Vitamin E decreases the occurrence of malformations in the offspring of diabetic rats

An association between excess oxygen radical activity and disturbed embryogenesis in diabetic pregnancy has been suggested. In the present study, the protective capacity of vitamin E with different treatment regimens was investigated in early and late pregnancy of streptozotocin-induced diabetic rats. Daily gavaging of 0.2 g/kg or 0.8 g/kg of vitamin E exerted moderate protective effects. In contrast, treatment with a diet enriched with 2% (wt/wt) of vitamin E, yielding an approximate daily dosage of 2 g/kg of vitamin E, clearly restored both embryonic and fetal morphology. High-performance liquid chromatography measurement showed that maternal diabetes decreased embryonic content of vitamin E. When pregnant diabetic animals were supplemented with vitamin E, increased concentrations of the vitamin were found in maternal, embryonic, and fetal tissues. Thus, despite marked accumulation of vitamin E in maternal tissues, the compound apparently reached the conceptus. Thiobarbituric acid reactive substances (TBARS) were estimated as a measure of lipid peroxidation, and no changes were observed in maternal tissue, embryonic tissue, placenta, and fetal brain in the untreated diabetic group. In contrast, a fivefold increase of TBARS was found in fetal liver, a rise that was reduced with vitamin E treatment of the diabetic pregnant rats and completely normalized with 2% vitamin E in the diet. Congenital malformations caused by experimental diabetes can be prevented by antioxidants in vivo. These findings further corroborate the notion that an imbalance in the metabolism of free oxygen radicals is involved in the embryonic maldevelopment of diabetic pregnancy, and suggest a direction for prophylactic treatment in the future.     

Congenital malformations in experimental diabetic pregnancy: aetiology and antioxidative treatment. Minireview based on a doctoral thesis

Diabetes mellitus in pregnancy causes congenital malformations in the offspring. The aim of this work was to characterize biochemical and morphologic anomalies in the conceptus of an animal model of diabetic pregnancy. In addition, a preventive treatment against diabetes-induced dysmorphogenesis was developed. Congenital cataract was often found in the offspring of diabetic rats. The fetal lenses had increased water accumulation, sorbitol concentration and aldose reductase activity compared to control lenses. The results suggest that the cataracts form via osmotic attraction of water due to sorbitol accumulation in the fetal lens. Another set of malformations, with possible neural crest cell origin, occurred frequently in offspring of diabetic rats. These included low set ears, micrognathia, hypoplasia of the thymus, thyroid and parathyroid glands, as well as anomalies of the heart and great vessels. Furthermore, diabetes caused intrauterine death and resorptions more frequently in the late part of gestation. When the pregnant diabetic rats were treated with the antioxidants butylated hydroxytoluene, vitamin E or vitamin C, the occurrence of gross malformations was reduced from approximately 25% to less than 8%, and late resorptions from 17% to 7%. This suggests that an abnormal handling of reactive oxygen species (ROS) is involved in diabetes-induced dysmorphogenesis in vivo. Indeed, an increased concentration of lipid peroxides, indicating damage caused by ROS, was found in fetuses of diabetes rats. In addition, embryos of diabetic rats had low concentrations of the antioxidant vitamin E compared to control embryos. These biochemical alterations were normalized by vitamin E treatment of the pregnant diabetic rats. The antioxidants are likely to have prevented ROS injury in the embryos of the diabetic rats, in particular in the neural crest cells, thereby normalizing embryonic development. These results provide a rationale for developing new anti-teratogenic treatments for pregnant women with diabetes mellitus.     

Maternal administration of superoxide dismutase and catalase in phenytoin teratogenicity

