Responses of antioxidants and lipid peroxidation in mussels to oxidative damage exposure

• 1.1. The aim of this work was to evaluate the relationships between free radical scavengers and lipid peroxidation in the common mussel Mytilus edulis.
• 2.2. Mussels were exposed to compounds known for their ability to produce free radicals (carbon tetrachloride, CCl₄) and reactive oxygen species via redox cycling (menadione), and the effects on digestive gland, gills and remaining tissues were studied.
• 3.3. Lipid peroxidation parameters and the status of free radical scavengers (glutathione, vitamins A, E and C) were affected more by exposure to menadione than to CCl₄.
• 4.4. The observed changes in the free radical scavengers content are indicative of a role in detoxication of damaging reactive species.

     

Carbon tetrachloride toxicity as a model for studying free-radical mediated liver injury

A single dose of CCl4 when administered to a rat produces centrilobular necrosis and fatty degeneration of the liver. These hepatotoxic effects of CCl4 are dependent upon its metabolic activation in the liver endoplasmic reticulum to reactive intermediates, including the trichloromethyl free radical. Positive identification of the formation of this free radical in vivo, in isolated liver cells and in microsomal suspensions in vitro has been achieved by e.s.r. spin-trapping techniques. The trichloromethyl radical has been found to be relatively unreactive in comparison with the secondarily derived peroxy radical CCl3O2., although each free radical species contributes significantly to the biological disturbances that occur. Major early perturbations produced to liver endoplasmic reticulum by exposure in vivo or in vitro to CCl4 include covalent binding and lipid peroxidation; studies of these processes occurring during CCl4 intoxication have uncovered a number of concepts of general relevance to free-radical mediated tissue injury. Lipid peroxidation produces a variety of substances that have high biological activities, including effects on cell division; many liver tumours have a much reduced rate of lipid peroxidation compared with normal liver. A discussion of this rather general feature of liver tumours is given in relation to the liver cell division that follows partial hepatectomy.     

NADPH- and NADH-dependent oxygen radical generation by rat liver nuclei in the presence of redox cycling agents and iron

Redox cycling agents such as paraquat and menadione increase the generation of reactive oxygen species in biological systems. The ability of NADPH and NADH to catalyze the generation of oxygen radicals from the metabolism of these redox cycling agents by rat liver nuclei was determined. The oxidation of hydroxyl radical scavenging agents by the nuclei was increased in the presence of menadione or paraquat, especially with NADPH as the reductant. Paraquat, even at high concentrations, was relatively ineffective with NADH. The highest rates of generation of .OH-like species occurred with ferric-EDTA as the iron catalyst. Certain ferric complexes such as ferric-ATP, ferric-citrate, or ferric ammonium sulfate, which were ineffective catalysts for .OH generation in the absence of paraquat or menadione, were reactive in the presence of the redox cycling agents. Oxidation of .OH scavengers was sensitive to catalase and competitive .OH-scavenging agents under all conditions. The redox cycling agents increased NADPH-dependent nuclear generation of H2O2; stimulation of H2O2 production may play a role in the increase in .OH generation by menadione and paraquat. Menadione inhibited nuclear lipid peroxidation, whereas paraquat and adriamycin were stimulatory. The nuclear lipid peroxidation with either NADPH or NADH plus the redox cycling agents was not sensitive to catalase or .OH scavengers. These results indicate that the interaction of rat liver nuclei with redox cycling agents and iron leads to the production of potent oxidants which initiate lipid peroxidation or oxidize .OH scavengers. Although NADPH is more effective, NADH can also participate in catalyzing the production of reactive oxygen intermediates from the interaction of quinone redox cycling agents with nuclei. The ability of redox cycling agents to interact with various ferric complexes to catalyze nuclear generation of potent oxidizing species with either NADPH or NADH as reductants may contribute to the oxidative stress, toxicity, and mutagenicity of these agents in biological systems.     

