Effect of Carnosine in Experimental Arthritis and on Primary Culture Chondrocytes.

Carnosine's (CARN) anti-inflammatory potential in autoimmune diseases has been but scarcely investigated as yet. The aim of this study was to evaluate the therapeutic potential of CARN in rat adjuvant arthritis, in the model of carrageenan induced hind paw edema (CARA), and also in primary culture of chondrocytes under H2O2 injury. The experiments were done on healthy animals, arthritic animals, and arthritic animals with oral administration of CARN in a daily dose of 150 mg/kg b.w. during 28 days as well as animals with CARA treated by a single administration of CARN in the same dose. CARN beneficially affected hind paw volume and changes in body weight on day 14 and reduced hind paw swelling in CARA. Markers of oxidative stress in plasma and brain (malondialdehyde, 4-hydroxynonenal, protein carbonyls, and lag time of lipid peroxidation) and also activity of gamma-glutamyltransferase were significantly corrected by CARN. CARN also reduced IL-1alpha in plasma. Suppression of intracellular oxidant levels was also observed in chondrocytes pretreated with CARN. Our results obtained on two animal models showed that CARN has systemic anti-inflammatory activity and protected rat brain and chondrocytes from oxidative stress. This finding suggests that CARN might be beneficial for treatment of arthritic diseases.

Mechanisms of iron-induced oxidative modifications of creatine kinase in rat brain in vitro. possible involvement of HNE.

The model of oxidative stress induced by Fe/ascorbate in rat brain in vitro was used to compare the antioxidant capacity of known antioxidants. Creatine kinase (CK) was selected as a marker of protein injury in such studies. Of the antioxidant enzymes (catalase, superoxide dismutase), oxygen radical scavengers (mannitol, glutathione), and the chelator (EDTA) tested in this work and this system, only catalase and glutathione prevented the injury induced by oxidative stress, indicating that H2O2 and the glutathione peroxidase reaction were involved in the preventive effect. Additionally, the preventive effect of glutathione may be caused also by the fact that glutathione easily reacts with 4-hydroxynonenal (HNE), generated in rat brain homogenate, thus protecting CK from inactivation by this aldehyde. To find out whether and if at which concentrations CK may be oxidatively modified by HNE, pure CK was incubated in the presence of 10 and 64 micromol/l HNE for 30 min at 37 degrees C. The activity of CK incubated with HNE decreased significantly. Simultaneously, the protein carbonyls, determined by electrophoresis and immunoblotting increased at 10 micromol/l HNE or disappeared probably due to crosslinking of CK at 64 micromol/l HNE. The concentration of HNE in rat brain homogenates after oxidative stress was determined by HPLC and was in the range of 10-16 nmol/mg prot., corresponding to a concentration of 10-16 micromol/l HNE. This indicates that CK of rat brain homogenates oxidized by Fe/ascorbate may be impaired not only directly by oxygen radicals but also secondarily by HNE.

Preventive effect of several antioxidants after oxidative stress on rat brain homogenates.

Brain homogenate was used as a model system to study antioxidant properties of several natural and synthetic antioxidants under oxidative stress. Oxidative stress was induced by Fe/ascorbate system and lipid peroxidation as well as protein modification were studied. Thiobarbituric acid reactive substances (TBARS) were used as a marker of lipid peroxidation. The preventive effect concerning lipid peroxidation decreased in the order: buthylated hydroxytoluene (BHT) (3.5), stobadine (ST) (35), serotonin (54), trolox (98), U 74389G (160), melatonin (3100), (the numbers in the brackets represent IC50 in micromol/l). Methylprednisolone had no effect, and spin traps interfered with TBARS determination. Concerning creatine kinase (CK) activity as a selected marker of oxidative modification of proteins, the preventive effect of antioxidants (30 micromol/l) decreased in the order: BHT (30), trolox (75), stobadine (ST) (77), alpha-phenyl-N-tert-buthylnitrone (PBN) (87), sodium salt of N-tert-buthyl-C-(phenyl-2-sulfone) nitrone (SPBN) (90), (the numbers in the brackets represent the loss of CK activity in percentages, when 100% was the loss of CK activity in the absence of any antioxidant). The nonglucocorticoid steroid U 74389G, methylprednisolone and serotonin had no preventive effects, while melatonin had antioxidant effect only in a higher concentration (1 mmol/l).

