Oxidative stress induced by the Fe/ascorbic acid system or model ischemia in vitro: effect of carvedilol and pyridoindole antioxidant SMe1EC2 in young and adult rat brain tissue.

New effective strategies and new highly effective neuroprotective agents are being searched for the therapy of human stroke and cerebral ischemia. The compound SMe1EC2 is a new derivative of stobadine, with enhanced antioxidant properties compared to the maternal drug. Carvedilol, a non-selective beta-blocker, possesses besides its cardioprotective and vasculoprotective properties also an antioxidant effect. We compared the effect of carvedilol and SMe1EC2, antioxidants with a similar chemical structure, in two experimental models of oxidative stress in young and adult rat brain tissue. SMe1EC2 was found to improve the resistance of hippocampal neurons to ischemia in vitro in young and even in 18-month-old rats and inhibited formation of protein carbonyl groups induced by the Fe(2+)/ascorbic acid pro-oxidative system in brain cortex homogenates of young rats. Carvedilol exerted a protective effect only in the hippocampus of 2-month-old rats and that at the concentration 10-times higher than did SMe1EC2. The inhibitory effect of carvedilol on protein carbonyl formation induced by the pro-oxidative system was not proved in the cortex of either young or adult rats. An increased baseline level of the content of protein carbonyl groups in the adult versus young rat brain cortex confirmed age-related changes in neuronal tissue and may be due to increased production of reactive oxygen species and low antioxidant defense mechanisms in the adult rat brain. The results revealed the new pyridoindole SMe1EC2 to be more effective than carvedilol in neuroprotection of rat brain tissue in both experimental models involving oxidative stress.

Carboxymethylated tetrahydropyridoindoles as aldose reductase inhibitors: in vitro selectivity study in intact rat erythrocytes in relation to glycolytic pathway.

Oxidative stress and polyol pathway hypotheses are generally accepted in the etiology of diabetic complications. Recently, novel carboxymethylated pyridoindoles, structural analogues of the efficient chain-breaking antioxidant stobadine, were designed, synthesised and characterised as prospective aldose reductase inhibitors endowed with antioxidant activity. Of them (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (compound 1) and (2-phenethyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (compound 2) were found to be the most efficient inhibitors of aldose reductase with the corresponding IC50 values in a micromolar region. The aim of this work was to study cellular uptake of the novel pyridoindole derivatives and their effect on the complex metabolism of glucose in isolated rat erythrocytes under euglycaemic conditions. Glycolysis was shown to be the sole process responsible for the observed clearance of glucose. The compounds studied were avidly taken up by the cells, yet they did not significantly affect glucose consumption and lactate production nor did they affect osmotic fragility of the erythrocytes. On balance, the present experimental findings indicate that compounds 1 and 2, efficient inhibitors of aldose reductase, are selective in relation to the glycolytic pathway of glucose elimination. This conclusion supports current preclinical development of novel carboxymethylated tetrahydropyridoindoles as promising aldose reductase inhibitors for pharmacological prevention and treatment of diabetic complications.

Testing of bacteria isolated from HIV/AIDS patients in experimental models.

OBJECTIVES: Bacteria purified from the intestinal tract of HIV/AIDS patients were tested for the capacity to be internalised by cells of the HL-60 cell line. Secondly, the bacteria have been applied to the rabbit's colon in order to test their pathogenic ability. RESULTS: The ability of the bacteria to be internalised by HL-60 cells was found to be very expressive. For a more complex biological characterisation of internalised bacteria, these were applied in 6-day intervals per rectum to NZB x CA rabbits during 8 months. The administered bacteria were detected by dot blot hybridisation using HIV-1 PCR probes in the rabbits' intestinal tract after 2 months. No histological and pathological changes were recorded in the gastrointestinal epithelial cells of rabbits. CONCLUSIONS: The applied bacteria of HIV/AIDS patients were not colonised in the rabbit's colon and disappeared after 2 months. Differences between HL-60 and rabbit model are discussed.

