Evidence for formation of superoxide and formate radicals in Methanobacterium formicicum.

Using spin labeling and spin trapping techniques in combination with electron paramagnetic resonance spectrometry, we have detected the formation of superoxide by whole cells of Methanobacterium formicicum under aerobic conditions in the presence and absence of sodium formate. Rates of superoxide generation have been estimated. The formation of additional free radical species, including formate, was observed. Production of these and other free radicals resulted in lipid peroxidation and concomitant cell damage.     

Metabolic activation of 1,2-dibromoethane to a free radical intermediate by rat liver microsomes and isolated hepatocytes

A one-electron reductive metabolism of 1,2-dibromoethane (DBE) is described that gives rise to a free radical intermediate, which can be stabilized by a spin trapping agent and detected by electron spin resonance spectroscopy. Using rat liver microsomes or isolated hepatocytes from phenobarbitone pretreated animals, under hypoxic conditions, it has been possible to trap a free radical intermediate and identify it by using ¹³C-DBE. Inhibition experiments have demonstrated that the site of activation is the microsomal drug metabolizing system.     

Polymorphonuclear leukocytes-induced injury in hypoxic cardiac myocytes

Growing evidence suggests that free radicals derived from polymorphonuclear leukocytes (PMNs) play an important role in myocardial ischemia-reperfusion injury. To elucidate the cellular mechanism by which activated PMNs exacerbate ischemic myocardial damage, we investigated the extent of cell injury, assessed by the morphological deterioration, free radical generation, and lipid peroxidation in mouse embryo myocardial cells coincubated with activated PMNs. The generation of PMN-derived free radicals was related to the extent of myocardial cell injury. When myocardial cell sheets were subjected to hypoxia and glucose-free media, myocardial cells were injured (cristalysis in the mitochondria and disruption of the sarcolemma) after adding various PMN activators, and the injury extended to the adjacent cells. Chemiluminescent emission and production of thiobarbituric acid-reactive substances in the coincubated cells increased markedly compared with myocardial cells or PMNs alone. The augmented lipid peroxidation coincided with the progression of myocardial cell injury. Catalase inhibited the myocardial cell injury by 52%, the chemiluminescence by 46%, and lipid peroxidation by 50%, whereas superoxide dismutase exhibited less pronounced inhibition. These results indicate that a chain reaction of lipid peroxidation in myocardial cells induced by PMN-derived free radicals closely correlates with membrane damage and contributes to the propagation of irreversible myocardial cell damage.     

Zinc suppression of free radicals induced in cultures of rat hepatocytes by iron, t-butyl hydroperoxide, and 3-methylindole

The effect of zinc on lipid peroxidation initiated by either ferric-nitrilotriacetate, t-butyl hydroperoxide, or 3-methylindole was studied using primary monolayer cultures of rat liver parenchymal cells. The malondialdehyde content of the cells and culture medium was used to estimate the extent of lipid peroxidation. As the zinc concentration of the culture medium was increased from 1 to 48 microM, peroxidation was diminished. Cellular zinc and metallothionein levels were proportionally increased by supplemental zinc. Zinc supplementation of the medium inhibited NADPH-cytochrome c reductase activity and stimulated glutathione peroxidase activity. The uptake of iron into the hepatocytes was significantly reduced as the level of zinc was raised, suggesting that zinc antagonizes uptake of chelated iron into isolated hepatocytes and in this way blocks iron-induced peroxidation. Furthermore, induction of metallothionein synthesis by zinc may contribute to the reduction in free radicals. Spectra from electron spin resonance studies, using phenylbutylnitrone as a spin-trapping reagent, demonstrated that free radical production was inversely related to the zinc concentration of the culture medium. Spin trap data suggest that metallothionein added to lysed cells in vitro decreases free radical production. Studies using the spin trap, 3,3,5,5-tetramethylpyrroline-N-oxide indicated that cumulatively the predominant radical present in the cultures was a phenyl radical with hydroperoxide or methylindole. Collectively, our data demonstrate that zinc inhibits free radical production and lipid peroxidation in cultured hepatocytes. The mode of action of zinc could occur via free radical scavenging by zinc-induced metallothionein and/or by processes related to cytochrome P-450 and glutathione peroxidase, since these were also found to be sensitive to zinc supplementation levels of the culture medium.     

