Fe2+-supported in vivo lipid peroxidation induced by compounds undergoing redox cycling

Treatment of male mice with the redox cycling compounds nitrofurantoin, paraquat, diquat or menadione failed to elicit in vivo lipid peroxidation as evidenced by ethane exhalation. The first three led to an enhanced ethane production, however, when the animals were pretreated with a low dose of Fe2+. While GSH-depletion by phorone pretreatment alone had no influence on the in vivo lipid peroxidation as evidenced by ethane expiration in the presence of either compound, the combined treatment with phorone, Fe2+ and nitrofurantoin, paraquat or diquat led to a further enhancement of ethane exhalation. These results indicate that redox cycling compounds do not initiate lipid peroxidation by themselves, but are well capable of stimulating the iron-induced LPO.     

The reductive metabolism of nifurtimox and benznidazole in Crithidia fasciculata is similar to that in Trypanosoma cruzi

1. Addition of nifurtimox or benznidazole to the NADH-containing mitochondrial fraction of Crithidia fasciculata led to the appearance of the characteristic ESR spectra corresponding to their nitro anion radicals, suggesting that the nitro anion radical is a necessary intermediate in the reduction of both nitro compounds. 2. Nifurtimox anion radical generation by the mitochondrial fraction was insensitive to rotenone and antimycin A but was enhanced by KCN. 3. The nifurtimox anion radical reacted with oxygen under aerobic conditions leading to an increase in the cyanide-insensitive respiration of the intact cells and in the rate of O2- and H2O2 production by the C. fasciculata mitochondrial fraction. 4. In contrast, generation of O2- and H2O2 was not stimulated with pharmacological concentrations of benznidazole. Furthermore, benznidazole inhibited the cyanide-insensitive respiration of the intact cells.     

Novel antioxidants isolated from plants of the genera Ferula, Inula, Prangos and Rheum collected in Uzbekistan

We examined the effects of 48 compounds isolated from Ferula pallida, F. penninervis, Inula macrophylla, Prangos pabularia, P. tschimganica and Rheum maximowiczii collected in Uzbekistan on ADP/Fe²⁺-induced lipid peroxidation of egg yolk phosphatidylcholine liposomes. Of those compounds, 23 inhibited ADP/Fe²⁺-induced lipid peroxidation and nine showed especially strong inhibition of lipid peroxidation. Most compounds that inhibited peroxidation scavenged the 1,1′-diphenyl-2-picrylhydrazyl (DPPH) radical, indicating that the inhibition was due to radical scavenging. However, some compounds did not scavenge DPPH but inhibited lipid peroxidation significantly, suggesting that their inhibitory effect was not due to radical scavenging but to some other mechanism, such as prevention of Fe²⁺ function. Thus, we found various new antioxidants, some of which had a unique mechanism of action, in Ferula, Inula, Prangos and Rheum plants collected in Uzbekistan as seeds used in medicine.     

Antioxidant properties of dehydrozingerone and curcumin in rat brain homogenates

The present study investigates the inhibition of lipid peroxidation by dehydrozingerone and curcumin in rat brain homogenates. Both the test compounds inhibited the formation of conjugated dienes and spontaneous lipid peroxidation. These compounds also inhibited lipid peroxidation induced by ferrous ions, ferric-ascorbate and ferric-ADP-ascorbate. In all these cases, curcumin was more active than dehydrozingerone and dl--tocopherol.     

Determination of Antibiotic Susceptibility of Rickettsia by the Plaque Assay Technique

Plaque formation by various rickettsiae was completely inhibited by commercial antibiotic discs impregnated with tetracycline, chloramphenicol, nitrofurantoin, and erythromycin; partial inhibition was observed around discs containing nalidixic acid and sulfisoxazole, but no inhibition was seen around discs containing cephalothin, ampicillin, oxacillin, kanamycin, polymyxin B, streptomycin, or penicillin.     

