Investigations into the loss of glutathione from lenses in organ culture

PURPOSE: To investigate possible causes and implications of the decrease in glutathione concentration in rat lenses during organ culture. METHODS: Freshly excised lenses were incubated in modified TC-199 medium. Ellman's Reagent or the GSH-400 assay were used to assay glutathione levels in lenses cultured for different times and under a variety of altered culture conditions. RESULTS: In lenses from young rats the glutathione decrease was not ameliorated by reduction of oxygen tension in the incubator, nor by supplementation of the culture medium with various antioxidants or sulfhydryl compounds, nor with the amino acid precursors of glutathione. Addition of 2-mercaptoethanol stimulated cysteine transport into the lens but had only a modest effect in maintaining the level of glutathione. The decrease in glutathione concentration was less in cultured lenses from older rats. Lenses from rhesus monkeys exhibited no decrease in glutathione levels when maintained in organ culture for up to 48 h. CONCLUSIONS: The basis for the decreased glutathione in cultured young rat lenses is still uncertain. The data from the present study indicate a definite relationship between glutathione loss and age for cultured rat lenses, with young lenses being much more susceptible. The resistance of cultured monkey lenses to loss of glutathione demonstrates species differences in this property which may be relevant to previously reported differences in susceptibility to oxidative damage.     

Increased susceptibility of late passage human diploid fibroblasts to oxidative stress

Three strains of human diploid fibroblasts, TIG-3, TIG-7, and MRC-5, were serially cultivated. The susceptibility of early-passage and late-passage cells at 20-30 and 60-70 population doubling levels, respectively, to hydrogen peroxide, the superoxide radical (exposure to the hypoxanthine-xanthine oxidase system), or linoleic acid hydroperoxide was examined for lactate dehydrogenase release. The susceptibility of late-passage cells to such oxidative stress was considerably enhanced compared with early-passage cells. The concentration of reduced glutathione in late-passage cells was lower by 24-44% on a per-cell-number basis and by 86.0-94.5% on a per-protein-quantity basis than in early-passage cells. In addition, the activity of catalase in late-passage cells was lower by 19-46% compared with early-passage cells. There was, however, no difference between the mRNA levels of catalase in early-passage and late-passage cells. The activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, and glutathione peroxidase in late-passage cells were all higher than in early-passage cells. These results suggest that late-passage cells are more susceptible to oxidative stress than early-passage cells presumably because of decreases in cellular reduced glutathione concentration and catalase activity, and that their primary defense against oxidative stress is reduced glutathione.     

Evaluation of oxidative processes in human pigment epithelial cells associated with retinal outer segment phagocytosis

To investigate the nature of the oxidative event that occurs during phagocytosis of retinal outer segments (ROS) by cultured human retinal pigment epithelial (RPE) cells, cells were incubated with isolated bovine ROS labeled with either the fluorescence probe carboxy-SNAFL-2 or the nonfluorescent, oxidizable probe 2',7'-dichlorodihydrofluorescein (H2DCF). The increase in fluorescence following phagocytosis was measured by a flow cytometer. Other measurements included: oxygen consumption using a Clark-type oxygen electrode, extracellular superoxide release by superoxide dismutase inhibitable lucigenin chemiluminescence, intracellular hydrogen peroxide (H2O2) production, and the effect of catalase inhibition on cellular thiobarbituric acid-reactive substances (TBARS) caused by phagocytosis. The activities of the enzymes NADPH oxidase and palmitoyl-CoA oxidase were also measured. H2DCF attached to bovine ROS was oxidized during phagocytosis with a time course suggesting oxidation subsequent to ROS uptake. Measurements of oxygen consumption showed a time-dependent increase of 10%, 4 h after ROS feeding, attributable to a doubling of the cyanide-resistant oxygen consumption. Intracellular H2O2 production also doubled 4 h after ROS phagocytosis. ROS uptake by RPE cells produced no significant extracellular superoxide, while extracellular superoxide production was readily demonstrated in a control macrophage cell line. Enzyme activity measurements showed that incubation of RPE cells with ROS doubled catalase activity without affecting superoxide dismutase or glutathione peroxidase activities. Inhibition of catalase during ROS uptake increased TBARS by 66%. Other enzyme activity measurements showed that human RPE cells possess both NADPH oxidase and palmitoyl-CoA oxidase activities. We conclude that ROS phagocytosis subjects RPE cells to an oxidative event on the same order of magnitude as measured in a macrophage. The event is not an extracellular macrophage-type respiratory burst and may be due to intracellular H2O2 resulting from an NADPH oxidase in the phagosome or from beta-oxidation of ROS lipids in peroxisomes. Irrespective of case, the enzyme catalase appears to be essential in protecting the RPE cell against reactive oxygen species produced during phagocytosis.     