An aqueous extract of Rubus chingii fruits protects primary rat hepatocytes against tert-butyl hydroperoxide induced oxidative stress

Different in vitro free radical generating systems were used to assess the antioxidative activity of aqueous extracts of the five herbal components of Wu-zi-yan-zong-wan, a traditional Chinese medicinal formula with a long history of use for tonic effects. Fructus Rubi [Rubus chingii (Rosaceae) fruits] was found to be the most potent. It was further investigated using the primary rat hepatocyte system. tert-Butyl hydroperoxide (t-BHP) was used to induce oxidative stress. Being a short chain analog of lipid hydroperoxide, t-BHP is metabolized into free radical intermediates by the cytochrome P450 system in hepatocytes, which in turn, initiate lipid peroxidation, glutathione depletion and cell damage. Pre-treatment of hepatocytes with Fructus Rubi extract (50 microg/ml to 200 microg/ml) for 24 h significantly reversed t-BHP-induced cell viability loss, lactate dehydrogenase leakage and the associated glutathione depletion and lipid peroxidation. The amount of reactive oxygen species formed was also decreased as visualized by the fluorescence probe 2',7'-dichlorofluorescin diacetate. These results suggested that Fructus Rubi was useful in protecting against t-BHP-induced oxidative damage and may also be capable of attenuating cytotoxicity of other oxidants.     

Over-expression of Bcl-2 protects against apoptosis induced by the bioreductive cytotoxic drug SR4233 (Tirapazamine)

The human B-cell lymphoma cell line PW undergoes radiation-induced programmed cell death (PCD). Bcl-2 transfected PW cells, that overexpressed Bcl-2, were significantly more radioresistant than parental or neomycin control transfected PW cells. The viability of Bcl-2 transfected cells was significantly greater than that of parental PW cells treated with the bioreductive cytotoxin SR4233 under aerobic conditions. Bcl-2 transfectants were also significantly more resistant to hypoxia-induced PCD. However, there was no significant difference in the viability of parental and Bcl-2 transfected cells exposed to SR4233 under hypoxic conditions (pO(2)<100 ppm). Incubation of parental PW cells with N-acetyl cysteine decreased the cytotoxicity of SR4233 under aerobic but not anaerobic conditions. Depletion of cellular glutathione with buthionine sulphoxamine killed nearly 100% of control PW cells, but none of the Bcl-2 transfectants under the same conditions. The TBARS assay for lipid peroxidation showed that Bcl-2 transfectants had a significantly lower level of lipid peroxidation than parental PW cells following a 24 hour constant exposure to SR4233 under aerobic conditions. These results suggest that Bcl-2 overexpression inhibits PCD induced by the bioreductive cytotoxin SR4233 under aerobic conditions as well as PCD induced by hypoxia, and that there are other pathways leading to PCD that are unaffected by Bcl-2 overexpression.     

Postanoxic damage of microglial cells is mediated by xanthine oxidase and cyclooxygenase

Brain ischemia and the following reperfusion are important causes for brain damage and leading causes of brain morbidity and human mortality. Numerous observations exist describing the neuronal damage during ischemia/reperfusion, but the outcome of such conditions towards glial cells still remains to be elucidated.

Microglia are resident macrophages in the brain. In this study, we investigated the anoxia/reoxygenation caused damage to a microglial cell line via determination of energy metabolism, free radical production by dichlorofluorescein fluorescence and nitric oxide production by Griess reagent. Consequences of oxidant production were determined by measurements of protein oxidation and lipid peroxidation, as well. By using site-specific antioxidants and inhibitors of various oxidant-producing pathways, we identified major sources of free radical production in the postanoxic microglial cells. The protective influences of these compounds were tested by measurements of cell viability and apoptosis. Although, numerous free radical generating systems may contribute to the postanoxic microglial cell damage, the xanthine oxidase- and the cyclooxygenase-mediated oxidant production seems to be of major importance.     