Synthesis and evaluation of N-substituted indole-3-carboxamide derivatives as inhibitors of lipid peroxidation and superoxide anion formation

The in vitro antioxidant effects of novel N-substituted indole-3-carboxamides (I3CDs) 1-10 on rat liver microsomal NADPH-dependent lipid peroxidation (LP) levels and their free radicals scavenging properties were determined by the inhibition of superoxide anion formation (SOD). Among the synthesized compounds, 4, 5, 8 and 9 significantly inhibited SOD with an inhibition range at 84-100% at 10(-3) M concentration. The presence of halo substituents both ortho- and para- positions of these compounds resulted 100% inhibition of SOD. Comparison the activity results of halogenated and non-halogenated derivatives suggested that the halogenated compounds are more active than the non-halogenated compounds. On the other hand, the introduction of a para fluoro benzyl in the 1-position of indole (compounds 7, 8) has more impact on the SOD inhibition when the benzamide ring was mono halogenated. However, none of other compounds had a significant inhibitory effects on the level of lipid peroxidation.     

In vitro and in vivo assessment of the antioxidant activity of melatonin and related indole derivatives

Effects of melatonin and some structurally related indole compounds were studied by in vitro methods such as (i) an inhibition of the hyaluronic acid degradation and (ii) a standard lipid peroxidation assay. In vivo approach was based on the alloxan model of hyperglycaemia. Reduction of the viscosity of a hyaluronic acid solution in the reaction mixture was inhibited by tryptamine (91% inhibition), as well as by indole-3-carboxylic acid and indomethacin (80% and 77% inhibition, respectively). Lipid peroxidation with tert-butyl hydroperoxide as a source of radicals was followed by the formation of thiobarbituric acid reactive substances. Tested drugs inhibited lipid peroxidation in the order: tryptamine (59%) > indole-2-carboxylic acid (38%) > indomethacin (26%) > melatonin and indole-3-carboxylic acid (13%). In vivo, alloxan-induced hyperglycaemia was reduced in mice pretreated with drugs tested. The highest protective effect was observed with indomethacin (52% inhibition), followed by tryptamine and melatonin (18% and 16% inhibition, respectively).     

Trypanosoma cruzi: In vitro effect of aspirin with nifurtimox and benznidazole

Nifurtimox and benznidazole are the only active drugs against Trypanosoma cruzi; however, they have limited efficacy and severe side effects. During primoinfection, T. cruzi infected macrophages mount an antiparasitic response, which the parasite evades through an increase of tumor growth factor β and PGE₂ activation as well as decreased iNOS activity. Thus, prostaglandin synthesis inhibition with aspirin might increase macrophage antiparasitic activity and increase nifurtimox and benznidazole effect.Aspirin alone demonstrated a low effect upon macrophage antiparasitic activity. However, isobolographic analysis of the combined effects of aspirin, nifurtimox and benznidazole indicated a synergistic effect on T. cruzi infection of RAW cells, with combinatory indexes of 0.71 and 0.61, respectively.The observed effect of aspirin upon T. cruzi infection was not related with the PGE₂ synthesis inhibition. Nevertheless, NO levels were restored by aspirin in T. cruzi-infected RAW cells, contributing to macrophage antiparasitic activity improvement.Thus, the synergy of aspirin with nifurtimox and benznidazole is due to the capability of aspirin to increase antiparasitic activity of macrophages.     

Effect of seminal plasma antioxidant on lipid peroxidation in spermatozoa, mitochondria and microsomes

Seminal plasma antioxidant inhibited ascorbate/iron-induced lipid peroxidation in spermatozoa, brain and liver mitochondria. The concentration required to produce inhibition in brain and liver mitochondria was high. Denaturation of spermatozoa resulted in complete loss of antioxidant action. Maintenance of native structure was essential for action of seminal plasma antioxidant in spermatozoal lipid peroxidation. The antioxidant inhibited NADPH, Fe3+-ADP induced lipid peroxidation in microsomes and consequences of lipid peroxidation such as glucose-6-phosphatase inactivation were prevented by presence of antioxidant. It did not inhibit microsomal lipid peroxidation induced by ascorbate and iron and xanthine-xanthine oxidase.     

Inhibition of Microsomal Lipid Peroxidation and Cytochrome P-450-Catalyzed Reactions by Nitrofuran Compounds

(5-Nitro-2-furfuryliden)amino compounds bearing triazol-4-yl, benzimidazol-l-yl, pyrazol-l-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-l-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-l-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-l-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-l-yl and substituted triazol-4-yl derivitives.     

