Free radicals of acetaminophen: their subsequent reactions and toxicological significance

The oxidation of acetaminophen to the corresponding phenoxyl free radical and N-acetyl-p-benzoquinone imine by mammalian peroxidases is discussed. The acetaminophen free radical is very reactive--forming dimers, and, ultimately, melanin-like polymeric products. A model compound, leading to more stable metabolites, can be obtained by introduction of methyl groups next to the oxygen, to produce 3,5-dimethylacetaminophen. The electron spin resonance spectrum of this free radical could be completely analyzed. The phenoxyl radical of the dimethyl analog does not form polymers or bind with nucleophiles. N-Acetyl-p-benzoquinone imine, a hepatic metabolite of acetaminophen, and its analog N-acetyl-3,5-dimethyl-p-benzoquinone imine are metabolized by rat liver microsomes and NADPH to their corresponding p-aminophenoxyl free radicals. The p-aminophenoxyl free radical formation could be suppressed by the deacetylase inhibitors sodium fluoride and paraoxon. Substitution of NADPH-cytochrome P-450 reductase for rat liver microsomes eliminates the deacetylase activity and results in the direct reduction of N-acetyl-3,5-dimethyl-p-benzoquinone imine to the 3,5-dimethylacetaminophen phenoxyl free radical. Neither the acetaminophen nor the 3,5-dimethylacetaminophen phenoxyl radical reduces oxygen to form superoxide or reacts with oxygen in any other detectable way.     

Glutathione and ascorbate reduction of the acetaminophen radical formed by peroxidase. Detection of the glutathione disulfide radical anion and the ascorbyl radical

The acetaminophen phenoxyl radical was generated by the oxidation of acetaminophen by horseradish peroxidase in a fast-flow ESR experiment, and its reaction with glutathione and ascorbate was studied. Glutathione reduces the phenoxyl radical of acetaminophen to regenerate acetaminophen and form the thiyl radical of glutathione. This thiyl radical reacts with the thiolate anion of glutathione to form the disulfide radical anion, which was detected and characterized by ESR spectroscopy. In the presence of ascorbate, the ascorbyl radical was produced by the reduction of the acetaminophen phenoxyl radical by ascorbate. This reaction results in the complete reduction of the free radical of acetaminophen, whereas the glutathione reduction of the phenoxyl radical of acetaminophen was not complete on the fast-flow ESR time scale of milliseconds. This suggests that ascorbate rather than glutathione is more likely to react with the acetaminophen phenoxyl free radical in vivo. In the presence of both ascorbate and higher concentrations of glutathione, the reaction with ascorbate is dominant. When cysteine was used in the place of reduced glutathione in the above assay system, the disulfide radical anion of cystine was observed in a manner similar to glutathione. These reactions may have significance in the detoxification of acetaminophen and the free radical metabolites of xenobiotics in general. Only in cells containing low levels of ascorbate can glutathione play a direct role in the detoxification of the acetaminophen phenoxyl radical.     

Mechanisms of acetaminophen oxidation to N-acetyl-P-benzoquinone imine by horseradish peroxidase and cytochrome P-450

Horseradish peroxidase rapidly catalyzed the H2O2-dependent polymerization of acetaminophen. Acetaminophen polymerization was decreased and formation of GSSG and minor amounts of GSH-acetaminophen conjugates were detected in reaction mixtures containing GSH. These data suggest that horseradish peroxidase catalyzed the 1-electron oxidation of acetaminophen and that GSH decreased polymerization by reducing the product, N-acetyl-p-benzosemiquinone imine, back to acetaminophen. Analyses of reaction mixtures that did not contain GSH showed N-acetyl-p-benzoquinone imine formation shortly after initiation of reactions. When GSH was added to similar reaction mixtures at various times, 3-(glutathion-S-yl)-acetaminophen was formed. The formation and disappearance of this product were very similar to N-acetyl-p-benzoquinone imine formation and were consistent with the disproportionation of 2 mol of N-acetyl-p-benzosemiquinone imine to 1 mol of N-acetyl-p-benzoquinone imine and 1 mol of acetaminophen followed by the rapid reaction of N-acetyl-p-benzoquinone imine with GSH to form 3-(glutathion-S-yl)acetaminophen. When acetaminophen was incubated with NADPH, oxygen and hepatic microsomes from phenobarbital-pretreated rats, 1.2 nmol 3-(glutathion-S-yl)acetaminophen/nmol cytochrome P-450/10 min was formed. Formation of polymers was not observed indicating that N-acetyl-p-benzoquinone imine was formed via an overall 2-electron oxidation rather than a disproportionation reaction. However, when cumene hydroperoxide was replaced by NADPH in microsomal incubations, polymerization was observed suggesting that cytochrome P-450 might also catalyze the 1-electron oxidation of acetaminophen.     

