Influence of cadmium on lipid peroxidation and antioxidant enzymes in isolated rat hepatocytes

Cadmium (Cd) influences lipid peroxidation (LPO) by enhancing peroxidation of membrane lipids and by disturbing the antioxidant system of cells. In isolated rat hepatocytes, LPO was observed in cells incubated with Cd (50-250 mu M) for various time periods up to 90 min. The antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were inhibited along with depletion of glutathione (GSH) in hepatocytes treated with Cd. The results show that Cd influences LPO in rat hepatocytes due to decrease in antioxidant status.

Occurrence of Lipid Peroxidation in Brain Microsomes in the Presence of NADH and Vanadate

Oxidation of NADH by rat brain microsomes was stimulated severalfold on addition of vanadate. During the reaction, vanadate was reduced, oxygen was consumed, and H2O2 was generated with a stoichiometry of 1:1 for NADH/O2, as in the case of other membranes. Extra oxygen was found to be consumed over that needed for H2O2 generation specifically when brain microsomes were used. This appears to be due to the peroxidation of lipids known to be accompanied by a large consumption of oxygen. Occurrence of lipid peroxidation in brain microsomes in the presence of NADH and vanadate has been demonstrated. This activity was obtained specifically with the polymeric form of vanadate and with NADH, and was inhibited by the divalent cations Cu2+, Mn2+, and Ca2+, by dihydroxy-phenolic compounds, and by hemin in a concentration-dependent fashion. In the presence of a small concentration of vanadate, addition of an increasing concentration of Fe2+ gave increasing lipid peroxidation. After undergoing lipid peroxidation in the presence of NADH and vanadate, the binding of quinuclidinyl benzylate, a muscarinic antagonist, to brain membranes was decreased.

Microsomal redox systems in brown adipose tissue: high lipid peroxidation, low cholesterol biosynthesis and no detectable cytochrome P-450

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C]-mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b 5 in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.

Ferrous-iron induces lipid peroxidation with little damage to energy transduction in mitochondria

Addition of ferrous sulfate, but not ferric chloride, in micromolar concentrations to rat liver mitochondria induced high rates of consumption of oxygen. The oxygen consumed was several times in excess of the reducing capacity of ferrous-iron (O: Fe ratios 5�8). This occurred in the absence of NADPH or any exogenous oxidizable substrate. The reaction terminated on oxidation of ferrous ions. Malondialdehyde (MDA), measured as thiobarbituric acid-reacting material, was produced indicating peroxidation of lipids. The ratio of O2: MDA was about 4: 1. Pretreatment of mitochondria with ferrous sulfate decreased the rate of oxidation (state 3) with glutamate (+malate) as the substrate by about 40% but caused little damage to energy tranduction process as represented by ratios of ADP: O and respiratory control, as well as calcium-stimulated oxygen uptake and energy-dependent uptake of [45Ca]-calcium. Addition of succinate or ubiquinone decreased ferrous iron-induced lipid peroxidation in intact mitochondria. In frozen-thawed mitochondria, addition of succinate enhanced lipid peroxidation whereas ubiquinone had little effect. These results suggest that ferrous-iron can cause peroxidation of mitochondrial lipids without affecting the energy transduction systems, and that succinate and ubiquinone can offer protection from damage due to such ferrous-iron released from the stores within the cells.

Role of lipid peroxidation in impairment of mitochondrial function at complex I by methyl isocyanate treatment of rats in vivo

The subcutaneous administration of methyl isocyanate (MIC) in 1.0 LD50 dose in rats caused a significant effect on hepatic mitochondrial function only at complex I region of the respiratory chain. MIC administration at 1.0 LD50 dose also resulted in significant increases in malondialdehyde and ferrous ion concentration in liver mitochondria. It is suggested that the augmented lipid peroxidation in hepatic mitochondria, catalyzed by iron, possibly mobilized from intracellular stores leads to the inhibition of enzymes of mitochondrial respiration at complex I region, in vivo, in rats receiving a lethal dose of MIC subcutaneously.

