Dušan Mladenović

Liver antioxidant capacity in the early phase of acute paracetamol-induced liver injury in mice

The aim of our study was to investigate the relationship between liver antioxidant capacity and hepatic injury in the early phase of acute paracetamol intoxication in mice. Male Swiss mice were divided into groups: (1) control, that received saline, (2) paracetamol-treated group (300 mg/kg intraperitoneally). Animals were sacrificed 6, 24 and 48 h after treatment. Oxidative stress parameters were determined in blood and liver samples spectrophotometrically. Liver malondialdehyde and nitrite + nitrate level were significantly increased 6 h after paracetamol administration in comparison with control group (p < 0.05). Paracetamol induced a significant reduction in total liver superoxide dismutase (SOD) and copper/zinc SOD activity at all time intervals (p < 0.01). However, manganese SOD activity was significantly increased within 6 h (p < 0.01), while its activity progressively declined 24 and 48 h after paracetamol administration in comparison with control group (p < 0.01). Content of sulfhydryl groups in the liver was increased 24 h after paracetamol administration (p < 0.05), while its level was decreased within next 24 h when compared to control animals (p < 0.01). Our data showed that liver antioxidant capacity increases in first 24 h of paracetamol-induced liver injury were in correlation with manganese SOD activity and increase in level of sulfhydryl groups.

Dynamics of oxidative/nitrosative stress in mice with methionine–choline-deficient diet-induced nonalcoholic fatty liver disease

Insulin resistance, oxidative stress, and proinflammatory cytokines play a key role in pathogenesis of nonalcoholic fatty liver disease (NAFLD). The aim of our study was to investigate the dynamics of oxidative/nitrosative stress in methionine–choline-deficient (MCD) diet -induced NAFLD in mice. Male C57BL/6 mice were divided into following groups: group 1: control group on standard diet; group 2: MCD diet for 2, 4, and 6 weeks (MCD2, MCD4, and MCD6, respectively). After treatment, liver and blood samples were taken for histopathology, alanine- and aspartate aminotransferase, acute phase reactants, and oxidative/nitrosative stress parameters. Liver malondialdehyde level was higher in all MCD-fed groups versus control group (p < 0.01), while nitrites + nitrates level showed a progressive increase. The activity of total superoxide dismutase and its isoenzymes was significantly lower in all MCD-fed groups (p < 0.01). Although catalase activity was significantly lower in MCD-fed animals at all intervals (p < 0.01), the lowest activity of this enzyme was evident in MCD4 group. Liver content of glutathione was lower in MCD4 (p < 0.05) and MCD6 group (p < 0.01) versus control. Ferritin and C-reactive protein serum concentration were significantly higher only in MCD6 group. Our study suggests that MCD diet induces a progressive rise in nitrosative stress in the liver. Additionally, the most prominent decrease in liver antioxidative capacity is in the fourth week, which implies that application of antioxidants would be most suitable in this period, in order to prevent nonalcoholic steatohepatitis but not the initial NAFLD phase.

Finasteride improves motor, EEG, and cellular changes in rat brain in thioacetamide-induced hepatic encephalopathy

Neurosteroids are involved in the pathogenesis of hepatic encephalopathy (HE). This study evaluated the effects of finasteride, inhibitor of neurosteroid synthesis, on motor, EEG, and cellular changes in rat brain in thioacetamide-induced HE. Male Wistar rats were divided into the following groups: 1) control; 2) thioacetamide-treated group, TAA (300 mg·kg(-1)·day(-1)); 3) finasteride-treated group, FIN (50 mg·kg(-1)·day(-1)); and 4) group treated with FIN and TAA (FIN + TAA). Daily doses of TAA and FIN were administered in three subsequent days intraperitoneally, and in the FIN + TAA group FIN was administered 2 h before every dose of TAA. Motor and reflex activity was determined at 0, 2, 4, 6, and 24 h, whereas EEG activity was registered about 24 h after treatment. The expressions of neuronal (NeuN), astrocytic [glial fibrilary acidic protein (GFAP)], microglial (Iba1), and oligodendrocyte (myelin oligodendrocyte glycoprotein) marker were determined 24 h after treatment. While TAA decreased all tests, FIN pretreatment (FIN + TAA) significantly improved equilibrium, placement test, auditory startle, head shake reflex, motor activity, and exploratory behavior vs. the TAA group. Vital reflexes (withdrawal, grasping, righting and corneal reflex) together with mean EEG voltage were significantly higher (P < 0.01) in the FIN + TAA vs. the TAA group. Hippocampal NeuN expression was significantly lower in TAA vs. control (P < 0.05). Cortical Iba1 expression was significantly higher in experimental groups vs. control (P < 0.05), whereas hippocampal GFAP expression was increased in TAA and decreased in the FIN + TAA group vs. control (P < 0.05). Finasteride improves motor and EEG changes in TAA-induced HE and completely prevents the development of hepatic coma.

