Cytotoxic and antiproliferative effects of thymoquinone on rat C6 glioma cells depend on oxidative stress.

Thymoquinone (TQ) is a highly perspective chemotherapeutic agent against gliomas and glioblastomas because of its ability to cross the blood-brain barrier and its selective cytotoxicity for glioblastoma cells compared to primary astrocytes. Here, we tested the hypothesis that TQ-induced mild oxidative stress provokes C6 glioma cell apoptosis through redox-dependent alteration of MAPK proteins. We showed that low concentrations of TQ (20-50 µM) promoted cell-cycle arrest and induced hydrogen peroxide generation as a result of NADH-quinone oxidoreductase 1-catalyzed two-electron reduction of this quinone. Similarly, low concentrations of TQ efficiently conjugated intracellular GSH disturbing redox state of glioma cells and provoking mitochondrial dysfunction. We demonstrated that high concentrations of TQ (70-100 µM) induced reactive oxygen species generation due to its one-electron reduction. TQ provoked apoptosis in C6 glioma cells through mitochondrial potential dissipation and permeability transition pore opening. The identified TQ modes of action on C6 glioma cells open up the possibility of considering it as a promising agent to enhance the sensitivity of cancer cells to standard chemotherapeutic drugs.

Interaction of tryptophan and related compounds with oxygen- and carbon-centered radicals.

Effects of tryptophan, 5-hydroxytryptophan, serotonin, and melatonin on final product formation during radiolysis of deaerated and oxygen-saturated ethanol and aqueous 1M ethanol solutions were studied. The named amino acids were found to be capable of adding α-hydroxyethyl radicals, thereby suppressing recombination reactions of these species. Unlike melatonin, tryptophan, 5-hydroxytryptophan, and serotonin were able to reduce oxygen-centered radicals being formed on radiation-chemical oxidation of ethanol in the presence of oxygen via electron transfer from the amine nitrogen lone pair.

[Antioxidant activity of hydroxy derivatives of coumarin].

The inhibition efficiency (antioxidant activity) of hydroxy derivatives of coumarin, such as esculetin, dicumarol, and fraxetin, was studied in the methemalbumin-H2O2-tetramethylbenzidine (TMB) pseudoperoxidase system at 20 degrees C in a buffered physiological solution (pH 7.4) containing 6% DMF and 0.25% DMSO. The inhibitor's efficiency was quantitatively characterized by the inhibition constants (K(i), microM) and the inhibition degree (%). The K(i) values for esculetin, dicumarol, and fraxetin were 9.5, 15, and 26 microM, respectively. Esculetin and fraxetin inhibited pseudoperoxidase oxidation of TMB in a noncompetitive manner; dicumarol, in a mixed manner. The inhibiting activity ofesculetin in peroxidase-catalyzed TMB oxidation at pH 6.4 is characterized by a K(i) value equal to 1.15 microM, and the inhibition process is competitive. Esculetin was found to be the most effective antioxidant of plant origin among all derivatives previously studied in model biochemical systems.

[Antioxidant activity of oxygen-containing aromatic compounds].

Inhibition efficiency (antioxidant activity) of 26 oxygen-containing aromatic compounds was studied in methemalbumin-H202-o-phenylenediamine (PDA) or tetramethylbenzidine (TMB) pseudoperoxidase system at 20 degrees C in buffered physiological solution (pH 7.4) containing 6% DM F and 0.25% DMSO. The inhibitor's efficiency was quantitatively characterized by the inhibition constants (Ki, microM) or the inhibition degree (%). Ki values varied in the range of4 to 500 microM and were influenced by a substrate, the structure of an inhibitor, hydroxyl groups, electron-donating substituents in aromatic ring, and steric hindrances. The type of inhibition at cooxidation of eight pairs was noncompetitive, and that of five pairs was mixed and determined by the substrate nature and the inhibitor structure. Lignin phenolic compounds ofguaiacyl and syringal series exhibited high antioxidant activity (Ki in the range of 10-300 microM), and their efficiency decreased in the following order: caffeic acid > synapaldehyde > syringic acid > coniferyl aldehyde > para-hydroxycou maric acid.