Embryonic bioactivation and formation of reactive oxygen species (ROS) are implicated in the mechanism of phenytoin teratogenicity. This in vivo study in pregnant CD-1 mice evaluated whether maternal administration of the antioxidative enzymes superoxide dismutase (SOD) and/or catalase conjugated with polyethylene glycol (PEG) could reduce phenytoin teratogenicity. Initial studies showed that pretreatment with PEG-SOD alone (0.5-20 KU/kg i.p. 4 or 8 h before phenytoin) actually increased the teratogenicity of phenytoin (65 mg/kg i.p. on gestational days [GD] 11 and 12, or 12 and 13) (p < .05), and appeared to increase embryonic protein oxidation. Combined pretreatment with PEG-SOD and PEG-catalase (10 KU/kg 8 or 12 h before phenytoin) was not embryo-protective, nor was PEG-catalase alone, although PEG-catalase alone reduced phenytoin-initiated protein oxidation in maternal liver (p < .05). However, time-response studies with PEG-catalase (10 KU/kg) on GDs 11, or 11 and 12, showed maximal 50-100% increases in embryonic activity sustained for 8-24 h after maternal injection (p < .05), and dose-response studies (10-50 KU/kg) at 8 h showed maximal respective 4-fold and 2-fold increases in maternal and embryonic activities with a 50 KU/kg dose (p < .05). In controls, embryonic catalase activity was about 4% of that in maternal liver, although with catalase treatment, enhanced embryonic activity was about 2% of enhanced maternal activity (p < .05). PEG-catalase pretreatment (10-50 KU/kg 8 h before phenytoin) also produced a dose-dependent inhibition of phenytoin teratogenicity, with maximal decreases in fetal cleft palates, resorptions and postpartum lethality at a 50 KU/kg dose (p < .05). This is the first evidence that maternal administration of PEG-catalase can substantially enhance embryonic activity, and that in vivo phenytoin teratogenicity can be modulated by antioxidative enzymes. Both the SOD-mediated enhancement of phenytoin teratogenicity, and the inhibition of phenytoin teratogenicity by catalase, indicate a critical role for ROS in the teratologic mechanism, and the teratologic importance of antioxidative balance.     

Teratogenicity of 3-deoxyglucosone and diabetic embryopathy

The increased rate of embryonic dysmorphogenesis in diabetic pregnancy is correlated with the severity and duration of the concurrent hyperglycemia during early gestation. Whole embryo culture was used to investigate a possible association of hyperglycemia-induced disturbances of embryo development with tissue levels of the three alpha-oxoaldehydes: glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG). Rat embryos exposed to high glucose levels in vitro showed severe dysmorphogenesis and a 17-fold increased concentration of 3-DG compared with control embryos cultured in a low glucose concentration. Exogenous 3-DG (100 micromol/l) added to the medium of control cultures yielded an increased embryonic malformation rate and a 3-DG concentration similar to that of embryos cultured in high glucose. Addition of superoxide dismutase (SOD) to the culture medium decreased the malformation rates of embryos exposed to either high glucose or high 3-DG levels, but it did not decrease the high embryonic 3-DG concentrations caused by either agent. Our results implicate the potent glycating agent 3-DG as a teratogenic factor in diabetic embryopathy. In addition, the anti-teratogenic effect of SOD administration appears to occur downstream of 3-DG formation, suggesting that 3-DG accumulation leads to superoxide-mediated embryopathy.     

Subcellular distribution of oxygen free radical scavenging enzymes in goat ovary

Oxygen free radical scavenging enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase were heterogeneously distributed in goat ovary. Activities of SOD and catalase were predominantly located in cytosolic fractions compared to mitochondrial and microsomal fractions. Most of the peroxidase activity was observed in microsomal fractions with little activity in cytosolic and mitochondrial fractions. Higher activities of all these enzymes were in luteal phase as compared to follicular phase. Most of the activities of these enzymes in luteal phase were restricted to luteal cells while in follicular phase these were mainly present in the follicles of the ovary.     

Superoxide dismutase restores impaired histamine-induced increases in venular macromolecular efflux during diabetes mellitus