Inhibition of Microsomal Lipid Peroxidation and Cytochrome P-450-Catalyzed Reactions by Nitrofuran Compounds

(5-Nitro-2-furfuryliden)amino compounds bearing triazol-4-yl, benzimidazol-l-yl, pyrazol-l-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-l-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-l-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-l-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-l-yl and substituted triazol-4-yl derivitives.     

Vitamin E dependent reduced glutathione inhibition of rat liver microsomal lipid peroxidation

Effects of reduced glutathione (GSH) were investigated on $\text{in}$$\text{vitro}$ lipid peroxidation of hepatic microsomes obtained from Long-Evans Hooded rats fed chemically defined, purified diets containing adequate or documented deficiencies of vitamin E (E), selenium (Se) or both. Glutathione inhibited lipid peroxidation mediated by both NADPH-dependent enzymatic and ascorbate-dependent non-enzymatic systems. The inhibitory effect of GSH was observed in microsomes obtained from E supplemented groups whereas it had no effect on microsomes from E deficient animals. Selenium status had no effect on GSH inhibition. Glutathione was found to be specific for the E dependent inhibition of lipid peroxidation and could not be substituted by other sulfhydryl compounds tested. Also, GSH did not inhibit non-enzymatic lipid peroxidation of heat-denatured microsomes from either E-supplemented groups or any of the other dietary regimens.     

Inhibition of carbon tetrachloride-induced lipid peroxidation by novel antioxidants in rat hepatic microsomes: dissociation from hepatoprotective effects in vivo

The ability of two novel antioxidants, U-74,006F and U-78,517G, as well as the known antioxidant N,N'-diphenyl-p-phenylenediamine to inhibit lipid peroxidation induced by carbon tetrachloride (CCl4) was investigated in Aroclor 1254-induced rat hepatic microsomes. All three compounds completely inhibited lipid peroxidation in microsomes as measured by the formation of thiobarbituric acid reactive substances (TBARS). Inhibition of lipid peroxidation was not a function of decreased bioactivation of CCl4, as the compounds did not substantially inhibit benzphetamine N-demethylase activity or covalent binding of [14-C]CCl4 to lipid or protein. Parallel studies examined the hepatoprotective effects of the compounds in vivo. Rats were pretreated with antioxidant or vehicle prior to administration of CCl4 (300 or 600 microL/kg i.p.). Sera were collected 24 h postadministration of CCl4 and analyzed for alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities and total bilirubin. Administration of CCl4 produced elevations in ALT, moderate changes in bilirubin, and no change in ALP activities. Histological examination of CCl4-treated livers revealed lipidosis and centrilobular necrosis. The antioxidants partially improved the clinical chemistry parameters, but had minimal effects on the histological lesion. In contrast to the complete inhibition of lipid peroxidation observed in the in vitro studies, none of the antioxidants markedly protected against CCl4-induced toxicity in vivo.     

Mechanism of cobalt (II) ion inhibition of iron-supported phospholipid peroxidation

Co2+ inhibited nonenzymatic iron chelate-dependent lipid peroxidation in dispersed lipids, such as ascorbate-supported lipid peroxidation, but not iron-independent lipid peroxidation. Histidine partially abolished the Co2+ inhibition of the iron-dependent lipid peroxidation. The affinity of iron for phosphatidylcholine liposomes in Fe(2+)-PPi-supported systems was enhanced by the addition of an anionic lipid, phosphatidylserine, and Co2+ competitively inhibited the peroxidation, while the inhibiting ability of Co2+ as well as the peroxidizing ability of Fe(2+)-PPi on liposomes to which other phospholipids, phosphatidylethanolamine, or phosphatidylinositol had been added was reduced. Co2+ inhibited microsomal NADPH-supported lipid peroxidation monitored in terms of malondialdehyde production and the peroxidation monitored in terms of oxygen consumption. The inhibitory action of Co2+ was not associated with iron reduction or NADPH oxidation in microsomes, suggesting that Co2+ does not affect the microsomal electron transport system responsible for lipid peroxidation. Fe(2+)-PPi-supported peroxidation of microsomal lipid liposomes was markedly inhibited by Co2+.     

