Levels Of Thiobarbituric Acid Reactants Research Articles

Detection and partial characterization of a putative closterovirus affecting Ficus carica: molecular, ultrastructural and physiological aspects of infected leaves

Fig mosaic disease (FMD) was systemically transmitted to susceptible fig plants by grafting and led to development of symptoms including a wide array of discoloration, green blisters, mottling, crinkling and deformation of leaves and fruits. A sensitive assay for the detection of FMV using specific enzyme-linked immune sorbent assay and reverse transcription-polymerase chain reaction was developed. A double-stranded RNA ca. 19 kbp in size was obtained from the fig tissue extract and used as a template. A cDNA fragment (350 bp) of phosphate motifs 1 and 2 of the heat shock-protein 70 homologue of the family Closteroviridae was amplified using a specific primer set. Virions were flexuous filaments up to 1,800 nm in length and ~21 nm in diameter. Viral infection formed new polypeptides in leaves that reflect pathogen-related proteins. Total photosynthetic pigment contents of virus-infected leaves decreased by 33.06%, with a raise in chl a/b ratio that was mainly caused by a decline in chl b content. Chloroplasts of virus-infected leaves lost their envelopes, and the internal structures of grana and stroma thylakoids were deformed and turned to spherical shapes. No virus inclusion bodies were detected in chloroplasts or other mesophyll cell organelles. Starch grains of chloroplasts were absent in the virus-infected leaves. Viral infection caused a decline in catalase and induction in peroxidase activities in leaves. Levels of thiobarbituric acid reactants in viral-infected leaves increased by 15%, while the content of total phenolics increased by 29%, in respect to healthy ones. The present results indicate a possible presence of Fig leaf mottle-associated virus-1 (FLMaV-1) in fig trees and provide an overview of the negative effects on fig leaves in response to FLMaV-1 infection from morphological, physiological and subcellular perspectives.

Healing Impairment Effect of Cyclooxygenase Inhibitors on Dextran Sulfate Sodium-Induced Colitis in Rats

We examined the effects of various cyclooxygenase (COX) inhibitors on the healing of colonic lesions induced by dextran sulfate sodium (DSS) in the rat. Colonic lesions were induced by 2.5% DSS in the drinking water for 7 days, and then the animals were fed with tap water for subsequent 7 days. Indomethacin (a nonselective COX inhibitor), SC-560 (a selective COX-1 inhibitor), or rofecoxib (a selective COX-2 inhibitor) was given orally twice daily after termination of the DSS treatment. DSS treatment caused severe colonic lesions with a decrease in body weight gain and colon length as well as an increase in myeloperoxidase activity and thiobarbituric acid reactant levels. The severity of colitis gradually reduced, with an improvement of morphological and histological alterations. Daily administration of indomethacin and rofecoxib significantly delayed the healing of colitis with deleterious influences on histological restitution as well as mucosal inflammation, while SC-560 had no effect. Although COX-1 mRNA was expressed in the colon without much alteration during the test period, the expression of COX-2 was upregulated with a peak on day 3 and decreased thereafter. The mucosal prostaglandin E₂ content in the colon showed a biphasic change, in parallel with that of the COX-2 expression. The increased prostaglandin E₂ production in the injured mucosa was attenuated by indomethacin and rofecoxib, but not by SC-560. These results suggest that endogenous prostaglandins produced by COX-2 play an important role in the healing of DSS-induced colonic lesions. Caution should be paid to the use of selective COX-2 inhibitors as well as nonsteroidal anti-inflammatory drugs in patients with colitis.

Iron toxicity and chelation therapy.