Free radical scavenging action of the medicinal herb Limonium wrightii from the Okinawa islands

Free radical scavenging action of Limonium wrightii O. kunthe was examined in vitro and in vivo by using electron spin resonance spectrometer and chemiluminescence analyzer. A water extract of L. wrightii showed a strong scavenging action for the 1,1-diphenyl-2-picrylhydrazyl, or superoxide anion and moderate for hydroxyl radical. The extract also depressed production of reactive oxygen species from polymorphonuclear leukocytes stimulated by phorbor-12-mysistate acetate and inhibited lipid peroxidation in rat liver microsomes. When the extract was given intraperitoneally to mice prior to carbon tetrachloride (CCl4) treatment, CCl4-induced liver toxicity, as seen by an elevation of serum aspartate aminotransferase and alanine aminotransferase activities, was significantly reduced. Gallic acid was identified as the active component of L. wrightii with a strong free radical scavenging action. Our results demonstrate the free radical scavenging action of L. wrightii and that gallic acid contributes to these actions.     

The in vitro NADPH-dependent inhibition by CCl4 of the ATP-dependent calcium uptake of hepatic microsomes from male rats. Studies on the mechanism of the inactivation of the hepatic microsomal calcium pump by the CCl3.radical

The hepatotoxicity of CCl4 is mediated through its initial reduction by cytochrome P-450 to the CCl3.radical. This radical then damages important metabolic systems such as the ATP-dependent microsomal Ca2+ pump. Previous studies from our laboratory on isolated microsomes have shown that NADPH in the absence of toxic agents inhibits this pump. We have now found in in vitro incubations that CCl4 (0.5-2.5 mM) enhanced the NADPH-dependent inhibition of Ca2+ uptake from 28% without CCl4 to a maximum of 68%. These concentrations are in the range found in the livers and blood of lethally intoxicated animals (Dambrauskas, T., and Cornish, H. H. (1970) Toxicol. Appl. Pharmacol. 17, 83-97; Long, R.M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306) and are toxic to cultured hepatocytes (Long, R. M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306). The inhibition of Ca2+ uptake was due both to a decrease in the Ca2(+)-dependent ATPase and to an enhanced release of Ca2+ from the microsomes. The NADPH-dependent CCl4 inhibition was greater under N2 and was totally prevented by CO. GSH (1-10 mM) added during the incubation with CCl4 prevented the inhibition. This protection was also seen when the incubations were performed under nitrogen. When samples were preincubated with CCl4, the CCl4 metabolism was stopped, and then the Ca2+ uptake was determined; GSH reversed the CCl4 inhibition of Ca2+ uptake. This reversal showed saturation kinetics for GSH with two Km values of 0.315 and 93 microM when both the preincubation and the Ca2+ uptake were performed under air, and 0.512 and 31 microM when both were performed under nitrogen. Cysteine did not prevent the NADPH-dependent CCl4 inhibition of Ca2+ uptake. CCl4 increased lipid peroxidation in air, but no lipid peroxidation was seen under nitrogen. Lipid peroxidation was only modestly reversed by GSH. GSH did not remove 14C bound to samples preincubated with the 14CCl4. Although EDTA (100 microM) decreased the CCl4 inhibition, the metal-complexing agents deferoxamine (100 microM) and diethyldithiocarbamate (100 microM) had no effect on the inhibition of the pump. Similarly, the reactive oxygen scavengers catalase (65 micrograms/ml), superoxide dismutase (15 micrograms/ml), mannitol (10 mM), and dimethyl sulfoxide (50 mM) also had no effect. Our results suggest that the initial toxicity of CCl4 for the Ca2+ pump results from the metabolism of CCl4 to the CCl3. radical. This radical then directly oxidizes the Ca2+ pump, leading to decreased Ca2+ uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Three models of free radical-induced cell injury

