Formation of Methemoglobin and Phenoxyl Radicals from P-Hydroxyanisole and Oxyhemoglobin

The reaction of p-hydroxyanisole with oxyhemoglobin was investigated using electron spin resonance spectroscopy (ESR) and visible spectroscopy. As a reactive reaction intermediate we found the p-methoxyphenoxyl radical, the one-electron oxidation product of p-hydroxyanisole. Detection of this species required the rapid flow device elucidating the instability of this radical intermediate. The second reaction product formed is methemoglobin. Catalase or SOD had no effect upon the reaction kinetics. Accordingly, reactive oxygen species such as hydroxyl radicals or superoxide could not be observed although the spin trapping agent DMPO was used to make these short-lived species detectable. When the sulfhydryl blocking agents N-ethylmaleimide or mersalyl acid were used, an increase of the methemoglobin formation rate and of the phenoxyl radical concentration were observed. We have interpreted this observation in terms of a side reaction of free radical intermediates with thiol groups.     

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.     

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.     

Stimulation of Monensin Biosynthesis in Streptomyces Cinnamonensis by Antioxidants Butylated Hydroxyanisole and Butylated Hydroxytoluene

Phenolic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) at concentration 0.55 mM and 2.25 mM, respectively, added to cultures of Streptomyces cinnamonensis C-100-5 caused up to 380% higher production of polyether antibiotic monensin. S. cinnamonensis exhibits high tolerance to BHT, but it is more sensitive towards BHA. 5.5 mM BHA practically stopped growth, while 45 mM BHT still stimulated antibiotic production. Another antioxidants probucol and tocoferol acetate did not exert such positive effects. BHA was predominantly metabolised to 5-hydroxy-BHA. When BHA and BHT were applied simultaneously, the main transformation metabolite was 3,3,5,5-tetra-tert-butyl-stilbene-4,4-quinone accompanied by 1,2-bis(3,5-di-tert-butyl-4-hydroxyphenyl)ethane.     

Butylated hydroxyanisole specifically inhibits tumor necrosis factor-induced cytotoxicity and growth enhancement.

The effect of commonly used food antioxidants on recombinant tumor necrosis factor alpha (rTNF-alpha)-induced cytotoxicity, growth enhancement and adhesion has been evaluated. Butylated hydroxyanisole (BHA) and 4-hydroxymethyl-2,6-di-t-butylphenol (HBP) were the only two of nine antioxidants that completely inhibited rTNF-alpha-induced cytotoxicity in L929 and WEHI 164 fibrosarcoma cells. Ethoxyquin, propyl gallate and butylated hydroquinone only partially inhibited rTNF-alpha-induced cytotoxicity, while the antioxidants butylated hydroxytoluene (BHT), alpha-tocopherol, ascorbic acid and thiodipropionic acid had minimal effects. The only difference between the molecular structure of the efficient HBP and the non-efficient BHT, is a hydroxymethyl group instead of a hydroxyl group on the phenolic ring. Neither BHA nor BHT inhibited the activation of NF kappa B after 10 or 60 min challenge with rTNF-alpha in L929 cells. BHA also inhibited rTNF-alpha-induced, but not rIL-1 beta-induced growth enhancement in FS-4 fibroblasts. Further, BHA blocked both rTNF-alpha-induced and rIL-1 beta-induced prostaglandin E2 synthesis in FS-4 fibroblasts. BHA inhibited the rTNF-alpha-induced release of arachidonic acid in both FS-4 and L929 cells, suggesting that BHA inhibits cellular phospholipase(s). Neither alpha-tocopherol nor BHA inhibited rTNF-alpha-induced adhesiveness of human endothelial cells. The results indicate that BHA is a specific and potent inhibitor of rTNF-alpha- and rTNF-beta-induced cytotoxicity, as well as of rTNF-alpha-induced growth enhancement.     