A vanadate-stimulated NADH oxidase in erythrocyte membrane generates hydrogen peroxide

Oxidation of NADH by rat erythrocyte plasma membrane was stimulated by about 50-fold on addition of decavanadate, but not other forms of vanadate like orthovanadate, metavanadate aad vanadyl sulphate. The vanadate-stimulated activity was observed only in phosphate buffer while other buffers like Tris, acetate, borate and Hepes were ineffective. Oxygen was consumed during the oxidation of NADH and the products were found to be NAD+ and hydrogen peroxide. The reaction had a stoichiometry of one mole of oxygen consumption and one mole of H2O2 production for every mole of NADH that was oxidized. Superoxide dismutase and manganous inhibited the activity indicating the involvement of superoxide anions. Electron spin resonance in the presence of a spin trap, 5, 5prime-dimethyl pyrroline N-oxide, indicated the presence of superoxide radicals. Electron spin resonance studies also showed the appearance of VIV species by reduction of VV of decavanadate indicating thereby participation of vanadate in the redox reaction. Under the conditions of the assay, vanadate did not stimulate lipid peroxidation in erythrocyte membranes. Extracts from lipid-free preparations of the erythrocyte membrane showed full activity. This ruled out the possibility of oxygen uptake through lipid peroxidation. The vanadate-stimulated NADH oxidation activity could be partially solubilized by treating erythrocyte membranes either with Triton X-100 or sodium cholate. Partially purified enzyme obtained by extraction with cholate and fractionation by ammonium sulphate and DEAE-Sephadex was found to be unstable.

Interplay of electrostatics and lipid packing determines the binding of charged polymer coated nanoparticles to model membranes

Understanding of nanoparticle-membrane interactions is useful for various applications of nanoparticles like drug delivery and imaging. Here we report on the studies of interaction between hydrophilic charged polymer coated semiconductor quantum dot nanoparticles with model lipid membranes. Atomic force microscopy and X-ray reflectivity measurements suggest that cationic nanoparticles bind and penetrate bilayers of zwitterionic lipids. Penetration and binding depend on the extent of lipid packing and result in the disruption of the lipid bilayer accompanied by enhanced lipid diffusion. On the other hand, anionic nanoparticles show minimal membrane binding although, curiously, their interaction leads to reduction in lipid diffusivity. It is suggested that the enhanced binding of cationic QDs at higher lipid packing can be understood in terms of the effective surface potential of the bilayers which is tunable through membrane lipid packing. Our results bring forth the subtle interplay of membrane lipid packing and electrostatics which determine nanoparticle binding and penetration of model membranes with further implications for real cell membranes.

Quantitation and characterization of glutathionyl haemoglobin as an oxidative stress marker in chronic renal failure by mass spectrometry

Objectives: Glutathionyl haemoglobin (GS-Hb) belonging to the class of glutathionylated proteins has been investigated as a possible marker of oxidative stress in different chronic diseases. The purpose of this study was to examine whether glutathionyl haemoglobin can serve as an oxidative stress marker in non-diabetic chronic renal failure patients on different renal replacement therapies (RRT) through its quantitation, and characterization of the specific binding site of glutathione in haemoglobin molecule by mass spectrometric analysis. Design and methods: The study group consisted of non-diabetic chronic renal failure patients on renal replacement therapy (RRT): hemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD) and renal allograft transplant (Txp) patients. Haemoglobin samples of these subjects were analyzed by liquid chromatography electrospray ionization mass spectrometry for GS-Hb quantitation. Characterization of GS-Hb was done by tandem mass spectrometry. Levels of erythrocyte glutathione (GSH) and lipid peroxidation (as thiobarbituric acid reacting substances) were measured spectrophotometrically, while glycated baernoglobin (HbA1c) was measured by HPLC. Results: GS-Hb levels were markedly elevated in the dialysis group and marginally in the transplant group as compared to the controls. GS-Hb levels correlated positively with lipid peroxidation and negatively with the erythrocyte glutathione levels in RRT groups indicating enhanced oxidative stress. De novo sequencing of the chymotryptic fragment of GS-Hb established that glutathione is attached to Cys-93 of the beta globin chain. Mass spectrometric quantitation of total glycated haemoglobin showed good agreement with HbA1c estimation by conventional HPLC method. Conclusions: Glutathionyl haemoglobin can serve as a clinical marker of oxidative stress in chronic debilitating therapies like RRT. Mass spectrometry provides a reliable analytical tool for quantitation and residue level characterization of different post-translational modifications of haemoglobin. (c) 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Selective inhibition of cyclooxygenase-2 by C-phycocyanin, a biliprotein from Spirulina platensis

