Chlorpyrifos induced alterations in levels of thiobarbituric acid reactive substances and glutathione in rat brain

Chlorpyrifos, O,O'-diethyl-O-3,5,6-trichloro-2-pyridyl phosphorothionate, exposure in rats caused significant inhibition of acetylcholinesterae activity in different regions of brain and generated oxidative stress as evidenced by increase in the level of thiobarbituric acid reactive substances and decrease in the ratio of reduced to oxidized glutathione in all the three regions of brain. Malondialdehyde level was increased significantly in all regions of brain and the increase was dose dependent. Mid brain showed highest level of lipid peroxidation.     

Effect of chlorpyrifos on thiobarbituric acid reactive substances, scavenging enzymes and glutathione in rat tissues

The present study showed that exposure of chlorpyrifos, O,O'-diethyl-O-3,5,6-trichloro-2-pyridyl phosphorothionate (CPF), a widely used pesticide in rats caused significant inhibition of acetylcholinesterase (AChE) activity in different tissues viz., liver, kidney and spleen. CPF exposure also generated oxidative stress in the body, as evidenced by increase in thiobarbituric acid reactive substances (TBARS), decrease in the levels of superoxide scavenging enzymes viz., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in liver, kidney and spleen at all doses. Malondialdehyde levels were increased by 14%, 31% and 76% in liver, 11%, 31% and 64% in kidney and 32%, 75% and 99.9% in spleen when 50 mg, 100 mg and 200 mg/kg body wt. CPF was administered for three days. SOD and CAT activities were decreased in liver, kidney and spleen, while GPx activity showed slight increase in kidney at 50 mg and 100 mg dose, and decreased on further increase in dose of CPF. Liver and spleen showed dose-dependent decrease in GPx activity. The levels of reduced glutathione (GSH) was decreased, while oxidized glutathione (GSSG) was increased, thus a marked fall in GSH/GSSG ratio was observed in all tissues. A maximum decrease of 83% was observed in liver, followed by kidney and spleen, which showed 78% and 57% decrease, respectively in group given 200 mg/kg CPF. The levels of glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) were also decreased in liver and kidney, while spleen showed increase at lower doses, but decrease at high dose of CPF. The data provide evidence for induction of oxidative stress on CPF exposure.     

GM1 ganglioside attenuates convulsions and thiobarbituric acid reactive substances production induced by the intrastriatal injection of methylmalonic acid

The effects of the administration of monosialoganglioside (GM1) on methylmalonic acid (MMA)-induced convulsions, production of thiobarbituric acid reactive substances (TBARS) and on the striatal content of ascorbic acid and total non-protein thiol (SH) groups were evaluated in adult male rats. Animals received two intraperitoneal injections of GM1 (50 mg/kg) or saline (0.85% NaCl) spaced 24h apart. Thirty minutes after the second GM1 or saline injection, L-MMA (6 micromol) or NaCl (9 micromol) was injected into the right striatum and the animals were observed for the appearance of convulsions for 15 min. The animals were sacrificed and their striatal content of ascorbic acid, SH groups and TBARS was measured. The effect of GM1 on MMA-induced TBARS production in striatal homogenates was also evaluated in vitro.MMA injection caused convulsions (Sal-MMA: 9.8+/-1.4 episodes, which lasted 271+/-48 s) and increased the striatal content of TBARS (Sal-MMA: 149.0+/-11.5 nmol MDA/g tissue), but did not alter total striatal SH or ascorbic acid contents. GM1 pretreatment decreased MMA-induced convulsions (GM1-MMA: 6.3+/-2.0 episodes, which lasted 115.1+/-42.2s) and TBARS increase (GM1-MMA: 102.4+/-19.5 nmol MDA/g tissue). GM1 pretreatment increased ascorbic acid content of the striata (saline-pretreated: 1514+/-75.9; GM1-pretreated: 1878.6+/-102.8 microg ascorbic acid/mg tissue). MMA increased TBARS production in vitro, and GM1 had no effect on such MMA-induced effect.This study provides evidence that GM1 increases striatal ascorbic acid content and decreases MMA-induced neurotoxicity assessed by behavioral and neurochemical parameters.     

