Oxidative damage is a common cellular event involved in numerous diseases and drug toxicities. An... more Oxidative damage is a common cellular event involved in numerous diseases and drug toxicities. Antioxidants prevent or delay oxidative damage, and therefore there has been extensive research into the discovery of natural and newly designed antioxidants. Initial excitement regarding the potential health benefits of antioxidants has diminished. Currently, it is even claimed that antioxidants increase mortality. The antioxidant pendulum appears to swing from healthy to toxic and from general panacea to insignificant ingredient. Owing to the polarity of views towards antioxidants, nutritional recommendation ranges from advice to increase antioxidant status in plasma to the notion that it is a useless measurement. Such views, lacking sufficient scientific support, lead to misconceptions, which in our opinion hinder the rational use of food supplements and impedes the design and development of new antioxidant drugs. As a result, good opportunities might easily be missed.
Short communications 7. Distlerath LM and Guengerich FP, Characterization 14. of a human liver cy... more Short communications 7. Distlerath LM and Guengerich FP, Characterization 14. of a human liver cytochrome P-450 involved in the oxidation of debrisoquine and other drugs by using antibodies raised to the analogous rat enzyme. Proc 15. Nad Acad Sci USA 81: 7348-7352, 1984. _ 8. Anthonv L. Koshakuii R and Wood AJJ. Multiole pathways oi propranofol's metabolism are inhibited-by debrisoquin. Clin Pharmacol Ther 46: 297-300, 1989. 16. 9. Boobis AR, Murray S, Hampden CE and Davis DS, Genetic polymorphism in drug oxidation: in vitro studies of human debrisoquine 4-hydroxylase and bufuralol 1 '-hydroxylase activities. Biochem Pharmacol 34: 65-71, 1985. 10. Matsunaga E, Zanger UM, Hardwick JP, Gelboin HV, Meyer US and Gonzalez FJ, The CYP2D gene 17. subfamily: analysis of the molecular basis of the debrisoquine 4-hydroxylase deficiency in DA rats. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275, 1951. Al-Dabbagh SG, Idle JR and Smith RL, Animal modelling of human polymorphic drug oxidationmetabolism of debrisoquine and phenacetin in rat inbred strains. J Pharm Pharmacol33: 161-164, 1981. Gonzalez FJ, Matsunaga T, Nagata K, Meyer US, Nebert DW. Pastewka J. Kozak CG. Gillette J. Gelboin HV and Hgrdwick JP,' Debrisoqiine 4-hydkoxylase: characterization of a new P450 gene subfamily, regulation, chromosomal mapping, and molecular analysis of the DA rat polymorphism. DNA 6: 149-161, 1987. Kahn GC, Rubenfield M, Davies DS, Murray S and Boobis AR, Sex and strain differences in hepatic debrisoquine 4-hydroxylase activity of the rat. Drug Metab Dispos 13: 510-515, 1985.
It has been reported that¯avonoids eciently protect against peroxynitrite toxicity. Two pharmacop... more It has been reported that¯avonoids eciently protect against peroxynitrite toxicity. Two pharmacophores have been identi®ed in avonoids, namely the catechol group in ring B and the hydroxyl (OH) group at the 3-position. In this study, this structure±activity relationship was further examined. It was found that catechol (1,2-dihydroxybenzene) is a potent peroxynitrite scavenger, whereas phenol (hydroxybenzene) is not. Of the¯avonols tested without a catechol group in ring B, kaempferol (OH groups at positions 3,5,7,4 H ) and galangin (OH groups at positions 3,5,7) are also potent scavengers, whereas apigenin (OH groups at positions 5,7,4 H ) and chrysin (OH groups at positions 5,7) are not. This con®rms the importance of the OH group at the 3-position. However, the synthetic¯avonol TUM 9761 and 3-hydroxy¯avone (OH group only at position 3) are poor scavengers. Based on these results, the structure±activity relationship on the peroxynitrite scavenging activity of¯avonols was re®ned. The catechol in ring B remains important. Also the 3-OH group remains important, but the activity of this pharmacophore is in¯uenced by the substituents at position 5 and at position 7. #
Biochimica Et Biophysica Acta (bba) - Lipids and Lipid Metabolism, 1988
, which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. B... more , which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. Biophys. 259,449-456). Lipoic acid exerts its therapeutic effect in pathologies in which free radicals are involved. We investigated the interplay between lipoic acid and glutathione in microsomal Fe'+ (10 pM)/ascorbate (0.2 mM)-indu~ lipid ~roxidation. Neither reduced nor oxidized lipoic acid (0.5 mM) displayed protection against microsomal lipid peroxidation, measnred as thiobarbituric acid-reactive material. Reduced lipoic acid even bad a pro-oxidant activity, which is probably due to reduction of Fe 3+. Notably, protection against lipid peroxidation was afforded by the combination of oxidized glutathione (GSSG) and reduced lipoic acid. It is shown that this effect can be ascribed completely to reduction of GSSG to GSH by reduced lipoic acid. This may provide a rationale for the therapeutic effectiveness of lipoit acid. ~5-2760/88/$03.50 Q 1988 Elsevier Science Pub&hers B.V. (Biomedic~ Division)