The oxidative stress in the cataract formation

The lens of the eye is an avascular tissue surrounded by fluids such as the aqueous humor and vitreous body, with one side facing toward the outside of the body. We investigated peroxidative reactions occurring in cataractous lenses, examining changes within the lens tissues as well as in the surrounding environment. 1. Peroxidative reactions in lenses. 1) Aging and peroxidative reactions. The activity of superoxide dismutase (SOD) began to decrease in the lenses of rats at six months of age. Moreover, the level of lipid peroxide increased significantly in the lenses of rats at 24 months of age. Lipoproteins became increasingly oxidized with age. The levels of Na+, K+, and Ca++, ions that are important to the maintenance of membrane function, also varied significantly with age. In the lenses of six-month-old Senescence Accelerated Mice (SAM), there was a marked decrease in the ability of scavenge active oxygen and a marked increase in the amount of lipid peroxide. In human lenses, the level of autofluorescence increased as the lens fiber structure changed with age. 2) Generation of free radicals inside the lens. We verified that HO. and ascorbic acid radicals were being generated inside cataractous lenses using electron spin resonance (ESR). 3) Changes in oxidation-related substances in cataractous lenses. Senile cataractous lenses and diabetic cataractous lenses were classified as four types, cortical, nuclear, posterior subcapsular, and mature. In cataractous lenses from all types of diabetic patients, the levels of glucose, glycated protein, and lipid peroxide were higher than in senile cataractous lenses. Among the four types of cataracts, the accumulation of peroxides was the greatest in the nuclear type both diabetic and senile cataractous lenses. 4) Transitional metals. Iron ions and copper ions existed in lens tissue. In particular, the subepithelial region of the lens stained strongly for copper ions. The increased level of copper ions in cataractous lenses is likely to be related to the increased peroxidation in this tissue. 5) Changes in membrane. Lowered levels of phospholipids and a higher degree of saturation of fatty acids were observed in senile cataractous lenses as compared with normal lenses. The increased saturation of fatty acids indicated that there was a damage to the membrane structure due to peroxidative reactions. The receptors for low density lipoprotein (LDL) were shown to exist on the epithelium of normal lenses. Acetyl-LDL, a denatured lipoprotein was incorporated into senile cataractous lenses but not into normal lenses, suggesting that the barrier function of the membrane deteriorates in cataractous lenses. Moreover, in diabetic cataractous lenses, the levels of very low density lipoprotein (VLDL) and LDL significantly increased. 2. Change in the environment surrounding the lens and peroxidative reactions. 1) Changes in the levels of oxidation-related substances in blood, aqueous humor, and vitreous body from diabetic patients: all had decreased levels of reduced glutathione and superoxide scavenging activity, and increased levels of lipid peroxide and glycated protein. This may have been due to a reduction in the anti-oxidative potential in the environment surrounding the lens due to the enhanced glycation. Changes in the level of oxidation related substances in the vitreous body in particular, will likely have a significant impact on the lens. 2) Changes in lenses as the surrounding environment deteriorates. Human lenses were cultured for three weeks under conditions similar to those found in vivo utilizing the culture system that we had originally designed and constructed. When protective activity against peroxidation was reduced, the amount of lipid peroxide increased significantly. In the presence of high levels of glucose, the levels of lipid peroxide increased and the amount and activity of SOD decreased. 3. Effects of changes in the external environment on peroxidative reactions.     

Effect of cis-unsaturated fatty acids on cellular oxidant stress in macrophage tumor (AK-5) cells in vitro

Cis-unsaturated fatty acids (c-UFAs) induced decreased survival of macrophage tumor (AK-5) cells in vitro. The cytotoxic action of c-UfAs was associated with an increase in free radical generation and lipid peroxidation process. In addition, exposure of AK-5 cells to various c-UFAs for a short period (1 h) decreased the cellular concentrations of anti-oxidants: superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase, glutathione and vitamin E. However, prolonged (24 h) exposure of AK-5 cells to c-UFAs enhanced the levels of SOD with little or no change in the concentrations of catalase, glutathione peroxidase and glutathione reductase. These results indicate that c-UFAs can enhance free radical generation and lower the concentrations of various anti-oxidants in the tumor cells which may explain the cytotoxic action of c-UFAs.     