Iron is an essential mineral for normal cellular physiology, but an excess can result in cell injury. Iron in low-molecular-weight forms may play a catalytic role in the initiation of free radical reactions. The resulting oxyradicals have the potential to damage cellular lipids, nucleic acids, proteins, and carbohydrates; the result is wide-ranging impairment in cellular function and integrity. The rate of free radical production must overwhelm the cytoprotective defenses of cells before injury occurs. There is substantial evidence that iron overload in experimental animals can result in oxidative damage to lipids in vivo, once the concentration of iron exceeds a threshold level. In the liver, this lipid peroxidation is associated with impairment of membrane-dependent functions of mitochondria and lysosomes. Iron overload impairs hepatic mitochondrial respiration primarily through a decrease in cytochrome C oxidase activity, and hepatocellular calcium homeostasis may be compromised through damage to mitochondrial and microsomal calcium sequestration. DNA has also been reported to be a target of iron-induced damage, and this may have consequences in regard to malignant transformation. Mitochondrial respiratory enzymes and plasma membrane enzymes such as sodium-potassium-adenosine triphosphatase (Na(+) + K(+)-ATPase) may be key targets of damage by non-transferrin-bound iron in cardiac myocytes. Levels of some antioxidants are decreased during iron overload, a finding suggestive of ongoing oxidative stress. Reduced cellular levels of ATP, lysosomal fragility, impaired cellular calcium homeostasis, and damage to DNA all may contribute to cellular injury in iron overload. Evidence is accumulating that free-radical production is increased in patients with iron overload. Iron-loaded patients have elevated plasma levels of thiobarbituric acid reactants and increased hepatic levels of aldehyde-protein adducts, indicating lipid peroxidation. Hepatic DNA of iron-loaded patients shows evidence of damage, including mutations of the tumor suppressor gene p53. Although phlebotomy therapy is effective in removing excess iron in hereditary hemochromatosis, chelation therapy is required in the treatment of many patients who have combined secondary and transfusional iron overload due to disorders in erythropoiesis. In patients with beta-thalassemia who undergo regular transfusions, deferoxamine treatment has been shown to be effective in preventing iron-induced tissue injury and in prolonging life expectancy. The use of the oral chelator deferiprone remains controversial, and work is continuing on the development of new orally effective iron chelators.

Vitamin E, lipid profile, and peroxidation in hemodialysis patients

Hypertriglyceridemia, lipid peroxidation, and abnormalities of the plasma fatty acid (PUFA) profile may be important risk factors for the atherosclerotic cardiovascular disease in hemodialysis (HD) patients. We investigated how these factors are affected by vitamin E supplementation carried out by oral administration (clinical study 1) and dialysis with vitamin E-modified dialyzers (clinical study 2). In the HD patients, conditions of relative vitamin E deficiency were observed [lowered vitamin E/triglyceride (TG) ratio] in the presence of high levels of thiobarbituric acid reactants (TBARs) and decreased levels of the polyunsaturated fraction of PUFAs paired with an increased amount of monounsaturated ones (MUFA). In both studies, vitamin E supplementation significantly increased the levels of vitamin E in the plasma without affecting TG levels and provided a partial correction of TBAR levels. Of note was the relative increase in the PUFA fraction, which gave solid proof of an anti(per)oxidant effect of vitamin E supplementation in HD patients. Vitamin E supplementation was also observed to increase plasma levels of reduced glutathione and NOx (NO2 + NO3). The results suggest that vitamin E supplementation may be an effective accessory therapy to combat oxidative stress-lowering lipid peroxidation in HD patients.

Role of inducible nitric oxide synthase in dextran sulphate sodium-induced colitis.

Different authors have postulated both toxic and protective effects for nitric oxide (NO) in the pathophysiology of active inflammation. To examine the role of NO, especially that produced by the inducible form of nitric oxide synthase (iNOS), by investigating the effects of NOS inhibitors and NO donors on inflammation in experimental acute colitis. Acute colitis was induced in rats by dextran sulphate sodium (DSS). White blood cell counts and levels of thiobarbituric acid reactants in the portal blood were determined, as were histological changes in the colonic mucosa. We then evaluated the effects of N(G)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine (AG) and an NO donor on DSS-induced changes in these inflammatory parameters. Inhibition of NO production by either L-NAME or AG worsened DSS-induced inflammation, suggesting a protective role for NO in acute colitis. On the other hand, a NO donor also exaggerated DSS-induced inflammatory parameters, suggesting that acute colitis may be aggravated by either too much or too little NO. These results suggest that medical treatment of ulcerative colitis must aim for maintenance of appropriate NO levels in the intestinal mucosa.