The effect of antioxidants is often executed in complex biological mixtures where various interac... more The effect of antioxidants is often executed in complex biological mixtures where various interactions may take place. Therefore, the antioxidant capacity of antioxidants in blood plasma is examined. The assay used is the trolox equivalent antioxidant capacity (TEAC). This method gives the antioxidant capacity of a compound by measuring spectrophotometrically the disappearance of the blue/green stable ABTS [2,2 0 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical, caused by scavenging. The results show that the antioxidant capacity of quercetin, rutin, catechin or 7-monohydroxyethylrutoside (monoHER) and blood plasma is not additive. This is partly due to interactions between the antioxidant and plasma proteins. However, the antioxidant capacity of a-tocopherol, which also binds to protein, is not affected by the interaction. This means that besides the antioxidant capacity of the compound itself, the environment in which the antioxidant has to execute his function is important. #
Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 2001
K-Tocopherol inhibits glutathione S-transferase P1-1 (GST P1-1) (R.I.M. van Haaften, C.T.A. Evelo... more K-Tocopherol inhibits glutathione S-transferase P1-1 (GST P1-1) (R.I.M. van Haaften, C.T.A. Evelo, G.R.M.M. Haenen, A. Bast, Biochem. Biophys. Res. Commun. 280 (2001)). In various cosmetic and dietary products K-tocopherol is added as a tocopherol ester. Therefore we have studied the effect of various tocopherol derivatives on GST P1-1 activity. It was found that GST P1-1 is inhibited, in a concentration dependent manner, by these compounds. Of the compounds tested, the tocopherols were the most potent inhibitors of GST P1-1; the concentration giving 50% inhibition (IC 50 ) is 6 1 WM. The esterified tocopherols and K-tocopherol quinone also inhibit the GST P1-1 activity at a very low concentration: for most compounds the IC 50 was below 10 WM. RRR-K-Tocopherol acetate lowered the V max values, but did not affect the K m for either 1-chloro-2,4-dinitrobenzene or GSH. This indicates that the GST P1-1 enzyme is non-competitively inhibited by RRR-K-tocopherol acetate. The potential implications of GST P1-1 inhibition by tocopherol and K-tocopherol derivatives are discussed. ß
Glutathione (GSH) protects rat liver microsomes against ascorbic acid (0.2 mM)/ferrous iron (10 p... more Glutathione (GSH) protects rat liver microsomes against ascorbic acid (0.2 mM)/ferrous iron (10 pM)induced lipid peroxidation for some time. The inhibitory effect of GSH is concentration-dependent (0.1-1.0 mM). Our data suggest that GSH acts by preventing initial radical formation rather than via radical scavenging or GSH-peroxidase activity. A labile GSH-dependent factor is involved in the inhibition of microsomal lipid peroxidation by GSH, inasmuch as heating the microsomes abolishes the GSH effect. We found that besides heating, lipid peroxidation also destroys the GSH-dependent factor. Consequently, continuous radical stress will produce lipid peroxidation, despite the presence of GSH. Moreover, a detrimental effect of in vivo-induced lipid peroxidation (CCL-treatment) on the GSHdependent factor was observed. The implications of the present data for the genesis of and the protection against peroxidative damage are discussed.
We studied the effects of frozen storage on (pro)vitamin concentrations in EDTA-plasma and whole ... more We studied the effects of frozen storage on (pro)vitamin concentrations in EDTA-plasma and whole blood. Aliquots from 55 samples were analyzed before storage and after 3,6, 12,24, 36 and 48 months at -20°C. Dramatic decreases occurred for EDTA-plasma concentrations of vitamin E between 6 and 12 months, vitamin A, total carotenoids and B-carotene after 1 year, and whole blood niacin. A smaller decrease was observed for folic acid at 1 year of storage, but the level remained constant thereafter. The vitamins D, Be, B,* (EDTA-plasma), B, and B, (whole blood) showed no decline during 4 years of storage. With the exception of folic acid, the observed decreases varied considerably among subjects. Therefore using EDTA-plasma stored longer than 1 year at -20°C will result in highly attenuated odds ratios when assessing the relationship between vitamin A, carotenoids, or vitamin E with a given disease. Attenuation will also occur when using niacin concentrations in whole blood stored for 4 years at -20°C.