Three models of free radical-induced cell injury are presented in this review. Each model is described by the mechanism of action of few prototype toxic molecules. Carbon tetrachloride and monobromotrichloromethane were selected as model molecules for alkylating agents that do not induce GSH depletion. Bromobenzene and allyl alcohol were selected as prototypes of GSH depleting agents. Paraquat and menadione were presented as prototypes of redox cycling compounds. All these groups of toxins are converted, during their intracellular metabolism, to active species which can be radical species or electrophilic intermediates. In most cases the activation is catalyzed by the microsomal mixed function oxidase system, while in other cases (e.g. allyl alcohol) cytosolic enzymes are responsible for the activation. Radical species can bind covalently to cellular macromolecules and can promote lipid peroxidation in cellular membranes. Of course both phenomena produce cell damage as in the case of CCl4 or BrCCl3 intoxication. However, the covalent binding is likely to produce damage at the molecular site where it occurs; lipid peroxidation, on the other hand, besides causing loss of membrane structure, also gives rise to toxic products such as 4-hydroxyalkenals and other aldehydes which in principle can move from the site of origin and produce effects at distant sites. Electrophilic intermediates readily reacts with cellular nucleophiles, primarily with GSH. The result is a severe GSH depletion as in the case of bromobenzene or allyl alcohol intoxication. When the depletion reaches some threshold values lipid peroxidation develops abruptly and in an extensive way. This event is accompanied by cellular death. The reason for which lipid peroxidation develops in a cell severely depleted of GSH remains to be clarified. Probably the loss of the defense systems against a constitutive oxidative stress is not compatible with cellular life. Some free radicals generated by one-electron reduction can react with oxygen to give superoxide anions which can be converted to other more dangerous reactive oxygen species. This is the case of paraquat and menadione. Damage to cellular macromolecules is due to the direct action of these oxygen radicals and, at least in the menadione-induced cytotoxicity, lipid peroxidation is not involved. All these initial events affect the protein sulfhydryl groups in the membranes. Since some protein thiols are essential components of the molecular arrangement responsible for the Ca2+ transport across cellular membranes, loss of such thiols can affect the calcium sequestration activity of subcellular compartments, that is the capacity of mitochondria and microsomes to regulate the cytosolic calcium level.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alternatives in hepatoprotection: cytoprotection--influences on mono-oxidase system--free radical scavengers (a review)

Three correlating fields of hepatoprotection have been reviewed: (1) "classic" gastrointestinal cytoprotection: (2) effects via the mixed function mono-oxidase enzyme system: (3) the role of the free radical scavengers and lipid peroxidation inhibiting agents. Gastrointestinal cytoprotection was originally defined in experimentally induced gastric mucosal injuries where several members of the prostaglandin group proved to be effective. Their capacity to increase the stability of biomembranes can be manifested also in hepatocytes, as PGE2 and PGI2 prevent the CCl4-toxicity in experimental animals. Induction of the mono-oxidase system (e.g. with barbiturates or with antioestrogens) may be useful by increasing bilirubin excretion and the biotransformation activity as well as possible by enhancing protein synthesis in liver cells. Inhibition of the mono-oxidases, on the other hand, can prevent the transformation of toxic substances into even more toxic metabolites and the production of free radicals. Free radical scavengers and inhibitors of lipid peroxidation play a central role in hepatoprotection. A flavonoid substance, (+)-cyanidanol-3 possessing many of the mentioned effects may be a promising alternative in the therapy of liver disease.     

Lipid peroxidation in purified plasma membrane fractions of rat liver in relation to the hepatoxicity of carbon tetrachloride

Preparations of rat liver sinusoidal plasma membrane have been tested for their ability to metabolize the hepatotoxin carbon tetrachloride (CCl4) to reactive free radicals in vitro and compared in this respect with standard preparations of rat liver microsomes. The sinusoidal plasma membranes were relatively free of endoplasmic reticulum-associated activities such as the enzymes of the cytochrome P450 system and glucose-6-phosphatase. CCl4 metabolism was measured as (i) covalent binding of [14C]-CCl4 to membrane protein, (ii) electron spin resonance spin-trapping of CCl3. radicals and (iii) CCl4-induced lipid peroxidation. By all of these tests, purified sinusoidal plasma membranes were found unable to metabolize CCl4. The fatty acid composition of the plasma membranes was almost identical to that of the microsomal preparation and both membrane fractions exhibited similar rates of the lipid peroxidation that was stimulated non-enzymically by gamma-radiation or incubation with ascorbate and iron. The absence of CCl4-induced lipid peroxidation in the plasma membranes seems to be due, therefore, to an absence of CCl4 activation rather than an inherent resistance to lipid peroxidation. We conclude that damage to the hepatocyte plasma membrane during CCl4 intoxication is not due to a significant local activation of CCl4 to CCl3. within that membrane.     

