Gordana Bjelakovic

Glucocorticoids and oxidative stress.

Glucocorticoids (GC) are used widely for the treatment of patients with various disorders, including autoimmune diseases, allergies, and lymphoproliferative disorders. Glucocorticoid therapy is often limited by several adverse reactions associated with GC excess. Excess GC can elicit a variety of symptoms and signs, including growth retardation in children; immunosuppression; cardiovascular disorders like hypertension and atherosclerosis; osteoporosis; myopathy; and diabetes mellitus. Currently, attention is focused on oxidative stress as one of the major determinants of endothelial dysfunction and cardiovascular senescence. The main reason for all unwanted effects of GC is that dexamethasone induces the overproduction of reactive oxygen species, causing dysregulation of physiological processes. Humans and animals with GC-induced hypertension exhibit reduced nitric oxide levels; patients with excess GC levels also suffer from depression as a consequence of low levels of serotonin and melatonin. The common cofactor for the production of these vasoactive molecules is tetrahydrobiopterin (BH4), which is required for nitric oxide synthesis.

Effect of caffeine on metabolism of L-arginine in the brain

Methylxanthines are widely consumed because of their stimulating effect primarily on the central nervous system. Their diuretic and respiratory stimulant action is used in clinical medicine. L-Arginine metabolism in the brain is very important for normal brain function. In addition to brain protein synthesis, arginine is a substrate for the production of urea, creatine, nitric oxide, agmatine, glutamic acid, ornithine, proline and polyamines. As known, many of these compounds are very important in brain function. There is no information relating to effects of caffeine on arginine metabolism in the brain, however, there is a lot of new information about arginine metabolism and caffeine action on the central nervous system. So, we have hypothesized the existence of a relationship that may be of interest in understanding mechanisms of caffeine effects on the central nervous system that may have utility in the clinical applications.In our experiment protocol we used male Wistar rats weighing about 200 g. Caffeine was added to the drinking water in gradually increasing amounts, from 2 g/l over the first 3 days, to 4 g/l over the last 7 days. A control group was given drinking water without caffeine. The level of lipid peroxidation, arginase and diamine oxidase (DAO) activity in the brain was measured. The results of our study show that arginase and diamine oxidase were decreased in animals treated with caffeine. The level of lipid peroxidation (MDA) was decreased also.The inhibitory effect of caffeine on arginase activity indicates that caffeine provides more arginine for consumption in other metabolic pathways. Considering the central stimulant effects of caffeine and the decreased lipid peroxidation level, it can be assumed that moderate short-term consumption of caffeine may be beneficial for brain function.

Metabolic correlations of glucocorticoids and polyamines in inflammation and apoptosis

Glucocorticoid hormones (GC) are essential in all aspects of human health and disease. Their anti-inflammatory and immunosuppressive properties are reasons for therapeutic application in several diseases. GC suppress immune activation and uncontrolled overproduction and release of cytokines. GC inhibit the release of pro-inflammatory cytokines and stimulate the production of anti-inflammatory cytokines. Investigation of GC’s mechanism of action, suggested that polyamines (PA) may act as mediators or messengers of their effects. Beside glucocorticoids, spermine (Spm) is one of endogenous inhibitors of cytokine production. There are many similarities in the metabolic actions of GC and PA. The major mechanism of GC effects involves the regulation of gene expression. PA are essential for maintaining higher order organization of chromatin in vivo. Spermidine and Spm stabilize chromatin and nuclear enzymes, due to their ability to form complexes with negatively charged groups on DNA, RNA and proteins. Also, there is an increasing body of evidence that GC and PA change the chromatin structure especially through acetylation and deacetylation of histones. GC display potent immunomodulatory activities, including the ability to induce T and B lymphocyte apoptosis, mediated via production of reactive oxygen species (ROS) in the mitochondrial pathway. The by-products of PA catabolic pathways (hydrogen peroxide, amino aldehydes, acrolein) produce ROS, well-known cytotoxic agents involved in programmed cell death (PCD) or apoptosis. This review is an attempt in the better understanding of relation between GC and PA, naturally occurring compounds of all eukaryotic cells, anti-inflammatory and apoptotic agents in physiological and pathological conditions connected to oxidative stress or PCD.

