The use of Xenopus oocytes to evaluate drugs affecting brain Ca2+ channels: effects of bifemelane and several nootropic agents

Effects of nootropic or cerebroprotective drugs on voltage-sensitive Ca2+ channels (VSCC) in the mammalian brain were evaluated comparatively by depolarization-evoked Ca2+ channel currents in Xenopus oocytes injected with brain mRNA and by a high K(+)-stimulated 45Ca uptake into synaptosomes. It was found that several anti-amnesic agents (bifemelane, idebenone and vinpocetine) inhibited these VSCC-related responses with stronger potency than phenytoin and flurazepam. On the inhibiting potency of various compounds, there was a significant but weak correlation between the results from synaptosomes and those from the injected oocytes, since nifedipine, verapamil and diltiazem at 100 microM did not reduce 45Ca influx in synaptosomes but partly inhibited VSCCs in the oocytes. The blockade of neuronal L- and N-type VSCCs may participate in the anti-ischemic/hypoxic actions of nootropic drugs.     

Characteristics of lipid peroxidation in various tissues during acute stress and its correction by pyracetam and cerebrolysin

The process of lipid peroxidation (LPO), the activity of antioxidant enzymes, and the effects of piracetam (300 mg/kg) and cerebrolysin (0.1 ml/kg) were studied in the brain, liver, thymus, spleen, and blood serum of white male rats under acute stress (immobilization) conditions. The results of analyses confirmed that the stress syndrome development involves the LPO acceleration and retardation stages not coinciding in various tissues. The antioxidant effect of the nootropic agents in CNS has a permanent character, being independent of the initial status of the animal organism. The effect of both drugs upon the LPO level in peripheral tissues has a modulative character and can be considered as a sum of the central antistressor action and the direct influence upon the functional activity of effectors.     

Model corasol-induced seizures are followed by increase of nitric oxide generation and are abolished by mexidol and alpha-tocopherol

The effect of mexidol and alpha-tocopherol on the onset and development of acute epilepsy model was studied in Wistar rats using penthylenetetrazole induced convulsions. The intensity of the nitric oxide (NO) production in the cerebral cortex was determined by a direct method using electron paramagnetic resonance. The rate of lipid peroxidation (LPO) was estimated by measuring the level of secondary products (thiobarbituric acid reactive species). The peak of penthylene-tetrazole convulsions is accompanied by a significant increase in the levels of both NO and LPO products. Mexidol (150 mg/kg) and alpha-tocopherol (100 mg/kg) hindered the development of model convulsions, prevented NO buildup, and inhibited LPO growth. It is suggested that suppression of the excess NO production in the cortex and inhibition of the LPO enhancement can be involved in the mechanism of action of antiepileptic drugs.     

Inhibition of liposomal lipid peroxidation by spermidine.

Spermidine was found to inhibit the in vitro formation of thiobarbituric acid-reactive material from sonicated vesicles of rat brain and individual phospholipids, especially in the presence of externally added iron. With arachidonic acid incorporated liposomes spermidine inhibited lipid peroxidation in the presence or the absence of Fe2+, to similar extent. Thus spermidine inhibition of lipid peroxidation may not be entirely iron-dependent.     

Lipid peroxidation modifies the effect of phenolic anti-inflammatory drugs on prostaglandin biosynthesis

The effects of phenolic anti-inflammatory drug, MK-447, on prostaglandin (PG) I2 and thromboxane (TX) A2 biosynthesis by rat dental pulp tissue were evaluated in the presence of 10 mM mannitol (MA) or 1 mM ascorbic acid with 0.3 mM Fe2+ (A + F). Although MK-447 alone at 1 and 10 microM had no significant effects, MK-447 at 100 microM stimulated both PGI2 and TXA2 biosynthesis, and suppressed the lipid peroxidation in the pulp tissue as estimated by thiobarbituric acid method. MA also reduced the lipid peroxidation, but had no effect on PG and TX production. However, in the presence of MA, the stimulatory effect of MK-447 was potentiated, and the significant effects were observed at concentrations higher than 1 microM. In contrast, A + F remarkably stimulated the lipid peroxidation, and inhibited both PG and TX biosynthesis. In the presence of A + F, MK-447 showed no stimulatory effect, and contrary, at 100 microM inhibited PG and TX production. These results suggest that the cellular levels of lipid peroxidation exert a significant influence on the effects of phenolic anti-inflammatory drugs like MK-447 on PG biosynthesis. The possible mechanism of action for such drugs has been discussed in view of the significance of lipid peroxidation in inflammatory condition.     