[Highly effective test-system for determination of the total antioxidant activity of human blood serum].

A highly effective test-system for quantitative characterization of the total antioxidant activity (TAA) of human blood serum (HBS), including methemalbumin (MetHa) as biocatalyst, H2O2 as the oxidant, o-phenylenediamine (PDA) as the acceptor of radicals and 2,2,5,7,8-pentamethylchroman-6-ol (PMC) as the inhibitor-calibrator, has been developed and proved under the laboratory environments. The test-system has been optimized for the concentrations of all the components, the reaction conditions and the PDA consumption monitoring at 37 degrees C in the medium of buffered physiological solution, pH 7.4 containing 5% DMFA and 0.5% DMSO. Under strongly standardized conditions by a comparison of the inhibiting action of the HBS and the calibrator PMC at 37 degrees C, the quantitative parameters of the HBS TAA were determined in microg of PMC, equivalent to 1 mg of HBS, or as a reverse value in mg of HBS, equivalent to the action of 1 microg of PMC. The values of the HBS TAA significantly varied for the group of healthy individuals and essentially decreased for the group of patients with thyroid gland pathologies. This underlies necessity of antioxidant therapy in these patients.

The effects of ascorbic acid on homolytic processes involving alpha-hydroxyl-containing carbon-centered radicals.

Effects of ascorbic acid and 5,6-O-isopropylidene-2,3-O-dimethylascorbic acid on final product formation in radiolysis of ethanol, aqueous solutions of ethanol, ethylene glycol, alpha-methylglycoside, maltose, alpha-glycerophosphate, and alpha-glucose phosphate were studied. It was found that ascorbic acid is able to suppress reactions involving various alpha-hydroxyl-containing carbon-centered radicals and depending on the experimental conditions can either oxidize or reduce alpha-hydroxyethyl radicals.

[Substituted aminophenols and flavonoids as potential components for test-systems of total antioxidant activity].

A comparative kinetic study of ortho-phenylenediamine (PDA) oxidation in the "pseudoperoxidase" system Methemalbumin-H2O2 in the presence of 2-amino-4-tret-butylphenol (ATBP), 2-amino-4,6-di-tret-butylphenol (ADTBP) and its four N-acyl derivates, as well as flavonoids (quercetin, morin, silibin, hesperidin and naringin) has been carried out under standart conditions at 20 degrees C in phosphate buffered saline, pH 7.4, containing 5.25% ethanol and DMFA. ATBP, ADTBP and two its N-acyl-derivatives as well as all five flavonoids inhibited with different efficiency the PDA oxidation characterized in terms of the inhibition constants, K(i), M, or the percent of inhibition degree at the maximal taking concentrations of these inhibitors. Most effective antioxidants were quercetin (K(i) = 7.7x10(-5) M) and ATBP (K(i) = 1.26x10(-4) M). Using these characteristics and other necessary criteria, the pairs PDA-quercetin and PDA-ATBP were proposed for a practical application in the test-systems for total antioxidant activity of biological objects.

Effects of quinones on free-radical processes of oxidation and fragmentation of hydroxyl-containing organic compounds.

The coenzymes Q and Vitamin K(3), as well as their synthetic analogues, have been found to inhibit free-radical processes of fragmentation of hydroxyl-containing organic compounds and oxidation of the latter by molecular oxygen. It has been established that the observed effects are due to the ability of quinones to oxidize the alpha-hydroxyl-containing carbon-centered radicals formed from the starting compounds.

Effects of vitamins, coenzymes and amino acids on reactions of homolytic cleavage of the O-glycoside bond in carbohydrates.

It has been established that vitamins B1, K3 and C, coenzyme Q0 and amino acids cysteine and histidine effectively inhibit reactions of homolytic cleavage of the O-glycoside bond, which are responsible for the destruction of di- and polysaccharides on gamma-irradiation or the action of other reactive radical initiators. This effect was shown to originate from either oxidation or reduction of the radicals of carbohydrates undergoing destruction.