The 1- and 2-electron oxidation of acetaminophen catalyzed by prostaglandin H synthase

Purified and microsomal preparations of prostaglandin H synthase catalyzed the arachidonic acid-dependent polymerization of acetaminophen and, in the presence of GSH, catalyzed the formation of 3-(glutathion-S-yl)acetaminophen. The formation of these products was inhibited by indomethacin and by purging reaction mixtures with argon. When H2O2 replaced arachidonic acid, neither indomethacin nor argon purging inhibited product formation. These results suggest that the peroxidase activity of prostaglandin H synthase catalyzed the oxidation of acetaminophen. Addition of GSH to reaction mixtures decreased acetaminophen polymerization; however, 3-(glutathion-S-yl)acetaminophen formation was maximal with 40 microM GSH, and higher concentrations of GSH did not substantially alter its formation. In the presence of GSH, either ascorbic acid or NADPH decreased polymerization by greater than 97% while 3-(glutathion-S-yl)acetaminophen formation was still observed. These data suggest that polymers and conjugates were formed by two different pathways. Since polymerization of acetaminophen involves radical termination of N-acetyl-p-benzosemiquinone imine whereas 3-(glutathion-S-yl)acetaminophen is formed by conjugation of N-acetyl-p-benzoquinone imine with GSH, the data suggest that prostaglandin H synthase catalyzed both the overall 1- and 2-electron oxidation of acetaminophen.     

Direct electron spin resonance detection of free radical intermediates during the peroxidase catalyzed oxidation of phenacetin metabolites

The oxidation of the phenacetin metabolites p-phenetidine and acetaminophen by peroxidases was investigated. Free radical intermediates from both metabolites were detected using fast-flow ESR spectroscopy. Oxidation of acetaminophen with either lactoperoxidase and hydrogen peroxide or horseradish peroxidase and hydrogen peroxide resulted in the formation of the N-acetyl-4-aminophenoxyl free radical. Totally resolved spectra were obtained and completely analyzed. The radical concentration was dependent on the square root of the enzyme concentration, indicating second-order decay of the radical, as is consistent with its dimerization or disproportionation. The horseradish peroxidase/hydrogen peroxide-catalyzed oxidation of p-phenetidine (4-ethoxyaniline) at pH 7.5-8.5 resulted in the one-electron oxidation products, the 4-ethoxyaniline cation free radical. The ESR spectra were well resolved and could be unambiguously assigned. Again, the enzyme dependence of the radical concentration indicated a second-order decay. The ESR spectrum of the conjugate base of the 4-ethoxyaniline cation radical, the neutral 4-ethoxyphenazyl free radical, was obtained at pH 11-12 by the oxidation of p-phenetidine with potassium permanganate.     

Vitamin E analogue Trolox C. E.s.r. and pulse-radiolysis studies of free-radical reactions.