The goal of this study was to determine the role of oxygen radicals in impaired histamine-induced increases in venular macromolecular efflux from the hamster cheek pouch. We used intravital fluorescent microscopy and fluorescein isothiocyanate dextran (FITC-dextran; MW = 70K) to examine macromolecular extravasation from post-capillary venules in nondiabetic and diabetic (2-4 weeks after injection of streptozotocin) hamsters in response to histamine. Increases in extravasation of macromolecules were quantitated by counting venular leaky sites and by calculating clearance (ml/s x 10(-6)) of FITC-dextran-70K. In nondiabetic hamsters, superfusion with histamine (1.0 and 5.0 microM) increased venular leaky sites from 0 +/- 0 to 17 +/- 6 and 35 +/- 6 per 0.11 cm2, respectively. In addition, clearance of FITC-dextran-70K increased during superfusion with histamine. In contrast, superfusion with histamine did not increase the formation of venular leaky sites (0 +/- 0) or clearance of FITC-dextran-70K in diabetic hamsters. Next, we examined whether alterations in histamine-induced increases in macromolecular efflux in diabetic hamsters may be related to the production of oxygen radicals. We examined whether exogenous application of superoxide dismutase (150 U/ml) could restore impaired histamine-induced increases in macromolecular extravasation in diabetic hamsters. Application of superoxide dismutase did not alter histamine-induced increases in venular leaky sites or clearance of FITC-dextran-70K in nondiabetic hamsters. However, application of superoxide dismutase restored histamine-induced increases in leaky site formation and clearance of FITC-dextran-70K in diabetic hamsters towards that observed in nondiabetic hamsters. These findings suggest that oxygen radical formation appears to contribute to impaired macromolecular efflux in response to histamine during short-term diabetes mellitus.     

Rorschach patterns of response in Vietnam veterans with posttraumatic stress disorder versus combat and normal controls

The aim of the present study was to determine whether maternal diabetes affects rat embryo and yolk sac apoptosis during the postimplantation period. Severely malformed and growth-retarded embryos of gestational day 12 from diabetic rats exhibited pronounced DNA laddering on agarose gels. On the other hand, no DNA laddering could be observed in any of the non-malformed embryos from control and diabetic rats, or in their corresponding yolk sacs. Analysis of embryos of gestational day 10 revealed only a few scattered TUNEL positive cells mainly located in the allantois, the foregut epithelium, the cranial neuroepithelium and in the cranial mesenchyme. Embryonic tissue of gestational day 12 showed numerous aggregates of TUNEL-positive cells, indicating developmental remodelling of multiple organs. Analysis of non-malformed embryos of day 10 and 12 revealed a distribution and frequency of TUNEL positive cells unaffected by the diabetic state of the mother on both days. In vitro incubation (2-8 hr) of normal day-12 yolk sacs resulted in strong DNA laddering, but not in the corresponding embryos. Dispersed yolk sac cells generated higher levels of reactive oxygen species than dispersed embryonic cells. Reactive oxygen species levels in both embryonic and yolk sac cells were unaffected by the diabetic state of the mother. Moreover, immunoblot analysis showed high Bcl-2 and undetectable caspase-1 levels in embryos from both normal and diabetic rats and low Bcl-2 and high caspase-1 levels in the corresponding yolk sacs. Immunohistochemical analysis of embryos demonstrated caspase-1-reactivity in a small subpopulation of cells located in proximity to TUNEL-positive cells. We conclude that the inherent capacity of embryonic cells to enter apoptosis in vitro is low as compared to yolk sac cells, and that wide-spread apoptosis is not likely to play a major role in diabetes-induced dysmorphogenesis but rather in early phases of resorption of severely malformed and developmentally retarded embryos.     

Role of polymorphonuclear leucocytes and oxygen-derived free radicals in the formation of gastric lesions induced by HCl/ethanol, and a possible mechanism of protection by anti-ulcer polysaccharide

This study examined the role of oxygen-derived free radicals in the pathogenesis of gastric mucosal lesions induced by HCl/ethanol. Superoxide dismutase, and catalase, and their combination reduced gastric lesion formation in mice. Gastric lesions were also reduced in mice treated with cyclophosphamide or anti-neutrophils, but not in mice treated with allopurinol or desulphated-carrageenan. Cobra venom factor did not reduce lesion formation. These results suggested that oxygen-free radicals may contribute to the formation of gastric mucosal lesions induced by HCl/ethanol, and that oxygen radicals were generated from neutrophils but not from xanthine oxidase. Anti-ulcer pectic polysaccharide, bupleuran 2IIc, which was recently isolated from the roots of Bupleurum falcatum L., showed potent inhibition of HCl/ethanol-induced gastric lesions in mice. Bupleuran 2IIc seemed to scavenge hydroxyl radical effectively. It was suggested that this anti-ulcer polysaccharide may provide protection to the gastric mucosa by scavenging oxygen-free radicals.     

Maternal adjustment and infant outcome in medically defined high-risk pregnancy.