Effects of alpha-tocopherol and related compounds on reactions involving various organic radicals.

Effects of alpha-tocopherol, PMC, and a number of the respective sulfur-containing analogues on reactions involving various organic radicals were studied. The test compounds were found to interact with alkyl radicals more effectively than with peroxyl radicals. The presence of a sulfur atom in structures of the respective analogues did not produce significant effects on reactivity. Derivatives of 5-hydroxy-1,3-benzoxathiol-2-one and 6-hydroxy-1,4-benzoxathiin-2(3H)-one displayed a high reactivity toward alpha-hydroxyalkyl radicals.

Reactions of alpha-hydroxyethyl radicals with flavonoids of various structures.

It has been found that flavonoids (FL) are able to reduce, add or oxidize alpha-hydroxyethyl radicals (HER). The probability of these processes to occur depends on the structure of the FL under study. Namely, to cause reduction of HER, the presence of hydroxyl groups is necessary, and to effect oxidation or addition of HER, the presence of a carbonyl group at C4 and a C2-C3 double bond in the C ring is required.

Effects of B group vitamins on reactions of various alpha-hydroxyl-containing organic radicals.

Effects of vitamins B1, B2, B6, and pyridoxal phosphate (PPh) on final product formation in radiolysis of aqueous solutions of ethanol, ethylene glycol, alpha-methylglycoside, and maltose were studied. It has been found that vitamin B2 and PPh effectively oxidize R*CHOH species, while suppressing their recombination and fragmentation reactions, thereby increasing the yields of the respective oxidation products. Vitamins B1 and B2 are capable of reducing alcohol radicals to the respective initial molecules, decreasing the yields of the radical transformation products.

Inhibition of peroxidase-catalyzed oxidation of 3,3 ,5,5 -tetramethylbenzidine by aminophenols.

Peroxidase-catalyzed oxidation of 3,3 ,5,5 -tetramethylbenzidine (TMB) was inhibited by o-aminophenol (AP), 2-amino-4-tert-butylphenol (ATBP), 2-amino-4,6-di-tert-butylphenol (ADTBP), and 4-tert-butylpyrocatechol (TBP). Inhibitors were characterized by inhibition constant K(i) and stoichiometric coefficient f, the number of radicals terminated by one inhibitor molecule. The most efficient inhibitor is ADTBP characterized by K(i) = 36 microM in 0.015 M phosphate citrate buffer, pH 6.0, at 20 degrees C. According to their antiradical efficiency, the studied inhibitors can be arranged as follows: ADTBP > ATBP > AP > TBP. The role of the NH(2) group in the inhibitory capacity of aminophenols is discussed. Using gas-liquid chromatography, kinetics of consumption of the initial components and accumulation of the reaction products on peroxidase-catalyzed oxidation of the TMB-TBP pair was studied; the data clarify the stages of a complex process of co-oxidation of amines and phenols.

Reactions of arylamine and aminophenol derivatives, and riboflavin with organic radicals.

Based on product yield data on radiolysis of hexane, ethanol and 3 M aqueous ethylene glycol solutions, the ability of a number of arylamine, aminophenol and quinonimine derivatives to affect processes involving peroxyl, alkyl or alpha-hydroxyalkyl radicals was assessed. It has been shown that the introduction of a hydroxyl group into aromatic amine structure enhances its antioxidant performance and makes it significantly more reactive with respect to carbon-centered organic radicals. Replacement of the hydrogen atom of a hydroxyl group by a methyl group decreases the anti-radical activity of aminophenols drastically. Compounds containing (or capable of forming) a quinonimine moiety interact with alkyl or alpha-hydroxyalkyl radicals most effectively, suppressing recombination and fragmentation reactions of the latter. In the sequence: aromatic amines--aminophenols--quinonimines, a trend towards enhancement of the ability of the compounds studied to react with carbon-centered radicals was noted. Also, this study presents for the first time evidence of riboflavin reactivity with respect to organic radicals.