Analysis of interactions between potent inhibitors of ATP sulfurylase via molecular dynamics

The dynamic behaviour of adenosine triphosphate sulfurylase (ATPs) is analysed to investigate its energetic interactions with inhibitors recently studied theoretically and tested experimentally. The interactions between ATPs and three inhibitors namely, 2,2′-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), butylated hydroxytoluene (BHT), and 3-tert-butyl-4-hydroxyanisole (BHA) in aqueous solution were studied via molecular dynamics simulations. The results of the absolute and relative free energies reveal the existence of synergism in the system. Additionally, the system demonstrates strong steric effect between ABTS and BHA that is not only due to the size of these inhibitors but also their surroundings.     

Butylated hydroxyanisole, butylated hydroxytoluene and tert.-butylhydroquinone are not mutagenic in the Salmonella/microsome assay using new tester strains

The phenolic antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert.-butylhydroquinone (TBHQ) were reassessed for mutagenic activity using the recently developed Salmonella tester strains TA97, TA102 and TA104, and in addition TA100. None of the phenolic antioxidants showed mutagenic activity, either with or without metabolic activation. At doses of 100 micrograms/plate and higher all 3 phenolic antioxidants exhibited toxic effects. A modification of the assay using the preincubation procedure with strain TA104 did not affect mutation frequencies. Combinations of BHA and BHT, tested to detect possible synergistic effects, did not exert mutagenic activity.     

Free radical formation and erythrocyte membrane alterations during MetHb formation induced by the BHA metabolite, tert-butylhydroquinone

Erythrocyte membranes are altered as a consequence of oxidative stress following the incubation of intact erythrocytes with one of the major metabolites of the antioxidant butylated hydroxyanisole (BHA), tert-butylhydroquinone(tBHQ). A ratherpersistentsemiquinone radical was observed by electron spin resonance (ESR) spectroscopy when tBHQ was incubated with either homogeneous oxyhemoglobin solutions or suspensions of intact erythrocytes. Erythrocyte ghosts prepared from fresh control erythrocytes and ghosts from erythrocytes preincubated with BHA and its metabolite, tBHQ, were subjected to polyacrylamide gel electrophoresis (SDS-PAGE). Only minor changes of the electrophoresis pattern relative to the control was observed in the BHA incubations whereas tBHQ significantly increased the amount of high molecular weight degradation products of erythrocyte membrane constituents. These changes were only observed when incubations were performed in the presence of oxygen. In control experiments where heme oxygen was replaced by carbon monoxide, no membrane degradation products appeared. These observations can be interpreted in terms of metabolic activation of the antioxidant BHA via tBHQ to the tert-butylsemiquinone free radical and finally to the corresponding quinone, thereby leading to harmful effects on erythrocyte membrane structures. Moreover, deleterious effects on other biological membranes are also likely to occur.     

Comparative effects of antioxidants on enzymes involved in glutathione metabolism

The present study was designed to examine changes in glutathione metabolism in the liver of mice as influenced by supplementation of their diet with 1 of 4 antioxidants: butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), vitamin E and selenium. In addition to determination of the acid-soluble thiol levels, 5 different enzymes involved with glutathione utilization and synthesis were measured: glutathione transferase, γ-glutamyl transpeptidase, selenium-dependent glutathione peroxidase, γ-glutamylcysteine synthetase and glutathione reductase. All 4 antioxidants produced significant increases in glutathione transferase activity, with BHA and BHT being much more effective than the other two. With the exception of vitamin E, BHA, BHT and selenium all resulted in a slight enhancement in the activity of glutathione reductase as well as in the acid-soluble thiol level. On the other hand, the induction of γ-glutamyl transpeptidase and γ-glutamylcysteine synthetase was responsive to only vitamin E and selenium supplementation, respectively. Although the influence of each of these antioxidants in glutathione metabolism appears to be specific and somewhat compartmentalized, the overall impression is that of an increased capacity for glutathione-conjugate formation and recovery of reduced glutathione. These biochemical changes in glutathione metabolism may be relevant to the anticarcinogenic effects observed with BHA, BHT and selenium.     

Butylated hydroxytoluene prevents cumene hydroperoxide-induced Ca2+ release from liver mitochondria by inhibiting pyridine nucleotide hydrolysis.