Lipid peroxidation inhibition and antiradical activities of some leaf fractions of Mangifera indica

This study was undertaken to assess in vitro lipid peroxidation inhibitions and anti-radical activities of methanolic, chloroform, ethyl acetate and water fractions of Mangifera indica leaf. Inhibition of Fe(2+)-induced lipid peroxidation (LPO) in egg, brain, and liver homogenates, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl (OH-) radical scavenging activities were evaluated. Total phenol was assessed in all fractions, and the reducing power of methanolic fraction was compared to gallic acid and ascorbic acid. The results showed that Fe2+ induced significant lipid peroxidation (LPO) in all the homogenates. Ethyl acetate fraction showed the highest percentage inhibition of LPO in both egg yolk (68.3%) and brain (66.3%), while the aqueous fraction exerted the highest inhibition in liver homogenate (89.1%) at a concentration of 10 microg/mL. These observed inhibitions of LPO by these fractions were higher than that of ascorbic acid used as a standard. The DPPH radical scavenging ability exhibited by ethyl acetate fraction was found to be the highest with IC50 value of 1.5 microg/mL. The ethyl acetate and methanolic fractions had the highest OH- radical scavenging ability with the same IC50 value of 5 microg/mL. The total phenol content of ethyl acetate fraction was the highest with 0.127 microg/mg gallic acid equivalent (GAE). The reductive potential of methanolic fraction showed a concentration-dependent increase. This study showed that inhibition of LPO and the DPPH and OH- radicals scavenging abilities of Mangifera indica leaf could be related to the presence of phenolic compounds. Therefore, the ethyl acetate fraction of the leaf may be a good source of natural antioxidative agent.     

Effects of Gomishi and Shosaiko-to on lipid peroxidation of rat brain]

Gomishi and Shosaiko-to were administered to the rats at a dose of 10-100 mg/kg daily for 2 weeks, and their effects on lipid peroxidation of rat brain were compared with that of alpha-tocopherol. Administration of Gomishi and Shosaiko-to showed almost the same suppressive action on the lipid peroxidation. Gomishi and Shosaiko-to exhibited a radical-trapping action on a stable free radical, 1, 1-diphenyl-2-picrylhydrazyl (DPPH), which was estimated photometrically. The effects of Gomishi or Shosaiko-to at concentrations of 10(-3) to 10(-5)g/ml on lipid peroxidation of rat brain homogenates were investigated. The lipid peroxidation was inhibited by the addition of these drugs, and the suppressive effect was also dependent on the concentration. These suppressive effects with Shosaiko-to were stronger than those of Gomishi. These results suggest that the radical trapping action of Gomishi or Shosaiko-to is the likely mechanism suppressing brain lipid peroxidation; Particularly, the suppressive effect on the brain's lipid peroxidation by Shosaiko-to is at least in part due to its radical trapping action and inhibition of O2-. production.     

Brain regional responses in antioxidant system to alpha-lipoic acid in arsenic intoxicated rat