The aim of the present study was to investigate acute DNA damage in epithelial lung cells from ra... more The aim of the present study was to investigate acute DNA damage in epithelial lung cells from rats exposed to quartz. Since surface reactivity is considered to play a crucial role in the toxicity of quartz, the effect of surface modifying agents polyvinylpyridine-N-oxide (PVNO) and aluminium lactate (AL) was evaluated. Therefore, rats were instilled with quartz (DQ12, 2 mg/rat) or quartz treated with PVNO or AL. After 3 days animals were killed and brochoalveolar lavage (BAL) was performed to evaluate inflammatory cell influx. BAL-fluid levels of lactate dehydrogenase (LDH), alkaline phosphatase (AP) and total protein were used as lung damage markers. Neutrophil activation was assessed by myeloperoxidase (MPO) measurement, and total antioxidant capacity of the BAL-fluid was determined using the TEAC (trolox equivalent antioxidant capacity) assay. Lung epithelial cells were isolated and DNA strand breakage was determined by single cell gel electrophoresis (comet assay). DNA damage was significantly increased in epithelial cells from rats instilled with DQ12, whereas no enhanced DNA strand breakage was observed when quartz was treated with PVNO or AL. Total protein, LDH and TEAC were increased in rats treated with native quartz, and this was inhibited by both coatings. A significant correlation between neutrophil numbers and MPO levels was observed, indicating neutrophil activation. Inhibition of DNA damage by both coatings was paralleled by a reduction of neutrophil influx as well as MPO activity. In this study we provide evidence that modification of the particle surface prevents DNA strand breakage in epithelial lung cells from quartzexposed rats. Furthermore, the present data show the feasibility of our in vivo model to evaluate the role of inflammation, antioxidant status, and cytotoxicity in particle-induced DNA damage.
The presumed protective e¡ect of folic acid on the pathogenesis of cardiovascular, hematological ... more The presumed protective e¡ect of folic acid on the pathogenesis of cardiovascular, hematological and neurological diseases and cancer has been associated with the antioxidant activity of folic acid. Peroxynitrite (PON) scavenging activity and inhibition of lipid peroxidation (LPO) of the physiological forms of folate and of structurally related compounds were tested. It was found that the fully reduced forms of folate, i.e. tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5-MTHF), had the most prominent antioxidant activity. It appeared that their protection against LPO is less pronounced than their PON scavenging activity. The antioxidant activity of these forms of folic acid resides in the pterin core, the antioxidant pharmacophore is 4-hydroxy-2,5,6-triaminopyrimidine. It is suggested that an electron donating e¡ect of the 5-amino group is of major importance for the antioxidant activity of 4-hydroxy-2,5,6-triaminopyrimidine. A similar electron donating e¡ect is probably important for the antioxidant activity of THF and 5-MTHF.
In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, i.e. the capacity of a compound to sc... more In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, i.e. the capacity of a compound to scavenge the ABTS radical (ABTS ), is assessed. The aim of the present study is to evaluate the applicability of the TEAC assay to predict the antioxidant effectivity of a compound. For this purpose the TEAC assay is compared with other screening assays, such as superoxide scavenging, peroxynitrite scavenging and lipid peroxidation. Of the structurally related compounds, catechol, resorcinol and hydroquinone, resorcinol has the highest TEAC. In contrast, resorcinol appears to have a much lower antioxidant activity than catechol and hydroquinone in other in vitro assays. Similar discrepancies were observed with the flavonoids, chrysin and galangin. The TEAC values of chrysin and galangin are comparable, whereas galangin appears to be a much better antioxidant in other assays. The relatively high TEAC values of chrysin and resorcinol are due to the ability of the reaction products, formed by the reaction of the parent compound with ABTS , to further react with ABTS . With catechol, hydroquinone and galangin, these reaction products do not react with ABTS and therefore make no contribution to the TEAC. The possible contribution of reaction products to the TEAC of a compound hampers the use of the TEAC assay for constructing structure-activity relationships (SAR). #
Racemic lipoic acid is therapeutically applied in pathologies in which free radicals are involved... more Racemic lipoic acid is therapeutically applied in pathologies in which free radicals are involved. * Corresponding author. Tel-31 20 4447583; FAX-31 20 4447610. t Abbreuiacions: DHLA, dihydrolipoic acid; DTNB, 5,5-dithiobis
Free radical scavenging antioxidants, such as quercetin, are chemically converted into oxidation ... more Free radical scavenging antioxidants, such as quercetin, are chemically converted into oxidation products when they protect against free radicals. The main oxidation product of quercetin, however, displays a high reactivity towards thiols, which can lead to the loss of protein function. The quercetin paradox is that in the process of offering protection, quercetin is converted into a potential toxic product. In the present study, this paradox is evaluated using rat lung epithelial (RLE) cells.