The mechanism by which the free radical scavenger butylated hydroxytoluene (BHT) prevents cumene hydroperoxide-induced Ca2+ release from rat liver mitochondria was studied. In Ca(2+)-loaded mitochondria cumene hydroperoxide induced a rapid oxidation and subsequent hydrolysis of the pyridine nucleotides. In the presence of BHT, pyridine nucleotide oxidation by cumene hydroperoxide occurred but was reversible as hydrolysis was prevented by BHT. However, the addition of BHT directly to rat liver submitochondrial particles did not inhibit NAD+ hydrolysis or the formation of ADP-ribose from NAD+. Thus, whilst BHT prevented NAD+ hydrolysis in isolated mitochondria, this appeared not to be due to a direct effect of BHT on the NADase. It is concluded that the mechanism of action of BHT on cumene hydroperoxide-induced Ca2+ release from mitochondria involves the inhibition of pyridine nucleotide hydrolysis by an indirect mechanism rather than the radical scavenging properties of BHT.     

Influence of Antioxidants on Arachidonic Acid Metabolism and Platelet Function

Studies from our laboratory demonstrated that the free radical scavenger, nitro blue tetrazolium, and iron chelators, such as dypyrydil, are potent inhibitors of arachidonic acid oxidation and platelet function. In the present study, we have evaluated the effects of known antioxidants, such as butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), and diphenylamine, on arachidonic acid metabolism and platelet function. Diphenylamine, a common dye intermediate used in hair color formulations, was the most potent inhibitor of arachidonic acid metabolism by platelet cyclooxygenases. Diphenyl and BHA were also potent inhibitors of arachidonic acid oxidation. Other diphenyl analogues and BHT were relatively poor inhibitors of arachidonic-mediated platelet activation. Results of this study, as well as those of our earlier studies, suggest that antioxidants and iron chelators prevent arachidonic acid metabolism and alter platelet function by interfering with the heme/arachidonic acid interaction and blocking cyclooxygenase metabolites essential for the formation of thromboxane A₂, a potent platelet agonist.     

Lipid peroxidation of phosphatidylcholine liposomes depressed by the calcium channel blockers nifedipine and verapamil and by the antiarrhythmic-antihypoxic drug stobadine

Nifedipine, verapamil and stobadine were tested and compared with butylated hydroxytoluene (BHT) as possible free radical scavengers inhibiting lipid peroxidation in phosphatidylcholine liposomes. Liposomes were peroxidized by incubation in air at 50 degrees C. Verapamil less than nifedipine less than BHT less than stobadine depressed the lipid peroxidation as detected spectroscopically for conjugate diene and thiobarbituric acid product formation. Verapamil and stobadine were tested as OH radical scavengers in a Fenton-type reaction against spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO), as detected by ESR spectroscopy. The tested drugs competed with DMPO in trapping OH radicals, with stobadine being more effective than verapamil. ESR spectra of nifedipine in the incubated liposomes revealed that nifedipine could be involved in free radical reactions in the liposomes leading to nifedipine-stable radical(s) which were immobilized in the membrane. The obtained results suggest that some of the beneficial effects of the studied drugs can be mediated in disease by their ability to scavenge free radicals and by their protective effect on lipid peroxidation.     

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.     

Antioxidant and radical scavenging properties of curcumin

Curcumin (diferuoyl methane) is a phenolic compound and a major component of Curcuma longa L. In the present paper, we determined the antioxidant activity of curcumin by employing various in vitro antioxidant assays such as 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH*) scavenging, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD) radical scavenging activity, total antioxidant activity determination by ferric thiocyanate, total reducing ability determination by the Fe(3+)-Fe(2+) transformation method, superoxide anion radical scavenging by the riboflavin/methionine/illuminate system, hydrogen peroxide scavenging and ferrous ions (Fe(2+)) chelating activities. Curcumin inhibited 97.3% lipid peroxidation of linoleic acid emulsion at 15 microg/mL concentration (20 mM). On the other hand, butylated hydroxyanisole (BHA, 123 mM), butylated hydroxytoluene (BHT, 102 mM), alpha-tocopherol (51 mM) and trolox (90 mM) as standard antioxidants indicated inhibition of 95.4, 99.7, 84.6 and 95.6% on peroxidation of linoleic acid emulsion at 45 microg/mL concentration, respectively. In addition, curcumin had an effective DPPH* scavenging, ABTS*(+) scavenging, DMPD*(+) scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, ferric ions (Fe(3+)) reducing power and ferrous ions (Fe(2+)) chelating activities. Also, BHA, BHT, alpha-tocopherol and trolox, were used as the reference antioxidant and radical scavenger compounds. According to the present study, curcumin can be used in the pharmacological and food industry because of these properties.     