[Inhibition of the peroxidase-catalyzed oxidation of chromogenic substrates by alkyl-substituted diphenols]. / Ingibirovanie peroksidaznogo okisleniia khromogennykh substratov alkilzameshchennymi difenolami.

A comparative kinetic study of the peroxidase oxidation of three chromogenic substrates--2,2'-azino-bis(3-ethyl-2,3-dihydrobenzothiazoline-6-sulfonic acid), o-phenylenediamine (PDA), and 3,3',5,5'-tetramethylbenzidine--inhibited by trimethylhydroquinone and six tert-butylated pyrocatechols (InH) was carried out at 20 degrees C in 0.015 M phosphate-citrate buffer (pH 6.0) containing organic cosolvents (0-10% ethanol or DMF). The inhibitors were quantitatively characterized by the inhibition constants (Ki), the duration of the lag period in the oxidation product formation (delta tau), and the stoichiometric coefficient of inhibition that specifies the number of radicals terminated by one InH molecule (f). The inhibition could be competitive, noncompetitive, mixed, or uncompetitive, which depended on the nature and structure of the chromogenic substrate-diatomic phenol pair. Various substrate-diatomic phenol pairs exhibited Ki values within the range of 11-240 microM and f values from 0.7 to 2.6. The absence of a lag period was characteristic of oxidation of the substituted o-phenylenediamine-substituted pyrocatechol. The total kinetic parameters and properties of the components allowed us to suggest six chromogenic substrate-substituted diatomic phenol pairs for use in test systems for the determination of antioxidant activity in human body fluids, natural biological preparations, and food. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

Radiation-induced fragmentation of cardiolipin in a model membrane.

PURPOSE: To obtain evidence for the possibility of free-radical fragmentation of cardiolipin under the action of ionizing radiation as measured by its aqueous dispersion from liposomes. MATERIALS AND METHODS: Liposomes of tetramyristoylcardiolipin (TMCL) were exposed to gamma-rays from 60Co or 137Cs sources at doses between 1 and 24kGy. Fragmentation products were identified using thin-layer chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Using MALDI-TOF MS and thin-layer chromatography, it was shown that gamma-irradiation of liposomes consisting of TMCL was accompanied by free-radical fragmentation of the lipid to form dimiristoylphosphatidic acid and dimiristoylphosphatidyl hydroxyacetone. The yields of dimiristoylphosphatidic acid were greater than those of dimiristoylphosphatidyl hydroxyacetone, and formation of the named compounds was inhibited by dissolved oxygen. CONCLUSION: It is shown for the first time that on gamma-irradiation, cardiolipin can undergo free-radical fragmentation in its polar component.

[Effect of phosphatidic acid on the radiation-initiated peroxidation of phosphatidylcholine in liposomes]. / Vliianie fosfatidnoi kisloty na radiatsionno-initsiirovannoe perekisnoe okislenie fosfatidilkholina v liposomakh.

It was found that the incorporation of anionic dipalmitoyl phosphatidic acid (DPPA) into phosphatidylcholine (PC) liposomes up to 15 mol % was accompanied by the intensification of accumulation of diene conjugates (DC), which are primary products of lipid peroxidation (LPO), if the LPO was initiated by gamma-irradiation of a 137Cs source. Monoethyl ester of DPPA, phosphatidylethanol (DPPEt), exerted a lesser influence at the same concentrations. Ca2+ ions inhibited the DC production not only in liposomes consisting of lipid mixture but in lipid membranes of PC alone as well. It was assumed that the electrostatic repulsion of negatively charged DPPA and DPPEt resulted in the loosening of polarside region of membrane hydrophobic layer and in consequence the access of hydroxyl radicals to hydrocarbon chains of PC. This assumption is in good agreement with the results of osmotic behavior of liposomes in hypertonic urea solution.

[Inhibition of peroxidase oxidation of aromatic amines by substituted phenols]. / Ingibirovanie peroksidaznogo okisleniia aromaticheskikh aminov zameshchennymi fenolami.