Peroxynitrite can oxidise and nitrosylate biomolecules and is associated with several diseases. T... more Peroxynitrite can oxidise and nitrosylate biomolecules and is associated with several diseases. The peroxynitrite scavenging of substituted phenols and several flavonoids was studied. The activity of phenol (poor scavenger) is positively influenced by electron donating substituents. A good correlation was found between the peroxynitrite scavenging activity of the substituted phenols and the Hammett | or the E HOMO . Flavonols containing a catechol group (3%-and 4%-OH) in ring B (rutin and monohydroxyethyl rutoside) or an AC-ring with three OH groups (3-, 5-and 7-OH) were potent scavengers. Evidence has been produced that in the AC-ring the 3-OH group was the reactive centre and that the reactivity of this group was positively influenced by electron donating groups at the 5 and/or 7 position (galangin, kaempferol, trihydroxyethyl quercetin).
Flavonoids are potent antioxidants. It is also known that flavonoids bind to proteins. The effect... more Flavonoids are potent antioxidants. It is also known that flavonoids bind to proteins. The effect of the interaction between tea flavonoids and proteins on the antioxidant capacity was examined. Their separate and combined antioxidant capacities were measured with the Trolox equivalent antioxidant capacity (TEAC) assay. It was observed that the antioxidant capacity of several components of green and black tea with R-, -, and κ-casein or albumin is not additive; that is, a part of the total antioxidant capacity is masked by the interaction. This masking depends on both the protein and the flavonoid used. Components in green and black tea, which show the highest masking in combination with -casein, are epigallocatechin gallate and gallic acid. The results demonstrate that the matrix influences the efficacy of an antioxidant.
The presumed protective e¡ect of folic acid on the pathogenesis of cardiovascular, hematological ... more The presumed protective e¡ect of folic acid on the pathogenesis of cardiovascular, hematological and neurological diseases and cancer has been associated with the antioxidant activity of folic acid. Peroxynitrite (PON) scavenging activity and inhibition of lipid peroxidation (LPO) of the physiological forms of folate and of structurally related compounds were tested. It was found that the fully reduced forms of folate, i.e. tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5-MTHF), had the most prominent antioxidant activity. It appeared that their protection against LPO is less pronounced than their PON scavenging activity. The antioxidant activity of these forms of folic acid resides in the pterin core, the antioxidant pharmacophore is 4-hydroxy-2,5,6-triaminopyrimidine. It is suggested that an electron donating e¡ect of the 5-amino group is of major importance for the antioxidant activity of 4-hydroxy-2,5,6-triaminopyrimidine. A similar electron donating e¡ect is probably important for the antioxidant activity of THF and 5-MTHF.
The TEAC (Trolox equivalent antioxidant capacity) assay is based on scavenging of 2,2 H -azinobis... more The TEAC (Trolox equivalent antioxidant capacity) assay is based on scavenging of 2,2 H -azinobis-(3-ethylbenzothiazoline-6sulfonate) radical anions (ABTS À ). In this report we describe a modi®cation based on pre-generation of the ABTS radical anions with a thermolabile azo compound, 2,2 H -azobis-(2-amidinopropane)HCl (ABAP). This modi®cation makes the assay less susceptible to artefacts, e.g. in¯uence on the radical generation process. For most antioxidants tested, a biphasic reaction pattern was seen, i.e. a fast and slow scavenging rate. We evaluated application of the assay with both lipophilic and hydrophilic compounds with antioxidant capacity. Several organic solvents, compatible with water, were tested with a-tocopherol, quercetin and b-carotene. It was found that the TEACs diered in various solvents. Under standardized conditions additivity of TEACs obtained from individual antioxidants could be demonstrated. This might enable application of the assay for the identi®cation of``unknown'' antioxidants. #
In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, i.e. the capacity of a compound to sc... more In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, i.e. the capacity of a compound to scavenge the ABTS radical (ABTS ), is assessed. The aim of the present study is to evaluate the applicability of the TEAC assay to predict the antioxidant effectivity of a compound. For this purpose the TEAC assay is compared with other screening assays, such as superoxide scavenging, peroxynitrite scavenging and lipid peroxidation. Of the structurally related compounds, catechol, resorcinol and hydroquinone, resorcinol has the highest TEAC. In contrast, resorcinol appears to have a much lower antioxidant activity than catechol and hydroquinone in other in vitro assays. Similar discrepancies were observed with the flavonoids, chrysin and galangin. The TEAC values of chrysin and galangin are comparable, whereas galangin appears to be a much better antioxidant in other assays. The relatively high TEAC values of chrysin and resorcinol are due to the ability of the reaction products, formed by the reaction of the parent compound with ABTS , to further react with ABTS . With catechol, hydroquinone and galangin, these reaction products do not react with ABTS and therefore make no contribution to the TEAC. The possible contribution of reaction products to the TEAC of a compound hampers the use of the TEAC assay for constructing structure-activity relationships (SAR). #
Oxidative damage is a common cellular event involved in numerous diseases and drug toxicities. An... more Oxidative damage is a common cellular event involved in numerous diseases and drug toxicities. Antioxidants prevent or delay oxidative damage, and therefore there has been extensive research into the discovery of natural and newly designed antioxidants. Initial excitement regarding the potential health benefits of antioxidants has diminished. Currently, it is even claimed that antioxidants increase mortality. The antioxidant pendulum appears to swing from healthy to toxic and from general panacea to insignificant ingredient. Owing to the polarity of views towards antioxidants, nutritional recommendation ranges from advice to increase antioxidant status in plasma to the notion that it is a useless measurement. Such views, lacking sufficient scientific support, lead to misconceptions, which in our opinion hinder the rational use of food supplements and impedes the design and development of new antioxidant drugs. As a result, good opportunities might easily be missed.