The reactions between Trolox C, a water-soluble vitamin E analogue, and several oxidizing free radicals including the hydroxyl radical and various peroxy radicals were examined by using the pulse-radiolysis technique. The results demonstrate that Trolox C may undergo rapid one-electron-transfer reactions as well as hydrogen-transfer processes; the resulting phenoxyl radical is shown to be relatively stable, in common with the phenoxyl radical derived from vitamin E. The reactions between the Trolox C phenoxyl radical and a variety of biologically relevant reducing compounds were examined by using both pulse radiolysis and e.s.r. The results demonstrate that the Trolox C phenoxyl radical is readily repaired by ascorbate (k = 8.3 x 10(6) dm3.mol-1.s-1) and certain thiols (k less than 10(5) dm3.mol-1.s-1) but not by urate, NADH or propyl gallate. Evidence from e.s.r. studies indicates that thiol-containing compounds may also enter into similar repair reactions with the alpha-tocopherol phenoxyl radical. Kinetic evidence is presented that suggests that Trolox C may 'repair' proteins that have been oxidized by free radicals.     

Direct evidence for recycling of myeloperoxidase-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxy chromane,by ascorbate/dihydrolipoate in living HL-60 cells

Myeloperoxidase (MPO)-catalyzed one-electron oxidation of endogenous phenolic constituents (e.g., antioxidants, hydroxylated metabolites) and exogenous compounds (e.g., drugs, environmental chemicals) generates free radical intermediates: phenoxyl radicals. Reduction of these intermediates by endogenous reductants, i.e. recycling, may enhance their antioxidant potential and/or prevent their potential cytotoxic and genotoxic effects. The goal of this work was to determine whether generation and recycling of MPO-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), by physiologically relevant intracellular reductants such as ascorbate/lipoate could be demonstrated in intact MPO-rich human leukemia HL-60 cells. A model system was developed to show that MPO/H(2)O(2)-catalyzed PMC phenoxyl radicals (PMC*) could be recycled by ascorbate or ascorbate/dihydrolipoic acid (DHLA) to regenerate the parent compound. Absorbance measurements demonstrated that ascorbate prevents net oxidation of PMC by recycling the phenoxyl radical back to the parent compound. The presence of DHLA in the reaction mixture containing ascorbate extended the recycling reaction through regeneration of ascorbate. DHLA alone was unable to prevent PMC oxidation. These conclusions were confirmed by direct detection of PMC* and ascorbate radicals formed during the time course of the reactions by EPR spectroscopy. Based on results in the model system, PMC* and ascorbate radicals were identified by EPR spectroscopy in ascorbate-loaded HL-60 cells after addition of H(2)O(2) and the inhibitor of catalase, 3-aminotriazole (3-AT). The time course of PMC* and ascorbate radicals was found to follow the same reaction sequence as during their recycling in the model system. Recycling of PMC by ascorbate was also confirmed by HPLC assays in HL-60 cells. Pre-loading of HL-60 cells with lipoic acid regenerated ascorbate and thus increased the efficiency of ascorbate in recycling PMC*. Lipoic acid had no effect on PMC oxidation in the absence of ascorbate. Thus PMC phenoxyl radical does not directly oxidize thiols but can be recycled by dihydrolipoate in the presence of ascorbate. The role of phenoxyl radical recycling in maintaining antioxidant defense and protecting against cytotoxic and genotoxic phenolics is discussed.     

The Antioxidant Trolox Enhances the Oxidation of 2', 7'-Dichlorofluorescin to 2', 7'-Dichlorofluorescein

The use of antioxidants to prevent intracellular free radical damage is an area currently attracting considerable research interest. The compound 2',7'-dichlorofluorescin diacetate (DCFH-DA) is a probe for intracellular peroxide formation commonly used in such studies. During our studies we unexpectedly found that incubation of Trolox, a water soluble vitamin E analog, with DCFH-DA in cell-free physiological buffers resulted in the deacetylation and oxidation of DCFH-DA to form the fluorescent compound, 2',7'-dichlorofluororescein (DCF). The reaction was time-, temperature-, and pH-dependent. Fluorescence intensity increased with an increase in either Trolox or DCFH-DA concentration. These results indicate that even at physiological pH, DCFH-DA can be deacetylated to form 2',7'-dichlorofluorescin (DCFH). DCFH can then be oxidized to DCF by abstraction of a hydrogen atom by the phenoxyl radical of Trolox. Exposure of the reaction mixture to 10 Gy of ⁶⁰Co gamma radiation greatly increased production of DCF. Antioxidant compounds reported to “repair” the Trolox phenoxyl radical (e.g., ascorbic acid, salicylate) can also prevent the Trolox-induced DCFH-DA fluorescence. However, compounds that cannot repair the Trolox phenoxyl radical (e.g., catechin) or can themselves form a radical (e.g., uric acid, TEMPOL) either have no effect or can increase levels of DCF. These results demonstrate that experimental design must be carefully considered when using DCFH-DA to measure peroxide formation in combination with certain antioxidants.     