Biological and psychosocial risk factors in high-risk pregnancy and their relation to infant developmental outcomes were explored in a sample of 153 pregnant Israeli women who had pregestational diabetes melfitus, gestational diabetes mellitus, or were nondiabetic. Questionnaires on coping and resources as well as well-being and distress during the 2nd trimester were administered. Estimates of maternal fuels (HbAlc and fructosamine) were obtained throughout pregnancy. At 1 year, offspring were administered the Bayley Scales of Infant Development and mother-infant interactions were observed. Infants of mothers in the diabetic groups scored lower on the Bayley Scales and revealed fewer positive and more negative behaviors than did infants of mothers in the nondiabetic group. Infant outcomes in the maternal diabetic groups were associated with maternal metabolism. Maternal coping and resources differed in the 3 groups and differentially predicted infant development. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The Diabetes in Early Pregnancy Study: beta-hydroxybutyrate levels in type 1 diabetic pregnancy compared with normal pregnancy. NICHD-Diabetes in Early Pregnancy Study Group (DIEP). National Institute of Child Health and Development

OBJECTIVE: The objective was to assess relationships between beta-hydroxybutyrate (beta-OHB) level and pregnancy outcome in human pregnancy in light of the fact that high levels of beta-OHB cause malformations and growth retardation in in vitro studies. RESEARCH DESIGN AND METHODS: We analyzed beta-OHB in prospectively collected specimens from the National Institute of Child Health and Human Development-Diabetes in Early Pregnancy Study, in gestational weeks 6-12 in diabetic (n = 204-239) and nondiabetic (n = 316-332) pregnant women. RESULTS: Levels of beta-OHB in diabetic women were 2.5-fold higher than in nondiabetic pregnant women at 6 weeks' gestation and declined to 1.6-fold above nondiabetic women by 12 weeks' gestation (P < 0.0001 at all times). beta-OHB was positively correlated with glucose levels (P < 0.0001) in diabetic mothers, probably reflecting degree of diabetic control. beta-OHB correlated inversely with glucose (P < 0.0003) (gestational week 6 only) in nondiabetic mothers, possibly reflecting caloric intake. beta-OHB tended to be lower (not higher) in diabetic and nondiabetic mothers with malformed infants or pregnancy losses, but the difference was not statistically significant. beta-OHB in diabetic mothers at 8, 10, and 12 weeks correlated inversely with birth weight (P = 0.004-0.02), even after adjusting for maternal glucose levels. beta-OHB levels were also generally lower in diabetic mothers of macrosomic infants, and week 12 ultrasound crown-rump measurements were inversely related to beta-OHB levels. CONCLUSIONS: The lst trimester beta-OHB is significantly higher in diabetic than nondiabetic pregnant women. In both groups, beta-OHB tended to be lower, not higher, in mothers who had a malformed infant or pregnancy loss. beta-OHB was inversely related to crown-rump length and birth weight. The modest beta-OHB elevation in the 1st trimester of reasonably well-controlled diabetic pregnancy is not associated with malformations, probably because beta-OHB levels causing malformations in embryo culture models are 20- to 40-fold higher. The mechanism of the beta-OHB association with impaired fetal growth is unknown.     

Multicenter comparison of first- and second-generation IMx tacrolimus microparticle enzyme immunoassays in liver and kidney transplantation

Foetuses born to mothers with gestational diabetes are at increased risk of developing respiratory distress, foetal macrosomia, foetal anomalies and platelet hyperaggregability. High blood glucose level induces oxidative stress and decreases antioxidant defences. The present study discusses the possibility of lipid peroxidation and protein oxidation in both maternal and foetal erythrocytes as an indicator of oxygen radical activity. The level of lipid peroxidation and protein oxidation in erythrocytes was estimated in 20 mothers with gestational diabetes and their newborns. The maternal age varied between 19 and 42 y and foetal age ranged between 34 and 39 weeks. The proteolytic activities in the erythrocyte lysates obtained from mothers with gestational diabetes and their newborns were significantly greater [(mean +/- SD) 24.41 +/- 9.05 and 16.70 +/- 3.36 microM of amino groups/g haemoglobin, n = 20, respectively] than those from control group (10.18 +/- 4.84 and 14.64 +/- 6.21 microM amino groups/g haemoglobin, n = 15, respectively; p < 0.05 in both cases). Similarly erythrocyte malondialdehyde levels were significantly elevated in babies born to mothers with gestational diabetes (10.11 +/- 2.21 nM/g haemoglobin) when compared to controls (6.8 +/- 3.75 nM/g haemoglobin) (p < 0.05). In the erythrocytes of mothers with gestational diabetes, malondialdehyde levels correlated significantly with glycated haemoglobin levels (p < 0.01). The results of this study indicate that the oxidative stress induced by gestational diabetes manifests as increased lipid peroxidation and protein oxidative damage in the erythrocytes of both mothers with gestational diabetes and their newborn infants.     

