Hydrogen peroxide affects contractile activity and anti-oxidant enzymes in rat uterus.

BACKGROUND AND PURPOSE: The effects of hydrogen peroxide (H(2)O(2)) on uterine smooth muscle are not well studied. We have investigated the effect and the mechanism of action of exogenous hydrogen peroxide on rat uteri contractile activity [spontaneous and calcium ion (Ca(2+))-induced] and the effect of such treatment on anti-oxidative enzyme activities. EXPERIMENTAL APPROACH: Uteri were isolated from virgin Wistar rats and suspended in an organ bath. Uteri were allowed to contract spontaneously or in the presence of Ca(2+) (6 mM) and treated with H(2)O(2) (2 microM-3 mM) over 2 h. Anti-oxidative enzyme activities (manganese superoxide dismutase-MnSOD, copper-zinc superoxide dismutase-CuZnSOD, catalase-CAT, glutathione peroxidase-GSHPx and glutathione reductase-GR) in H(2)O(2)-treated uteri were compared with those in uteri immediately frozen after isolation or undergoing spontaneous or Ca(2+)-induced contractions, without treatment with H(2)O(2). The effect of inhibitors (propranolol, methylene blue, L-NAME, tetraethylamonium, glibenclamide and 4-aminopyridine) on H(2)O(2)-mediated relaxation was explored. KEY RESULTS: H(2)O(2) caused concentration-dependent relaxation of both spontaneous and Ca(2+)-induced uterine contractions. After H(2)O(2) treatment, GSHPx and MnSOD activities were increased, while CuZnSOD and GR (In Ca(2+)-induced rat uteri) were decreased. N(omega)-nitro-L-arginine methyl ester antagonized the effect of H(2)O(2) on Ca(2+)-induced contractions. H(2)O(2)-induced relaxation was not affected by propranolol, potentiated by methylene blue and antagonized by tetraethylamonium, 4-aminopyridine and glibenclamide, with the last compound being the least effective. CONCLUSIONS AND IMPLICATIONS: H(2)O(2) induced dose-dependent relaxation of isolated rat uteri mainly via changes in voltage-dependent potassium channels. Decreasing generation of reactive oxygen species by stimulation of anti-oxidative pathways may lead to new approaches to the management of dysfunctional uteri.

The anti-oxidative defence system in the isolated rat uterus during spontaneous rhythmic activity.

Possible interactions between nitric oxide donors, reactive oxygen species and anti-oxidative defence enzymes led us to determine the activities of anti-oxidative defence enzymes in isolated uterine smooth muscle before and after spontaneous rhythmic activity ex vivo. For our experiments we used isolated uteri from female Wistar rats. Our results showed an increase in total superoxide dismutase (SOD) and Mn SOD activities in uterine smooth muscle after spontaneous contractions when compared with nonexercised uterine smooth muscle. The activity of catalase (CAT) and glutathione preoxidase (GSH-Px) were also increased. No statistically significant changes in the activities of glutathione reductase (GR) and CuZn SOD were found. It is known that an organism's anti-oxidative defence system (guarding against excessive reactive oxygen species generation) requires balanced increments in its individual anti-oxidative enzyme activities rather than increases in the activity of only some enzymes without increases in others. Thus, we may conclude that some adaptive responses are found in exercised uterine smooth muscle but are not complete. Therefore, our results indicate that changes in anti-oxidative enzyme activities may influence the results of the examination of substances ex vivo.

The effects of nitroglycerine on the redox status of rat erythrocytes and reticulocytes.

The effects of nitroglycerine (NTG) are mediated by liberated nitric oxide (NO) after NTG enzymatic bio-transformation in cells. The aim of this study was to evaluate some products of NTG bio-transformation and their consequences on the redox status of rat erythrocytes and reticulocytes, considering the absence and presence of functional mitochondria in these cells, respectively. Rat erythrocyte and reticulocyte-rich red blood cell (RBC) suspensions were aerobically incubated (2 h, 37 degrees C) without (control) or in the presence of different concentrations of NTG (0.1, 0.25, 0.5, 1.0 and 1.5 mM). In rat erythrocytes, NTG did not elevate the concentrations of any reactive nitrogen species (RNS). However, NTG robustly increased concentration of methemoglobin (MetHb), suggesting that NTG bio-transformation was primarily connected with hemoglobin (Hb). NTG-induced MetHb formation was followed by the induction of lipid peroxidation. In rat reticulocytes, NTG caused an increase in the levels of nitrite, peroxinitrite, hydrogen peroxide, MetHb and lipid peroxide levels, but it decreased the level of the superoxide anion radical. Millimolar concentrations of NTG caused oxidative damage of both erythrocytes and reticulocytes. These data indicate that two pathways of NTG bio-transformation exist in reticulocytes: one generating RNS and the other connected with Hb (as in erythrocytes). In conclusion, NTG bio-transformation is different in erythrocytes and reticulocytes due to the presence of mitochondria in the latter.