Short communications 7. Distlerath LM and Guengerich FP, Characterization 14. of a human liver cy... more Short communications 7. Distlerath LM and Guengerich FP, Characterization 14. of a human liver cytochrome P-450 involved in the oxidation of debrisoquine and other drugs by using antibodies raised to the analogous rat enzyme. Proc 15. Nad Acad Sci USA 81: 7348-7352, 1984. _ 8. Anthonv L. Koshakuii R and Wood AJJ. Multiole pathways oi propranofol's metabolism are inhibited-by debrisoquin. Clin Pharmacol Ther 46: 297-300, 1989. 16. 9. Boobis AR, Murray S, Hampden CE and Davis DS, Genetic polymorphism in drug oxidation: in vitro studies of human debrisoquine 4-hydroxylase and bufuralol 1 '-hydroxylase activities. Biochem Pharmacol 34: 65-71, 1985. 10. Matsunaga E, Zanger UM, Hardwick JP, Gelboin HV, Meyer US and Gonzalez FJ, The CYP2D gene 17. subfamily: analysis of the molecular basis of the debrisoquine 4-hydroxylase deficiency in DA rats. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275, 1951. Al-Dabbagh SG, Idle JR and Smith RL, Animal modelling of human polymorphic drug oxidationmetabolism of debrisoquine and phenacetin in rat inbred strains. J Pharm Pharmacol33: 161-164, 1981. Gonzalez FJ, Matsunaga T, Nagata K, Meyer US, Nebert DW. Pastewka J. Kozak CG. Gillette J. Gelboin HV and Hgrdwick JP,' Debrisoqiine 4-hydkoxylase: characterization of a new P450 gene subfamily, regulation, chromosomal mapping, and molecular analysis of the DA rat polymorphism. DNA 6: 149-161, 1987. Kahn GC, Rubenfield M, Davies DS, Murray S and Boobis AR, Sex and strain differences in hepatic debrisoquine 4-hydroxylase activity of the rat. Drug Metab Dispos 13: 510-515, 1985.
It has been reported that¯avonoids eciently protect against peroxynitrite toxicity. Two pharmacop... more It has been reported that¯avonoids eciently protect against peroxynitrite toxicity. Two pharmacophores have been identi®ed in avonoids, namely the catechol group in ring B and the hydroxyl (OH) group at the 3-position. In this study, this structure±activity relationship was further examined. It was found that catechol (1,2-dihydroxybenzene) is a potent peroxynitrite scavenger, whereas phenol (hydroxybenzene) is not. Of the¯avonols tested without a catechol group in ring B, kaempferol (OH groups at positions 3,5,7,4 H ) and galangin (OH groups at positions 3,5,7) are also potent scavengers, whereas apigenin (OH groups at positions 5,7,4 H ) and chrysin (OH groups at positions 5,7) are not. This con®rms the importance of the OH group at the 3-position. However, the synthetic¯avonol TUM 9761 and 3-hydroxy¯avone (OH group only at position 3) are poor scavengers. Based on these results, the structure±activity relationship on the peroxynitrite scavenging activity of¯avonols was re®ned. The catechol in ring B remains important. Also the 3-OH group remains important, but the activity of this pharmacophore is in¯uenced by the substituents at position 5 and at position 7. #
Biochimica Et Biophysica Acta (bba) - Lipids and Lipid Metabolism, 1988
, which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. B... more , which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. Biophys. 259,449-456). Lipoic acid exerts its therapeutic effect in pathologies in which free radicals are involved. We investigated the interplay between lipoic acid and glutathione in microsomal Fe'+ (10 pM)/ascorbate (0.2 mM)-indu~ lipid ~roxidation. Neither reduced nor oxidized lipoic acid (0.5 mM) displayed protection against microsomal lipid peroxidation, measnred as thiobarbituric acid-reactive material. Reduced lipoic acid even bad a pro-oxidant activity, which is probably due to reduction of Fe 3+. Notably, protection against lipid peroxidation was afforded by the combination of oxidized glutathione (GSSG) and reduced lipoic acid. It is shown that this effect can be ascribed completely to reduction of GSSG to GSH by reduced lipoic acid. This may provide a rationale for the therapeutic effectiveness of lipoit acid. ~5-2760/88/$03.50 Q 1988 Elsevier Science Pub&hers B.V. (Biomedic~ Division)