Direct measurement of oxygen free radicals during in utero hypoxia in the fetal guinea pig brain

The present study tested the hypothesis that maternal hypoxia induces oxygen free radical generation in the fetal guinea pig brain utilizing techniques of electron spin resonance spectroscopy and alpha-phenyl-tert-butyl nitrone (PBN) spin trapping. Pregnant guinea pigs of 60 days gestation were divided into normoxic and hypoxic groups and exposed to 21% or 7% oxygen for 60 min. Free radical generation was documented by measuring the signal of PBN spin adducts. Fluorescent compounds were determined as an index of lipid peroxidation and the activity of Na+,K+-ATPase was determined as an index of brain cell membrane function. Hypoxic fetal cerebral cortical tissue showed a significant increase in spin adducts (normoxic: 33.8+/-9.3 units/g tissue vs. hypoxic: 57.9+/-9.2 units/g tissue, p<0.01) and fluorescent compounds (normoxic: 0.639+/-0.054 microg quinine sulfate/g brain vs. 0.810+/-0.102 microg quinine sulfate/g brain, p<0.01) and a decrease in Na+,K+-ATPase activity (normoxic: 43.04+/-2.50 micromol Pi/mg protein/h vs. hypoxic: 33. 80+/-3.51 micromol Pi/mg protein/h, p<0.001). These results demonstrate an increased free radical generation during hypoxia in the fetal guinea pig brain. The spectral characteristics of the radicals were consistent with those of alkoxyl radicals. The increased level of fluorescent compounds and decreased activity of Na+,K+-ATPase indicated hypoxia induced brain cell membrane lipid peroxidation and dysfunction, respectively. These results directly demonstrate an increased oxygen free radical generation during hypoxia and suggest that hypoxia-induced increase in lipid peroxidation and decrease in membrane function, as indicated by a decrease in Na+,K+-ATPase activity, are consequences of increased free radicals. The nature of predominantly present alkoxyl radical indicates ongoing lipid peroxidation during hypoxia. The direct demonstration of oxygen free radical generation during hypoxia is the critical missing link in the mechanism of hypoxia-induced brain cell membrane dysfunction and damage.     