Oxidative stress in liver and red blood cells in acute lindane toxicity in rats

The aim of our study was to determine the role and dynamics of oxidative and nitrosative stress, as well as superoxide dismutase (SOD) and catalase activity in the hepatocytes and erythrocytes in early phase of acute lindane intoxication. Male Wistar rats were divided into groups: control, dimethylsulfoxide and lindane-treated groups (L, 8 mg/kg, intraperitoneally). Animals were sacrificed 0.5 and 4 hours after treatment (L0.5 and L4 groups, respectively). Oxidative and nitrosative stress parameters and antioxidant enzymes were determined spectrophotometrically. Liver and plasma thiobarbituric acid reactive substances (TBARS) concentration were significantly increased 0.5 after lindane administration (p < .01), with subsequent additional rise within 4 hours (p < .01), while plasma nitrite + nitrate level was significantly higher only 4 hours after lindane treatment. Total liver SOD activity was significantly increased in L4 group in comparison with control group (p < .01). In conclusion, oxidative and nitrosative stress play an important role in early phase of acute lindane hepatotoxicity. Antioxidant capacity of hepatocytes is partly increased, due to an adaptive increase in SOD activity.

Different Sensitivity of Various Brain Structures to Thioacetamide-Induced Lipid Peroxidation

Thioacetamide (TAA) exerts hepatotoxic, neurotoxic and carcinogenic effects. The aim of our study was to investigate the effects of TAA on lipid peroxidation and catalase activity in various rat brain regions. Male Wistar rats were divided into following groups: 1. control, saline-treated; 2. thioacetamide-treated groups, TAA300 (300 mg/kg), TAA600 (600 mg/kg) and TAA900 (900 mg/kg). Daily dose of TAA (300 mg/kg) was administered intraperitoneally once (TAA300), twice (TAA600) and three times (TAA900) in consecutive days. Brain samples were collected 24 h after the last dose of TAA and malondialdehyde (MDA) level and catalase activity were determined in cortex, brainstem and hippocampus. MDA level was significantly increased while catalase activity was significantly lower in all brain regions in TAA900 group in comparison with control group. In TAA600 MDA level was increased in the brainstem and cortex when compared to control (p < 0.01). The same dose of TAA 600 mg/kg induced a significant decline in catalase activity in the brainstem and cortex and an increase in its activity in the hippocampus when compared to control (p < 0.01). In TAA300 an increase in MDA level was evident only in the brainstem. Catalase activity was significantly higher in the cortex and hippocampus in TAA300 group in comparison with control (p < 0.01). Based on these results, it may be concluded that various rat brain regions have different sensitivity to TAA-induced lipid peroxidation with hippocampus being less sensitive than cerebral cortex and brainstem.