Postischemic reperfusion of the spinal cord: prolonged reperfusion alleviates the metabolic alterations induced by 25 min ischemia in the cervical and thoracolumbal segments.

In recent years, increasing amount of information has indicated that in some tissues the main damage due to oxidative stress does not occur during reperfusion but during the ischemic episode of the ischemia/reperfusion event. In this respect, serious doubts were also expressed about the origin of the increased amounts of free radicals which were believed to form and reported to appear in the perfusate during the first minutes of reperfusion. Moreover, speculative explanations were only available for a second increase in lipid peroxidation which was reported to occur after postischemic reperfusions exceeding 60 min. For this reasons, the present paper reports the results of investigation of ischemia/reperfusion injury to the cervical (CE) and thoracolumbal (ThL) segments of the spinal cord (SP) after an acute 25 min occlusion of the abdominal aorta, followed by 60-120 min reperfusion of the ischemic areas in rabbits. In CE and ThL segments of the SP, the ischemia induced: 1) a decrease in activities of superoxide dismutase (SOD), from 57.35+/-6.36 to 45.27+/-5.45 U x mg(-1) x min(-1) (S.E.M., 20.92%), p < 0.01, and from 58.36+/-5.45 to 33.00+/-4.55 U x mg(-1) x min(-1) (S.E.M., 43.46%), p < 0.001; 2) a significant decrease in gamma-glutamyl transpeptidase (gamma-GTP), from 114.66+/-1.45 to 99.88+/-4.4 micromol p-nitroaniline x mg(-1) x h(-1) (S.E.M. 12.89 %), p < 0.05 and from 112.24+/-1.20 to 95.09+/-2.40 micromol p-nitroaniline x mg(-1) x h(-1) (S.E.M., 16.26%), p < 0.05; 3) a considerable depression in Na,K-ATPase activity, from 7.14+/-0.58 to 5.08+/-0.32 micromol Pi x mg(-1) x h(-1) (S.E.M., 28.86%), p < 0.01, and from 7.23+/-0.11 to 5.09+/-0.31 micromol Pi x mg(-1) x h(-1) (S.E.M., 30.00%), p < 0.01. The Na,K-ATPase activity became decreased by ischemia and remained depressed significantly (all p < 0.01) throughout the experiment. After 60 min of reperfusion, SOD activity in the CE segment and that of gamma-GTP in the CE as well as ThL segments recovered, even slightly surpassing the control values, wheras SOD activity in the ThL segment became stabilized again close to its post-ischemic value. Prolonged, reperfusion for 120 min resulted in a further increase in gamma-GTP activity in the CE and ThL segments (to 132.79 and 132.30%, p < 0.01), and this was accompanied by a slight (p > 0.05) elevation in the content of conjugated dienes as well as by a new wave of depression of the SOD activity (p < 0.05) in both the CE and the ThL segment. From our results it could be concluded that all considerable damage to the spinal cord occurred during the ischemic period. In the period of reperfusion reparative changes started to predominate. This is in accordance with the recent discoveries indicating that, when coupled with an increase in tissue gamma-GTP activity, the post-ischemic reparative changes comprise a replenishment of the cell glutathione pool. This process is accompanied with a gradual increase in H2O2 production that results in repeatead inhibition of the SOD activity and a tendency to conjugated dienes formation.

Effect of stobadine on lipid peroxidation in brain and heart after ischemia and reperfusion of the brain.