CO2.- radical induced cleavage of disulfide bonds in proteins. A gamma-ray and pulse radiolysis mechanistic investigation

Disulfide bond reduction by the CO2.- radical was investigated in aponeocarzinostatin, aporiboflavin-binding protein, and bovine immunoglobulin. Protein-bound cysteine free thiols were formed under gamma-ray irradiation in the course of a pH-dependent and protein concentration dependent chain reaction. The chain efficiency increased upon acidification of the medium, with an apparent pKa around 5, and decreased abruptly below pH 3.6. It decreased also at neutral pH as cysteine accumulated. From pulse radiolysis analysis, CO2.- proved able to induce rapid one-electron oxidation of thiols and of tyrosine phenolic groups in addition to one-electron donation to exposed disulfide bonds. The bulk rate constant of CO2.- uptake by the native proteins was 5- to 10-fold faster at pH 3 than at pH 8, and the protonated form of the disulfide radical anion, [symbol: see text], appeared to be the major protein radical species formed under acidic conditions. The main decay path of [symbol: see text] consisted of the rapid formation of a thiyl radical intermediate [symbol: see text] in equilibrium with the closed, cyclic form. The thiyl radical was subsequently reduced to the sulfhydryl level [symbol: see text] on reaction with formate, generating 1 mol of the CO2.- radical, thus propagating the chain reaction. The disulfide radical anion [symbol: see text] at pH 8 decayed through competing intramolecular and/or intermolecular routes including disproportionation, protein-protein cross-linking, electron transfer with tyrosine residues, and reaction with sulfhydryl groups in prereduced systems. Disproportionation and cross-linking were observed with the riboflavin-binding protein solely. Formation of the disulfide radical cation [symbol: see text], phenoxyl radical Tyr-O. disproportionation, and phenoxyl radical induced oxidation of preformed thiol groups should also be taken into consideration to explain the fate of the oxygen-centered phenoxyl radical.     

The peroxidase-dependent activation of butylated hydroxyanisole and butylated hydroxytoluene (BHT) to reactive intermediates. Formation of BHT-quinone methide via a chemical-chemical interaction

The food antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are shown to be metabolized to covalent binding intermediates and various other metabolites by prostaglandin H synthase and horseradish peroxidase. BHA was extensively metabolized by horseradish peroxidase (80% conversion of parent BHA into metabolites) resulting in the formation of three dimeric products. Only two of these dimers were observed in prostaglandin H synthase-catalyzed reactions. In contrast to BHA, BHT proved to be a relatively poor substrate for prostaglandin synthase and horseradish peroxidase, resulting in the formation of a small amount of polar and aqueous metabolites (23% conversion of parent BHT into metabolites). With arachidonic acid as the substrate, prostaglandin H synthase catalyzed the covalent binding of [14C]BHA and [14C]BHT to microsomal protein which was significantly inhibited by indomethacin and glutathione. The covalent binding of BHA and its metabolism to dimeric products were also inhibited by BHT. In contrast, the addition of BHA enhanced the covalent binding of BHT by 400%. Moreover, in the presence of BHA, the formation of the polar and aqueous metabolites of BHT was increased and two additional metabolites, BHT-quinone methide and stilbenequinone, were detected. The increased peroxidase-dependent oxidation of BHT in the presence of BHA is proposed to occur via the direct chemical interaction of BHA phenoxyl radical with BHT or BHT phenoxyl radical. These results suggest a potential role for phenoxyl radicals in the activation of xenobiotic chemicals to toxic metabolites.     