Inhibition of adriamycin-stimulated microsomal lipid peroxidation by mitoxantrone and ametantrone,two new anthracenedione antineoplastic agents

Ametantrone and mitoxantrone, two new anthracenedione antineoplastic agents, produced a concentration-dependent inhibition of hepatic microsomal lipid peroxidation. Malondialdehyde production was diminished from 10.6 nmoles/mg/60 min to 3.3 and 5.4 nmoles/mg/60 min, in the presence of 100 μM mitoxantrone and ametantrone, respectively. Under similar conditions, Adriamycin stimulated lipid peroxidation over twofold. In addition, both mitoxantrone and ametantrone inhibited Adriamycin-stimulated lipid peroxidation, with 50% inhibition occurring at concentrations of 4 and 6 μM, respectively. Microsomal superoxide production was not significantly inhibited at anthracenedione concentrations which markedly decreased lipid peroxidation, suggesting that inhibition of lipid peroxidation was not the result of inhibition of superoxide generation. These results correlate with the lack of anthracenedione cardiotoxicity and also demonstrate anthracenedione inhibition of lipid peroxidation at micromolar concentrations; an observation with potential therapeutic significance.     

Bioactive terpenoids and guggulusteroids from Commiphora mukul gum resin of potential anti-inflammatory interest

Guggulu, the gum resin from Commiphora mukul, is one of the components of various formulations of traditional Ayurvedic medicine to treat inflammation, obesity, and lipid disorders. In most preparations of Ayurvedic medicine in India, guggulu is boiled prior to its use. Therefore, guggulu was boiled with H2O prior to extractions in our study. Bioassay-guided isolation of compounds from the hexane-soluble portion of the MeOH extract of guggulu yielded cembrenoids, 1-6, a bicyclic diterpene, 7, guggulusterone derivatives, 8-11, myrrhanone derivatives, 12, myrrhanol derivative, 13, and a lignan, 14. The structures of these compounds were confirmed by spectroscopic methods. Compounds 5, 6, 7, 10, and 12-14 are novel. These compounds were assayed for lipid peroxidation and cyclooxygenase (COX) enzyme inhibitory activities. At 100 ppm, compounds 3, 6, and 14 inhibited the lipid peroxidation by 79, 57, and 58%, respectively, and the rest of isolated compounds showed 20-40% inhibitory activity with respect to the controls. In COX-1 and COX-2 enzyme inhibitory assays, compound 3 showed 79 and 83%, and compound 8 gave 67 and 54% of inhibition, respectively, at 100 ppm. All fourteen compounds inhibited COX-1 enzyme at 100 ppm. The lipid peroxidation and COX enzyme inhibitory activities exhibited by compounds isolated from C. mukul may substantiate its use in traditional medicine.     

Inhibition of rat liver microsomal lipid peroxidation by N-acyldehydroalanines: an in vitro comparative study

Captodative substituted olefins are radical scavengers which react with free radicals to form stabilized radical adducts. One of those compounds, N-(paramethoxyphenylacetyl)dehydroalanine (AD-5), may react and scavenge both superoxide anion (O-2) and alk-oxyl radicals (RO.), and in this way prevent the appearance of their mediated biological effects. Nitrofurantoin and tert-butyl hydroperoxide were used as model compounds to stimulate free radical production and their mediated lipid peroxidation in rat liver microsomes. In addition, lipid peroxidation was also initiated by exposure of rat liver microsomal suspensions to ionizing radiation (gamma rays). The microsomal lipid peroxidation induced by these chemicals and physical agents was inhibited by the addition of AD-5. These effects were dose-dependent in a millimolar range of concentration. In addition, AD-5 has no effect on microsomal electron transport, showing that NADPH-cytochrome P450 reductase activity was not modified. These data, together with the comparisons of the effects of AD-5 and some antioxidant molecules such as superoxide dismutase, uric acid, and mannitol, support the conclusion that inhibition of lipid peroxidation by AD-5 is the result of its free radical scavenger activity. In addition, the inhibitory effect of AD-5 on microsomal lipid peroxidation was dependent of the nature of the free radical species involved in the initiation of the process, suggesting that O-2 is scavenged more efficiently than RO.     