Cyclooxygenase-Mediated Generation of Free Radicals During Hypoxia in the Cerebral Cortex of Newborn Piglets

Previous studies have demonstrated that free radicals are formed under hypoxic conditions in newborn piglet brain. To test the hypothesis that the cyclooxygenase pathway serves as a source of free radical generation during hypoxia studies were performed on 24 piglets divided into four groups. Six saline (group 3) and six indomethacin treated (group 4) were exposed to hypoxia (FiO2 0.05-0.07) for 60 min. Cerebral hypoxia was documented biochemically by determination of ATP and phosphocreatine. Fluorescent compounds and conjugated dienes were determined as indices of lipid peroxidation. Free radical formation was determined by using n-tert butyl phenyl nitrone (PBN) as a spin trap agent and measuring spin adduct formation in duplicate using a Varian E-109 spectrometer. Groups 1 and 2 (normoxic) showed no spin adduct formation. Group 3 showed a significant increase in spin adduct formation compared to normoxia (372+/-125 vs. 63+/-15, P<0.001). Hypoxic animals pretreated with indomethacin had a spin adduct level of 197+/-132 and were similar to normoxic animals. ATP/PCr levels were the same in groups 3 and 4 denoting the same degree of cerebral hypoxia in all hypoxic animals. Conjugated dienes increased significantly during hypoxia as compared to normoxia (0.142+/-0.017 vs. 0.0+/-0.0) and were decreased insignificantly with indomethacin treatment. Fluorescent compounds were not significantly different among the four groups. Na+,K+-ATPase activity decreased during hypoxia but was not preserved in hypoxic animals pretreated with indomethacin. These data provide direct evidence of the presence of free radicals during hypoxia and the contribution of cyclooxygenase metabolism to their formation.     

The effect of cadmium chloride and hydrogen peroxide on the lipid peroxidation and fractional composition of lipids in hepatocytes of rats

The isolated hepatocytes were incubated in the medium, containing cadmium chloride or hydrogen peroxide. Influence of the latter on the intensity of lipid peroxidation and contents of some lipids fractions, as well as viability of hepatocytes in these conditions has been studied. It is shown that under such cultivation conditions the activation of lipid peroxidation in the hepatocytes takes place. Its activation in presence of cadmium chloride was one of the factors of the membranes damage. The changes in the content of some fractions of lipids were similar both under the incubations of the cells with cadmium chloride and hydrogen peroxide. This allows one to suppose that cadmium chloride causes changes in the lipid composition of membranes as a result of intensification of lipid peroxidation.     

Protection against cell damage due to hypoxia and reoxygenation: The role of taurine and the involved mechanisms

Summary The effect of taurine on cell viability and metabolism of human colon and porcine renal cells was investigated during and after hypoxia. Taurine administered during hypoxia markedly reduced cellular deterioration due to hypoxia and reoxygenation and led to a significantly greater recovery of cellular function following the hypoxic insult. The responsible mechanisms for the beneficial effects were an improvement in osmotic status and calcium homeostasis and an induction in cellular growth despite oxygen deficiency and reoxygenation. Free oxygen radical generation and lipid membrane peroxidation were not reduced by taurine. Taurine acted as a potent endogenous agent with multifactorial effects against cellular damage due to hypoxia and reoxygenation.     

Spin-trapping of free radicals formed during in vitro and in vivo metabolism of 3-methylindole

Electron spin resonance spin-trapping techniques were used to investigate the in vitro and in vivo formation of free radicals during 3-methylindole (3MI) metabolism by goat lung. Utilizing the spin trap phenyl-t-butylnitrone, a nitrogen-centered free radical was detected 3 min after the addition of 3MI to an in vitro incubation system containing goat lung microsomes in the presence of NADPH and O2. The spectrum of the spin adduct was identical to that observed when 3MI was irradiated with ultraviolet light. A carbon-centered radical was also observed which increased in concentration with increasing incubation time. Microsomal incubations containing ferrous sulfate in the absence of 3MI to initiate lipid peroxidation produced the same carbon-centered free radical as obtained by spin-trapping. Malondialdehyde, and end product of lipid peroxidation, was also found to increase in concentration with increasing incubation time of 3MI. The concept that 3MI causes lipid peroxidation in the lung was supported by the in vivo study in which a carbon-centered radical was spin-trapped by phenyl-t-butylnitrone in lungs of intact goats infused with 3MI. This carbon-centered radical had hyperfine splitting constants identical to those carbon-centered free radicals trapped in in vitro incubations of 3MI. These data demonstrate that microsomal metabolism of 3MI produces a nitrogen-centered radical from 3MI which initiates lipid peroxidation in vitro and in vivo causing the formation of carbon-centered radicals from microsomal membranes.     