Acetaminophen stimulates the peroxidative metabolism of anthracyclines

Acetaminophen, a common analgesic and antipyretic drug, is frequently administered to individuals undergoing anthracycline chemotherapy. Here, the effect of acetaminophen on the metabolism of daunorubicin and doxorubicin by isolated enzymes lactoperoxidase and myeloperoxidase, and by myeloperoxidase-containing human leukemia HL-60 cells was investigated using spectrophotometric and EPR techniques. We report that at pharmacological concentrations acetaminophen strongly stimulates oxidation of the anthracyclines by lactoperoxidase and myeloperoxidase systems, which results in irreversibly altered (colorless) products. The initial rate and efficacy of daunorubicin oxidation depends on pH. While at pH approximately 7 the oxidation is rapid and extensive, almost no oxidation occurs at pH approximately 5. In the absence of daunorubicin, oxidation of acetaminophen by lactoperoxidase/hydrogen peroxide is only weakly dependent on pH, however, at pH 7.4 it strongly depends on [daunorubicin]. Ascorbate and reduced glutathione strongly inhibited oxidation of anthracyclines by lactoperoxidase and HL-60 systems. Using EPR, a daunorubicin-derived radical was detected in a daunorubicin/acetaminophen/peroxidase/hydrogen peroxide system as a narrow single line (0.175 mT) with g = 2.0047. When daunorubicin was omitted, only an acetaminophen-melanin EPR signal was detected (g = 2.0043, line width approximately 0.5 mT). Similar results were obtained with doxorubicin. We suggest that the stimulation by acetaminophen is primarily due to its preferential oxidation by peroxidases to the corresponding phenoxyl radical, which subsequently reacts with daunorubicin (doxorubicin). Because biological properties of oxidatively transformed anthracyclines will certainly be different from those of their parent compounds, the possible acetaminophen-enhanced degradation of the anthracyclines in vivo is likely to interfere with anticancer and/or cardiotoxic activities of these agents.     

Phenoxyl free radical formation during the oxidation of the fluorescent dye 2',7'-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements.

The oxidation of the fluorescent dye 2',7'-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF(.)). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF(.) oxidizes GSH, generating the glutathione thiyl radical (GS(.)), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS(.) radical. Similarly, DCF(.) oxidized NADH, generating the NAD(.) radical that reduced oxygen to superoxide (O-(2)), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H(2)O(2), which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H(2)O(2) production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.     

Methemoglobin Formation from Butylated Hydroxyanisole and Oxyhemoglobin. Comparison with Butylated Hydroxytoluene and P-Hydroxyanisole

The widely used food additives butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) react with oxyhemoglobin, thereby forming methemoglobin. The reaction rates were measured using visible spectroscopy, and second order rate constants were established for BHA and compared with p-hydroxyanisole. Using ESR we investigated the involvement of free radical reaction intermediates. The expected one-electron oxidation product of BHA and BHT, the phenoxyl radical, could only be detected with pure 3-t-butyl-4-hydroxyanisole and oxyhemoglobin. With the commercial mixture of 2- and 3-t-butyI-4-hydroxyanisole a very strong ESR signal of a secondary free radical species was observed, similar to the one observed earlier with p-hydroxyanisole and dependent on the presence of free thiol groups, so that we assumed the intermediate existence of a perferryl species, the MetHb-H₂O₂ adduct. In a second series of experiments we investigated the reactivity of this postulated intermediate with BHA and BHT, starting with a pure MetHb/H₂O₂-phenol mixture in a stopped-flow apparatus linked to the ESR spectrometer, detecting the expected phenoxyl radicals from BHA and p-hydroxyanisole. Due to the low solubility and decreased reactivity of BHT only traces of the phenoxyl type radical were found together with a high concentration of unreacted perferryl species. The reactivity of BHA, BHT and p-hydroxyanisole with free thiol groups is demonstrated by an increased reaction rate in the presence of the thiol group blocking substance NEM.     