Peroxidase-catalyzed oxidation of o-phenylene diamine (OPD) was competitively inhibited by trimethylhydroquinone (TMHQ), 4-tert-butylpyrocatechol (In5), and 4,6-di-tert-butyl-3-sulfanyl-1,2-dihydroxybenzene (In6). In6 was the most efficient inhibitor (Ki = 11 microM at 20 degrees C in 0.015 M phosphate-citrate buffer, pH 6.0). The effects of In5 and In6 were not preceded by periods of induction of OPD oxidation products (contrary to TMHQ). Peroxidase-catalyzed oxidation of tetramethylbenzidine (TMB) was non-competitively inhibited by In6 and 3-(2-hydroxyethylthio)-4,6-di-tert-butylpyrocatechol (In4), whereas o-aminophenol (OAP) acted as a mixed-type inhibitor. The effects of all three inhibitors were preceded by an induction period, during which TMB oxidation products were formed. Again, In6 was the most efficient inhibitor (Ki = 16 microM at 20 degrees C in 0.015 M phosphate-citrate buffer supplemented with 5% ethanol, pH 6.0). Judging by the characteristics of the inhibitors, taken in aggregate, it is advisable to use the pairs OPD-In5 and OPD-In6 in systems for testing the total antioxidant activity of biological fluids of humans.

C-N bond cleavage reactions on the radiolysis of amino-containing organic compounds and their derivatives in aqueous solutions.

PURPOSE: To investigate regularities observed in the course of reactions involving cleavage of C-N bonds on the radiolysis of amino-containing organic compounds and their derivatives in aqueous solutions. MATERIALS AND METHODS: Aqueous solutions of amino-containing compounds, saturated with Ar or O(2), were exposed to gamma-radiation from a (137)Cs source. The absorbed dose-rate was 0.33 Gy s(-1), the absorbed dose range was 0.1-7.5 kGy. Analyses for ammonia and amino-containing organic compounds were performed using an amino acid analyser, and analyses for other radiolysis products were made by gas-liquid chromatography. RESULTS: The presence of a hydroxyl group in the beta-position to the amino group promotes deamination of amino alcohols, amino acids, di- and tripeptides, these processes being associated with the possible occurrence of monomolecular fragmentation of the respective radicals. Oxygen inhibits the deamination of alpha,beta-amino alcohols and hydroxyl-containing amino acids. The presence of alkyl substituents in the amino group favours C-N bond cleavage on the radiolysis of amino alcohols, whereas the presence of an acyl group sharply suppresses this process. Replacement of the hydroxyl group in molecules of the initial amino alcohols by a hydrogen atom (i.e. a changeover to alkylamines) or by a methoxy group decreases the efficiency of deamination sharply. The presence of hydroxyl groups in side residues of di- and tripeptides promotes destruction of the peptide chain with formation of amides of amino acids, while suppression of deamination and main chain destruction processes in these compounds is observed in the presence of oxygen. CONCLUSIONS: The most effective processes leading to the C-N bond cleavage on the radiolysis of aqueous solutions of amino-containing organic compounds are those in which the key role is played by reactions of monomolecular decomposition of radicals of the initial compounds.

Effects of phenolic compounds on reactions involving various organic radicals.

Investigation of effects produced by 26 various phenol and diphenol derivatives, including industrial and natural antioxidants (ionol, bis-phenol 2246, alpha-tocopherol), on final product yields of radiation-induced free-radical processes involving peroxyl, alkyl, alpha-hydroxyalkyl and alpha,beta-dihydroxyalkyl radicals has been performed. Ionol and bis-phenol 2246 have been shown to be more effective than alpha-tocopherol or diphenol derivatives in suppressing hydrocarbon oxidation processes. At the same time, alpha-tocopherol and its water-soluble analogues, as well as diphenol-based substances, are more effective than phenol derivatives in regulating various homolytic processes involving carbon-centered radicals. This fact can be accounted for by taking into consideration the contribution to formation of the final product set and the respective yields made by semiquinone radicals and compounds with quinoid structure arising in the course of homolytic transformations in systems containing diphenol derivatives.