Impaired antioxidant defense mechanisms and oxidative stress are implicated in the pathogenesis of arsenic toxicity. Our study was designed to determine whether alpha-lipoic acid, which has been shown to have substantial antioxidant properties, when administered (70 mg/kg body weight) once daily for 60 days along with arsenic (100 ppm sodium arsenite mixed in drinking water) would prevent arsenic-induced changes in antioxidant defense system, superoxide dismutase (SOD-total SOD, Mn SOD, Cu/Zn SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) in rat brain regions such as cortex, hypothalamus, striatum, cerebellum and hippocampus. The present study also examined the effect of alpha-lipoic acid over arsenic-induced oxidant production and lipid peroxidation level (LPO) in discrete brain regions of rats. The cortex, striatum and hippocampus showed greater decreases in GSH-Px enzyme activity than cerebellum and hypothalamus with arsenic exposure. Striatum had the greatest percentage of decreased activities of total SOD and Mn SOD, whereas cortex had the greatest percentage decrease in the activity of Cu/Zn SOD in arsenic-alone treated rats. Hypothalamus and cerebellum exhibited the lowest catalase activity among all tested regions in arsenic-only treated rats. Rate of dichlorofluorescin oxidation, an indication of reactive oxygen species and other intracellular oxidants production was increased with arsenic exposure in all brain regions studied. Cortex, hippocampus and striatum exhibited greater increase of LPO levels than cerebellum and hypothalamus. SOD, CAT, GSH-Px activities were upregulated in arsenic plus lipoic acid treated versus arsenic-only treated rats. Also, simultaneous lipoic acid treatment along with arsenic proved to be sufficient in reducing oxidant production and LPO level in all rat brain regions. Our results demonstrate that arsenic-induced deficits in antioxidant enzyme activities and increase in oxidant production and lipid peroxidation level in brain regions can be overcome through simultaneous treatment with lipoic acid.     

Inhibition of arachidonic acid release by nordihydroguaiaretic acid and its antioxidant action in rat alveolar macrophages and Chinese hamster lung fibroblasts

The ability of nordihydroguaiaretic acid (NDGA) to inhibit arachidonic acid (AA) release from rat alveolar macrophages treated with t-butyl hydroperoxide (tBOOH) or from Chinese hamster lung fibroblasts (V79 cells) treated with linoleic acid hydroperoxide (LOOH) was examined. Treatment of alveolar macrophages with 100 microM tBOOH significantly increased arachidonic acid release and its conversion to metabolites. Pretreatment of macrophages with NDGA (greater than or equal to 2.5 microM) inhibited the release of AA and its subsequent metabolism following addition of tBOOH. Treatment of V79 cells with 1 microM LOOH stimulated the release of AA. Pretreatment with either 1 or 10 microM NDGA prior to the addition of LOOH inhibited the release of AA. A23187 (2 microM)-stimulated release of AA from V79 cells was less sensitive to NDGA inhibition. Pretreatment with 10 microM NDGA, but not with 1 microM NDGA, inhibited A23187-stimulated release of AA. PLA2-dependent hydrolysis of micelle preparations of disaturated phosphatidylcholine was not inhibited by NDGA. Previous studies have suggested that the addition of peroxides alters cells by inducing lipid peroxidation so that the action of phospholipases upon their membranes is enhanced. The results suggest that NDGA, a lipid-soluble antioxidant which traps free radicals, indirectly blocked the action of phospholipases upon cell membranes by inhibiting lipid peroxidation.     

Cadmium-induced lipid peroxidation in rat liver slices: A possible involvement of hydroxyl radicals

Cadmium-induced lipid peroxidation (LPO) in the presence of various hydroxyl radical-generating media was studied in rat liver slices. In the absence of such media, Cd showed an inhibitory action on LPO in vitro. Cd-induced LPO in liver slices in the presence of OH radical-generating media was inhibited by antioxidants. The findings indicate that Cd induced LPO as a result of the presence of transition metal ions and the generation of hydroxyl radicals.     

Interactions between paraquat and ferric complexes in the microsomal generation of oxygen radicals