Stobadine (ST), a novel drug with pyridoindol structure, was recently found to prevent reperfusion injury in rat brain. The aim of the present study was to reveal whether ST may prevent peroxidative changes in the heart and brain that were triggered by postischemic reperfusion of the brain. In the brain, reperfusion significantly increased the contents of malondialdehyde (MDA) by 43.8% and conjugated diens (CD) by 24.5% when compared with the end of ischemia. In the heart, contents of MDA and CD in reperfusion became elevated three fold and by 41.7%, respectively, when comparing to the values at the end of ischemia. In the heart, no significant changes in activities of the superoxide dismutase (SOD) and glutathione peroxidase (GPx) induced by ischemia or reperfusion were detected. In contrast, reperfusion induced a slight decrease in GPx activity in the brain. In accordance with our previous results, an application of ST (2 mg/kg) to the femoral artery shortly prior to reperfusion of the ischemic brain, prevented significantly MDA and CD accumulation in brain. Nevertheless, ST was not able to prevent the brain-ischemia/reperfusion-induced elevation of MDA and CD contents in the heart.

Effects of histamine H1 antagonist dithiaden on acetic acid-induced colitis in rats.

To assess the possible involvement of mast cells and/or their mediators in inflammatory bowel diseases, the effect of the histamine H1 antagonist Dithiaden was studied on a model of acetic acid-induced colitis in rats. Dithiaden pretreatment by intracolonic administration was found to reduce the extent of acute inflammatory colonic injury. This was manifested by a decrease in the score of gross mucosal injury, by lowered colonic wet weight and by diminished myeloperoxidase activity reflecting reduced leukocyte infiltration. Vascular permeability and gamma-glutamyl transpeptidase activity, elevated by acetic acid exposure, were decreased after Dithiaden pretreatment. The results indicate that locally administered Dithiaden may protect the colonic mucosa against an acute inflammatory attack by interfering with the action of the major mast cell mediator histamine.

Free radicals in rabbit spinal cord ischemia: electron spin resonance spectroscopy and correlation with SOD activity.

1. In nonanesthetized rabbits temporal occlusion of the abdominal aorta was used to induce oxidative stress in the lower part of the body including distal segments of the spinal cord. 2. Spinal cord samples were taken from the animals exposed to 25-min aortic occlusion (AO) or to occlusion followed by 1- or 2-hr reperfusion (AO/R1 or AO/R2, respectively) or from sham-operated animals (C). The presence of free radicals (FR) in the spinal cord samples frozen in liquid N2 was assessed by ESR spectroscopy without spin trapping. Moreover, superoxide dismutase (SOD) activity and conjugated diene (CD) levels were measured in the samples. 3. In the AO group FR were detected in the spinal cord regions close to the occlusion (lower thoracic and distal segments) along with a decrease in SOD activity. The calculated g value (g = 2.0291) indicated that the paramagnetic signal recorded might be attributed to superoxide radicals. FR were absent in the AO/R1 group. Concurrently, the SOD activity revealed a significant tendency to return to the control level. FR appeared again in the AO/R2 group, mostly in the upper and middle lumbar regions, along with a decrease in SOD activity. No sample from the C group revealed FR. A significant increase in CD levels was observed in the thoracolumbar region only in the AO/R2 group. The temporary absence of FR in the AO/R1 group suggests activation of defense antioxidant mechanisms (e.g., specific enzymatic systems such as SOD), which might have been exhausted later. 4. Changes in SOD activity similar to those observed in the thoracolumbar region, though less noticeable, occurred in the obviously noncompromised tissue (upper cervical region). This points to a kind of generalized response of the animal to aortic occlusion. 5. Direct ESR spectroscopy revealed the presence of FR as well as their time course in the spinal cord during the early phase of ischemia/reperfusion injury and the inverse relationship between FR and SOD activity.

Effect of BDM, verapamil, and cardiac work on mitochondrial membrane potential in perfused rat hearts.