Effect of gossypol on tissue levels of glutathione and thiobarbituric acid reactive products in rats.

Gossypol was administered in pubertal and adult rats and lipid peroxide formation and GSH levels were estimated in different tissues like liver, testis, heart and kidney. Gossypol caused low generation of lipid peroxides, measured as thiobarbituric acid reactive products (TBAR), without causing significant changes in tissue glutathione (GSH) levels. This effect was more pronounced in liver and testis as compared to other tissues. In vitro effect of gossypol to inhibit lipid peroxidation as observed in vivo suggested that binding of gossypol to plasma membranes may result in inhibition of lipid peroxide generation.     

Up-regulation of inducible nitric oxide synthase and nitric oxide in Helicobacter pylori-infected human gastric epithelial cells: possible role of interferon-gamma in polarized nitric oxide secretion

Background. Nitric oxide (NO) generated by nitric oxide synthase (NOS) is known to be an important modulator of the mucosal inflammatory response. In this study, we questioned whether Helicobacter pylori infection could up‐regulate the epithelial cell inducible NOS (iNOS) gene expression and whether NO production could show polarity that can be regulated by immune mediators. Materials and Methods. Human gastric epithelial cell lines were infected with H. pylori, and the iNOS mRNA expression was assessed by quantitative RT‐PCR. NO production was assayed by determining nitrite/nitrate levels in culture supernatants. To determine the polarity of NO secretion by the H. pylori‐infected epithelial cells, Caco‐2 cells were cultured as polarized monolayers in transwell chambers, and NO production was measured. Results. iNOS mRNA levels were significantly up‐regulated in the cells infected with H. pylori, and expression of iNOS protein was confirmed by Western blot analysis. Increased NO production in the gastric epithelial cells was seen as early as 18 hours postinfection, and reached maximal levels by 24 hours postinfection. The specific MAP kinase inhibitors decreased H. pylori‐induced iNOS and NO up‐regulation. After H. pylori infection of polarized epithelial cells, NO was released predominantly into the apical compartment, and IL‐8 was released predominantly into basolateral compartment. The addition of IFN‐γ to H. pylori‐infected polarized epithelial cells showed a synergistically higher apical and basolateral NO release. Conclusion. These results suggest that apical NO production mediated by MAP kinase in H. pylori‐infected gastric epithelial cells may influence the bacteria and basolateral production of NO and IL‐8 may play a role in the tissue inflammation.     

Exhalation of H2O2 and thiobarbituric acid reactive substances (TBARs) by healthy subjects

Enhanced exhalation of H2O2 and TBARs have been reported in various inflammatory lung diseases. This may reflect activated phagocytes influx and free radical generation in the airways. However, to apply these compounds as markers of oxidative stress it is necessary to understand factors influencing their exhalation in healthy subjects. We investigated the concentration of H2O2 and TBARs in expired breath condensate (EBC) of 58 healthy volunteers. EBC was collected seven times every 4 h during 24 h and three times every 7 d during 2 consecutive weeks. The H2O2 exhalation revealed diurnal variation with two-peak values 0.45 +/- 0.29 microM and 0.43 +/- 0.22 microM at 12:00 and 24:00 h. The lowest concentrations, 0.26 +/- 0.13 microM and 0.25 +/- 0.26 microM, were found at 20:00 and 8:00 h. Cigarette smokers exhaled about 2.4 times more H(2)O(2) than never smoked subjects. Moreover, in contrast to nonsmokers, cigarette smokers' H2O2 exhalation was stable over 2 week observation. The mean H2O2 concentration estimated over the whole 2 week period was higher in subjects above 40 years regardless of smoking habit, and it positively correlated with age in never smoked subjects (p <.004). Smoking of one cigarette caused 1.8-fold rise in H2O2 exhalation (p <.01). The baseline H2O2 levels correlated with cumulative cigarette consumption (p <.05) and MEF 25% of predicted (p <.05). Neither moderate exercise nor one puff of salbutamol nor ipratropium influenced significantly the concentration of H2O2 and TBARs in EBC. Only 4 of 120 EBC specimens from never smoked subjects revealed detectable levels of TBARs. Cigarette smokers exhaled more TBARs (p <.05) than never smoked volunteers. Our results indicate that healthy never smoked subjects exhale H2O2 with diurnal variation and significant changes over 2 week observation. Cigarette smoking enhanced H2O2 generation in the airways. These results could be useful for planning studies with exhaled H2O2 as a marker of airway inflammation. Occasional detection of TBARs in EBC of never smoked persons may be a result of sufficient antioxidant activity in the airways that protects tissues from peroxidative damage.     