Transition metals may play a central role in the toxicity associated with paraquat. Studies were carried out to evaluate the interaction of paraquat with several ferric complexes in the promotion of oxygen radical generation by rat liver microsomes. In the absence of added iron, paraquat produced some increase in low level chemiluminescence by microsomes; there was a synergistic increase in light emission in the presence of paraquat plus ferric-ATP or ferric-citrate, but not paraquat plus either ferric-EDTA or ferric-diethylenetriamine pentaacetic acid (ferric-DETAPAC). Synergistic interactions could be observed at a paraquat concentration of 100 microM and a ferric-ATP concentration of 3 microM. In the absence or presence of paraquat, microsomal light emission was not affected by catalase or dimethyl sulfoxide (DMSO), indicating no significant role for hydroxyl radicals. Superoxide dismutase (SOD) did not affect chemiluminescence in the absence of paraquat but produced some inhibition in the presence of paraquat; this inhibition by SOD was most prominent in the absence of added iron and less pronounced in the presence of ferric-ATP or ferric-citrate. Although microsomal chemiluminescence is closely associated with lipid peroxidation, paraquat did not increase malondialdehyde production as reflected by production of thiobarbituric acid-reactive components. However, lipid peroxidation was sensitive to inhibition by SOD in the presence, but not in the absence, of paraquat, analogous to results with chemiluminescence. Paraquat synergistically increased microsomal hydroxyl radical production as measured by the production of ethylene from 2-keto-4-thiomethylbutyrate in the presence of ferric-EDTA or ferric-citrate. The interaction of paraquat with microsomes and ferric complexes resulted in an increase in oxygen radical generation. Various ferric complexes can increase the catalytic effectiveness of paraquat in promoting microsomal generation of oxygen radicals, although, depending on the reaction being investigated, the nature of the ferric complex is important.     

Effects of aluminum on brain lipid peroxidation

Excessive dietary aluminum (Al) has been proposed to be a factor contributing to several neurological disorders in humans. Six 8-week-old female Swiss Webster mice were fed for 10 weeks purified diets containing 100 (control, 100 Al), 500 (500 Al) or 1000 (1000 Al) micrograms Al/g diet. Brain and liver lipid peroxidation was determined by evaluating the production of 2-thiobarbituric acid reactive substances (TBARS) in brain and liver homogenates in the presence or absence of 50 microM ferrous iron. TBARS production in the absence of iron in brain homogenates from mice fed the 1000 Al diet was higher (30%) than that in the 100 Al control group (3.1 vs. 2.4 nmol TBARS/mg protein). The addition of ferrous iron increased TBARS production in brain homogenates from all 3 dietary groups. The iron-induced TBARS production was 26% higher in the 1000 Al brain homogenates than in the 100 Al group (4.9 vs. 3.9 nmol TBARS/mg protein). Brain TBARS production in the presence and absence of iron was similar between the 100 and 500 Al groups. TBARS production in liver homogenates measured either with or without iron was similar for the 3 groups. These results show that, in mice, dietary Al intoxication leads to increased brain TBARS production, suggesting that enhanced lipid peroxidation may be one possible mechanism underlying the neurological damage associated with increased tissue Al.     

The pyrimido-pyrimidine derivative RA-642: a potent inhibitor of ferrous-induced lipid peroxidation in cell membranes

The effects of three pyrimido-pyrimidine derivatives (RA-642, dipyridamole and mopidamol) on hydroxyl anion-induced lipid peroxidation in cell membranes from liver, brain, kidney, lung and heart rat tissue were studied using d-alpha-tocopherol as standard for lipid peroxidation. Ferrous sulfate and ascorbic acid (FeAs) were used to induce lipid peroxidation via the formation of hydroxyl anions. The products resulting from the reaction with thiobarbituric acid were taken to be indicators of lipid peroxidation. Thiobarbituric acid reactive substances (TBARS) were produced by different rat tissues in the following sequence: brain greater than liver greater than kidney greater than heart greater than lung. Dose-response and time-response curves were plotted for all compounds. Inhibiting concentrations, 50% (IC50), ranged from 0.3-1.4 microM for RA-642, and 2.5 and 4.6 microM for dipyridamole. In liver mitochondrial membranes, IC50s of these compounds were 0.4 +/- 0.2 and 5.8 +/- 1.2 microM, respectively. At 15 min after beginning TBARS production, dipyridamole and RA-642 did not exert any inhibitory effect.     