OH-induced free radicals in 3'-UMP and poly(U): spin-trapping and radical chromatography

Characterization of OH-induced free radicals using 3'-UMP and poly(U) was performed by a method combining spin-trapping and radical chromatography. A N2O-saturated aqueous solution containing 3'-UMP and 2-methyl-2-nitrosopropane as a spin-trap was X-irradiated. The spin adducts generated by the reactions of OH radicals with 3'-UMP were separated by paired-ion HPLC and the separated spin adducts were identified by ESR spectroscopy. In the case of poly(U), the spin adducts were digested to oligonucleotides with RNase A and then separated and identified in the same manner as 3'-UMP. The free radicals observed for poly(U) were identical to those for 3'-UMP. The 5-yl radical and the 6-yl radical were identified as precursors of various oxidized products of the base moiety, and the 4'-yl radical and 5'-yl radical, formed by H-abstraction at the C-4' and C-5' positions of the sugar moieties, respectively, were identified as precursors of strand breaks. The 1'-yl radical, produced by H-abstraction at the C-1' position of the sugar moiety, was also identified. From the similarity of the free radicals of 3'-UMP and poly(U), it is suggested that the reactivities of OH radicals with nucleotides are identical to those in polynucleotides.     

Acetaminophen and analogs as cosubstrates and inhibitors of prostaglandin H synthase

Previous studies have shown that acetaminophen (APAP) is converted by prostaglandin H synthase (PGHS) to both one-electron oxidized products and the two-electron oxidized product, N-acetyl-p-benzoquinone imine (NAPQI). The present study further characterizes this reaction and shows that relatively low concentrations (20-200 microM) of APAP stimulate PGHS activity in ram seminal vesicle microsomes, whereas high concentrations (greater than 10 mM) inhibit the conversion of arachidonic acid (AA) to 15-hydroperoxy-9,11-peroxidoprosta-5,13-dienoic acid (PGG2). Stimulatory and inhibitory activities apparently involve the reduction of oxidized complexes of PGHS, and stimulatory and inhibitory activities roughly correlate with the electrochemical half-wave oxidation potentials of a series of hydroxyacetanilides. Using APAP as a probe, it was found that at low concentrations, APAP is converted in a cooxidation reaction with arachidonic acid to a dimer, 4'4"'-dihydroxy-3', 3"'-biacetanilide (bi-APAP), and other polymeric products. Moreover, an electrophilic metabolite of acetaminophen, NAPQI, was detected directly and also detected indirectly by its reaction with glutathione (GSH) to form 3'-(S-glutathionyl)acetaminophen (GS-APAP). The formation of all products was inhibited by indomethacin and the reductants, ascorbic acid and butylated hydroxyanisole (BHA). However, in the presence of GSH, ascorbic acid only partially inhibited the formation of GS-APAP while almost completely inhibiting the formation of bi-APAP. The same products of APAP (bi-APAP and NAPQI) were formed by PGHS and hydrogen peroxide in reactions that were not inhibited by indomethacin. At high concentrations of APAP that inhibit PGHS, the formation of products in the presence of arachidonic acid but not H2O2 was inhibited. These findings are generally consistent with a mechanism of acetaminophen oxidation by PGHS that involves common intermediate enzyme forms for both cyclooxygenase- and hydroperoxidase-catalyzed reactions. At least one of the intermediate complexes is reduced by relatively low concentrations of APAP and stimulates PGHS, whereas another intermediate complex is reduced by APAP at higher concentrations to inhibit the enzyme.     