The effect of antioxidants is often executed in complex biological mixtures where various interac... more The effect of antioxidants is often executed in complex biological mixtures where various interactions may take place. Therefore, the antioxidant capacity of antioxidants in blood plasma is examined. The assay used is the trolox equivalent antioxidant capacity (TEAC). This method gives the antioxidant capacity of a compound by measuring spectrophotometrically the disappearance of the blue/green stable ABTS [2,2 0 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical, caused by scavenging. The results show that the antioxidant capacity of quercetin, rutin, catechin or 7-monohydroxyethylrutoside (monoHER) and blood plasma is not additive. This is partly due to interactions between the antioxidant and plasma proteins. However, the antioxidant capacity of a-tocopherol, which also binds to protein, is not affected by the interaction. This means that besides the antioxidant capacity of the compound itself, the environment in which the antioxidant has to execute his function is important. #
Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 2001
K-Tocopherol inhibits glutathione S-transferase P1-1 (GST P1-1) (R.I.M. van Haaften, C.T.A. Evelo... more K-Tocopherol inhibits glutathione S-transferase P1-1 (GST P1-1) (R.I.M. van Haaften, C.T.A. Evelo, G.R.M.M. Haenen, A. Bast, Biochem. Biophys. Res. Commun. 280 (2001)). In various cosmetic and dietary products K-tocopherol is added as a tocopherol ester. Therefore we have studied the effect of various tocopherol derivatives on GST P1-1 activity. It was found that GST P1-1 is inhibited, in a concentration dependent manner, by these compounds. Of the compounds tested, the tocopherols were the most potent inhibitors of GST P1-1; the concentration giving 50% inhibition (IC 50 ) is 6 1 WM. The esterified tocopherols and K-tocopherol quinone also inhibit the GST P1-1 activity at a very low concentration: for most compounds the IC 50 was below 10 WM. RRR-K-Tocopherol acetate lowered the V max values, but did not affect the K m for either 1-chloro-2,4-dinitrobenzene or GSH. This indicates that the GST P1-1 enzyme is non-competitively inhibited by RRR-K-tocopherol acetate. The potential implications of GST P1-1 inhibition by tocopherol and K-tocopherol derivatives are discussed. ß
Glutathione (GSH) protects rat liver microsomes against ascorbic acid (0.2 mM)/ferrous iron (10 p... more Glutathione (GSH) protects rat liver microsomes against ascorbic acid (0.2 mM)/ferrous iron (10 pM)induced lipid peroxidation for some time. The inhibitory effect of GSH is concentration-dependent (0.1-1.0 mM). Our data suggest that GSH acts by preventing initial radical formation rather than via radical scavenging or GSH-peroxidase activity. A labile GSH-dependent factor is involved in the inhibition of microsomal lipid peroxidation by GSH, inasmuch as heating the microsomes abolishes the GSH effect. We found that besides heating, lipid peroxidation also destroys the GSH-dependent factor. Consequently, continuous radical stress will produce lipid peroxidation, despite the presence of GSH. Moreover, a detrimental effect of in vivo-induced lipid peroxidation (CCL-treatment) on the GSHdependent factor was observed. The implications of the present data for the genesis of and the protection against peroxidative damage are discussed.
We studied the effects of frozen storage on (pro)vitamin concentrations in EDTA-plasma and whole ... more We studied the effects of frozen storage on (pro)vitamin concentrations in EDTA-plasma and whole blood. Aliquots from 55 samples were analyzed before storage and after 3,6, 12,24, 36 and 48 months at -20°C. Dramatic decreases occurred for EDTA-plasma concentrations of vitamin E between 6 and 12 months, vitamin A, total carotenoids and B-carotene after 1 year, and whole blood niacin. A smaller decrease was observed for folic acid at 1 year of storage, but the level remained constant thereafter. The vitamins D, Be, B,* (EDTA-plasma), B, and B, (whole blood) showed no decline during 4 years of storage. With the exception of folic acid, the observed decreases varied considerably among subjects. Therefore using EDTA-plasma stored longer than 1 year at -20°C will result in highly attenuated odds ratios when assessing the relationship between vitamin A, carotenoids, or vitamin E with a given disease. Attenuation will also occur when using niacin concentrations in whole blood stored for 4 years at -20°C.