Oxidant induced injury of erythrocyte—Role of green tea leaf and ascorbic acid

Oxidant and free radical-generating system were used to promote oxidative damage in erythrocytes. Among the oxidants used, phenylhydrazine represents one of the most investigated intracellular free radical-generating probes, which in the presence of haemoglobin autooxidises and give rise to hydroxyl radical, a marker for cellular damage. Erythrocyte, as a single cell, is a good model to be used for studying the haemolytic mechanism of anaemia. Our present investigations reveal increased lipid peroxidation of erythrocyte using phenylhydrazine as well as other oxygen-generating systems (hydrogen peroxide, iron with hydrogen peroxide). It has further been observed that not only lipid peroxidation, phenylhydrazine causes significant elevation in methemoglobin formation, catalase activity and turbidity, in the above system, which are the typical characteristics of haemolytic anaemia. However, exogenous administration of green tea leaf extract and ascorbic acid as natural antioxidants and free radical scavengers were shown to protect separately increased lipid peroxidation caused by phenylhydrazine, though the degree of protection is more in case of green tea leaf extract than ascorbic acid. Results suggest that oxidative damage in vivo due to haemolytic disease may be checked to some extent by using natural antioxidants. (Mol Cell Biochem 276: 205–210, 2005)     

Increase in cellular pool of low-molecular-weight iron during ethanol metabolism in rat hepatocyte cultures

Ethanol-induced lipid peroxidation was studied in primary rat hepatocyte cultures supplemented with ethanol at the concentration of 50 mM. Lipid peroxidation was assessed by two indices: (1) conjugated dienes by second-derivative UV spectroscopy in lipid extract of hepatocytes (intracellular content), and (2) free malondialdehyde (MDA) by HPLC-UV detection and quantitation for the incubation medium (extracellular content). In cultures supplemented with ethanol, free MDA increased significantly in culture media, whereas no elevation of conjugated diene level was observed in the corresponding hepatocytes. The cellular pool of low-mol-wt (LMW) iron was also evaluated in the hepatocytes using an electron spin resonance procedure. An early increase of intracellular LMW iron (≤1 hr) was observed in ethanol-supplemented cultures; it was inhibited by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, whereas α-tocopherol, which prevented lipid peroxidation, did not inhibit the increase of LMW iron. Therefore, the LMW iron elevation was the result of ethanol metabolism and was not secondarily induced by lipid hydroperoxides. Thus, ethanol caused lipid peroxidation in rat hepatocytes as shown by the increase of free MDA, although no conjugated diene elevation was detected. During ethanol metabolism, an increase in cellular LMW iron was observed that could enhance conjugated diene degradation.     

Spin trapping agents (Tempol and POBN) protect HepG2 cells overexpressing CYP2E1 against arachidonic acid toxicity

Polyunsaturated fatty acids such as arachidonic acid were previously shown to be toxic to HepG2 cells expressing CYP2E1 by a mechanism involving oxidative stress and lipid peroxidation. This study investigated the effects of the spin trapping agents Tempol and POBN on the arachidonic acid toxicity. Arachidonic acid caused toxicity and induced lipid peroxidation and mitochondrial membrane damage in cells overexpressing CYP2E1 but had little or no effect in control cells not expressing CYP2E1. The toxicity appeared to be both apoptotic and necrotic in nature. 4-Hydroxy-[2,2,6,6-tetramethylpiperidine-1-oxyl] (Tempol) and alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN) protected against the decrease in cell viability and the apoptosis and necrosis. These spin traps prevented the enhanced lipid peroxidation and the loss of mitochondrial membrane potential. Tempol and POBN had little or no effect on cellular viability or on CYP2E1 activity at concentrations which were protective. It is proposed that elevated production of reactive oxygen intermediates by cells expressing CYP2E1 can cause lipid peroxidation, which subsequently damages the mitochondrial membrane leading to a loss in cell viability when the cells are enriched with arachidonic acid. Tempol and POBN, which scavenge various radical intermediates, prevent in this way the enhanced lipid peroxidation, mitochondrial dysfunction, and the cell toxicity. Since oxidative stress appears to play a key role in ethanol hepatotoxicity, it may be of interest to evaluate whether such spin trapping agents are useful candidates for the prevention or improvement of ethanol-induced liver injury.     