The role of L-arginine in toxic liver failure: interrelation of arginase, polyamine catabolic enzymes and nitric oxide synthase.

Summary.The existing interrelation in metabolic pathways of L-arginine to polyamines, nitric oxide (NO) and urea synthesis could be affected in sepsis, inflammation, intoxication and other conditions. The role of polyamines and NO in the toxic effect of mercury chloride on rat liver function was studied. Administration of mercury chloride for 24 h led to significantly elevated plasma activities of Alanine transaminase (ALT) and Aspartate transaminase (AST). Malondyaldehyde (MDA) levels were unaffected (p > 0.05) and arginase activity was significantly decreased (p < 0.05) while nitrate/nitrite production was significantly elevated (p < 0.001) in liver tissue. Polyamine oxidase (PAO) and diamine oxidase (DAO) activities, enzymes involved in catabolism of polyamines, were decreased. L-arginine supplementation to intoxicated rats potentiated the effect of mercury chloride on NO production and it was ineffective on arginase activity.Results obtained in this study show that mercury chloride-induced toxicity leads to abnormally high levels of ALT and AST that may indicate liver damage with the involvement of polyamine catabolic enzymes and NO.

Spermidine Influence on the Nitric Oxide Synthesis and Arginase Activity Relationship During Experimentally Induced Seizures

Nitric oxide (NO), a potential candidate for a modulator of convulsive activity, is a mediator in several pathological events in the central nervous system. The polyamines, spermidine (Spd) and spermine, are neuromodulators influencing the metabolism of L-arginine and NO production. Here we examined the effects of Spd on NO production and arginase activity during convulsions induced by pentylenetetrazol (PTZ). Male Wistar rats were allocated into four experimental groups of 8 animals each and received the following treatments: I (control)--saline, intraperitoneally (i.p.); II (PTZ)--seizures induced by pentylenetetrazol (100mg/kg bw i.p); III (Spd)--Spd (1 mg/kg bw i.p.) 50 min before PTZ application; IV (Mid)--antiepileptic Midazolam (100 mg/kg bw) 45 min before PTZ. In brain cortex, striatum, hippocampus, cerebellum, and brainstem homogenates, nitrite + nitrate levels and arginase activity were determined. Spermidine showed proepileptic effects. shortening seizure latency and inducing a more profound increase of NO production than PTZ in all brain structures. PTZ reduced arginase activity, whereas Spd pretreatment increased enzyme activity, with the most profound effects in cerebellum and brainstem. The results point out the importance of polyamine and arginine metabolism in the brain during seizures, suggesting a regulatory role for polyamines and arginase in NO production.

Effects of glucocorticoids on polyamine metabolism in liver and spleen of guinea pig during sensitization

Summary.Glucocorticoids are potent anti-inflammatory and immunosuppressive agents. As endogenous inhibitors of cytokine synthesis, glucocorticoids suppress immune activation and uncontrolled overproduction of cytokines, preventing tissue injury. Also, polyamine spermine is endogenous inhibitor of cytokine production (inhibiting IL-1, IL-6 and TNF synthesis). The idea of our work was to examine dexamethasone effects on the metabolism of polyamines, spermine, spermidine and putrescine and polyamine oxidase activity in liver and spleen during sensitization of guinea pigs. Sensitization was done by application of bovine serum albumin with addition of complete Freund’s adjuvant. Our results indicate that polyamine amounts and polyamine oxidase activity increase during immunogenesis in liver and spleen. Dexamethasone application to sensitized and unsensitized guinea pigs causes depletion of polyamines in liver and spleen. Dexamethasone decreases polyamine oxidase activity in liver and spleen of sensitized guinea pigs, increasing at the same time PAO activity in tissues of unsensitized animals.

Possible impact of plasma RNase activity on immune dysfunction in juvenile diabetes mellitus

Aim:  The ribonuclease (RNase) family represents important enzymes used widely in biomedical and biotechnological applications, as well as for diagnostic and therapeutic purposes. This study was undertaken to test the possibility that plasma alkaline RNase (free or inhibitory bound) determination may be useful in studying the dysregulation of nucleic acid and oligonucleotide metabolism as a possible pathogenetic mechanism in development of immune dysfunction in juvenile diabetes mellitus.