The biochemical link providing effective coordination between the mitochondrial ATP synthetic machinery and the contractile apparatus following transitions in cardiac work remains enigmatic. Studies were designed to determine whether activation of the actomyosin adenosinetriphosphatase (ATPase) is a necessary part of the signaling mechanism to the mitochondrial ATP synthase or whether a rise in cytosolic free Ca2+ is sufficient to activate the synthase. With the use of Langendorff-perfused rat hearts, cardiac work was varied via changes in perfusion pressure and by the inclusion of a beta-adrenergic agent. Furthermore, 2,3-butanedione monoxime and verapamil were used to vary independently either the activity of the actomyosin ATPase or the level of cytosolic free Ca2+. Determinations of the in vivo mitochondrial membrane potential [delta psi m; see Wan et al. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H445-H452, 1993] and its vectorial displacement during work transitions provide valuable information concerning direct activation of the ATP synthase and proton movement through the membrane domain of the synthase. Increased cardiac work in the presence of the beta-adrenergic agent resulted in a decrease in delta psi m. Addition of 2,3-butanedione monoxime decreased cardiac work but did not change delta psi m. The inclusion of verapamil resulted in similar decreases in cardiac work. However, delta psi m reversed back to a value observed under control, low-work conditions. These results in conjunction with data regarding levels of high-energy phosphates, free Mg2+, and adenosine 3',5'-cyclic monophosphate suggest a Ca(2+)-mediated increase in the activity of the ATP synthase.

Doxorubicin, daunorubicin, and mitoxantrone cytotoxicity in yeast.

We have investigated the effect of doxorubicin (Adriamycin) on the yeast Saccharomyces cerevisiae. Drug treatment was found to be cytotoxic to wild-type strains, in a concentration-dependent manner, whereas a petite mutant lacking the cytochrome oxidase (EC 1.9.3.1) subunit IV gene was resistant to doxorubicin. Transformation of the doxorubicin-resistant mutant with a yeast in vivo expression vector harboring the cytochrome oxidase subunit IV gene restored both respiration and sensitivity to doxorubicin. Another petite strain, with a mutation in the mitochondrial adenine nucleotide translocator (pet9), did not display doxorubicin resistance. However, in contrast to the subunit IV mutant, it possesses a functional respiratory chain. We also compared the cytotoxic effect of doxorubicin with those of daunorubicin and mitoxantrone in yeast. We found comparable levels of cytotoxicity for doxorubicin and daunorubicin, which were significantly greater than that for mitoxantrone. Finally, we constructed a yeast strain that overexpresses manganese superoxide dismutase (EC 1.15.1.1), an antioxidant enzyme present in mitochondria. Overexpression of manganese superoxide dismutase protected significantly against doxorubicin and daunorubicin cytotoxicity but only slightly against mitoxantrone cytotoxicity. Collectively, our results provide direct in vivo evidence that superoxide radicals participate in doxorubicin- and daunorubicin-induced cytotoxicity in yeast. Furthermore, these results indicate that mitochondrial respiration is a crucial factor in anthracycline, and perhaps mitoxantrone, cytotoxicity in yeast.

Evaluation of ischemia-reperfusion injury by malondialdehyde, glutathione and gamma-glutamyl transpeptidase: lack of specific local effects in diverse parts of the dog heart following acute coronary occlusion.

Alterations in the levels of glutathione, glutathione disulfide, malondialdehyde, and the activity of gamma-glutamyl transpeptidase in nonischemic and ischemic parts of the left ventricle and in the right ventricle were studied in canine hearts after occlusion of the left anterior descending coronary artery for 60 minutes and subsequent reperfusion for 20 minutes. Ischemia caused no significant change in malondialdehyde concentration and gamma-glutamyl transpeptidase activity in ischemic or nonischemic parts of the left ventricle, but it increased the activity of gamma-glutamyl transpeptidase in the continuously perfused right ventricle. Reperfusion of the ischemic areas of the left ventricle was accompanied by accumulation of malondialdehyde and an increase in gamma-glutamyl transpeptidase activity, not only in the reperfused and adjacent areas of the left ventricle, but also in the continuously perfused right ventricle. An increase in the level of glutathione disulfide and decrease in glutathione occurred in all parts of the myocardium during coronary occlusion; these changes were maintained in reperfusion. The findings indicate that the effects of acute occlusion and reperfusion of the left anterior descending coronary artery on myocardial concentrations of glutathione, glutathione disulfide and malondialdehyde or gamma-glutamyl transpeptidase activity are not confined to the local area.