Effect of piperonyl butoxide or allopurinol on cyanide-induced lipid peroxidation in mouse brain

The effect of piperonyl butoxide or allopurinol on cyanide-induced lipid peroxidation was investigated using homogenates from whole brain of mice. The brain homogenate exposed to a low concentration of potassium cyanide (10, 50, or 100 μM) was significantly increased in their concentration of malondialdehyde (MDA)+4-hydroxyalkenals (4-MDA) as compared to control samples, in a concentration-dependent manner. The increased lipid peroxidation induced by cyanide was inhibited by piperonyl butoxide (1 mM), an inhibitor of mixed function oxidase, but not by allopurinol (0.1 mM), an inhibitor of xanthine oxidase. Furthermore, when a brain homogenate heated at 86°C for 1 min was incubated with or without cyanide at 37°C for 20 min, MDA+4-MDA levels in the homogenate were not changed between cyanide treatment and untreated. An intraperitoneal injection of piperonyl butoxide (1 g/kg) significantly inhibited cyanide-induced seizures in mice. These results suggest that cyanide-induced seizures may be partly involved in the lipid peroxidation produced by the heat unstable and piperonyl butoxide dependent factors in brain.     

Effects of cadmium on superoxide dismutase and lipid peroxidation in liver and kidney of growing rats: in vivo and in vitro studies

Intraperitoneal administration of cadmium acetate (Cd2+, 0.4 mg/kg) to rats daily for 30 days was found to inhibit the activity of superoxide dismutase (SOD), to increase the endogenous levels of lipid peroxides and lipid peroxidation in the liver and the kidney tissues. Addition of varying concentrations of Cd2+ (10-100 microM) in vitro also inhibited SOD in both the tissues. It appears that the inhibition of SOD could be due to direct interaction of Cd2+ with the enzyme molecule. Lipid peroxidation reaction was also increased after addition of Cd2+ to fresh homogenate of these tissues, however, it did not produce any effect in heated homogenates in in vitro experiments. It indicated that Cd-induced elevation in lipid peroxidation may not be only due to the possibility of higher level of superoxide radicals resulting from inhibited superoxide dismutase but could also be as a result of direct action of Cd2+ on the peroxidation reaction. Thus, the possibility of involvement of free radical damage to the membrane structures in Cd toxicity has been demonstrated in these experiments.     

Broussochalcone A, a potent antioxidant and effective suppressor of inducible nitric oxide synthase in lipopolysaccharide-activated macrophages

The antioxidant properties of broussochalcone A (BCA) and its effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated macrophages were investigated in this study. BCA, isolated from Broussonetia papyrifera Vent., inhibited iron-induced lipid peroxidation in rat brain homogenate in a concentration-dependent manner with an IC(50) of 0.63 +/- 0.03 microM. It was as potent as butylated hydroxytoluene, a common antioxidant used for food preservation. In a diphenyl-2-picrylhydrazyl assay system, the radical-scavenging activity of BCA seemed to be more potent than that of alpha-tocopherol, its IC(0.200) being 7.6 +/- 0.8 microM. BCA could directly scavenge superoxide anion and hydroxyl radicals. These results indicated that BCA was a powerful antioxidant with versatile free radical-scavenging activity. On the other hand, we found that BCA suppressed NO production concentration-dependently, with an IC(50) of 11.3 microM in LPS-activated macrophages. This effect was not the consequence of a direct inhibitory action on the enzyme activity of inducible NO synthase (iNOS). Our results indicated that BCA exerts potent inhibitory effects on NO production, apparently mediated by its suppression of IkappaBalpha phosphorylation, IkappaBalpha degradation, nuclear factor-kappa B activation, and iNOS expression. Therefore, we conclude that the antioxidant activities of BCA and its inhibition of IkappaBalpha degradation and iNOS protein expression may have therapeutic potential, given that excessive free radicals and NO production have been associated with various inflammatory diseases.     