Simultaneous production of carbon monoxide and thiobarbituric acid reactive substances in rat tissue preparations by an iron-ascorbate system.

Most of the carbon monoxide (CO) produced by mammals is a product of the heme oxygenase (HO) reaction, the rate-limiting step in the heme degradation pathway leading to the generation of bilirubin in man. However, some CO is derived from other sources. We studied the association of CO production with lipid peroxidation in tissue preparations from adult male Wistar rats. Supernatants, from 20% tissue homogenates in potassium phosphate buffer, centrifuged for 1 min at 13,000 x g, were incubated for 30 min at 37 degrees C in septum-sealed vials in the dark with ascorbate (100 microM) and Fe(II) (6 microM) and (or) Fe(III) (60 microM). Butylated hydroxytoluene (BHT, 100 microM) was added for the blank reaction. CO produced into the headspace was quantitated by gas chromatography. Thiobarbituric acid reactive substances (TBARS), conjugated dienes (CD), and lipid hydroperoxides (LOOH) in the reaction medium were quantitated by spectrophotometry. Of the tissues studied, CO and TBARS formation was greatest for brain, followed by kidney, lung, spleen, and blood, but no CO or TBARS formation was detected for testes, intestine, liver, and heart. Cell fractionation studies indicated that these differences might be due to the presence of endogenous soluble antioxidants in the latter tissues. Furthermore, these studies demonstrated that CO was exclusively generated by subcellular fractions that contained membranes. The magnitude of the rate of product formation in brain supernatants depended on the concentration of Fe(II) and (or) Fe(III). The formation of CO, TBARS, CD, and LOOH increased linearly with time for up to 30 min, but the rates of product formation were different. Product formation was completely inhibited by BHT (100 microM), biliverdin (50 microM), bilirubin (50 microM), citrate (100 microM), and the Fe(II) chelators, desferrioxamine mesylate (100 microM) and diethylenetriaminepentaacetate, but not by 10 microM of the HO inhibitor, zinc deuteroporphyrin bis glycol. We conclude that CO generation is associated with the process of in vitro lipid peroxidation in tissues with limited antioxidant reserves.     

Intestinal, hepatic and renal production of thiobarbituric acid reactive substances and myeloperoxidase activity after temporary aortic occlusion and reperfusion.

Ischemia induced oxygen free radical damage was formerly attributed only to xanthine oxidase in intestine, liver, kidney and heart. A reevaluation indicated neutrophils as one of the major sources of postischemic oxidative tissue damage, chiefly in the intestine. Our data, obtained from the same occlusion time period for intestine, liver and kidney, showed a certain oxidative damage in intestine and kidney already during ischemia, expressed by an increase of thiobarbituric acid reactive substances (TBARS), whereas the liver sustained damage of this kind only during reperfusion. Oxidative stress was expressed by a comparison of the increase of TBARS, though this test is not a measure of a specific product of lipid peroxidation, but rather comprises several breakdown products of free radical damage. Myeloperoxidase as measure of neutrophil stimulation increased in the intestine and liver. The kidney sustained damage without an increase of myeloperoxidase activity, but showed a similar pattern of increase of TBARS as in the intestine. Our data suggest a major role of neutrophils in intestinal ischemia induced damage, where neutrophils can effect initiation and propagation. In the liver neutrophils may play a minor role concerning propagation, but they may act as an important initiating mechanism. Hepatic tissue shows a high ischemic tolerance, which is demonstrated by a missing increase of TBARS in spite of a certain increase of myeloperoxidase activity during ischemia. This can be interpreted by the high capacity of antioxidative mechanisms of liver tissue and the ability of a higher oxygen extraction ratio under nearly ischemic conditions. In the kidney there appears a smaller contribution of neutrophils. The similar pattern of increase of TBARS in kidney and intestine demonstrates a comparable low ischemic tolerance of these two tissues, whereas different initiating and propagating systems may occur.     