Promethazine inhibits the formation of aldehydic products of lipid peroxidation but not covalent binding resulting from the exposure of rat liver fractions to CCl4.

Promethazine is known to have protective activity in relation to CCl4-induced liver necrosis. This hepatoprotective property has been investigated with regard to the free radical scavenging and antioxidant properties of promethazine using isolated hepatocytes and microsomal suspensions. CCl4 is activated in both systems to free radical metabolites that bind covalently to lipid and protein, and initiate lipid peroxidation. A large number of carbonyl products is produced during CCl4-induced lipid peroxidation; promethazine strongly inhibits the production of all classes of carbonyl compounds in both microsomal suspensions and isolated hepatocytes. In contrast, promethazine is a very weak inhibitor of the covalent binding of metabolites of CCl4. We conclude that promethazine acts by scavenging the trichloromethylperoxyl radical and lipid peroxyl radicals, and is a weak scavenger of the trichloromethyl radical. These data, when considered together with the hepatoprotective effects of promethazine, suggest that lipid peroxidation is of relatively more importance than covalent binding in the pathogenesis of CCl4-induced liver necrosis.     

Spin trapping study of reactive oxygen species formation during bopindolol peroxidation

The generation of singlet molecular oxygen ((1)O(2)) and hydroxyl radicals (HO*) during peroxidation of bopindolol in the presence of Co(II) ions was studied using electron spin resonance (ESR) and spectrophotometry methods. 2,2,6,6-Tetramethyl-4-piperidone and 5,5-dimethyl-1-pyrroline-1-oxide were used as traps. The spectrophotometry determination of (1)O(2) was based on bleaching of p-nitrosodimethylaniline (RNO), which was caused by the product of the reaction of (1)O(2) with imidazole and was followed by monitoring the decrease in optical density at 440 nm. The effect of (1)O(2) quenchers and oxygen free radical scavengers on the ESR signal and the bleaching of RNO was studied. The data presented here give new evidence for generation of the reactive oxygen species during peroxidation of bopindolol.     

Visible chemiluminescence induced by t-butyl hydroperoxide in red blood cell suspensions

Red blood cells from Wistar rats were exposed to milimolar concentrations of t-butyl hydroperoxide. Extensive hemoglobin oxidation (methemoglobin formation), t-butyl hydroperoxide cleavage (t-butanol formation) and peroxidation (measured by oxygen consumption and thiobarbituric acid reactive substances) was observed. Significant chemiluminescence was emitted by the system. Hemoglobin oxidation and t-butanol production were independent of oxygen pressure and free radical scavengers, however, luminescence was enhanced as oxygen pressure increased and it was reduced by addition of free radical scavengers. The spectral distribution of the light emitted suggests that the luminescence detected is not due to singlet oxygen dimol emission. The results are in agreement with a lipid peroxidative mechanism initiated by t-butoxy radicals produced in the interaction of hemoglobin and t-butyl hydroperoxide.     

In vitro anti-oxidant property of protein-A of Staphylococcus aureus.

Protein-A (PA) is a cell-surface glycoprotein of S. aureus Cowan I with immunomodulatory and anti-tumor activities, and ability to ameliorate cyclophosphamide and carbontetrachloride (CCl4) induced toxicity in rodents. The likely mechanism of this effect appears to be the anti-oxidant property of PA, evidenced in the present study by inhibition of CCl4 and Fe2-ascorbate induced lipid peroxidation in rat liver homogenates and inhibition of deaminative-oxidative degradation of L-glutamate into 2-thiobarbituric acid reactive products in a constituted chemical system. The anti-oxidant property of PA seem to arise from its molecular characteristics and the ability to interact with a superoxide derived free-radical species without any affinity for superoxide anion, hydroxyl radical and singlet oxygen species.     