Screening of antiradical and antioxidant activity of monodesmosides and crude extract from Leontice smirnowii tuber.

Leontice smirnowii is a member of the Berberidaceae family. In the current study we investigated the possible antiradical and antioxidant activity of the monodesmosides (MLS) and crude extract (CELS) of Leontice smirnowii using different antioxidant tests: 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, scavenging of superoxide anion radical-generated non-enzymatic system, ferric thiocyanate (FTC) method, reducing power, hydrogen peroxide scavenging and metal chelating activities. Experiment revealed that MLS and CELS have an antioxidant effect concentration-dependently. Total antioxidant activity was performed according to FTC method. At the 30mug/ml concentration, the inhibition effects of MLS and CELS on peroxidation of linoleic acid emulsion were found to be 95.3% and 95.6%, respectively. On the other hand, percentage inhibition of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol and trolox were found to be 98.2%, 98.5%, 84.0% and 87.9% inhibition of peroxidation of linoleic acid emulsion, respectively, at the same concentration. In addition, MLS and CELS had effective DPPH radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, reducing power and metal chelating activities. Also, these various antioxidant activities were compared with BHA, BHT, alpha-tocopherol and trolox which were accepted as references antioxidants.     

The interaction between nitrogen oxides and hemoglobin and endothelium-derived relaxing factor

Among nitrogen oxides, NO and NO₂ are free radicals and show a variety of biological effects. NO₂ is a strongly oxidizing toxicant, although NO, not oxidizing as NO₂, is toxic in that it interacts with hemoglobin to form nitrosyl-and methemoglobin. Nitrosylhemoglobin shows a characteristic electron spin resonance (ESR) signal due to an odd electron localized on the nitrogen atom of NO and reacts with oxygen to yield nitrate and methemoglobin, which is rapidly reduced by methemoglobin reductase in red cells. NO was found to inhibit the reductase activity. Part of NO inhaled in the body is oxidized by oxygen to NO₂, which easily dissolves in water and converts to nitrite. The nitrite oxyhemoglobin autocatalytically after a lag. The mechanism of the oxidation, particularly the involvement of superoxide, was controversial. The stoichiometry of the reaction has now been established using nitrate ion electrode and a methemoglobin free radical was detected by ESR during the oxidation. Complete inhibition of the autocatalysis by aniline or aminopyrine suggests that the radical catalyzes conversion of nitrite to NO₂, which oxidizes oxyhemoglobin. Recently NO was shown to be one of endothelium- derived relaxing factors and the relaxation induced by the factor was inhibited by hemoglobin and potentiated by superoxide dismutase.     

Visible-light promoted degradation of the commercial antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT): a kinetic study

Visible-light photo-irradiation of the commercial phenolic antioxidants (PhAs) butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), in the presence of vitamin B2 (riboflavin, Rf), in methanolic solutions and under aerobic conditions, results in the photo-oxidation of the PhAs. The synthetic dye photosensitiser Rose Bengal was also employed for auxiliary experiments. With concentrations of riboflavin and PhAs of ca. 0.02 mM and < 1 mM, respectively, the excited triplet state of the vitamin (3Rf*) is quenched by BHT in a competitive fashion with dissolved ground state triplet oxygen. From the quenching of 3Rf*, the semireduced form of the pigment is generated through an electron transfer process from BHT, with the subsequent production of superoxide anion radical (O2*-) by reaction with dissolved molecular oxygen. In parallel, the species singlet molecular oxygen, O2(1delta(g)), is also generated. Both reactive oxygen species produce the photodegradation of BHT. In the case of BHA, the lack of any effect exerted by superoxide dismutase drives out a significant participation of a O2(*-)-mediated mechanism. BHA mainly interacts with O2(1delta(g)) and exhibits a desirable property as an antioxidant--a relatively high capacity for O2(1delta(g)) de-activation and a low photodegradation efficiency by the oxidative species. Electrochemical determinations support the proposed photodegradative mechanism.     