Acute toxicity of magnetic nanoparticles in mice.

OBJECTIVES: The acute toxicity of magnetic nanoparticles was effectively lowered by their encapsulation with poly(D,L lactide). In relation to the idea to use magnetic nanoparticles in development of new delivery systems suitable for targeted drug administration, the toxicological profile of five types of magnetic fluids was assessed in mice. METHODS: The nanoprecipitation method was used to prepare magnetic fluids containing nanoparticles of Fe(3)O(4) encapsulated with biodegradable substances. The acute toxicity testing was performed according to OECD Test Guideline 425. In the pilot distribution study a special staining method was examined for the detection of Fe ions in body tissues of mice after intravenous administration of magnetic fluids. RESULTS: The p.o. LD(50) values were greater than 2,000.0 mg/kg of body weight and i.v. LD(50) values were in the range of 231.7-558.9 mg/kg of body weight. CONCLUSIONS: Of the magnetic nanoparticles tested, those encapsulated with poly(D,L lactide) were the most prospective for further in vivo testing.

Effect of the pyridoindole antioxidant stobadine on development of experimental diabetic cataract and on lens protein oxidation in rats: comparison with vitamin E and BHT.

PURPOSE: The aim of this study was to investigate the effect of dietary supplementation with the pyridoindole antioxidant stobadine on the development of diabetic cataract in rats. The findings were compared with the effect of the natural antioxidant vitamin E and the well known phenolic synthetic antioxidant butylated hydroxytoluene. METHODS: Streptozotocin induced diabetic male Wistars rats were fed for 18 weeks a standard diet or a diet supplemented with stobadine (0.05% w/w), vitamin E (0.1% w/w), butylated hydroxytoluene (BHT, 0.4% w/w), or a mixture of stobadine (0.05% w/w) and vitamin E (0.1% w/w). The progress of cataract was monitored biweekly by ophthalmoscopic inspection. Plasma glucose and body weight were recorded regularly. At the end of the experiment, the content of free sulfhydryl and carbonyl was determined in total lens proteins and in the stobadine group plasma levels of malondialdehyde were also measured. RESULTS: Long term treatment of diabetic animals with stobadine (STB), vitamin E, or BHT led to a marked delay in the development of advanced stages of cataract. At the end of the experiment, the visual cataract score was significantly decreased in the diabetic groups treated with stobadine or BHT, while vitamin E had no significant effect. Unexpectedly, combined treatment with STB+vitamin E advanced the progression of the higher stages of cataract, though without affecting the overall visual cataract score. Neither of the antioxidants exerted an effect on the glycemic state or body weight of the animals. Biochemical analyses of eye lens proteins showed significant diminution of sulfhydryl groups and elevation of carbonyl groups in diabetic animals in comparison to healthy controls. Dietary supplementation with any of the antioxidants studied did not influence the levels of these biomarkers significantly. Nevertheless, in diabetic animals, stobadine supplementation significantly attenuated plasma levels of malondialdehyde, an index of systemic oxidative damage. CONCLUSIONS: The results are in accordance with the postulated pro-oxidant role of chronic hyperglycemia, however, the direct oxidative free radical damage of eye lens proteins does not seem to be the key mechanism effective in the development of diabetic cataract. Sugar cataractogenesis appears to be a complex process, in which multiple mechanisms may be involved, including consequences of the overt oxidative stress in diabetes (e.g., protein modifying potential of toxic aldehydes generated as byproducts of carbohydrate autoxidation and lipid peroxidation). The ability of stobadine to attenuate lipoxidation reactions in diabetes may account, at least partly, for its observed anticataract action. Mechanisms involving reduction of mitochondrial damage by stobadine are also discussed.

Pentose phosphate pathway, glutathione-dependent enzymes and antioxidant defense during oxidative stress in diabetic rodent brain and peripheral organs: effects of stobadine and vitamin E.