Retinoic acid decreases nitric oxide production in endothelial cells: a role of phosphorylation of endothelial nitric oxide synthase at Ser(1179)

The effects of retinoic acid (RA) on nitric oxide (NO) production are controversial. Furthermore, it has never been studied whether these effects are mediated by direct modulation of phosphorylation of endothelial nitric oxide synthase (eNOS). Using bovine aortic endothelial cells, we found that all-trans RA (atRA) dose- and time-dependently decreased NO production without alteration in eNOS expression. This decrease was accompanied by reduction in eNOS-Ser(1179) phosphorylation. However, atRA did not alter the phosphorylation of eNOS-Ser(116) or eNOS-Thr(497). Concurrently, atRA also decreased the expressions of vascular endothelial growth factor (VEGF) and its receptor KDR/Flk-1, and Akt phosphorylation. Co-treatment with troglitazone, an activator of VEGF expression, reversed the atRA-induced reductions in eNOS-Ser(1179) phosphorylation and NO production, with concomitant restoration in VEGF expression. Direct treatment with VEGF also reversed these inhibitory effects, suggesting an important role for VEGF. Nonetheless, the RARalpha antagonist Ro 41-5253 did not block all the inhibitory effects of atRA, indicating that these inhibitory effects are not mediated by the RA response element (RARE). Thus, atRA decreases eNOS-Ser(1179) phosphorylation through a mechanism that depends on VEGF-KDR/Flk-1-mediated Akt phosphorylation but is independent of RARE, leading to reduction in NO production.     

Heat stress stimulates nitric oxide production in Symbiodinium microadriaticum: a possible linkage between nitric oxide and the coral bleaching phenomenon

Nitric oxide (NO) is a gas displaying multiple physiologicalfunctions in plants, animals and bacteria. The enzymes nitratereductase and NO synthase have been suggested to be involvedin the production of NO in plants and algae, but the implicationof those enzymes in NO production under physiological conditionsremains obscure. Symbiodinium microadriaticum, commonly referredto as zooxanthellae, is a marine microalga commonly found insymbiotic association with a cnidarian host including reef-buildingcorals. Here we demonstrate NO production in zooxanthellae uponsupplementation of either sodium nitrite or L-arginine as asubstrate. The nitrite-dependent NO production was detectedelectrochemically and confirmed by the application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(cPTIO), a specific NO scavenger. Cells stained with the diaminofluorescein,DAF-2 DA, an NO fluorescent probe, showed an increase in fluorescenceintensity upon supplementation of both sodium nitrite and L-arginine.Microscopic observations of DAF-stained cells verified thatNO was produced inside the cells. NO production in S. microadriaticumwas found to increase upon exposure of cells to an acute heatstress which also caused a decline in the photosynthetic efficiencyof PSII (F_v/F_m). This study provides substantial evidence toconfirm that zooxanthellae can synthesize NO even when theyare not in a symbiotic association with a coral host. The increasein NO production at high temperatures suggests that heat stressstimulates the microalgal NO production in a temperature-dependentmanner. The implications of these findings are discussed inthe light of the coral bleaching phenomenon which is associatedwith elevated sea surface temperature due to global warming.     