Increase in dopamine turnover and tyrosine hydroxylase enzyme in hippocampus of rats fed on low selenium diet

We have studied the turnover of dopamine, noradrenaline, and serotonin and their metabolites in hippocampus of adult female rats that were fed control or selenium-deficient diets during 15 days. Under these circumstances, there was an increase of dopamine turnover (4-fold) in rats fed with selenium-deficient diet with respect to controls and also an increase in the tyrosine hydroxylase activity (75.8%), which was the result of the increase of the amount of the enzyme (2-fold), without significant change in the phosphorylation of the tyrosine hydroxylase. In addition the glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase activities have been studied. After selenium-deficient diet, the enzymatic activities of superoxide dismutase and catalase did not show change with respect to the controls; however glutathione reductase and glutathione peroxidase significantly decreased 15% and 29%, respectively. It is concluded that the increase in dopamine turnover seems to be associated with the induction of tyrosine hydroxylase enzyme. In these conditions the decrease in antioxidant capacity may produce a cascade of events, which accelerates the degenerative process, since the increase in dopamine turnover produces an increase in oxygen radical by monoamine oxidase activity.     

Protective effects of chlorogenic acid on paraquat-induced oxidative stress in rats

The protective effects of chlorogenic acid on paraquat-induced oxidative stress were examined in rats. The activities of erythrocytes and liver glutathione peroxidase, and of both liver catalase and glutathione reductase, which were increased by feeding paraquat, declined to the levels in the control rats by supplementing chlorogenic acid to the paraquat diet. The activity of superoxide dismutase was not changed by dietary paraquat or by supplementing chlorogenic acid to the paraquat diet. Paraquat in the diet markedly decreased the liver triacylglycerol and phospholipid concentrations, as well as the food intake and body weight gain, while chlorogenic acid protected against these decreases. These in vivo results and the in vitro superoxide anion scavenging activity of chlorogenic acid suggest that chlorogenic acid acted preventively against paraquat-induced oxidative stress.     

Spin trapping of azidyl and hydroxyl radicals in azide-inhibited rat brain submitochondrial particles

Succinate-driven respiration in azide-inhibited rat brain submitochondrial particles (smps) produces azidyl and hydroxyl radicals that were detected by spin trapping with 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Production of radicals required succinate and oxygen and was eliminated by heat denaturation, which indicates that radical production is a result of respiration. The concentrations of both DMPO/.OH and DMPO/.N3 were decreased by addition of catalase to the smps, which indicates that H2O2 is involved in radical production. In the absence of azide anion, DMPO/.OH was not detected in the same system, even after five additions of succinate over a period of 24 h. It is proposed that azide inhibition of cytochrome c oxidase results in increased production of superoxide, which is efficiently converted to hydrogen peroxide by membrane-bound superoxide dismutase. Hydrogen peroxide activates endogenous peroxidase to react with azide anion forming azidyl radical, which damages the peroxidase, resulting in decreased production of azidyl radical with successive additions of succinate. Hydroxyl radical is produced from the hydrogen peroxide that is not removed by peroxidase. The increased production of superoxide in the azide-inhibited system suggests that loss of cytochrome c oxidase activity can lead to increased radical production if other proteins in the respiratory chain remain active. In the azide-inhibited system, reaction of azide anion with H2O2-activated endogenous peroxidase and spin-trapping of the resulting azidyl radical is a convenient monitor of H2O2 production.     

Developmental changes of antioxidant enzymes in kidney and liver from rats

The five principal antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione reductase in the kidney and liver, and the total hepatic glutathione were determined in rats of different ages (1, 2, 3, 6, and 12 months). Variance analysis proved the effect of age on the measured enzymes in the respective organ with the exception of glutathione S-transferase. The behavior of the enzymes was not uniform, and there were both increased and decreased changes in the two organs. A clear correlation between cellular antioxidative capacity and the age-specific processes of growing or aging could not be seen. A far more complicated network of interactions has to be assumed.     