The concentrations of antioxidant compounds in the hepatopancreas, the gills and muscle of some freshwater crayfish species.

We examined the concentrations of vitamin E (vit E), sulphydryl groups (-SH), total protein and protein electrophoretic profiles in the hepatopancreas, the gills and muscle of three freshwater crayfish species: Noble crayfish (Astacus astacus), stone crayfish (Austropotamobius torrentium) and spiny cheek crayfish (Orconectes limosus). Vit E concentration in the hepatopancreas of O. limosus was lower compared to A. astacus, while in the gills of O. limosus it was lower compared to both A. astacus and A. torrentium. The concentration of -SH groups was lower in the hepatopancreas of A. astacus compared to A. torrentium and O. limosus. In the gills of A. astacus and A. torrentium the concentration of -SH groups was higher compared to O. limosus. Protein concentration was higher in the hepatopancreas of A. torrentium compared to A. astacus and O. limosus. A lower protein concentration in muscle of A. torrentium was found compared to O. limosus and A. astacus. Electrophoretic analysis of proteins indicated species and tissue specifities between investigated crayfish species. Our results represent the first study of its kind and provide the basis for future studies that will consider our reported parameters as potential biomarkers for biomonitoring of basic environmental conditions and some anthropogenic impacts.

Protective influence of vitamin E on antioxidant defense system in the blood of rats treated with cadmium.

The effects of acute exposure to cadmium (Cd) on the blood antioxidant defense system, lipid peroxide concentration and hematological parameters, as well as the possible protective role of vitamin E were studied. Male Wistar albino rats (3 months old) were treated with cadmium (0.4 mg Cd/kg b.m., i.p., 24 h before the experiment) or with vitamin E + Cd (20 IU Vit E/kg b.m., i.m., 48 h + 0.4 mg Cd/kg b.m., i.p., 24 h before the experiment). The hematological parameters were assessed: red blood cell counts, hematocrit value and hemoglobin concentration were significantly decreased in the blood of Cd-treated rats. Intoxication with cadmium was also followed by significantly increased lipid peroxide concentrations. We also observed increased activity of antioxidant defense enzymes: copper zinc containing superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase as well as concentrations of non-enzymatic components of antioxidant defense system: reduced glutathione, vitamin C and vitamin E. Pretreatment with vitamin E exhibited a protective role on the toxic effects of cadmium on the hematological values, lipid peroxide concentration as well as on enzymatic and non-enzymatic components of antioxidant defense system.

Seasonal changes in the antioxidative defense in ground squirrels (Citellus citellus): possible role of GSH-Px.

As seasonal hibernators, ground squirrels decrease their body temperature to 7 degrees C and hibernate during the winter. Maintenance at 30 degrees C prevents seasonal changes of body temperature and animals remain euthermic and active. We measured selenium (Se)-dependent glutathione peroxidase (GSH-Px), as well as the activity of other antioxidative components such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione-S-transferase (GST) and the amount of low-molecular-weight antioxidants glutathione (GSH), ascorbic acid (AsA), and vitamin E (vit E) in spring, summer, and winter in ground squirrels continuously kept at a temperature of 30 degrees C. We examined liver and interscapular brown adipose tissue (IBAT) as thermogenic tissues, as well as the brain and the kidneys. During the winter, we found a decrease in enzymatic activity and an increase in the level of low molecular antioxidants in all tissues. Correlation analysis revealed a similarity in the composition of antioxidative defense (AD) among the tissues examined. The results obtained clearly demonstrated numerous correlative expressions of antioxidative components in this experimental model, especially of GSH-Px, suggesting the complexity of the system responsible for the maintenance of physiological homeostasis.

Effect of selenium-enriched yeast pretreatment on the antioxidative defense in the skin of rats exposed to heat shock.