Even free radicals should follow some rules: A Guide to free radical research terminology and methodology

Free radicals and oxidants are now implicated in physiological responses and in several diseases. Given the wide range of expertise of free radical researchers, application of the greater understanding of chemistry has not been uniformly applied to biological studies. We suggest that some widely used methodologies and terminologies hamper progress and need to be addressed. We make the case for abandonment and judicious use of several methods and terms and suggest practical and viable alternatives. These changes are suggested in four areas: use of fluorescent dyes to identify and quantify reactive species, methods for measurement of lipid peroxidation in complex biological systems, claims of antioxidants as radical scavengers, and use of the terms for reactive species.     

Differential role of CYP2E1 binders and isoniazid on CYP2E1 protein modification in NADPH-dependent microsomal oxidative reactions: free radical scavenging ability of isoniazid

We evaluated the effect of "weak" CYP2E1 binders (ethanol, acetone and glycerol) "tight" CYP2E1 binders (4-methylpyrazole, imidazole, isoniazid and pyridine) and CCl4 (suicide substrate of CYP2E1) on the NADPH-dependent production of microsomal reactive oxygen species (ROS), lipid peroxidation (LPO), and subsequent modification of microsomal and CYP2E1 proteins. The oxidation of 2',7'-dichlorofluorescin diacetate (DCFHDA) was used as an index of formation of microsomal ROS and LPO-derived reactive species. Microsomal LPO was determined by malondialdehyde (MDA) HPLC measurement. Addition of NADPH to rat liver microsomes initiated DCFHDA oxidation and MDA formation, leading to further selective modification of microsomal proteins and proteases-independent degradation of CYP2E1 protein. Iron chelators prevented these processes whereas hydroxyl radical scavengers showed weak effects, suggesting an important role of LPO. Among the tested CYP2E1 binders, only isoniazid strongly inhibited NADPH-dependent DCFHDA oxidation, LPO and modification of microsomal proteins. Other CYP2E1 binders showed weak inhibitory effects of these processes. Concerning NADPH-dependent modification of CYP2E1 protein, all of the tested CYP2E1 binders, except glycerol, prevented this process with a different potency (isoniazid > 4-methylpyrazole = imidazole = pyridine 3 > acetone > ethanol). "Tight" binders were more effective than "weak" binders. The CCl4 stimulated the DCFHDA oxidation, LPO and CYP2E1 protein modification. Among the tested CYP2E1 binders, only isoniazid effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. In microsomes isolated from CYP2E1 transfected HepG2 cells, isoniazid inhibited the CYP2E1-dependent DCFHDA oxidation whereas other CYP2E1 binders did not inhibit this reaction although these compounds strongly inhibited CYP2E1 activity. The present study demonstrates that CYP2E1 binders and isoniazid differentially inhibit LPO-catalyzed oxidative modification of CYP2E1 protein in NADPH-dependent microsomal reactions. It seems that CYP2E1 binders protect CYP2E1 from the oxidative modification mainly by binding to the active site of the enzyme, rather than by blocking the reactive species production. The strong protective effect of isoniazid can be attributed to its ability to scavenge free radicals. These effects of CYP2E1 binders are considered to contribute to the regulation of hepatic CYP2E1 protein levels via stabilization of the protein.     

Effect of benzophenones from Hypericum annulatum on carbon tetrachloride-induced toxicity in freshly isolated rat hepatocytes

Five benzophenones and a xanthone, isolated from Hypericum annulatum Moris, were investigated for their protective effect against carbon tetrachloride toxicity in isolated rat hepatocytes. The benzophenones and the xanthone gentisein were administered alone (100 microM) and in combination with CCl4 (86 microM). CCl4 undergoes dehalogenation in the liver endoplasmic reticulum. This process leads to trichlormethyl radical (*CCl3) formation, initiation of lipid peroxidation, and measurable toxic effects on the hepatocytes. The levels of thiobarbituric acid reactive substances (TBARS) were assayed as an index of lipid peroxidation (LPO). Lactate dehydrogenase (LDH) leakage, cell viability and reduced glutathione (GSH) depletion were used as signs of cytotoxicity. CCl4 significantly decreased hepatocyte viability, GSH level and increased TBARS level and LDH leakage as compared to the control. Our data indicate that 2,3',5',6-tetrahydroxy-4-methoxybenzophenone, 2-O-alpha-L-arabinofuranosyl-3',5',6-trihydroxy-4-methoxybenzophenone and 2-O-alpha-L-3'-acetylarabinofuranosyl-3',5',6-trihydroxy-4-methoxybenzophenone showed weaker toxic effects compared to CCl4 and in combination showed statistically significant protection against the toxic agent.     