Peroxidase-catalyzed oxidation of eugenol: formation of a cytotoxic metabolite(s)

The oxidation of eugenol (4-allyl-2-methoxyphenol) by horseradish peroxidase was studied. Following the initiation of the reaction with hydrogen peroxide, eugenol was oxidized via a one-electron pathway to a phenoxyl radical which subsequently formed a transient, yellow-colored intermediate which was identified as a quinone methide. The eugenol phenoxyl radical was detected using fast-flow electron spin resonance. The radicals and/or quinone methide further reacted to form an insoluble complex polymeric material. The stoichiometry of the disappearance of eugenol versus hydrogen peroxide was approximately 2:1. The addition of glutathione or ascorbate prevented the appearance of the quinone methide and also prevented the disappearance of the parent compound. In the presence of glutathione, a thiyl radical was detected, and increases in oxygen consumption and in the formation of oxidized glutathione were also observed. These results suggested that glutathione reacted with the eugenol phenoxyl radical and reduced it back to the parent compound. Glutathione also reacted directly with the quinone methide resulting in the formation of a eugenol-glutathione conjugate(s). Using 3H-labeled eugenol, extensive covalent binding to protein was observed. Finally, the oxidation products of eugenol/peroxidase were observed to be highly cytotoxic using isolated rat hepatocytes as target cells.     

ESR study on the structure-antioxidant activity relationship of tea catechins and their epimers

The purpose of this study is to examine the relationship between the free radical scavenging activities and the chemical structures of tea catechins ((-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC)) and their corresponding epimers ((-)-gallocatechin gallate (GCG), (-)-gallocatechin (GC) and (+)-catechin ((+)-C)). With electron spin resonance (ESR) we investigated their scavenging effects on superoxide anions (O-.2) generated in the irradiated riboflavin system, singlet oxygen(1O2) generated in the photoradiation-hemoporphyrin system, the free radicals generated from 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical. The results showed that the scavenging effects of galloylated catechins (EGCG and GCG) on the four free radicals were stronger than those of nongalloylated catechins (EGC, GC, EC, (+)-C), and the scavenging effects of EGC and GC were stronger than those of EC and (+)-C. Thus, it is suggested that the presence of the gallate group at the 3 position plays the most important role in their free radical-scavenging abilities and an additional insertion of the hydroxyl group at the 5' position in the B ring also contributes to their scavenging activities. Moreover, the corresponding phenoxyl radicals formed after the reaction with O-.2 were trapped by DMPO and the ESR spectra of DMPO/phenoxyl radical adducts were observed (aN=15.6 G and aHbeta=21.5 G). No significant differences were found between the scavenging effects of the catechins and their epimers when their concentrations were high. However, significant differences were observed at relatively low concentrations, and the lower their concentrations, the higher the differences. The scavenging abilities of GCG, GC and (+)-C were stronger than those of their corresponding epimers (EGCG, EGC and EC). The differences between their sterical structures played a more important role in their abilities to scavenge large free radicals, such as the free radicals generated from AAPH and the DPPH radical, than to scavenge small free radicals, such as O-.2 and 1O2, especially in the case with EGCG and GCG with more bulky steric hindrance.     

Inhibitory effect of quercetin metabolites and their related derivatives on copper ion-induced lipid peroxidation in human low-density lipoprotein