Oxidative stress and antioxidant status in mouse kidney: effects of dietary lipid and vitamin E plus iron

The purpose of this study was to determine the effects of dietary fat, vitamin E, and iron on oxidative damage and antioxidant status in kidneys of mice. Sixty 1-month-old male Swiss-Webster mice were fed a basal vitamin E-deficient diet that contained either 8% fish oil + 2% corn oil or 10% lard with or without 1 g all-rac-α-tocopherol acetate or 0.74 g ferric citrate per kilogram of diet for 4 weeks. Significantly ( P < 0.05) higher levels of lipid peroxidation products, thiobarbituric acid reactants (TBAR), and conjugated dienes were found in the kidneys of mice fed with fish oil compared with mice fed lard irrespective of vitamin E status. Mice maintained on a vitamin E-deficient diet had significantly higher renal levels of TBAR, but not conjugated dienes, than the supplemented group. Fish oil fed mice receiving vitamin E supplementation had lower levels of α-tocopherol than did mice in the lard fed group. Significantly higher levels of ascorbic acid were also found in the kidneys of mice fed with fish oil than were found in mice fed lard. The levels of protein carbonyls and glutathione (GSH), and activities of catalase, superoxide dismutase, selenium (Se)-GSH peroxidase, and non-Se-GSH peroxidase were not significantly altered by dietary fat or vitamin E. Dietary iron had no significant effect on any of the oxidative stress and antioxidant indices measured. The results obtained provide experimental evidence for the pro-oxidant effect of high fish oil intake in mouse kidney and suggest that dietary lipids play a key role in determining cellular susceptibility to oxidative stress.

High-Performance Liquid Chromatography-Electrochemical Determination of Salicylate Hydroxylation Products as an in Vivo Marker of Oxidative Stress

The in vivo measurement of highly reactive free radicals, such as hydroxyl radical ( . OH), in humans is very difficult if not impossible. Specific markers are currently under investigation (amino acid hydroxylatin, protein, DNA adducts, and aromatic probes). They are based on the ability of . OH to attack aromatic molecules to produce hydroxylated compounds that can be measured directly. In vivo, radical metabolism of salicylic acid produces two main hydroxylated derivatives, i.e., 2,3- and 2,5-dihydroxybenzoic acid (2,3- and 2,5-DHBA). The measurement of 2,3-DHBA, following oral administration of salicylate or its acetylated form (aspirin), has been proposed for assessment of in vivo oxidative stress. In this work, a sensitive method for the detection of in vivo . OH generation is presented. The methodology employs a high-pressure liquid chromatography with electrochemical detection for the identification and quantification of the hydroxylation products from the reaction of . OH with salicylate. A detection limit of less than 0.1 pmol for the hydroxylation products has been achieved with electrochemical detector responses which were linear over at least five orders of magnitude. Using this technique, we measured plasma levels of 2,3- and 2,5-DHBA and dihydroxylated derivatives/salicylic acid ratios following the administration of 1000 mg aspirin in 20 healthy subjects. In the same individuals, plasma levels of thiobarbituric acid reactants (TBARs), a major index of lipid peroxidation, were also measured and correlation with hydroxylated products was sought. The plasma level of TBARs was positively correlated with the 2,5-DHBA/salicylic acid ratio, but not with the absolute plasma level of 2,3-DHBA.

Free Radicals in Reperfusion Injury After Acute Aortic Occlusion

This investigation aims to evaluate the role of free radicals and the influence of radical scavengers in reperfusion injury of the skeletal muscle in male Wistar rats. Various parameters, including tissue blood flow, serum and calf muscle tissue levels of thiobarbituric acid (TBA) reactants, and the water content of calf muscles, were measured in the experimental setting, where infrarenal abdominal aorta was occluded for two hours followed by reperfusion with and without intraperitoneal administration of radical scavengers (superoxide dismutase or catalase). The rats were divided into four groups: group A: single laparotomy; group B: two-hour occlusion; group C: two-hour occlusion with superoxide dismutase; group D: two-hour occlusion with catalase. The increases in both serum and calf muscle tissue levels of TBA reactants following reperfusion were diminished in groups C and D as compared with those in group B. The water content of calf muscles showed a significant increase after reperfusion in group B, whereas it did not change in group D. The calf muscle tissue blood flow after reperfusion was significantly lower in group C than that in group B. These results indicate that oxygen-derived free radicals may be responsible for reperfusion injury in the lower extremity and that the attenuated blood flow during reperfusion produced by these scavengers seems to play an important role in reducing lipid peroxidation . In a clinical setting, limiting the rate of reperfusion blood flow may reduce the reperfusion injury in skeletal muscle.