Oxidative damage in chemical teratogenesis

The teratogenicity of many xenobiotics is thought to depend at least in part upon their bioactivation by embryonic cytochromes P450, prostaglandin H synthase (PHS) and lipoxygenases (LPOs) to electrophilic and/or free radical reactive intermediates that covalently bind to or oxidize cellular macromolecules such as DNA, protein and lipid, resulting in in utero death or teratogenesis. Using as models the tobacco carcinogens benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the anticonvulsant drug phenytoin, structurally related anticonvulsants (e.g. mephenytoin, nirvanol, trimethadione, dimethadione) and the sedative drug thalidomide, we have examined the potential teratologic relevance of free radical-initiated, reactive oxygen species (ROS)-mediated oxidative molecular target damage, genotoxicity (micronucleus formation) and DNA repair in mouse and rabbit models in vivo and in embryo culture, and in vitro using purified enzymes or cultured rat skin fibroblasts. These teratogens were bioactivated by PHS and LPOs to free radical reactive intermediary metabolites, characterized by electron spin resonance spectrometry, that initiated ROS formation, including hydroxyl radicals, which were characterized by salicylate hydroxylation. ROS-initiated oxidation of DNA (8-hydroxy-2'-deoxyguanosine formation), protein (carbonyl formation), glutathione (GSH) and lipid (peroxidation), and embryotoxicity were shown for phenytoin, its major hydroxylated metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin [HPPH], thalidomide, B[a]P and NNK in vivo and/or in embryo culture, the latter indicating a teratologically critical role for embryonic, as distinct from maternal, processes. DNA oxidation and teratogenicity of phenytoin and thalidomide were reduced by PHS inhibitors. Oxidative macromolecular lesions and teratogenicity also were reduced by the free radical trapping agent phenylbutylnitrone (PBN), and the antioxidants caffeic acid and vitamin E. In embryo culture, addition of superoxide dismutase (SOD) to the medium enhanced embryonic SOD activity, and SOD or catalase blocked the oxidative lesions and embryotoxicity initiated by phenytoin and B[a]P, suggesting a major contribution of ROS, as distinct from covalent binding, to the teratologic mechanism. In in vivo studies, other antioxidative enzymes like GSH peroxidase, GSH reductase and glucose-6-phosphate dehydrogenase (G6PD) were similarly protective. Even untreated G6PD-deficient mice had enhanced embryopathies, indicating a teratological role for endogenous oxidative stress. In cultured fibroblasts, B[a]P, NNK, phenytoin and HPPH initiated DNA oxidation and micronucleus formation, which were inhibited by SOD. Oxidation of DNA may be particularly critical, since transgenic mice with +/- or -/- deficiencies in the p53 tumor suppressor gene, which facilitates DNA repair, are more susceptible to phenytoin and B[a]P teratogenicity. Even p53-deficient mice treated only with normal saline showed enhanced embryopathies, suggesting the teratological importance of endogenous oxidative stress, as observed with G6PD deficiency. These results suggest that oxidative macromolecular damage may play a role in the teratologic mechanism of xenobiotics that are bioactivated to a reactive intermediate, as well in the mechanism of embryopathies occurring in the absence of xenobiotic exposure.     

Oxygen-derived free radicals contribute to baroreceptor dysfunction in atherosclerotic rabbits

The goal of the present study was to determine whether oxygen-derived free radicals contribute to baroreceptor dysfunction in atherosclerosis. Baroreceptor activity was measured from the carotid sinus nerve during pressure ramps in isolated carotid sinuses of anesthetized rabbits. Rabbits fed a 0.5% to 1.0% cholesterol diet for 7.9 +/- 0.4 months (mean +/- SE; range, 5.5 to 10) developed atherosclerotic lesions in the carotid sinuses. Maximum baroreceptor activity measured at 140 mm Hg and the slope of the pressure-activity curve were reduced in atherosclerotic (n = 15) compared with normal (n = 13) rabbits (425 +/- 34 versus 721 +/- 30 spikes per second and 6.2 +/- 0.6 versus 10.8 +/- 0.8 spikes per second per mm Hg, respectively, P < .05). The level of activity was inversely related to plasma cholesterol concentration (r = .86, P < .001) and total cholesterol load (plasma concentration x duration of diet, r = .92). Mean arterial pressure was normal in both groups. Exposure of the carotid sinus to the free-radical scavengers superoxide dismutase (SOD) and catalase significantly increased maximum baroreceptor activity by 25 +/- 4% in atherosclerotic rabbits (n = 6) but caused only small and irreversible changes in activity in normal rabbits (n = 8). Catalase alone but not SOD also increased baroreceptor activity in atherosclerotic rabbits (n = 7). Exposure of the carotid sinus of normal rabbits to exogenous free radicals generated from the reaction between xanthine and xanthine oxidase inhibited baroreceptor activity in a dose-dependent and reversible manner (n = 8, P < .05). The inhibition of activity was attenuated by SOD and catalase but was not attenuated by the inhibitor of hydroxyl radical formation, deferoxamine. Neither restoration of baroreceptor activity in atherosclerotic rabbits by catalase nor inhibition of activity by xanthine/xanthine oxidase could be explained by changes in the carotid pressure-diameter relation or prostacyclin formation. These results indicate that oxidant stress inhibits baroreceptor activity and that endogenous oxyradicals produced in atherosclerotic carotid sinuses contribute to baroreceptor dysfunction.     