Activity of superoxide dismutase (SOD) and concentration of thiobarbituric acid reactive substances (TBARS) in liver and muscles of some fish

In this study we examined superoxide dismutase (SOD) activity and thiobarbituric acid reactive substances (TBARS) concentration in liver and muscles of four fish species: the carp, the brown trout, the white cod and the flounder. Higher SOD activity and higher TBARS concentration was revealed in the tissues of marine fish in comparison to freshwater fish. The highest SOD activity was observed in the cod while the highest TBARS concentration was in the flounder. The observed differences are probably an effect of the different living mode of the compared fish.     

Lipid peroxidation assessed by serum thiobarbituric acid reactive substances in healthy subjects and in patients with pathologies known to affect trace element status

Serum thiobarbituric acid reactive substances (TBARS), Zn, Cu, and Se concentrations were determined in 47 healthy adults and in patients with diseases, such as renal in sufficiency, insulin-dependent diabetes mellitus, chronic pancreatitis, liver cirrhosis, or cancer, in order to clarify the relationship between this indicator of lipid peroxidation and antioxidative trace element status. TBARS levels were higher than control values in all pathological cases, except in cancer patients. Cu levels in patients highly correlated with ferroxidase ceruloplasmin activity (r=0.86), but were only statistically different from controls in diabetics. Zn levels were lower than normal in dialysis, liver cirrhosis, and cancer patients. Se levels were significantly decreased in all pathological cases. Half of the subjects with liver cirrhosis or renal insufficiency and 3/4 of chronic pancreatitis or cancer patients had an active inflammatory process. Despite intense modifications in determined indicators, no clear correlation could be demonstrated between the different parameters. Basic antioxidative trace element status and inflammation are therefore not major determinants of TBARS levels in normal and in pathological conditions, despite of the frequent association of low serum Zn and mainly low serum Se with high TBARS levels.     

Effect of nitric oxide on phagocytic activity of lipopolysaccharide-induced macrophages: possible role of exogenous L-arginine

Among the antimicrobial mechanisms associated with macrophages, NO produced by iNOS plays a major role in intracellular killing, but the relationship between NO and phagocytic activity after injection of inflammatory agents into the peritoneal cavity is not clear. The aim of the present study was to investigate the effect of nitric oxide (NO) on macrophage function after treatment with intraperitoneal lipopolysaccharide (LPS) and the role of exogenous L-arginine administration in this event. Six experimental groups and one control group, each consisting of seven Wistar rats were used: Group I: Control; Group II: LPS; Group III: LPS+L-arginine; Group IV: LPS+L-arginine+Aminoguanidine; Group V: LPS+Aminoguanidine; Group VI: L-arginine; Group VII: Aminoguanidine. Macrophage phagocytic activity and total plasma nitrite levels were increased in the LPS group. In the LPS+L-arginine group, both the phagocytic activity and total plasma nitrite levels showed large increases. Administration of aminoguanidine (AG), a specific iNOS inhibitor, abolished macrophage phagocytic activity and total plasma nitrite levels in the LPS and LPS+L-arginine groups. As a result, we showed that NO produced by macrophages has a role not only in intracellular killing, but also in phagocytic activity.     

Impact of stress, gender and menstrual cycle on immune system: possible role of nitric oxide

Stress is a factor found to be involved in the etiology of many diseases. Gender and menstrual cycle phases are other factors affecting the predisposition of individuals for certain diseases. Results from animal and human studies suggest that the distribution of immune system cells may change at different phases of the menstrual cycle. Acute mental stress in humans alters immune variables, too. The increase in the number of natural killer (NK) cells is the most consistent finding among the immune variables, though there are controversies for the other lymphocyte groups. Nitric oxide (NO) as an immune mediator has an unsettled role whether it causes the redistribution of the immune cells, or is an end product of lymphocyte activation. This study was planned to investigate the effect of mental stress on lymphocyte subtypes and the role of NO, for men and women at different phases of the cycle. For this purpose, healthy men (n = 10) and women (n = 10), during the follicular and luteal phases underwent Stroop colour-word interference and cold pressor tests. The immune system responses before and after the tests were determined by cell counts with the flowcytometer. Menstrual cycle phase was ascertained by plasma estrogen and progesterone measurements. Stress response was evaluated by blood pressure (BP) and heart rate (HR) measurements throughout the tests and plasma cortisol and urinary metanephrine and vanillylmandelic acid (VMA) measurements before and after the tests. Plasma and urinary NO determinations were performed before and after the test was completed. All the results were analysed with the appropriate statistical methods. The luteal phase differed from the other groups due to the presence of suppressed immune response to acute stress, including decreased CD4/CD8 ratio and NK cell percentage. On the other hand, acute stress caused a shift from cellular to humoral immunity in men. As indicated by these results, individual reaction towards stress is affected by gender and menstrual cycle phase. NO appears to be a possible effector molecule for these differences.     