Toxicity of L-ascorbic acid to L929 fibroblast cultures: relevance to biocompatibility testing of materials for use in wound management

Fibroblast cultures are often used to evaluate materials intended for medical use, cytotoxicity being taken as an indicator of bioincompatibility. Such an approach has previously been taken with ascorbic acid in determining its value in wound healing. We have now reexamined the toxicity of L-ascorbic acid to L929 fibroblast cells in culture. Concentrations of ascorbic acid between 0.5 mM and 11 mM were tested. At concentrations above 2 mM, ascorbic acid was found to inhibit cell proliferation, with cell viability decreasing as the concentration was increased. This effect could be prevented by the addition of either superoxide dismutase or catalase to the culture medium. Assays of glutathione and glutathione disulfide were carried out on 8 day old cultures exposed for 24 h to the same concentrations of ascorbic acid. A dose-related depletion of glutathione occurred whilst glutathione disulfide levels remained essentially constant. Lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities were induced by ascorbic acid at all concentrations tested but the ratio of NADP to NADPH nevertheless increased as the concentration of ascorbic acid increased. Finally, ATP in cells from 8-day-old cultures became depleted in the presence of ascorbic acid at concentrations in excess of about 5 mM when assayed after 24 h incubation. These biochemical changes and the concomitant cytostatic/cytotoxic effects may be ascribed to the reactive oxygen species produced by the autoxidation of ascorbic acid in the culture medium. Ascorbic acid breakdown products appeared not to be directly involved. In addition, our results suggested that superoxide acted cooperatively with hydroxyl to elicit these effects on the fibroblasts. It is evident from this study that the microenvironment surrounding fibroblasts in culture may differ fundamentally from that surrounding fibroblasts in a healing wound, making it impossible to extrapolate directly to an in vivo situation and hence to make any recommendations from these results concerning the use of ascorbic acid in wound healing.     

Presynaptic and postsynaptic subcellular localization of substance P receptor immunoreactivity in the neostriatum of the rat and rhesus monkey (Macaca mulatta)

Earlier studies with Arabidopsis thaliana exposed to ultraviolet B (UV-B) and ozone (O3) have indicated the differential responses of superoxide dismutase and glutathione reductase. In this study, we have investigated whether A. thaliana genotype Landsberg erecta and its flavonoid-deficient mutant transparent testa (tt5) is capable of metabolizing UV-B- and O3-induced activated oxygen species by invoking similar antioxidant enzymes. UV-B exposure preferentially enhanced guaiacol-peroxidases, ascorbate peroxidase, and peroxidases specific to coniferyl alcohol and modified the substrate affinity of ascorbate peroxidase. O3 exposure enhanced superoxide dismutase, peroxidases, glutathione reductase, and ascorbate peroxidase to a similar degree and modified the substrate affinity of both glutathione reductase and ascorbate peroxidase. Both UV-B and O3 exposure enhanced similar Cu,Zn-superoxide dismutase isoforms. New isoforms of peroxidases and ascorbate peroxidase were synthesized in tt5 plants irradiated with UV-B. UV-B radiation, in contrast to O3, enhanced the activated oxygen species by increasing membrane-localized NADPH-oxidase activity and decreasing catalase activities. These results collectively suggest that (a) UV-B exposure preferentially induces peroxidase-related enzymes, whereas O3 exposure invokes the enzymes of superoxide dismutase/ascorbate-glutathione cycle, and (b) in contrast to O3, UV-B exposure generated activated oxygen species by increasing NADPH-oxidase activity.     

Protein-energy malnutrition and oxidative injury in growing rats

1. Weaning rats were fed ad libitum isocaloric diets containing 5% and 20% casein based proteins. 5% protein diet was protein deficient diet. Pair fed rats with the 5% protein group were maintained simultaneously on 20% protein diet but the amount restricted to the amount taken up by PEM group. 2. Glutathione, antioxidative enzymes, lipid peroxidation and histopathological studies in liver and only glutathione and antioxidative enzymes in blood were carried out. 3. Rats fed the 5% protein diet developed a severe protein energy malnutrition (PEM) whereas those on pair-fed diet developed mild to moderate PEM. 4. Glutathione related thiols superoxide dismutase, glutathione peroxidase, catalase and glutathione-Stransferase with (1 Chloro 2,4-dinitro benzene (CDNB) substrate) were decreased in liver with concomitant increase of lipid peroxidation in severe PEM. In blood glutathione, glutathione peroxidase and catalase were decreased while superoxide dismutase was increased in severe PEM group. 5. Mild to moderate PEM (pair-fed group) also resulted in similar changes in liver except glutathione peroxidase, lipid peroxidation in liver and superoxide dismutase in blood. 6. Hepatic injury was detectable only in the severe PEM group. 7. Oxidative-stress and hepatic injury occurred in severe PEM and to a lesser degree in mild to moderate PEM.     