Effect of sprint cycle training on activities of antioxidant enzymes in human skeletal muscle

The effect of intermittent sprint cycle training on the level of muscle antioxidant enzyme protection was investigated. Resting muscle biopsies, obtained before and after 6 wk of training and 3, 24, and 72 h after the final session of an additional 1 wk of more frequent training, were analyzed for activities of the antioxidant enzymes glutathione peroxidase (GPX), glutathione reductase (GR), and superoxide dismutase (SOD). Activities of several muscle metabolic enzymes were determined to assess the effectiveness of the training. After the first 6-wk training period, no change in GPX, GR, or SOD was observed, but after the 7th week of training there was an increase in GPX from 120 +/- 12 (SE) to 164 +/- 24 mumol.min-1.g dry wt-1 (P < 0.05) and in GR from 10.8 +/- 0.8 to 16.8 +/- 2.4 mumol.min-1.g dry wt-1 (P < 0.05). There was no significant change in SOD. Sprint cycle training induced a significant (P < 0.05) elevation in the activity of phosphofructokinase and creatine kinase, implying an enhanced anaerobic capacity in the trained muscle. The present study demonstrates that intermittent sprint cycle training that induces an enhanced capacity for anaerobic energy generation also improves the level of antioxidant protection in the muscle.     

Blood activities of antioxidant enzymes in alcoholics before and after withdrawal

Since free radicals and peroxides seem to be involved in the toxicity of alcohol, several authors have examined the variations of blood activities of antioxidant enzymes in alcoholics, but published results are somewhat conflicting. In this study, erythrocyte (E) activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT), and plasma (P) activities of SOD and GPX were measured in 58 male alcoholics without evidence of severe liver disease before and after a 21-day weaning period, and in a control group of 78 healthy men. Before abstinence, E-SOD and E-GPX activities were, respectively, 6.8% and 13.0% lower in alcoholics than in controls (p < or = .05 and p < or = .01, respectively), whereas the slight increases of E-CAT, P-SOD and P-GPX were not statistically significant. After 21 days of abstinence, no change in activities of the erythrocyte enzymes was noticed; conversely, P-SOD activity was reduced by 8.3% (p < or = .01) and P-GPX by 23.3% (p < or = .001). Variations of blood antioxidant enzymes observed in patients were of limited amplitude and do not allow the use of either of them as markers of alcohol abuse.     

Diminution of mouse epidermal superoxide dismutase and catalase activities by tumor promoters

The effects of phorbol ester tumor promoters and related compounds on superoxide dismutase (SOD) and catalase were examined. The treatment of adult mouse skin with 2 micrograms 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a sustained decrease in the basal levels of both SOD and catalase activities in the epidermis. A decline in SOD activity occurred within 3 h after application and the maximum effect was seen at 16--17 h. The decrease in SOD activity was always accompanied by a similar decline in the epidermal catalase activity. The alterations in both enzymes occurred against a high background of enhanced protein synthesis which indicates that the effect of TPA is selective for SOD and catalase. Other tumor promoters such as phorbol 12,13-dibutyrate and the non-phorbol tumor promoter anthraline also lowered the activities of both the enzymes. Mezerein, a resiniferonol derivative with weak promoting activity but a potent stage-II promoter, appeared to be more potent than TPA in lowering the basal levels. These results indicate that damage which favors neoplastic progression could occur in TPA-treated mouse skin due to the accumulation of free radicals resulting from low levels of SOD and catalase activity. In addition, the TPA-caused decrease in the levels of SOD and catalase was not prevented by either retinoic acid, fluocinolone acetonide, tosyl amino-2-phenylethyl chloromethyl ketone, or butylated hydroxytoluene, suggesting that inhibition of tumor promotion by these agents is not mediated through alterations in the levels of enzymatic activities which decrease free radical concentrations.     

