioxidant properties similar to DF but does not show its polyamine-like structure, ATTC lysis was also prevented although paraquat uptake was not reduced. These in vitro data indicate that the mechanism of protection by DF against paraquat toxicity in lung epithelial type II cells is two-fold: inhibition of paraquat uptake through its compliance with the structural requirements necessary for transport, and inhibition of paraquat-induced iron-catalysed free radical generation.