Short cerebral ischemia and subsequent reperfusion and treatment with stobadine.

Lipid peroxidation and activities of antioxidative enzymes were studied in the brain cortex after short (15 min) cerebral ischemia and reperfusion (10 min) in rats. Conjugated dienes (CD) and thiobarbituric acid-reactive substances (TBARS) were significantly elevated in the group of rats with ischemia followed by reperfusion in comparison to the ischemic animals. Superoxide dismutase (SOD) activity significantly increased in the group of animals with ischemia and reperfusion. No significant changes in the activities of glutathione peroxidase (GP) were observed. Stobadine administered before ischemia or before reperfusion decreased the level of TBARS. Stobadine probably prevents malondialdehyde (MDA) formation from hydroperoxide or might elevate the activity of aldehyde dehydrogenase. In contradiction to the findings after long-lasting (4 h) ischemia and subsequent reperfusion, no decrease in the concentration of CD or in the activity of SOD or GP was found.

Effect of stobadine on brain lipid peroxidation induced by incomplete ischemia and subsequent reperfusion.

The ability of stobadine (ST) to prevent lipid peroxidation was tested in incomplete rat cerebral ischemia induced by 4 hour ligation of the common carotid arteries with a subsequent 10 min reperfusion. The extent of lipid peroxidation was determined by the measurement of the level of conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS). The levels of CD and TBARS were significantly elevated in brain cortex samples from animals subjected to ischemia followed by reoxygenation in comparison with ischemic samples without reperfusion, samples from sham operated or control animals. The concentration of CD and TBARS significantly decreased in animals treated with therapeutic doses of ST (2 mg/kg) administered i.v. immediately before reperfusion or 10 min after the onset of reperfusion. Stobadine was more effective than the known lipid antioxidant vitamin E, given in a dose of 30 mg/kg.day i.m. over 3 consecutive days prior to ischemia. The beneficial effect of ST on survival of rats was more effective in comparison with vitamin E. Significant changes were found in the activities of the antioxidative enzymes, i.e. increase in superoxide dismutase (SOD) and decrease in glutathione peroxidase (GP) in brain cortex samples from animals subjected to ischemia followed by reoxygenation. Stobadine prevented these changes. Catalase (CAT) activity was not detectable. It may be concluded from the increased SOD activity that oxygen radicals play a significant role in cerebral ischemia followed reperfusion. In addition to its antioxidant effect, stobadine probably prevents superoxide radical generation. The mechanism of xanthine oxidase inhibition is not involved in preventing superoxide radical generation by stobadine. Stobadine maintained high GP activity, probably by preventing glutathione oxidation.

Processes linked to the formation of reactive oxygen species are not necessarily involved in the development of isoproterenol-induced hypertrophy of the heart. The effect of stobadine.

Administration of stobadine, a cardioprotective substance in investigation prevents a decrease in the content of protein SH groups and glutathione in hearts of rats treated with high doses of isoproterenol (ISO) (30 mg/kg). Moreover, stobadine also attenuated the increase in the content of malondialdehyde and activities of catalase and glutathione reductase as well as a diminution in the GSH/GSSG ratio observed in heart mitochondria isolated from ISO-treated animals. Since stobadine may be considered as a scavenger of reactive oxygen species (ROS), the above effects of the latter substance support the assumption about a possible involvement of reactive oxygen species (ROS) in some processes initiated by administration of ISO in doses inducing cardiac hypertrophy. However our results also indicate that ROS-mediated processes are not necessarily involved in the mechanism of induction of cardiac hypertrophy itself.