Inhibition of S-(1,2-dichlorovinyl)-L-cysteine-induced lipid peroxidation by antioxidants in rabbit renal cortical slices: Dissociation of lipid peroxidation and toxicity

Precision-cut, rabbit renal slices were used to examine the effects of three novel antioxidants (U-74006, U-74500, and U-78517) on S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced lipid peroxidation and toxicity. Slices exposed to DCVC showed a dose- and time-dependent increase in lipid peroxidation (TBARS) and a decrease in cellular viability, as evidenced by the loss of intracellular potassium, during the course of a 3 hour incubation. Subsequent studies employed DCVC concentrations of 100 μM. Microemulsion formulations of U-78517, U-74500, and U-74006 (100 μM) inhibited DCVC-induced lipid peroxidation by 100±, 50±, and <5% (not significant), respectively. However, none of these antioxidants had a significant effect on DCVC-dependent cytotoxicity, as indicated by intracellular potassium release. The effects of U-78517, the most potent of the three antioxidants, were similar to those observed with two model antioxidants, diphenyl-p-phenylenedi-amine (DPPD) and the iron chelator, deferoxamine. Aminooxyacetic (AOAA), an inhibitor of renal cysteine conjugate β-lyase, had only a minimal effect on DCVC-induced lipid peroxidation, and no effect on toxicity. These data represent the first report of DCVC-induced lipid peroxidation in rabbit renal cortical slices, a system which has been widely used to investigate mechanisms of nephrotoxicity, including that induced by DCVC. Our results demonstrate that DCVC-induced lipid peroxidation in renal slices can be inhibited by a variety of antioxidant compounds operating by different mechanisms. Because inhibition of lipid peroxidation had minimal effect on DCVC-dependent cytotoxicity, the data suggest that DCVC-induced lipid peroxidation is not a major mechanism in the cytotoxicity induced by this compound.     

Microsomal glutathione-dependent protection against lipid peroxidation acts through a factor other than glutathione peroxidase and glutathione S-transferase in rat liver

Ascorbate-Fe3+-induced and NADPH-induced lipid peroxidation of rat liver microsomes were inhibited by glutathione (GSH). This inhibition was due to microsomal GSH-dependent factor. This factor was heat labile, and storage of microsomes at 4 degrees C for 1 week diminished the activity. GSH could not be substituted by other sulfhydryl compounds tested. Deoxycholate (1 mM) and bromosulfophthalein (0.1 mM) inhibited GSH-dependent protection but did not inhibit microsomal GSH peroxidase activity. Iodoacetate (10 mM) inhibited GSH-dependent protection but did not inhibit microsomal GSH S-transferase. N-Ethylmaleimide (0.1 mM) and oxidized glutathione (10 mM) inhibited GSH-dependent protection but activated microsomal GSH S-transferase activity. These results indicate the existence of a heat-labile, microsomal GSH-dependent protective factor against lipid peroxidation that acts through a factor other than GSH-peroxidase and GSH S-transferase.     

Effects of chloramphenicol isomers and erythromycin on enzyme and lipid synthesis induced by oxygen in wild-type and petite yeast

The synthesis of mitochondrial enzymes induced by exposure of anaerobically grown, lipid-depleted Saccharomyces cerevisiae to oxygen is inhibited by d(-)-threo-chloramphenicol and erythromycin. The concentration of these antibiotics required to cause 50% inhibition of this synthesis is less than 1 mm; this is also approximately the concentration required to inhibit by the same amount mitochondrial protein synthesis in situ. The synthesis of unsaturated fatty acids, ergosterol, and phospholipid induced by aeration is inhibited by d(-)-threo-chloramphenicol at high concentrations (12 mm) but is unaffected by erythromycin. l(+)-threo-Chloramphenicol affects neither enzyme nor lipid synthesis and is without effect on mitochondrial protein synthesis in situ. All three compounds inhibit the oxidative activity of isolated mitochondria; the chloramphenicol isomers also inhibit phosphorylation. In a euflavine-derived petite mutant, lacking mitochondrial protein synthesis and respiration, aeration results in the normal development of lipid in the cells, but no synthesis of mitochondrial enzymes. d(-)-threo-Chloramphenicol does not inhibit lipid synthesis in these cells. Thus inhibition of mitochondrial protein synthesis with erythromycin or genetic deletion of mitochondrial protein synthesis results in loss of the capacity to synthesize enzymes during aeration. d(-)-threo-Chloramphenicol, as well as inhibiting induced enzyme formation, inhibits lipid synthesis induced by oxygen. It is unlikely that the latter effect of chloramphenicol is due to inhibition of energy production and transformation, to direct effects on lipid synthesis, or to an inhibition of mitochondrial protein synthesis. It is, however, an effect not shared with the l isomer.