Comparison of Antioxidant Activity Between Aromatic Indolinonic Nitroxides and Natural and Synthetic Antioxidants

In view of the possible employment of nitroxide compounds in various fields, it is important to know how they compare with other synthetic antioxidant compounds currently used in several industries and with naturally occurring antioxidants. To address this issue, the antioxidant activity of two aromatic indolinonic nitroxides synthesized by us was compared with both commercial phenolic antioxidants (BHT and BHA) and with natural phenolic antioxidants (α-hydroxytyrosol, tyrosol, caffeic acid, α-tocopherol). DPPH radical scavenging ability and the inhibition of both lipid and protein oxidation induced by the peroxyl-radical generator, AAPH, were evaluated. The results obtained show that overall: (i) the reduced forms of the nitroxide compounds are better scavengers of DPPH radical than butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) but less efficient than the natural compounds; (ii) the nitroxides inhibit both linolenic acid micelles and bovine serum albumin (BSA) oxidation to similar extents as most of the other compounds in a concentration-dependent fashion. Since the aromatic nitroxides tested in this study are less toxic than BHT, these compounds may be regarded as potential, alternative sources for several applications. The mechanisms underlying the antioxidant activity of nitroxides were further confirmed by UV–Vis absorption spectroscopy experiments and macroscale reactions in the presence of radicals generated by thermolabile azo-compounds. Distribution coefficients in octanol/buffer of the nitroxides and the other compounds were also determined as a measure of lipophilicity.     

Determination of stoichiometry of radioiodination of proteins

A sensitive method for the detection of small quantities of hydrophobic antioxidant free radical scavengers such as butylatedhydroxytoluene (BHT) and butylatedhydroxyanisole (BHA) in aqueous samples is described. The procedure involves extraction of the hydrophobic free radical scavenger into an organic solvent phase, followed by the subsequent reaction of an aliquot of this extract with the stable cation radical tris(p-bromophenyl)amminium hexachloroantimonate (TBACA). In experiments with BHT and BHA, the loss of TBACA absorbance at 730 nm was found to be linearly proportional to the amount of antioxidant added, with quantities of BHT as small as 200 pmol being easily detectable. In aqueous suspensions of dimyristoylphosphatidylcholine vesicles, assays of the aqueous BHT concentration showed that BHT partitioned strongly into the membrane phase, achieving very high BHT/phospholipid ratios. For a given concentration of BHT, partitioning into the membrane phase was greater in large, multilamellar liposomes than in either small, single-walled vesicles or in purified rat brain synaptic vesicle membranes. Direct assay of BHT and BHA in phospholipid membranes, however, was complicated by a nonspecific interaction between TBACA and the phospholipid.     

Free Radical Formation from the Antineoplastic Agent VP 16-213

Free radical formation from VP 16-213 was studied by ESR spectroscopy. Incubation of VP 16-213 with the one-electron oxidators persulphate-ferrous, myeloperoxidase (MPO)/hydrogen peroxide and horseradish peroxidase (HRP)/hydrogen peroxide readily led to the formation of a free radical. The ESR spectra obtained in the last two cases, were in perfect accord with that of a product obtained by electrochemical oxidation of VP 16-213 at +550 mV. The half-life of the free radical in 1 mM Tris (pH 7.4), 0.1 MNaClat 20°C, was 257 ± 4 s. The signal recorded on incubation with HRP/H₂O₂ or MPO/H₂O₂ did not disappear on addition of 0.3 - 1.2 mg/ml microsomal protein. From incubations with rat liver microsomes in the presence of NADPH, no ESR signals were obtained.