The aim of the present study was to investigate the effects of treatment with antioxidant stobadine (ST) on the activities of enzymes related with pentose phosphate pathway and glutathione-dependent metabolism and the other markers of oxidative stress in brain and peripheral organs of diabetic rats, and to compare the effects of ST treatment alone with the effects of treatments with another antioxidant vitamin E and ST plus vitamin E. Rats were made diabetic by the injection of streptozotocin (STZ; 55 mg/kg IP), and, 2 days later, some control and diabetic rats were left untreated or treated with ST (24.7 mg/kg/day, orally), vitamin E (400-500 U/kg/day, orally), or both substances together. In the brain, although 6-phosphogluconate dehydrogenase activity (6-PGD) did not change, glucose-6-phosphate dehydrogenase activity (G-6PD) was markedly increased in diabetic rats compared with controls; only combined treatment with ST and vitamin E produced a partial prevention on this alteration. The aorta G-6PD and 6-PGD of diabetic rats were 52% and 36% of control values, respectively. Neither single treatments with each antioxidant nor their combination altered the G-6PD and 6-PGD in aorta of diabetic rats. Glutathione peroxidase (GSHPx) activity was increased by STZ-diabetes in brain, heart, and kidney. In diabetic brain, vitamin E alone or combination with ST kept GSHPx at normal levels. Diabetes-induced stimulation in GSHPx did not decrease in response to the treatment with vitamin E in heart and kidney, but was greatly prevented by ST alone. The activity of glutathione reductase (GR) was decreased in brain and heart of diabetic rats. The treatment with each antioxidant or with a combination of both agents completely prevented this deficiency and resulted in further activation of GR in diabetic tissues. Glutathione S-transferase (GST) activity did not significantly change in diabetic brain and aorta. GST was stimulated by all treatment protocols in the brain of diabetic rats and was depressed in aorta of control rats. Catalase (CAT) was activated in diabetic heart but depressed in diabetic kidney. Diabetes-induced abnormalities in CAT activity did not respond to vitamin E alone in heart, was moderately ameliorated by the treatment with this vitamin in kidney, and was completely prevented by ST alone in both tissues. Superoxide dismutase (SOD) activity of brain and heart was unchanged by the diabetes but inhibited in diabetic kidney after the treatment ST alone or ST plus vitamin E. The lipid peroxidation (MDA) was increased in diabetic brain and heart. ST or vitamin E alone partly prevented diabetes-induced increase in MDA in brain and heart; however, antioxidant combination achieved a completely amelioration in MDA of these tissues of diabetic rats. Kidney MDA levels were similar in control and untreated diabetic animals. ST and vitamin E treatments, when applied separately or together, significantly reduced kidney MDA in both control and diabetic rats; and the combined effect of antioxidants was greater than that of each alone. These results are consistent with the degenerative role of hyperglycemia on cellular reducing equivalent homeostasis and antioxidant defense, and provide further evidence that pharmacological intervention of different antioxidants may have significant implications in the prevention of the prooxidant feature of diabetes and protects redox status of the cells.

The pyridoindole antioxidant stobadine attenuates histochemical changes in kidney of streptozotocin-induced diabetic rats.

The aim of the present study was to investigate the effects of dietary supplementation with the pyridoindole antioxidant stobadine on histochemical parameters in kidney of streptozotocin-induced diabetic rats. Diabetic male Wistar rats were fed a standard diet or a diet supplemented with stobadine (0.05% w/w) for 24 weeks. The diabetic state was characterized by significantly elevated plasma levels of glucose and glycated hemoglobin, severe reduction of total body weight and relatively enlarged kidneys. Kidney alkaline phosphatase activity was not changed by diabetes. Activity of 5'-nucleotidase, K(+)-dependent p-nitrophenylphosphatase, ATPase and mitochondrial succinic dehydrogenase were markedly decreased in kidneys of diabetic rats. In contrast, activity of beta-hydroxybutyrate dehydrogenase was moderately increased in kidney of diabetic rats as compared to controls. Long-term treatment of diabetic animals with stobadine attenuated histochemical changes in kidney tissue.