Burn-induced gastric mucosal hemodynamic disturbance in the rat. Role of platelet-activating factor.

The role of platelet-activating factor (PAF) as a mediator of gastric damage associated with burn injury was examined in the rat. Gastric mucosal hemodynamics was recorded continuously by means of laser-Doppler flowmetry and reflectance spectrophotometry. Burn injury induced prolonged hypotension and rapid-onset and long-lasting gastric mucosal hemodynamic derangement-that is, ischemia with congestion. This hemodynamic disturbance was significantly inhibited by CV-6209, a specific PAF antagonist. Elevated tissue levels of thiobarbituric acid reactants and pathologic changes in the gastric mucosa subsequent to burn injury were also significantly alleviated by the preadministration of CV-6209. These results strongly suggest a role of PAF as an important mediator of gastric damage after burn injury through its potent vasoactive effect on gastric microcirculation.

Biochemical effects of combined gases of nitrogen dioxide and ozone II. Species differences in lipid peroxides and antioxidative protective enzymes in the lungs

Changes in lipid peroxide (thiobarbituric acid reactant) levels, in the content of non-protein sulfhydryls (NPSH) and total proteins, and in the activities of antioxidative protective enzymes were examined in the lungs of four animal species exposed to a mixture of NO 2 and O 3 for 2 weeks. Male mice, hamsters, rats and guinea pigs were used. Thiobarbituric acid (TBA) reactant levels were increased significantly in the lungs of mice and guinea pigs, but not in hamsters and rats. NPSH contents were increased markedly in hamsters, mice and rats, but not in guinea pigs. The activities of antioxidative protective enzymes also changed with the exposure. The most characteristic change was the significant increase in glutathione peroxidase (GPx-H 2O 2) activity in hamsters and rats — species which did not exhibit increases in their TBA reactant levels. The increase in this enzyme activity in mice was significant, but not very large. Furthermore, guinea pigs were genetically deficient in this enzyme, and the increase in glycolytic enzymes for regenerating NADPH was also lowest in guinea pigs. The glutathione S-transferase (GSH-Tase) activity in mice and guinea pigs was decreased by exposure to the combined gases. These results suggest that the increases in lipid peroxide levels in mice and guinea pigs may be due to a lesser ability to regenerate protective reducing substances, such as NPSH and NADPH, than that of hamsters and rats. Induction of protective enzyme activities on exposure to the combined gases was also poor in mice and guinea pigs.

Lipid Peroxidation and Experimental Disseminated Intravascular Coagulation in Rats Induced by Endotoxin

The experimental model of disseminated intravascular coagulation (DIC) in rats induced by a 4 hr sustained infusion of 100 mg/kg endotoxin was used to study the relationship between lipid peroxidation and DIC. Serum thiobarbituric acid (TBA) reactive substances, important and damaging products of lipid peroxidation, increased gradually during the infusion of endotoxin. The levels of TBA reactants in serum, abdominal aortic wall and ileum mucosa were significantly increased in rats infused with 100 mg/kg of endotoxin for 4 hr, as compared with the control rats continuously given 11.4 ml of physiological saline for 4 hr. However, the levels in liver and kidney tissues, and in gastric, jejunum and colon mucosa did not increase significantly.

Influence of dietary vitamin E on the red cells of ozone-exposed rats

The effect of dietary vitamin E on the susceptibility of red blood cells to ozone exposure was studied in rats. One- and two-month-old male Sprague-Dawley rats were fed a basal vitamin E-deficient diet with or without 45 ppm vitamin E for 4 and 3 months, respectively, and were exposed to 0 or 0.8 ppm ozone continuously for 7 days. Ozone exposure resulted in a significant increase in the activities of glutathione (GSH) peroxidase, pyruvate kinase, and lactate dehydrogenase, and a decrease in GSH level in the red cells of vitamin E-deficient rats, but not in those of the supplemented group. The activities of glucose-6-phosphate dehydrogenase, catalase, and superoxide dismutase and levels of thiobarbituric acid reactants, methemoglobin, hemoglobin, and reticulocytes were not significantly altered by ozone exposure or by the nutritional status of vitamin E. The results suggest that depletion of dietary vitamin E renders animals more susceptible to ozone exposure.