We report data from two related assay systems (isolated enzyme assays and whole blood assays) that C-phycocyanin a biliprotein from Spirulina platensis is a selective inhibitor of cyclooxygenase-a (COX-2) with a very low IC50 COX-2/IC50 COX-1 ratio (0.04). The extent of inhibition depends on the period of preincubation of phycocyanin with COX-2, but without any effect on the period of preincubation with COX-1. The IC50 value obtained for the inhibition of COX-2 by phycocyanin is much lower (180 nM) as compared to those of celecoxib (255 nM) and rofecoxib (401 nM), the well-known selective COX-2 inhibitors. In the human whole blood assay, phycocyanin very efficiently inhibited COX-2 with an IC50 value of 80 nM. Reduced phycocyanin and phycocyanobilin, the chromophore of phycocyanin are poor inhibitors of COX-2 without COX-2 selectivity. This suggests that apoprotein in phycocyanin plays a key role in the selective inhibition of COX-2. The present study points out that the hepatoprotective, anti-inflammatory, and anti-arthritic properties of phycocyanin reported in the literature may be due, in part, to its selective COX-2 inhibitory property, although its ability to efficiently scavenge free radicals and effectively inhibit lipid peroxidation may also be involved. (C) 2000 Academic Press.

Oxidative activities in mitochondria-like particles from Setaria digitata, a filarial parasite

The oxidative metabolic potential of Setaria digitata, a filarial parasite found in the intraperitoneal cavity of cattle, was investigated. These worms showed active wriggling movements which were not affected by respiratory poisons such as cyanide, rotenone and malonate. They also possessed cyanide-insensitive and glucose-independent oxygen consumption pathways. By differential centrifugation of sucrose homogenates, a fraction containing mitochondria-like particles was obtained in which the activity of the marker enzyme, succinate dehydrogenase, was recovered. This fraction catalysed succinate- and NADH-dependent reduction of both cytochrome c and dyes. Oxygen uptake found with succinate, NADH and ascorbate as substrates was not sensitive to cyanide. Cytochromes could not be detected in either this fraction or homogenates of the worms. H2O2 generation with a number of substrates and lipid peroxidation by measuring malondialdehyde formed as well as by accompanying oxygen uptake were demonstrated in the mitochondria-like particles. A lipid quinone, possibly with a short side chain and related to ubiquinone, was detected in the worms. The results suggested the existence of two cyanide-insensitive oxygen-consuming reactions in Setaria: one respiratory substrate-independent lipid peroxidation, and a second substrate-dependent reaction that requires an auto-oxidizable quinone but not a cytochrome system.

Triacylglycerol lipolysis is linked to sphingolipid and phospholipid metabolism of the yeast Saccharomyces cerevisiae

Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast triacylglycerol (TAG) lipase resulted in decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG fraction K. Athenstaedt and G. Daum, J. Biol. Chem. 278 (2003) 23317-23323]. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we carried out metabolic labeling experiments with wild type and deletion strains bearing defects in the three major yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Using H-3]inositol. P-32]orthophosphate, 3H]palmitate and C-14]acetate as precursors for complex lipids we demonstrated that tgl mutants had a lower level of sphingolipids and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG accumulation in these mutant cells resulted in reduced amounts of phospholipids and sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected the metabolic flux of long chain fatty acids and very long chain fatty acids required for sphingolipid and glycerophospholipid synthesis. Activity and expression level of fatty acid elongases, Elo1p and Elo2p were enhanced as a consequence of reduced TAG lipolysis. Finally, the pattern of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine molecular species was altered in tgl deletion strain underlining the important role of TAG turnover in maintaining the pool size of these compounds and the remodeling of complex membrane lipids. (C) 2010 Elsevier B.V. All rights reserved.

In vitro and in vivo effect of methyl isocyanate on rat liver mitochondrial respiration

Previous work has shown that irrespective of the route of exposure methyl isocyanate (MIC) caused acute lactic acidosis in rats (Jeevaratnam et al., Arch. Environ. Contam. Toxicol. 19, 314�319, 1990) and the hypoxia was of stagnant type due to tissue hypoperfusion resulting from hypovolemic hypotension in rabbits administered MIC subcutaneously (Jeevarathinam et al., Toxicology 51, 223�240, 1988). The present study was designed to investigate whether MIC could induce histotoxic hypoxia through its effects on mitochondrial respiration. Male Wistar rats were used for liver mitochondrial and submitochondrial particle (SMP) preparation. Addition of MIC to tightly coupled mitochondria in vitro resulted in stimulation of state 4 respiration, abolition of respiratory control, decrease in ADP/O ratio, and inhibition of state 3 oxidation. The oxidation of NAD+-linked substrates (glutamate + malate) was more sensitive (fiveto sixfold) to the inhibitory action of MIC than succinate while cytochrome oxidase remained unaffected. MIC induced twofold delay in the onset of anerobiosis, and cytochrome b reduction in SMP with NADH in vitro confirms inhibition of electron transport at complex I region. MIC also stimulated the ATPase activity in tightly coupled mitochondria while lipid peroxidation remained unaffected. As its hydrolysis products, methylamine and N,N?-dimethylurea failed to elicit any change in vitro; these effects reveal that MIC per se acts as an inhibitor of electron transport and a weak uncoupler. Administration of MIC sc at lethal dose caused a similar change only with NAD+-linked substrates, reflecting impairment of mitochondrial respiration at complex I region and thereby induction of histotoxic hypoxia in vivo.