Rimonabant Improves Oxidative/Nitrosative Stress in Mice with Nonalcoholic Fatty Liver Disease

The present study deals with the effects of rimonabant on oxidative/nitrosative stress in high diet- (HFD-) induced experimental nonalcoholic fatty liver disease (NAFLD). Male mice C57BL/6 were divided into the following groups: control group fed with control diet for 20 weeks (C; n = 6); group fed with HFD for 20 weeks (HF; n = 6); group fed with standard diet and treated with rimonabant after 18 weeks (R; n = 9); group fed with HFD and treated with rimonabant after 18 weeks (HFR; n = 10). Daily dose of rimonabant (10 mg/kg) was administered to HFR and R group by oral gavage for two weeks. Treatment induced a decrease in hepatic malondialdehyde concentration in HFR group compared to HF group (P < 0.01). The concentration of nitrites + nitrates in liver was decreased in HFR group compared to HF group (P < 0.01). Liver content of reduced glutathione was higher in HFR group compared to HF group (P < 0.01). Total liver superoxide dismutase activity in HFR group was decreased in comparison with HF group (P < 0.01). It was found that rimonabant may influence hepatic iron, zinc, copper, and manganese status. Our study indicates potential usefulness of cannabinoid receptor type 1 blockade in the treatment of HFD-induced NAFLD.

The effects of cold-induced stress on liver oxidative injury during binge drinking

The aim of our study was to evaluate the effects of cold stress on hepatic oxidative damage during binge drinking in rats. Male Wistar rats were divided into the following groups: group 1: control; group 2: ethanol-treated; group 3: stress-exposed; group 4: stress-exposed and ethanol-treated group. Oxidative and nitrosative stress parameters in the liver were determined spectrophotometrically, 12 h after treatment. Liver malondialdehyde concentration was significantly higher in group 4 when compared with groups 2 and 3. The highest increase in nitric oxide concentration was demonstrated in group 4 in comparison with groups 2 and 3. Superoxide dismutase (SOD) activity was significantly lower in group 4 when compared with groups 2 and 3. Ethanol administration induced a larger decrease in the activity of copper-/zinc-SOD in group 4 in comparison with group 2. Activity of manganese-SOD (Mn-SOD) was significantly higher in groups 3 and 4, when compared with control values, but the greatest increase in the activity of Mn-SOD was demonstrated in group 2. We also evaluated statistically significant decrease in the level of reduced gluthatione in the liver of group 4 in comparison with group 3. Based on our study, it can be concluded that cold-exposed stress and binge ethanol drinking have additive effects in imbalance between pro-oxidant and antioxidant defense system in liver.

Comparison between carotid artery wall thickness measured by multidetector row computed tomography angiography and intimae-media thickness measured by sonography.

The increased thickness of the carotid wall >1 mm is a significant predictor of coronary and cerebrovascular diseases. The purpose of our study was to assess the agreement between multidetector row computed tomography angiography (MDCTA) in measuring carotid artery wall thickness (CAWT) and color Doppler ultrasound (CD-US) in measuring intimae-media thickness (IMT). Eighty-nine patients (aged 35–81) were prospectively analyzed using a 64-detector MDCTA and a CD-US scanner. Continuous data were described as the mean value ± standard deviation, and were compared using the Mann–Whitney U test. A p value <0.05 was considered significant. Bland–Altman statistics were employed to measure the agreement between MDCTA and CD-US. CAWT ranged from 0.62 to 1.60 mm, with a mean value of 1.09 mm. IMT ranged from 0.60 to 1.55 mm, with a mean value of 1.06 mm. We observed an excellent agreement between CD-US and MDCTA in the evaluation of the common carotid artery thickness, with a bias between methods of 0.029 mm (which is a highly statistically important difference of absolute values [t = 43.289; p < 0.01] obtained by paired T test), and limits of agreement from 0.04 to 0.104. Pearson correlation coefficient was 0.9997 (95% CI 0.9996–0.9998; p < 0.01). We conclude that there is an excellent correlation between CAWT and IMT measurements obtained with the MDCTA and CD-US.

[The role of oxidative/nitrosative stress in pathogenesis of paracetamol-induced toxic hepatitis].