Attenuation of 4-hydroxynonenal-induced cataractogenesis in rat lens by butylated hydroxytoluene

PURPOSE: We have previously shown that 4-hydroxynonenal (4-HNE) causes opacification of cultured rat lenses and that a novel group of glutathione S-transferases (GSTs) exhibit high specific activity towards 4-HNE. Previous studies have shown that t-butylated hydroxy toluene (BHT) induced GSTs in cultured rat lens. Therefore, the purpose of the present studies was to investigate if the opacification of rat lenses exposed to 4-HNE is ameliorated by pre-culturing the lenses in media containing BHT. METHODS: Rat lenses were divided into four groups. Group I and II were controls and groups III and IV were cultured in the presence of 100 microM 4-HNE. Groups II and IV were pre-cultured in the media containing 10 microM BHT for 24 hrs which was designated as 0 time point. Lenses were withdrawn at 24 and 72 h and evaluated for opacification by digital image analysis. Induction of the specific GST isozyme (gammaGST8-8) was studied in the lens epithelium by immunohistochemical studies. Results. Digital image analysis revealed amelioration in opacification induced by 4-HNE, when the lenses were precultured with BHT. Immunohistochemical studies show that BHT induced GST8-8 several folds in the epithelium. CONCLUSIONS: These studies indicate that pretreatment with BHT would increase the lens capacity to detoxify 4-HNE by conjugating it with GSH, thus assigning an important detoxication role to this specific GST isozyme in oxidative cataract.     

Activities of conjugating and antioxidant enzymes following endotoxin exposure

Endotoxin exposure elicits various responses in mammals including the acute phase response that has been shown to cause changes in the activity of several forms of cytochrome P450s and other enzymes. Therefore, the hepatic conjugating enzyme, glutathione S-transferase (GST), and UDP-glucuronosyltransferase (UDPGT), the antioxidant enzymes, glutathione peroxidase (GSHPx), catalase, and superoxide dismutase (SOD), as well as lipid peroxidation were investigated following the administration of endotoxin to male Sprague-Dawley rats (8 mg/kg body weight). Rats were euthanized at various times following endotoxin administration and the livers removed and processed to assess various enzyme activities. Glutathione S-transferase, UDPGT, and GSHPx activity showed statistically significant decreases after 24 hours and remained lower than controls for the duration of the study. Decreases in total SOD and catalase activities were seen at 24, 48, and 72 hours following endotoxin administration; however, only catalase activity showed statistically significant differences between control and treated samples at those time points, and total SOD activity showed a statistically significant decrease at 24 hours. No statistically significant changes were seen in the level of lipid peroxidation in the liver microsomes from endotoxin-treated animals. Changes in the conjugative enzymes and the free-radical scavenging enzymes following endotoxin exposure may alter the host's metabolism and response to free radicals.     

Detoxicating enzymes of Entamoeba histolytica and their detoxifying roles

OBJECTIVE: To investigate the Entamoeba histolytica living in the low oxygen concentration colon of the host and how does it survive in the circumstance after invading the tissues with high oxygen concentration while obtaining oxygen without being damaged by the toxins. MATERIAL AND METHODS: E. histolytica cultured for 48 hours was collected, centrifuged, rinsed, ultrasonically shattered and again centrifuged, and the activities of superoxide dismutase and the catalase in the supernatant were determined. The catalase and peroxidase were identified by the electron microscopic enzyme cytochemical reaction technique. RESULTS: E. histolytica contained 122.42 +/- 15.47 U/mgpr of superoxide dismutase, 126.05 +/- 17.04 K/mgpr of catalase and peroxidase, and all of them are detoxifying enzymes. Catalase and peroxidase were located within microsomes and lysosome-like organelles respectively. CONCLUSIONS: E. histolytica contains the detoxifying enzymes as superoxide dismutase, catalase and peroxidase, that may prevent the aerobic metabolism from being poisoned by the activated oxygen free radical (superoxide anion radical and hydrogen peroxide) produced in this process, suggesting that the detoxifying function of these enzymes play an important defensive role in the survival of E. histolytica.     