The aim of the present study was to investigate acute DNA damage in epithelial lung cells from ra... more The aim of the present study was to investigate acute DNA damage in epithelial lung cells from rats exposed to quartz. Since surface reactivity is considered to play a crucial role in the toxicity of quartz, the effect of surface modifying agents polyvinylpyridine-N-oxide (PVNO) and aluminium lactate (AL) was evaluated. Therefore, rats were instilled with quartz (DQ12, 2 mg/rat) or quartz treated with PVNO or AL. After 3 days animals were killed and brochoalveolar lavage (BAL) was performed to evaluate inflammatory cell influx. BAL-fluid levels of lactate dehydrogenase (LDH), alkaline phosphatase (AP) and total protein were used as lung damage markers. Neutrophil activation was assessed by myeloperoxidase (MPO) measurement, and total antioxidant capacity of the BAL-fluid was determined using the TEAC (trolox equivalent antioxidant capacity) assay. Lung epithelial cells were isolated and DNA strand breakage was determined by single cell gel electrophoresis (comet assay). DNA damage was significantly increased in epithelial cells from rats instilled with DQ12, whereas no enhanced DNA strand breakage was observed when quartz was treated with PVNO or AL. Total protein, LDH and TEAC were increased in rats treated with native quartz, and this was inhibited by both coatings. A significant correlation between neutrophil numbers and MPO levels was observed, indicating neutrophil activation. Inhibition of DNA damage by both coatings was paralleled by a reduction of neutrophil influx as well as MPO activity. In this study we provide evidence that modification of the particle surface prevents DNA strand breakage in epithelial lung cells from quartzexposed rats. Furthermore, the present data show the feasibility of our in vivo model to evaluate the role of inflammation, antioxidant status, and cytotoxicity in particle-induced DNA damage.
The presumed protective e¡ect of folic acid on the pathogenesis of cardiovascular, hematological ... more The presumed protective e¡ect of folic acid on the pathogenesis of cardiovascular, hematological and neurological diseases and cancer has been associated with the antioxidant activity of folic acid. Peroxynitrite (PON) scavenging activity and inhibition of lipid peroxidation (LPO) of the physiological forms of folate and of structurally related compounds were tested. It was found that the fully reduced forms of folate, i.e. tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5-MTHF), had the most prominent antioxidant activity. It appeared that their protection against LPO is less pronounced than their PON scavenging activity. The antioxidant activity of these forms of folic acid resides in the pterin core, the antioxidant pharmacophore is 4-hydroxy-2,5,6-triaminopyrimidine. It is suggested that an electron donating e¡ect of the 5-amino group is of major importance for the antioxidant activity of 4-hydroxy-2,5,6-triaminopyrimidine. A similar electron donating e¡ect is probably important for the antioxidant activity of THF and 5-MTHF.
In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, i.e. the capacity of a compound to sc... more In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, i.e. the capacity of a compound to scavenge the ABTS radical (ABTS ), is assessed. The aim of the present study is to evaluate the applicability of the TEAC assay to predict the antioxidant effectivity of a compound. For this purpose the TEAC assay is compared with other screening assays, such as superoxide scavenging, peroxynitrite scavenging and lipid peroxidation. Of the structurally related compounds, catechol, resorcinol and hydroquinone, resorcinol has the highest TEAC. In contrast, resorcinol appears to have a much lower antioxidant activity than catechol and hydroquinone in other in vitro assays. Similar discrepancies were observed with the flavonoids, chrysin and galangin. The TEAC values of chrysin and galangin are comparable, whereas galangin appears to be a much better antioxidant in other assays. The relatively high TEAC values of chrysin and resorcinol are due to the ability of the reaction products, formed by the reaction of the parent compound with ABTS , to further react with ABTS . With catechol, hydroquinone and galangin, these reaction products do not react with ABTS and therefore make no contribution to the TEAC. The possible contribution of reaction products to the TEAC of a compound hampers the use of the TEAC assay for constructing structure-activity relationships (SAR). #
Racemic lipoic acid is therapeutically applied in pathologies in which free radicals are involved... more Racemic lipoic acid is therapeutically applied in pathologies in which free radicals are involved. * Corresponding author. Tel-31 20 4447583; FAX-31 20 4447610. t Abbreuiacions: DHLA, dihydrolipoic acid; DTNB, 5,5-dithiobis
Free radical scavenging antioxidants, such as quercetin, are chemically converted into oxidation ... more Free radical scavenging antioxidants, such as quercetin, are chemically converted into oxidation products when they protect against free radicals. The main oxidation product of quercetin, however, displays a high reactivity towards thiols, which can lead to the loss of protein function. The quercetin paradox is that in the process of offering protection, quercetin is converted into a potential toxic product. In the present study, this paradox is evaluated using rat lung epithelial (RLE) cells.