Role of reactive oxygen species and phosphatidylinositol 3-kinase in cardiomyocyte differentiation of embryonic stem cells

Cardiotypic development in embryonic stem cell-derived embryoid bodies may be regulated by reactive oxygen species (ROS). ROS were generated by a NADPH oxidase-like enzyme which was transiently expressed during the time course of embryoid body development. Incubation with either H(2)O(2) or menadione enhanced cardiomyogenesis, whereas the radical scavengers trolox, pyrrolidinedithiocarbamate and N-acetylcysteine exerted inhibitory effects. The phosphatidylinositol 3-kinase (PI-3-kinase) inhibitors LY294002 and wortmannin abolished cardiac commitment and downregulated ROS in embryoid bodies. Coadministration of LY294002 with prooxidants resumed cardiomyocyte differentiation, indicating a role for PI-3-kinase in the regulation of the intracellular redox state.     

Increased NADH-dependent production of reactive oxygen intermediates by microsomes after chronic ethanol consumption: comparisons with NADPH.

Microsomes from chronic ethanol-fed rats were previously shown to catalyze the NADPH-dependent production of reactive oxygen intermediates at elevated rates compared to controls. Recent studies have shown that NADH can also serve as a reductant and promote the production of oxygen radicals by microsomes. The current study evaluated the influence of chronic ethanol consumption on NADH-dependent microsomal production of reactive oxygen intermediates, and compared the results with NADH to those of NADPH. Microsomal oxidation of chemical scavengers, taken as a reflection of the production of hydroxyl radical (.OH)-like species was increased about 50% with NADH as cofactor and about 100% with NADPH after chronic ethanol consumption. The potent inhibition of the production of .OH-like species by catalase suggests a precursor role for H2O2 in .OH production. Rates of NADH- and NADPH-dependent H2O2 production were increased by about 50 and 70%, respectively, after chronic ethanol consumption. A close correlation between rates of H2O2 production and generation of .OH-like species was observed for both NADH and NADPH, and increased rates of H2O2 production appear to play an important role in the elevated generation of .OH-like species after chronic ethanol treatment. Microsomal lipid peroxidation was elevated about 60% with NADH, and 120% with NADPH, after ethanol feeding. With both types of microsomal preparations, the characteristics of the NADH-dependent reactions were similar to the NADPH-dependent reactions, e.g., sensitivity to antioxidants and free radical scavengers and catalytic effectiveness of ferric complexes. However, rates with NADPH exceeded the NADH-dependent rates by 50 to 100%, and the increased production of reactive oxygen intermediates by microsomes after ethanol treatment was greater with NADPH (about twofold) than with NADH (about 50%). Oxidation of ethanol results in an increase in hepatic NADH levels and interaction of NADH, iron, and microsomes can produce potent oxidants capable of initiating lipid peroxidation and oxidizing .OH scavengers. These acute metabolic interactions produced by ethanol-derived NADH are increased, not attenuated, in microsomes from chronic ethanol-fed rats, and it is possible that such increases in NADH (and NADPH)-dependent production of reactive oxygen species play a role in the development of oxidative stress in the liver as a consequence of ethanol treatment.     