Relationships between chronic oral infectious diseases and systemic diseases

Contact-inhibited catalase-deficient fibroblast cell strain has been established from the homozygous hypocatalasemic C3H/Csb mutant mouse. This cell strain has low level of catalase enzyme activity and has normal level of enzyme activities of both glutathione peroxidase and superoxide dismutase. Catalase-deficient C3H/Csb mutant cell strain is markedly more sensitive to the toxicity of hydrogen peroxide compared to wild-type C3H/Csa cell strain. In addition, mutant cell strain is sensitive to X-rays and near-UV compared to wild-type cell strain, but shows the same sensitivities to topoisomerase II inhibitors, adriamycin and 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA), and the DNA cross-linking agents, cisdiamminedichloroplatinum (II) (cis-Pt) and trans-diamminedichloroplatinum (II) (trans-Pt). These cell strains will be of use in the study of the roles which catalase plays in the intracellular prevention of DNA damage induced by oxidative stress.     

Fine-needle aspiration of the macrofollicular and microfollicular subtypes of the follicular variant of papillary carcinoma of the thyroid

The activity of antioxidant and detoxifying enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase, glutathione-S-transferase (GST), the contents of thiobarbituric acid reactive substances, and the superoxide dismutase and glutathione-S-transferase isoenzyme patterns, were determined in the liver and kidney of pheasants after acute intoxication by herbicides MCPA and ANITEN I. In the liver, the activity of antioxidant enzymes was significantly decreased in the group given ANITEN I. New superoxide dismutase isoforms (pI 6.30, 6.85, 7.00) and higher intensity of isoform with pI 6.60 were observed after isoelectrofocusing in all experimental groups. In the kidney, the activity of superoxide dismutase was significantly decreased, and a higher intensity of superoxide dismutase isoforms (pI 6.00 and 6.60) was observed in all experimental groups. The contents of thiobarbituric acid reactive substances were significantly increased in the group with ANITEN I. The glutathione-S-transferase isoenzyme pattern was studied by using subunit-specific substrates and by Western blotting. The activity of glutathione-S-transferase with ethacrynic acid and cross-reactivity with rat subunit 7 was lower in all experimental groups in the kidney and liver, except in the liver of the group given a higher dose of ANITEN I. In this group, we have found a 2.10-fold higher activity to ethacrynic acid and a strong induction of subunit 7.     

Endurance training improves the resistance of rat diaphragm to exercise-induced oxidative stress

The current study was designed to test the hypothesis that endurance training improves the ability of the diaphragm muscle to resist exercise-induced oxidative stress. Twenty-eight male Wistar rats were assigned to either untrained or trained groups. Trained rats were treadmill-trained for 9 wk. Each group was subdivided into acutely exercised or nonexercised groups. Diaphragm muscle from each rat was analyzed to determine the levels of certain antioxidant enzymes: Mn-superoxide dismutase (Mn-SOD), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), glutathione peroxidase, and catalase. In addition, interleukin-1 and myeloperoxidase levels were determined. Endurance training upregulated all of the antioxidant enzymes. Conversely, acute exercise increased glutathione peroxidase and catalase in untrained rats, while it had no overt effect on any antioxidant enzymes in trained rats. Both Mn-SOD and Cu,Zn-SOD contents and activities were increased with endurance training. However, the mRNA expressions of both forms of SOD did not show any significant change with endurance training. Acute exercise also increased the levels of interleukin-1 and myeloperoxidase in untrained rats but not in trained rats. Moreover, acute exercise significantly increased the ability of neutrophils to produce superoxide, especially in untrained rats. The results from this study demonstrate that endurance training can upregulate certain antioxidant enzyme activities in rat diaphragm muscle, indicating the potential for improvement of the resistance to intracellular reactive oxygen species. The results of this study also suggest that acute exercise may cause oxidative damage in rat diaphragm through the activation of the inflammatory pathway and that endurance training may minimize such an extracellular oxidative stress by acute exercise.     

Cigarette smoke extract is a modulator of mitogenic action in vascular smooth muscle cells

Cigarette smoking is associated with an increased incidence of atherosclerotic disease. In this study, we examined the mechanism underlying the growth-modulating effects of cigarette smoke extract (CSE) in confluent vascular smooth muscle cells (VSMCs). The treatment of VSMC by CSE decreased the activities of superoxide dismutase (SOD), catalase and glutathione peroxidase of VSMC in a time-dependent manner. In mitogenesis assays using the confluent cells, CSE was not a direct mitogen for VSMC, but potentiated the stimulatory effect of hydrogen peroxides. The reduction of activities of catalase and glutathione peroxidase was partially prevented by SH-containing compounds. In summary, CSE enhanced the mitogenic effect response of hydrogen peroxides, largely depending on the dysregulation of the activities of SOD, catalase and glutathione peroxidase by CSE.