Morphometric and biochemical characteristics of short-term effects of ethanol on rat cardiac muscle

Alcoholism is a very important cause of congestive cardiomyopathy in man. The aim of this study was to examine a short-term effect of ethanol in rat cardiac muscle, using histologic, morphometric and biochemical methods. Experiments were carried out in Wistar male albino rats, divided into two groups: the control group consisting of eight animals receiving tap water, and the experimental group comprising eight animals received ethyl alcohol for ten days, in a single daily dose of 3 g ethanol/kg body weight, per os, using esophageal intubation. The mean volume weighted nuclear volume of cardiac myocytes was estimated by point sampled intercept method, by objective x 100. The mean cubed nuclear intercept length was multiplied by pi and divided by 3. For biochemical analysis, a 10% water tissue homogenate from the left ventricle was made. In the experimental group, the mean volume-weighted nuclear volume (15.08 +/- 5.20 microm3) was significantly lower than in the control group (51.32 +/- 7.83 microm3) (p < 0.001). The treatment of experimental animals with ethanol caused significant increase of aldolase (p < 0.0001) and aspartate transaminase (p < 0.05) activity in the rat cardiac tissue; at the same time, the enzyme activity of creatine phosphokinase, alanine transaminase and alkaline phosphatase were not changed in the experimental group compared to the control values. The amount of the glucose in the cardiac muscle was greater in the experimental group compared to the control animals. Our results suggest that there is depression of cardiomyocyte nuclei in experimental animals treated with ethanol. Alcohol intake results in the loss of Krebs cycle enzymes and as a consequence there is greater utilization of fatty acids for energy production.

Protective effect of interferon-? on the DNA- and RNA-degrading pathway in anti-Fas-antibody induced apoptosis

Aim:  Fas membrane‐associated polypeptide antigen is a receptor molecule responsible for apoptosis‐mediated signals. In animal models of acute viral hepatitis, apoptosis of hepatocytes is mediated by Fas‐death receptors; therefore, the aim of this study was to evaluate the effect of interferon (IFN)‐α on apoptotic markers and nuclease activity against different coding and non‐coding single and double stranded RNAs during Fas‐induced liver apoptosis.

Polyamine oxidase activity in peripheral blood of newborn infants with neonatal hyperbilirubinemia: is bilirubin an antioxidant?

BACKGROUND Neonatal hyperbilirubinemia can be physiological and pathological and most frequently is a consequence of faster erythrocytes (RBC) hemolysis. Free unconjugated bilirubin is a highly toxic compound, especially for the central nervous system. The most abundant polyamines circulating in blood are spermidine (Spd) and spermine (Sp), which are mainly localized in RBC, where they control membrane permeability. Polyamine oxidase (PAO) exerts an important activity in the plasma and erythrocytes of newborn infants with hyperbilirubinemia, catalyzing the oxidative deamination of Sp and Spd, producing potentially toxic agents that induce apoptosis of mammalian cells. The present study investigated polyamine metabolism by measuring PAO activity in the blood of newborn infants with hyperbilirubinemia and explored the possible antioxidant function of bilirubin through monitoring malondialdehyde (MDA) levels. METHODS The study included 43 newborns, 10 in the control and 33 in the diseased group. Blood PAO activity and bilirubin and MDA levels were measured using spectrophotometric methods. RESULTS/DISCUSSION Our results indicate that bilirubin, at physiologic concentrations, protects neonatal erythrocytes against oxidative stress. The positive correlation between PAO activity and MDA levels with high bilirubin concentrations (> 200 micromol/L) in newborn infants indicates that in pathological conditions, bilirubin cannot exert its antioxidant function. CONCLUSION Investigating the function of polyamines in erythrocytes and the importance of PAO related to hemolysis and bilirubin synthesis is necessary to shed light on the functions of PAO and its metabolites on the permeability of the erythrocyte membrane.