Receptor independent stimulatory effect of noradrenaline on Na,K-ATPase in rat brain homogenate. Role of lipid peroxidation

The effect of different adrenoceptor agonists on Na,K-ATPase activity and lipid peroxidation of rat brain homogenate was studied. Drugs which enhanced Na,K-ATPase activity--noradrenaline, adrenaline and oxymethazoline--were found to inhibit endogenous membrane lipid peroxidation. Other drugs--phenylephrine, xylazine and clonidine--which did not cause any change in the enzyme activity did not influence lipid peroxidation either. No increase of Na,K-ATPase activity by noradrenaline could be detected after preincubation of the homogenate for 5 min at 37 degrees. During this time endogenous lipid peroxidation of considerable extent could be observed. It is concluded that there is no correlation between the adrenoceptor agonist feature of noradrenaline and its stimulatory effect on Na,K-ATPase activity of rat brain homogenate. However, it seems likely that in rat brain homogenate the increase of Na,K-ATPase activity and inhibition of endogenous lipid peroxidation by noradrenaline are related.     

5种抗肿瘤药对小鼠脂质过氧化作用的影响

研究抗肿瘤药甲氨蝶呤、阿糖胞苷、环磷酰胺、5—氟尿嘧啶和丝裂霉素C对小鼠脂质过氧化作用的影响、结果显示:5种抗肿瘤药使小鼠血清和肝、脑中MDA含量显著升高.但对肝组织体外生成MDA的速度无明显影响。提示这些药物对过氧化脂质的代谢有严重干扰。     

Metabolism of 4-hydroxy-trans-2-nonenal by central nervous system mitochondria is dependent on age and NAD+ availability

Lipid peroxidation and mitochondrial dysfunction are associated with multiple neurodegenerative disorders including Alzheimer's disease and Parkinson's disease. 4-Hydroxy-trans-2-nonenal (HNE) is a major, neurotoxic product of lipid peroxidation whose levels are elevated in these diseases. Previous data from this laboratory demonstrate that mitochondria play an important role in the detoxification of HNE particularly through the oxidation of HNE to 4-hydroxy-trans-2-nonenoate (HNEAcid). In this work, we examined the disposition of HNE when incubated with intact, well-coupled, rat brain mitochondria. Our results demonstrated that HNE loss occurred in a time- and concentration-dependent, saturable manner with a K(M) of 28.0 +/- 11.8 microM HNE and a V(Max) of 10.0 +/- 1.7 nmol/min/mg. HNEAcid formation occurred in a saturable manner with a K(M) of 25.3 +/- 6.3 microM HNE and a V(Max) of 4.4 +/- 0.43 nmol/min/mg. The formation of HNE-glutathione adducts and HNE-protein adducts comprised only a small percentage of HNE consumption. HNE metabolism was significantly diminished in rat brain mitochondria isolated from older animals. We then tested the hypothesis that the mitochondrial NADH/NAD(+) ratio regulated matrix aldehyde dehydrogenase activity. Our results demonstrate that HNE oxidation was significantly inhibited to a greater extent with pyruvate and malate as substrates vs succinate. Complex I inhibition with respiratory substrates further blocked HNE detoxification. Rotenone (100 nM) inhibited respiration by 15% whereas HNEAcid formation was decreased to 72% of control levels. These results demonstrate that in situ mitochondrial aldehyde detoxification is affected by decrements in NAD(+) availability and complex I activity.     

丹酚酸B镁盐对自由基的清除作用和对脂质过氧化的抑制作用(英文)

AIM: To study the effect of magnesium lithospermate B (MLB) on the lipid peroxidation and on its free radical scavenging activity. METHODS: MLB was incubated in rat tissue homogenate or in a free radical generating system. MLB induced inhibition of lipid peroxidation and its scavenging activity on superoxide anions and hydroxyl radicals was studied using colorimetric estimation. RESULTS: MLB inhibited the lipid peroxidation induced by either an auto-oxidant or Fe2+/VitC in vitro, in the liver homogenate, the inhibitory rate of MLB (10 mg/L) being 69.2% and 57.7%, respectively. MLB (25 and 50 mg/kg) decreased the amount of thiobarbituric acid reactive substances (TBARS) in rat serum, liver, kidney, and heart. However, it did not inhibit the lipid peroxidation of brain homogenate ex vivo. MLB scavenged superoxide anions generated from xanthine/xanthine oxidase system and iron-dependent hydroxyl radicals. CONCLUSION: MLB is an inhibitor of lipid peroxidation and scavenge superoxide anions and hydroxyl radicals both in vitro and ex vivo.