Peroxynitrite can oxidise and nitrosylate biomolecules and is associated with several diseases. T... more Peroxynitrite can oxidise and nitrosylate biomolecules and is associated with several diseases. The peroxynitrite scavenging of substituted phenols and several flavonoids was studied. The activity of phenol (poor scavenger) is positively influenced by electron donating substituents. A good correlation was found between the peroxynitrite scavenging activity of the substituted phenols and the Hammett | or the E HOMO . Flavonols containing a catechol group (3%-and 4%-OH) in ring B (rutin and monohydroxyethyl rutoside) or an AC-ring with three OH groups (3-, 5-and 7-OH) were potent scavengers. Evidence has been produced that in the AC-ring the 3-OH group was the reactive centre and that the reactivity of this group was positively influenced by electron donating groups at the 5 and/or 7 position (galangin, kaempferol, trihydroxyethyl quercetin).
Flavonoids are potent antioxidants. It is also known that flavonoids bind to proteins. The effect... more Flavonoids are potent antioxidants. It is also known that flavonoids bind to proteins. The effect of the interaction between tea flavonoids and proteins on the antioxidant capacity was examined. Their separate and combined antioxidant capacities were measured with the Trolox equivalent antioxidant capacity (TEAC) assay. It was observed that the antioxidant capacity of several components of green and black tea with R-, -, and κ-casein or albumin is not additive; that is, a part of the total antioxidant capacity is masked by the interaction. This masking depends on both the protein and the flavonoid used. Components in green and black tea, which show the highest masking in combination with -casein, are epigallocatechin gallate and gallic acid. The results demonstrate that the matrix influences the efficacy of an antioxidant.
The presumed protective e¡ect of folic acid on the pathogenesis of cardiovascular, hematological ... more The presumed protective e¡ect of folic acid on the pathogenesis of cardiovascular, hematological and neurological diseases and cancer has been associated with the antioxidant activity of folic acid. Peroxynitrite (PON) scavenging activity and inhibition of lipid peroxidation (LPO) of the physiological forms of folate and of structurally related compounds were tested. It was found that the fully reduced forms of folate, i.e. tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5-MTHF), had the most prominent antioxidant activity. It appeared that their protection against LPO is less pronounced than their PON scavenging activity. The antioxidant activity of these forms of folic acid resides in the pterin core, the antioxidant pharmacophore is 4-hydroxy-2,5,6-triaminopyrimidine. It is suggested that an electron donating e¡ect of the 5-amino group is of major importance for the antioxidant activity of 4-hydroxy-2,5,6-triaminopyrimidine. A similar electron donating e¡ect is probably important for the antioxidant activity of THF and 5-MTHF.
The TEAC (Trolox equivalent antioxidant capacity) assay is based on scavenging of 2,2 H -azinobis... more The TEAC (Trolox equivalent antioxidant capacity) assay is based on scavenging of 2,2 H -azinobis-(3-ethylbenzothiazoline-6sulfonate) radical anions (ABTS À ). In this report we describe a modi®cation based on pre-generation of the ABTS radical anions with a thermolabile azo compound, 2,2 H -azobis-(2-amidinopropane)HCl (ABAP). This modi®cation makes the assay less susceptible to artefacts, e.g. in¯uence on the radical generation process. For most antioxidants tested, a biphasic reaction pattern was seen, i.e. a fast and slow scavenging rate. We evaluated application of the assay with both lipophilic and hydrophilic compounds with antioxidant capacity. Several organic solvents, compatible with water, were tested with a-tocopherol, quercetin and b-carotene. It was found that the TEACs diered in various solvents. Under standardized conditions additivity of TEACs obtained from individual antioxidants could be demonstrated. This might enable application of the assay for the identi®cation of``unknown'' antioxidants. #
In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, i.e. the capacity of a compound to sc... more In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, i.e. the capacity of a compound to scavenge the ABTS radical (ABTS ), is assessed. The aim of the present study is to evaluate the applicability of the TEAC assay to predict the antioxidant effectivity of a compound. For this purpose the TEAC assay is compared with other screening assays, such as superoxide scavenging, peroxynitrite scavenging and lipid peroxidation. Of the structurally related compounds, catechol, resorcinol and hydroquinone, resorcinol has the highest TEAC. In contrast, resorcinol appears to have a much lower antioxidant activity than catechol and hydroquinone in other in vitro assays. Similar discrepancies were observed with the flavonoids, chrysin and galangin. The TEAC values of chrysin and galangin are comparable, whereas galangin appears to be a much better antioxidant in other assays. The relatively high TEAC values of chrysin and resorcinol are due to the ability of the reaction products, formed by the reaction of the parent compound with ABTS , to further react with ABTS . With catechol, hydroquinone and galangin, these reaction products do not react with ABTS and therefore make no contribution to the TEAC. The possible contribution of reaction products to the TEAC of a compound hampers the use of the TEAC assay for constructing structure-activity relationships (SAR). #
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