Antioxidant and antihepatotoxic effect of Spirulina laxissima against carbon tetrachloride induced hepatotoxicity in rats

The vast biodiversity of nature provides bioactive compounds that may be useful in the fight against chronic diseases. This study was designed to investigate the protective effects of the ethanol extract of Spirulina laxissima West (Pseudanabaenaceae) (EESL) against carbon tetrachloride (CCl4) induced hepatotoxicities in rats. Male albino rats of Sprague-Dawley strain were treated orally with the ethanol extract of S. laxissima (50, 100 mg kg(-1) body wt.) 1 h before each CCl4 administration. The ethanol extract of S. laxissima showed the maximum antioxidant property in vitro. There were statistically significant losses in the activities of antioxidant enzymes and an increase in TBARS and liver function marker enzymes in the serum of the CCl4-treated group compared with the control group. However, all the tested groups were able to counteract these effects. The antioxidant activity of the extracts might be attributable to its proton-donating ability, as evidenced by DPPH. In the present study, the decline in the level of antioxidant observed in CCl4-treated rats is a clear manifestation of excessive formation of radicals and activation of the lipid peroxidation system resulting in tissue damage. The significant increases in the concentration of antioxidant enzymes in tissues of animals treated with CCl4 + EESL indicate the antioxidant effect of EESL. This study suggests that EESL can protect the liver against CCl4-induced oxidative damage in rats, and the hepatoprotective effect might be correlated with its antioxidant and radical-scavenging effects.

Protection against UVB inactivation (in vitro) of rat lens enzymes by natural antioxidants

Oxidative damage, through increased production of free radicals, is believed to be involved in UV-induced cataractogenesis (eye lens opacification). The possibility of UVB radiation causing damage to important lenticular enzymes was assessed by irradiating 3 months old rat lenses (in RPMI-1640 medium) at 300 nm (100 mu Wcm(-2)) for 24 h, in the absence and presence of ascorbic acid, alpha-tocopherol acetate and beta-carotene. UVB irradiation resulted in decreased activities of hexokinase, glucose-6-phosphate dehydrogenase, aldose reductase, and Na, K- ATPase by 42, 40, 44 and 57% respectively. While endopeptidase activity (229%) and lipid peroxidation (156%) were increased, isocitrate dehydrogenase activity was not altered on irradiation. In the presence of externally added ascorbic acid, tocopherol and beta-carotene (separately) to the medium, the changes in enzyme activities (except endopeptidase) and increased lipid peroxidation, due to UVB exposure, were prevented. These results suggest that UVB radiation exerts oxidative damage on lens enzymes and antioxidants were protective against this damage.

Total peroxidase activity and peroxidase isoforms as modified by salt stress in two cultivars of fox-tail millet with differential salt tolerance

The effect of NaCl on total peroxidase activity, induction of isoperoxidases and lipid peroxidation in 5-day-old seedlings of two contrasting genotypes of Setaria italica L. (Prasad, a salt tolerant cultivar and Lepakshi, a salt susceptible cultivar), was studied. Total peroxidase activity increased under NaCl salinity and the degree of elevation in the activity was salt concentration dependent. Nevertheless, a greater activity was recorded in the tolerant cultivar (cv Prasad) compared to the susceptible (cv Lepakshi) one in all days of sampling. Further, the pattern of isoperoxidases was modified during stress conditions as evident from the electrophoregrams. Although, five acidic isoforms were detected in both cultivars, differences were found between the cultivars. Furthermore, it was observed that acidic isoperoxidases were strongly expressed and an acidic isoperoxidase, A(3p) (27 kDa) is specifically found in the tolerant cultivar (cv Prasad) under NaCl stress. This isoform was partially purified and found to be thermostable with pr 5.5 and the optimum pH 7.4. A close correlation exists between the rate of lipid peroxidation in terms of malonaldehyde (MDA) content and total peroxidase activity per gram fresh weight with salt tolerance of the two cultivars. The tolerant cultivar (cv Prasad) had low MDA content and high total peroxidase activity than the susceptible variety (cv Lepakshi) during salinity stress. (C) 1999 Published by Elsevier Science Ireland Ltd. All rights reserved.