Protective effects of antioxidants on deoxynivalenol-induced damage in murine lymphoma cells

As contradictory results have been reported on the immunotoxic properties of deoxynivalenol (DON) in animal studies, we introduced a lymphoblast cell culture model in order to examine the effects of DON on lymphoblastic cell growth and metabolism as well as the preventive properties of free radical scavenger molecules against the DON-induced cell damage. Murine YAC-1 lymphoma cells were used because lymphoblasts have been shown to be sensitive to DON-induced immunotoxicity. Cells were quantified and their proliferative activity was measured by a proliferation test. Lipid peroxidation and protein oxidation were determined using assays quantifying thiobarbituric acid reactive substances (TBARS) and carbonylated proteins. Severely reduced cell counts were detected in DON-treated samples, confirmed by a 5–10 times lower proliferative activity. Significant increases in lipid peroxidation and protein oxidation were found in parallel incubated samples. The pre-incubation with free radical scavengers significantly reduced DON-induced changes to proteins and lipids as well as the tarnished cell viability and cell proliferation. These results suggest that YAC-1 lymphoma cells are a suitable model to investigate and elucidate the basic molecular and cellular mechanisms for possible immunotoxic effects of DON. With regard to the impact of free radical scavengers, the applied in-vitro model might enable the investigation of potential prophylactic and therapeutic effects before or even without harmful animal experiments and cost- and time-intensive expenses.     

Cell-biology of ageing. III. Malondialdehyde as an index of free radical reactions in the early senescent mutants of Neurospora crassa and study of the effect of free radical scavengers on malondialdehyde contents

Malondialdehyde - a product of lipid peroxidation due to free radical reaction was estimated in the culture filtrates of early senescent mutants of Neurospora crassa and the effects of vitamin E, vitamin C and Sodium selenite (free radical scavengers) in malondialdehyde contents were studied. From the results obtained, it could not be established that increased free radical reaction was the sole factor for the early senescence of all the mutants; and the free radical scavengers had very little effect on the malondialdehyde content of the selected early senescent mutants.     

Differential Role of CYP2E1 Binders and Isoniazid on CYP2E1 Protein Modification in NADPH-dependent Microsomal Oxidative Reactions: Free Radical Scavenging Ability of Isoniazid

We evaluated the effect of "weak" CYP2E1 binders (ethanol, acetone and glycerol) "tight" CYP2E1 binders (4-methylpyrazole, imidazole, isoniazid and pyridine) and CCl 4 (suicide substrate of CYP2E1) on the NADPH-dependent production of microsomal reactive oxygen species (ROS), lipid peroxidation (LPO), and subsequent modification of microsomal and CYP2E1 proteins. The oxidation of 2',7'-dichlorofluorescin diacetate (DCFHDA) was used as an index of formation of microsomal ROS and LPO-derived reactive species. Microsomal LPO was determined by malondialdehyde (MDA) HPLC measurement. Addition of NADPH to rat liver microsomes initiated DCFHDA oxidation and MDA formation, leading to further selective modification of microsomal proteins and proteases-independent degradation of CYP2E1 protein. Iron chelators prevented these processes whereas hydroxyl radical scavengers showed weak effects, suggesting an important role of LPO. Among the tested CYP2E1 binders, only isoniazid strongly inhibited NADPH-dependent DCFHDA oxidation, LPO and modification of microsomal proteins. Other CYP2E1 binders showed weak inhibitory effects of these processes. Concerning NADPH-dependent modification of CYP2E1 protein, all of the tested CYP2E1 binders, except glycerol, prevented this process with a different potency (isoniazid > 4-methylpyrazole=imidazole=pyridine &#100 acetone > ethanol). "Tight" binders were more effective than "weak" binders. The CCl 4 stimulated the DCFHDA oxidation, LPO and CYP2E1 protein modification. Among the tested CYP2E1 binders, only isoniazid effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. In microsomes isolated from CYP2E1 transfected HepG2 cells, isoniazid inhibited the CYP2E1-dependent DCFHDA oxidation whereas other CYP2E1 binders did not inhibit this reaction although these compounds strongly inhibited CYP2E1 activity. The present study demonstrates that CYP2E1 binders and isoniazid differentially inhibit LPO-catalyzed oxidative modification of CYP2E1 protein in NADPH-dependent microsomal reactions. It seems that CYP2E1 binders protect CYP2E1 from the oxidative modification mainly by binding to the active site of the enzyme, rather than by blocking the reactive species production. The strong protective effect of isoniazid can be attributed to its ability to scavenge free radicals. These effects of CYP2E1 binders are considered to contribute to the regulation of hepatic CYP2E1 protein levels via stabilization of the protein.