Skin protection against heat shock and the specificity in the organization of antioxidative defenses were examined in rats given oral antioxidative pretreatment with selenium (Se)-enriched yeast and vitamins E, C, and A for 15 days and then exposed to hyperthermia. The activity of antioxidative enzymes in the skin and the liver was monitored 1 hour and 3 hours after heat shock. Glutathione peroxidase (GSH-Px) activity was increased in the skin after heat shock in the groups supplemented with antioxidants, but not in the controls. In contrast, the activity of liver GSH-Px was increased only in the controls receiving antioxidants. Heat shock led to a decrease in liver superoxide dismutase (SOD) activity at 1 hour in the antioxidant-supplemented group, but this was unchanged in the liver of all other groups and in the skin. The activity of thioredoxin reductase (TR) in the skin was increased in the antioxidant supplemented group 1 hour after heat shock, whereas the hepatic thioredoxin reductase activity was decreased. The activities of catalase (CAT), glutathione reductase (GR), and glutathione-S-transferase (GST) were unaffected by either treatment. These results suggest that supplementation with antioxidants protects the skin against heat shock, especially with respect to the GSH-Px and TR activity. The different response of the skin in comparison with the liver probably reflects differences in organization and regulation of antioxidative defenses.

Activities of antioxidant enzymes and monoamine oxidase-A in the rat interscapular brown adipose tissue: effects of insulin and 6-hydroxydopamine.

The effects of different doses of insulin (INS) (0.4 or 4.0 IU/kg body mass, i.p., for 3 hr) and 6-hydroxydopamine (6-HDA) (100 mg/kg., i.p.) on the activities of antioxidant enzymes--copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase (CAT) and catecholamine degrading enzyme monoamine oxidase (MAO-A)--in the rat interscapular brown adipose tissue (IBAT) were studied. In vivo 6-HDA administration, which induces the destruction of sympathetic nerves, markedly reduced IBAT CuZnSOD activity but did not change MnSOD and CAT activities. However, the low dose of INS, which did not induce hypoglycemia, significantly increased the activity of both IBAT mitochondrial enzymes (MnSOD and MAO-A) of control rats. This INS effect on MnSOD was abolished by 6-HDA. On the contrary, CuZnSOD activity was markedly reduced under the influence of INS in both control and 6-HDA-treated rats, whereas for the maintenance of the control level of this enzyme activity, the intact sympathetic nervous system (SNS) is necessary. INS, independent of the dose applied, did not affect CAT activity in control rats, whereas only low INS dose increased the activity of this enzyme in 6-HDA-treated rats. The results indicate that the stimulatory effect of INS on the IBAT mitochondrial enzymes studied is dose dependent and in the case of MnSOD is mediated by SNS. However, the depression in the activity of CuZnSOD is independent of the above-mentioned factors.

Chronic effect of insulin on monoamine oxidase and antioxidant enzyme activities in the rat brainstem.

It was shown that hydrogen peroxide (H2O2) is a possible intracellular second messenger in specific insulin action. Because its concentration in the cell depends on the activity of both antioxidant enzymes and monoamine oxidase (MAO), we studied the influence of different insulin doses (0.4 and 4.0 IU/kg body mass, i.p., daily injected over 3 days) on the activity of MAO, types A and B, copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and catalase in the rat brainstem. Chronic insulin treatment significantly increased Vmax of MAO-A and B activities (P < 0.05, P < 0.025, respectively) independent of the dose applied. CuZnSOD activity was also increased (P < 0.025), but only when higher dose of hormone was injected. However, insulin had the opposite effect on MnSOD and catalase causing a decrease in their activities (P < 0.005). The observed changes in the activities of the enzymes studied are possible compensations that potentially maintain an optimal H2O2 level in the brainstem, which might be important for insulin action.

The effect of glucocorticoids on the activity of monoamine oxidase, copper-zinc superoxide dismutase and catalase in the rat hypothalamus.

The activities of biogenic amine deaminating enzymes, monoamine oxidase (MAO), types A and B, copper-zinc superoxide dismutase (CuZnSOD) and hydrogen peroxide (H2O2) scavenging enzyme, catalase, were studied in the hypothalamus of rats treated with dexamethasone (DEX) and corticosterone (CORT) for 2 and 7 days. Only chronic treatment (7 days) with both glucocorticoids (DEX -0.2 mg/kg b.w., i.p. and CORT -5 mg/kg b.w., i.p.) produced a significant decrease in hypothalamic MAO-A and MAO-B activities. Under the same conditions DEX induced significant decrease in hypothalamic catalase and CORT in CuZnSOD activities. The results suggest that the effect of both glucocorticoids on MAO activities seems to be mediated by the same mechanisms, while this is not the case with antioxidant enzymes, catalase and CuZnSOD.

The effect of glucocorticoids on the activity of monoamine oxidase and superoxide dismutase in the rat interscapular brown adipose tissue.