Simultaneous measurement of intracellular nitric oxide and free calcium levels in chordate eggs demonstrates that nitric oxide has no role at fertilization

At fertilization in sea urchin, the free radical nitric oxide (NO) has recently been suggested to cause the intracellular Ca(2+) rise responsible for egg activation. The authors suggested that NO could be a universal activator of eggs and the present study was set up to test this hypothesis. Intracellular NO and Ca(2+) levels were monitored simultaneously in eggs of the mouse or the urochordate ascidian Ascidiella aspersa. Eggs were either fertilized or sperm extracts microinjected. Sperm-induced Ca(2+) rises were not associated with any global, or local, change in intracellular NO, although we were able to detect NO produced by the addition of a NO donor. Furthermore, the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester had no effect on sperm-induced Ca(2+) release but did block completely ionomycin-induced NO synthase activation. Therefore, we suggest that the current data provide evidence that NO has no role in the fertilization of these two chordate eggs.     

The possible role of essential fatty acids in the pathophysiology of malnutrition: a review

Biochemical evidence of essential fatty acid deficiency (EFAD) may exist in protein-energy malnutrition (PEM). EFAD is characterised by low 18:2omega6, often in combination with low 20:4omega6 and 22:6omega3, and high 18:1omega9 and 20:3omega9. Some PEM symptoms, notably skin changes, impaired resistance to infections, impaired growth rate and disturbed development may at least partly be explained by EFAD. One or more of the following factors could induce EFAD in PEM: low EFA intake, poor lipid digestion, absorption, transport, desaturation and increased EFA beta-oxidation and peroxidation. EFAD may perpetuate itself by decreasing lipid absorption and transport, and aggravate PEM by impairing nutrient absorption and dietary calorie utilisation. Micronutrient deficiencies may contribute to the impaired EFA bioavailability and metabolism. Nutritional rehabilitation strategies in PEM may consider adequate intakes of EFA and micronutrients, e.g. by promoting breastfeeding. More research is required to gain detailed insight into the role of EFAD in PEM.     

Fatty acid transduction of nitric oxide signaling. Nitrolinoleic acid is a hydrophobically stabilized nitric oxide donor

The aqueous decay and concomitant release of nitric oxide (*NO) by nitrolinoleic acid (10-nitro-9,12-octadecadienoic acid and 12-nitro-9,12-octadecadienoic acid; LNO2) are reported. Mass spectrometric analysis of reaction products supports a modified Nef reaction as the mechanism accounting for the generation of *NO by the aqueous reactions of fatty acid nitroalkene derivatives. Nitrolinoleic acid is stabilized by an aprotic milieu, with LNO2 decay and *NO release strongly inhibited by phosphatidylcholine/cholesterol liposome membranes and detergents when present at levels above their critical micellar concentrations. The release of *NO from LNO2 was induced by UV photolysis and triiodide-based ozone chemiluminescence reactions currently used to quantify putative protein nitrosothiol and N-nitrosamine derivatives. This reactivity of LNO2 complicates the qualitative and quantitative analysis of biological oxides of nitrogen when applying UV photolysis and triiodide-based analytical systems to biological preparations typically abundant in nitrated fatty acids. The results reveal that nitroalkene derivatives of linoleic acid are pluripotent signaling mediators that act not only via receptor-dependent mechanisms, but also by transducing the signaling actions of *NO via pathways subject to regulation by the relative distribution of LNO2 to hydrophobic versus aqueous microenvironments.