Effects of tumor necrosis factor-alpha on glucose metabolism in cultured human muscle cells from nondiabetic and type 2 diabetic subjects

The effects of tumor necrosis factor-alpha (TNF alpha) on glucose uptake and glycogen synthase (GS) activity were studied in human skeletal muscle cell cultures from nondiabetic and type 2 diabetic subjects. In nondiabetic muscle cells, acute (90-min) exposure to TNF alpha (5 ng/ml) stimulated glucose uptake (73 +/- 14% increase) to a greater extent than insulin (37 +/- 4%; P < 0.02). The acute uptake response to TNF alpha in diabetic cells (51 +/- 6% increase) was also greater than that to insulin (31 +/- 3%; P < 0.05). Prolonged (24-h) exposure of nondiabetic muscle cells to TNF alpha resulted in a further stimulation of uptake (152 +/- 31%; P < 0.05), whereas the increase in cells from type 2 diabetics was not significant compared with that in cells receiving acute treatment. After TNF alpha treatment, the level of glucose transporter-1 protein was elevated in nondiabetic (4.6-fold increase) and type 2 (1.7-fold) cells. Acute TNF alpha treatment had no effect on the fractional velocity of GS in either nondiabetic or type 2 cells. Prolonged exposure reduced the GS fractional velocity in both nondiabetic and diabetic cells. In summary, both acute and prolonged treatment with TNF alpha up-regulate glucose uptake activity in cultured human muscle cells, but reduce GS activity. Increased skeletal muscle glucose uptake in conditions of TNF alpha excess may serve as a compensatory mechanism in the insulin resistance of type 2 diabetes.     

Direct current shocks to the heart generate free radicals: an electron paramagnetic resonance study

OBJECTIVES: We sought to demonstrate that direct current (DC) shocks to the heart generate free radicals. BACKGROUND: Although it is a lifesaving maneuver, defibrillation is known to have myocardial toxicity. The mechanism of this toxicity is unknown. If DC shocks generate free radicals, free radicals could be a mechanism of myocardial injury. METHODS: In a canine model, DC shocks of 10 to 100 J were delivered to the epicardium of both beating and fibrillating hearts, and 200-J transthoracic shocks were administered in dogs with beating hearts. Ascorbate free radical (AFR) concentration was measured in arterial blood and blood continuously withdrawn from the coronary sinus. In some dogs, the antioxidant enzymes superoxide dismutase (15,000 U/kg) and catalase (55,000 U/kg) (SOD/Cat) were administered before shocks. RESULTS: Ascorbate free radicals were generated by DC shocks. A peak AFR increase of 14 +/- 2% (mean +/- SEM) was seen 5 to 6 min after 100-J epicardial shocks. A peak AFR increase of 7 +/- 5% occurred after transthoracic shocks. There was a significant linear relation between the shock energy and peak percent AFR increase: %AFR increase = 0.18 (Shock energy) + 2.9 (r = 0.73, p < 0.0001). Shocks delivered to hearts in ventricular fibrillation (30 s) resulted in generation of AFR equal to but not greater than that observed during similar shocks delivered to beating hearts in sinus rhythm. Multiple successive shocks (100 J delivered twice or five times) did not result in a greater AFR increase than single 100-J shocks, indicating that peak, not cumulative, energy is the principal determinant of AFR increase. Animals receiving SOD/Cat before shock administration showed significant attenuation of the AFR increase. CONCLUSIONS: Direct current epicardial and transthoracic shocks generate free radicals; antioxidant enzymes reduce the free radical generation by shocks.     

Neurovascular dysfunction in diabetic rats. Potential contribution of autoxidation and free radicals examined using transition metal chelating agents

Oxygen free radical activity is elevated in diabetes mellitus and has been implicated in the etiology of vascular complications. Recent studies have shown that impaired perfusion of nerve endoneurium is a major cause of nerve fiber dysfunction in experimental diabetes. Free radical scavenger treatment prevents the development of nerve conduction abnormalities in diabetic rats. In vitro experiments suggest that autoxidation reactions of glucose, catalyzed by free transition metal ions, are a potential source of free radicals in diabetes. We investigated whether chronic treatment with deferoxamine and trientine, transition metal chelating agents which can prevent autoxidation, could correct nerve conduction and blood flow changes in streptozotocin-diabetic rats. A 20% reduction in sciatic nerve motor conduction velocity after 2 mo diabetes was 90% ameliorated by 2 wk of treatment with deferoxamine or trientine. Sciatic endoneurial nutritive blood flow was 45% reduced by diabetes, but was completely corrected by treatment. In contrast, transition metal chelation had no effect on blood flow or conduction velocity in nondiabetic rats. Thus, the data support the hypothesis that increased free radical activity by glucose autoxidation as a result of impaired transition metal handling is a major cause of early neurovascular deficits in diabetes.