The effect of dexamethasone (DEX) and corticosterone (COR) on the activity of monoamine oxidase (MAO), copper-zinc superoxide dismutase (CuZn SOD) and manganese superoxide dismutase (Mn SOD) in the rat interscapular brown adipose tissue (IBAT) were studied. DEX (1 mg/kg, i.p. for two days) significantly increased MAO activity in the IBAT as compared to the corresponding controls. On the contrary, COR, in the corresponding dose (5 mg/kg), did not affect MAO activity in the IBAT. DEX also markedly enhanced the activity of both SODs in the tissue studied, while COR was ineffective. The results suggest that there exist the differences in the effect between the synthetic glucocorticoid, such as DEX, and COR, which is a natural glucocorticoid in the rat, on the activity of IBAT enzymes studied.

Seasonal dependence of the activity of antioxidant defence enzymes in the ground squirrel (Citellus citellus): the effect of cold.

1. The activity of antioxidant defense enzymes (SOD, CAT, GSH-Px and GST) was analysed during the autumn and winter in the ground squirrel adapted to 30 degrees C and subsequently exposed to cold for 6 and 24 hr. 2. The liver CAT activity as well as the IBAT CAT and GSH-Px activities differed between animals adapted to 30 degrees C, studied in autumn, and those studied in winter. 3. MnSOD activity in the liver was increased in autumn but decreased in winter after 6 hr cold exposure reaching the control level 24 hr later. Cold exposure induced a decrease in CAT activity (except after 24 hr cold exposure in winter) and an increase in GSH-Px activity. Lower GST activity was found after 24 hr exposure to cold in winter. 4. The IBAT SOD activity decreased under the influence of cold during both seasons with a tendency to return to the control level only in winter. Cold exposure produced a decrease in GST in both seasons and CAT activity in autumn. GSH-Px activity was increased in winter only. 5. The results indicate a seasonal dependence of the activity of antioxidant defence enzymes in the ground squirrel. Seasonal influence was evidenced in animals exposed to cold as well.

Antioxidant defenses in the ground squirrel Citellus citellus. 1. A comparison with the rat.

The antioxidant defenses of the liver, erythrocytes, blood plasma, and interscapular brown adipose tissue (IBAT) of male ground squirrels were compared with those of male rats kept under identical conditions and fed the same diet. Superoxide dismutase (SOD), ascorbate, vitamin E, catalase, glutathione, and enzymes of glutathione metabolism were measured. In general, antioxidant defenses in erythrocytes were lower in ground squirrels than in rats. The same was true in liver, except that catalase-specific activity was higher. In IBAT, ascorbate, vitamin E, catalase, and glutathione reductase were higher than in rat and more of the SOD activity present was cyanide-insensitive (MnSOD). It is suggested that IBAT in ground squirrels may need a relatively greater antioxidant defense because of its important role in thermogenesis, especially in reawakening from hibernation. No major differences in antioxidant defenses between male and female ground squirrels were observed, except that the SOD activity of IBAT was higher in females.

Antioxidant defenses in the ground squirrel Citellus citellus. 2. The effect of hibernation.

In spring and autumn, the ground squirrel Citellus citellus is awake and active but in winter it usually hibernates. Reawakening from hibernation involves intense metabolic activity in the interscapular brown adipose tissue (IBAT). The IBAT of hibernation animals showed significant increases in the activities of superoxide dismutase (both copper-zinc and manganese-containing enzymes), glutathione peroxidase, and in the amount of ascorbate present. Glutathione peroxidase also increased in the liver, as did ascorbate in the plasma. These changes were not merely a consequence of exposure to low environmental temperatures. It is proposed that antioxidant defenses are increased in the IBAT of ground squirrels at the onset of hibernation in order to protect the tissue from reactive oxygen species generated as a result of the intense metabolic activity sustained by this tissue during reawakening.

Distribution of superoxide dismutase in the ground squirrel (Citellus citellus): effect of the hibernation and arousal.

The activity of superoxide dismutase (SOD) was studied in the liver, kidney, interscapular brown adipose tissue (IBAT), lung, heart and spleen of the active and hibernating ground squirrel (Citellus citellus). One group was examined immediately after the arousal from the hibernation. A considerable activity of this enzyme was found in homogenates of all tissues studied except the lung. This activity was lower in the liver and lung of the ground squirrel than in the rat (P less than 0.01). In the other tissues studied the enzyme activity was about the same level in both animals. In the ground squirrel hibernation didn't produce the significant change in SOD activity, as compared with the active state, except in the spleen. Tested immediately after the arousal, SOD activity was significantly higher in all tissues studied except in the IBAT, as compared with the hibernating ones (P less than 0.01).