Electron spin resonance spectroscopic detection of oxygen-centred radicals in human serum following exhaustive exercise

Free radicals or oxidants are continuously produced in the body as a consequence of normal energy metabolism. The concentration of free radicals, together with lipid peroxidation, increases in some tissues as a physiological response to exercise – they have also been implicated in a variety of pathologies. The biochemical measurement of free radicals has relied in the main on the indirect assay of oxidative stress by-products. This study presents the first use of electron spin resonance (ESR) spectroscopy in conjunction with the spin-trapping technique, to measure directly the production of radical species in the venous blood of healthy human volunteers pre- and post-exhaustive aerobic exercise. Evidence is also presented of increased lipid peroxidation and total antioxidant capacity post-exercise. Accepted: 30 October 1997     

Activation of chloroform and related trihalomethanes to free radical intermediates in isolated hepatocytes and in the rat in vivo as detected by the ESR-spin trapping technique

When hepatocytes isolated from phenobarbital-induced rats were incubated with chloroform and the spin trap phenyl-t-butyl nitrone (PBN) under anaerobic conditions, a free radical-spin trap adduct was detectable by ESR spectroscopy. A similar incubation of hepatocytes in the presence of air resulted in an ESR signal that was eight times less intense than that seen under anaerobic conditions; incubation mixtures exposed to pure oxygen had no detectable adduct signal. A significant reduction in the signal intensity was also produced by the addition of cytochrome P-450 inhibitors such as SKF-525A, metyrapone and carbon monoxide, indicating that free radical formation depended upon the reductive metabolism of chloroform mediated by the mixed oxidase system. The origin of the CHCl3-derived free radical has been confirmed by using [13C]CHCl3, while the comparison between the ESR spectra obtained in the presence of deuterated chloroform (CDCl3) and bromodichloro-methane (CHBrCl2) suggests that the free radical derived from CHCl3 may be CHCl2. Free radical intermediates were also detected during the aerobic and anaerobic incubation of isolated hepatocytes with bromoform (CHBr3), and iodoform (CHI3). The intensity of the ESR signal obtained with the various trihalomethanes increases in the order CHCl3 less than CHBrCl2 less than CHBr3 less than CHI3. The formation of PBN-free radical adducts has also been observed in phenobarbital-induced rats in vivo when intoxicated with chloroform, bromoform or iodoform, suggesting that the reductive metabolism of trihalomethanes might be of relevance to their established toxicity in the whole animal.     

Electron Spin Resonance Studies on Isolated Hepatocytes Treated with Ferrous Or Ferric Iron

Isolated rat hepatocytes incubated with iron salts in the presence of the spin trapping agent tx-4-pyridyl-l-oxide N-tert-butyl nitrone (4-POBN) generate a clear electron spin resonance signal; this signal is not detectable in the absence of exogenous iron. The hyperfine splitting constants are identical whether ferrous or ferric iron is used. The free radical trapped does not appear to be an active oxygen species but rather a carbon-centred radical, which we here ascribe to a lipodienyl radical on the basis of its hyperfine splitting features. Support to this interpretation is lent by the fact that no such radical could be generated in hepatocytes fully protected against lipid peroxidation by pretreating the donor rats with α-tocopherol.     

Mechanism of carbon tetrachloride-induced hepatotoxicity. Hepatocellular damage by reactive carbon tetrachloride metabolites