To determine the antioxidant activity of dietary quercetin (3,3',4', 5,7-pentahydroxyflavone) in the blood circulation, we measured the inhibitory effect of quercetin metabolites and their related derivatives on copper ion-induced lipid peroxidation of human low-density lipoprotein (LDL). Conjugated quercetin metabolites were prepared from the plasma of rat 1 h after oral administration of quercetin aglycone (40 micromol/rat). The rate of cholesteryl ester hydroperoxide (CE-OOH) accumulation and the rate of alpha-tocopherol consumption in mixtures of LDL solution (0.4 mg/ml) with equal volumes of this preparation were slower than the rates in mixtures of LDL with preparations from control rats. The concentrations of CE-OOH after 2 h oxidation in the mixtures of LDL with preparations of conjugated quercetin metabolites were significantly lower than those in the control preparation. It is therefore confirmed that conjugated quercetin metabolites have an inhibitory effect on copper ion-induced lipid peroxidation in human LDL. Quercetin 7-O-beta-glucopyranoside (Q7G) and rhamnetin (3,3',4', 5-tetrahydroxy-7-methoxyflavone) exerted strong inhibition and their effect continued even after complete consumption, similarly to quercetin aglycone. The effect of quercetin 3-O-beta-glucopyranoside (Q3G) did not continue after its complete consumption, indicating that the antioxidant mechanism of quercetin conjugates lacking a free hydroxyl group at the 3-position is different from that of the other quercetin conjugates. The result that 4'-O-beta-glucopyranoside (Q4'G) and isorhamnetin (3,4',5, 7-tetrahydroxy-3'-methoxyflavone) showed little inhibition implies that introduction of a conjugate group to the position of the dihydroxyl group in the B ring markedly decreases the inhibitory effect. The results of azo radical-induced lipid peroxidation of LDL and the measurement of free radical scavenging capacity using stable free radical, 1,1,-diphenyl-2-picrylhydrazyl, demonstrated that the o-dihydroxyl structure in the B ring is required to exert maximum free radical scavenging activity. It is therefore likely that conjugation occurs at least partly in positions other than the B ring during the process of metabolic conversion so that the inhibitory effect of dietary quercetin is retained in blood plasma after absorption.     

DPPH free radical scavenger components from the fruits of Alpinia rafflesiana Wall. ex. Bak. (Zingiberaceae)

The methanol extract of the dried ripe fruits of Alpinia rafflesiana was investigated for its DPPH free radical scavenger constituents. 2',3',4',6'-Tetrahydroxychalcone (7), which has never been isolated from natural sources was found to be most active as a DPPH free radical scavenger with the IC50 value of 55 microM. Other known compounds isolated from this species include 5,6-dehydrokawain (1), flavokawin B (2). 1,7-diphenyl-5-hydroxy-6-hepten-3-one (3), (-)-pinocembrin (4), cardamonin (5) and (-)-pinostrobin (6). The DPPH free radical scavenger compounds were detected using TLC autographic analysis. The percentage inhibition of DPPH free radical scavenging activity was measured on isolates (5-7) using colorimetric analysis.     

Generation and recycling of radicals from phenolic antioxidants

Hindered phenols are widely used food preservatives. Their pharmacological properties are usually attributed to high antioxidant activity due to efficient scavenging of free radicals. Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) also cause tissue damage. Their toxic effects could be due to the production of phenoxyl radicals. If phenoxyl radicals can be recycled by reductants or electron transport, their potentially harmful side reactions would be minimized. A simple and convenient method to follow phenoxyl radical reactions in liposomes and rat liver microsomes based on an enzymatic (lipoxygenase + linolenic acid) oxidation system was used to generate phenoxyl radicals from BHT and its homologues with substitutents in m- and p-positions. Different BHT-homologues display characteristic ESR signals of their radical species. In a few instances the absence of phenoxyl radical ESR signals was found to be due to inhibition of lipoxygenase by BHT-homologues. In liposome or microsome suspensions addition of ascorbyl palmitate resulted in disappearance of the ESR signal of phenoxyl radicals with concomittant appearance of the ascorbyl radical signal. After exhaustion of ascorbate, the phenoxyl radical signal reappears. Comparison of the rates of ascorbyl radical decay in the presence or absence of BHT-homologues showed that temporary elimination of the phenoxyl radical ESR signal was due to their reduction by ascorbate. Similarly, NADPH or NADH caused temporary elimination of ESR signals as a result of reduction of phenoxyl radicals in microsomes. Since ascorbate and NADPH might generate superoxide in the incubation system used, SOD was tested. SOD shortened the period, during which the phenoxyl radicals ESR signal could not be observed. Both ascorbyl palmitate and NADPH exerted sparing effects on the loss of BHT-homologues during oxidation. These effects were partly diminished by SOD. These data indicate that reduction of phenoxyl radicals was partly superoxide-dependent. It is concluded that redox recycling of phenoxyl radicals can occur by intracellular reductants like ascorbate and microsomal electron transport.