INTRODUCTION Paracetamol is an effective analgesic/antipyretic drug when used at therapeutic doses. However, the overdose of paracetamol can cause severe liver injury and liver necrosis. The mechanism of paracetamol-induced liver injury is still not completely understood. Reactive metabolite formation, depletion of glutathione and alkylation of proteins are the triggers of inhibition of mitochondrial respiration, adenosine triphosphate depletion and mitochondrial oxidant stress leading to hepatocellular necrosis. ROLE OF OXIDATIVE STRESS IN PARACETAMOL-INDUCED LIVER INJURY: The importance of oxidative stress in paracetamol hepatotoxicity is controversial. Paracetamol-induced liver injury cause the formation of reactive oxygen species. The potent sources of reactive oxygen are mitochondria, neutrophils. Kupffer cells and the enzyme xatnine oxidase. Free radicals lead to lipid peroxidation, enzymatic inactivation and protein oxidation. ROLE OF MITOCHONDRIA IN PARACETAMOL-INDUCED OXIDATIVE STRESS: The production of mitochondrial reactive oxygen species is increased, and the glutathione content is decreased in paracetamol overdose. Oxidative stress in mitochondria leads to mitochondrial dysfunction with adenosine triphosphate depletion, increase mitochondrial permeability transition, deoxyribonucleic acid fragmentation which contribute to the development of hepatocellular necrosis in the liver after paracetamol overdose. ROLE OF KUPFFER CELLS IN PARACETAMOL-INDUCED LIVER INJURY: Paracetamol activates Kupffer cells, which then release numerous cytokines and signalling molecules, including nitric oxide and superoxide. Kupffer cells are important in peroxynitrite formation. On the other hand, the activated Kupffer cells release anti-inflammatory cytokines. ROLE OF NEUTROPHILS IN PARACETAMOL-INDUCED LIVER INJURY: Paracetamol-induced liver injury leads to the accumulation of neutrophils, which release lysosomal enzymes and generate superoxide anion radicals through the enzyme nicotinamide adenine dinucleotide phosphate oxidase. Hydrogen peroxide, which is influenced by the neutrophil-derived enzyme myeloperoxidase, generates hypochlorus acid as a potent oxidant. ROLE OF PEROXYNITRITE IN PARACETAMOL-INDUCED OXIDATIVE STRESS: Superoxide can react with nitric oxide to form peroxynitrite, as a potent oxidant. Nitrotyrosine is formed by the reaction of tyrosine with peroxynitrite in paracetamol hepatotoxicity. CONCLUSION Overdose of paracetamol may produce severe liver injury with hepatocellular necrosis. The most important mechanisms of cell injury are metabolic activation of paracetamol, glutathione depletion, alkylation of proteins, especially mitochondrial proteins, and formation of reactive oxygen/nitrogen species.

Effect of acute lindane and alcohol intoxication on serum concentration of enzymes and fatty acids in rats

This study examines possible synergistic effects of lindane and ethanol on inducing liver injury and serum fatty acid derangement in adult male Wistar rats. When administered together, ethanol and lindane-induced even more pronounced increase of alanine aminotransferase (165 +/- 10 U/L) and gamma-glutamyltranspeptidase activity (10.3 +/- 0.6 U/L) than after isolated administration of either substance. In addition, separate administration of lindane and ethanol was followed by a significant decrease of linoleic acid level in the serum (301 +/- 38 mg/L, 276 +/- 35 mg/L vs. 416 +/- 48 mg/L). However, when ethanol administration was followed by lindane injection, serum linoleic acid was at the similar level found in the control group (516 +/- 62 mg/L). Ethanol-treated rats that received lindane 30 min after ethanol administration have shown a marked increase of palmitic (421 +/- 27 mg/L) and linolic acid level (43 +/- 5 mg/L) in comparison with rats that have been treated only with ethanol (316+/-26 mg/L for palmitic and 32 +/- 2 mg/L for linolic acid) or lindane (295 +/- 26 mg/L for palmitic and 301 +/- 38 mg/L for linolic acid). Linolic acid level was significantly greater in comparison with control group (29 +/- 1 mg/L). In conclusion, this study found enough evidence to support the hypothesis that acute ethanol intoxication potentiates lindane-induced liver injury and enhances lipid derangement.