CCl4-induced liver damage was modeled in monolayer cultures of rat primary hepatocytes with a focus on involvement of covalent binding of CCl4 metabolites to cell components and/or peroxidative damage as the cause of injury. (1) Covalent binding of 14C-labeled metabolites was detected in hepatocytes immediately after exposure to CCl4. (2) Low oxygen partial pressure increased the reductive metabolism of CCl4 and thus covalent binding. (3) [14C]-CCl4 was bound to lipids and to proteins throughout subcellular fractions. Binding occurred preferentially to triacylglycerols and phospholipids, with phosphatidylcholine containing the highest amount of label. (4) The lipid peroxidation potency of CCl4 revealed subtle differences compared to other peroxidative substances, viz., ADP-Fe3+ and cumol hydroperoxide, respectively. (5) CCl4, but not the other peroxidative substances, decreased the rate of triacylglycerol secretion as very low density lipoproteins. (6) The anti-oxidant vitamin E (alpha-tocopherol) blocked lipid peroxidation, but not covalent binding, and secretion of lipoproteins remained inhibited. (7) The radical scavenger piperonyl butoxide prevented CCl4-induced lipid peroxidation as well as covalent binding of CCl4 metabolites to cell components, and also restored lipoprotein metabolism. The results confirm that covalent binding of the CCl3* radical to cell components initiates the inhibition of lipoprotein secretion and thus steatosis, whereas reaction with oxygen, to form CCl3-OO*, initiates lipid peroxidation. The two processes are independent of each other, and the extent to which either process occurs depends on partial oxygen pressure. The former process may result in adduct formation and, ultimately, cancer initiation, whereas the latter results in loss of calcium homeostasis and, ultimately, apoptosis and cell death.     

A possible role of cAMP dependent phosphorylation of hepatic microsomal cytochrome P450: a mechanism to increase lipid peroxidation in response to hormone

Enzymatic lipid peroxidation in hepatocytes is believed to involve cytochrome P450. cAMP dependent phosphorylation of cytochrome P450 was found to increase the NADPH dependent production of malondialdehyde (lipid peroxidation) by about 30%. The cytochrome P450 inhibitor cyanide abolished this activity. The presence of spermine decreased the cytochrome P450 dependent lipid peroxidation in non-phosphorylated microsomes, phosphorylation partially reversed this effect. Thus, phosphorylation of cytochrome P450 and the associated increased lipid peroxidation may be a hormone dependent response to pathological conditions e.g. stress Phosphorylation was observed to subtly alter other properties of cytochrome P450. The rate of 7-ethoxycoumarin deethylase activity was reduced and the microwave power required to saturate the EPR spectrum of the low spin cytochrome P450 was decreased. It is hypothesized that phosphorylation of cytochrome P450 alters the interaction between the components of the cytochrome P450 system, which may enhance production of free radical species, initiating lipid peroxidation.     

Oxidative Processes Induced by <Emphasis Type="Italic">tert</Emphasis>-Butyl Hydroperoxide in Human Red Blood Cells: Chemiluminescence Studies

The erythrocyte is a good model for investigation of the mechanisms of cell damage induced by oxidizing agents. Oxidative damage to cell components and cellular metabolism results in impaired rheological properties of circulating red blood cells and is involved in the development of some pathologies. The aim of the present study was to elucidate further the oxidative processes induced by tert-butyl hydroperoxide (tBOOH) in erythrocytes, identify cellular targets damaged by the oxidant, as well as estimate the energy and stoichiometry of the reactions that occur. The generation of free radicals in the cell was registered using the chemiluminescence technique. The products of oxyhemoglobin (oxyHb) oxidation, changes in intracellular glutathione (GSH) pool, and accumulation of the stable products of membrane lipid peroxidation were concurrently measured. The oxidative processes induced by tBOOH in red blood cells can be described as follows: 1) rapid GSH oxidation (30-60 sec) by glutathione peroxidase; 2) formation of radicals in the reaction between tBOOH and cellular Hb, which are then immediately consumed in lipid peroxidation reactions; 3) generation of chemiluminescence by the radicals formed. Several stages of the oxidative processes can be revealed. The order of the chemiluminescence reaction (n) with respect to oxidant was estimated to be equal to 2.5 at oxidant concentrations less than 0.5 mM and equal to 1.0 at higher oxidant concentrations. The order of the reaction of membrane lipid peroxidation was found to be n = 2.2 at 0.25-0.6 mM tBOOH and n = 0.5 at higher oxidant concentrations. The apparent activation energy of membrane lipid peroxidation was 55.8 +/- 6.4 kJ/mol, and that of oxyHb oxidation was 108 +/- 16 kJ/mol. It is shown that the interaction of tBOOH and HOCl in erythrocytes is accompanied by changes in both the total number of radicals generated in the cell and the time corresponding to the maximal rate of radical generation.