Identification of the free radical formed by addition of hydroxyl radical to dehydroalanine compounds.

N-substituted dehydroalanines, a class of compounds with both acceptor and donor substituents (ADs), react with and scavenge oxygen radicals. Interest in these compounds is based on their potential to lessen the cardiotoxicity of drugs with antineoplastic activity such as Adriamycin. The reactivity of these compounds with hydroxyl radical is evident from their inhibition of hydroxyl radical adduct formation. ESR spin trapping studies of the species formed by reaction of the AD series of compounds with the hydroxyl radical are reported here for the first time. ESR results show that hydroxyl radical attack on the capto-dative site of the AD compounds produces the predicted carbon-centered free radical.

N-acyl dehydroalanines scavenge oxygen radicals and inhibit in vitro free radical mediated processes.

N-substituted dehydroalanines react with and scavenge oxygen radicals. One of those compounds, the para-methoxyphenylacetyl dehydroalanine derivative, indexed as AD-5, inhibits the reduction of ferricytochrome c by superoxide anion (O2-.). It can also inhibit the oxidation of linolenic acid, another chemical process, which is mediated by hydroxyl radical (HO.). Furthermore, microsomal lipid peroxidation induced by iron salts was also inhibited by AD 5, but with a different degree of efficacy. In fact, lipid peroxidation initiated by a ferrous-oxygen complex (as in iron/NADPH-dependent peroxidation) was inhibited by AD 5 in a range of concentration of 2-4 mM. On the contrary, iron/NADPH-independent lipid peroxidation, where alkoxy radicals (RO.) have principally been involved, was inhibited in a range of concentration of 6-10 mM. The ESR studies by using the spin trapping agent DMPO, show that AD-5 reacts with HO. with a second order constant of 2.8 X 10(9)-4.5 X 10(9) M-1 s-1.

The one-electron oxidation of porphyrins to porphyrin pi-cation radicals by peroxidases: an electron spin resonance investigation.

For the first time, the enzymatic one-electron oxidation of several naturally occurring and synthetic water-soluble porphyrins by peroxidases was investigated by ESR and optical spectroscopy. The ESR spectra of the free radical metabolites of the porphyrins were singlets (g = 2.0024, delta H = 2-3 G), which we assigned to their respective porphyrin pi-cation free radicals. Several porphyrins were investigated and ranked by the intensity of their ESR spectra (coproporphyrin III greater than coproporphyrin I greater than deuteroporphyrin IX greater than mesoporphyrin IX greater than Photofrin II greater than protoporphyrin IX greater than uroporphyrin I greater than uroporphyrin III greater than hematoporphyrin IX). The porphyrins were oxidized by several peroxidases (horseradish peroxidase, lactoperoxidase, and myeloperoxidase), yielding the same type of ESR spectra. From these results, we conclude that porphyrins are substrates for peroxidases. The changes in the visible absorbance spectra of the porphyrins during enzymatic oxidation were monitored. The two-electron oxidation product, which was assigned to the dihydroxyporphyrin, was detected as an intermediate of the oxidation process. The optical spectrum of the porphyrin pi-cation free radical was not detected, probably due to its low steady-state concentration.

In vitro free radical metabolism of phenolphthalein by peroxidases.

Phenolphthalein, a widely used laxative, is the active ingredient in more than a dozen commercial nonprescription formulations. Fast-flow EPR studies of the reaction of phenolphthalein with horseradish peroxidase (HRP) and hydrogen peroxide permit the direct detection of two free radicals. One has EPR parameters characteristic of phenoxyl radicals. The other has a broad unresolved spectrum, possibly arising from free radical polymeric products of the initial phenoxyl radical. EPR spin-trapping studies of incubations of phenolphthalein with lactoperoxidase, reduced glutathione (GSH), and hydrogen peroxide with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) demonstrate stimulated production of DMPO/.SG compared with an identical incubation lacking phenolphthalein. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed by a sequence of reactions initiated by the glutathione thiyl radical. Enhanced production of DMPO superoxide radical adduct is also found in a system of phenolphthalein, NADH, and lactoperoxidase. In this system the phenolphthalein phenoxyl radical abstracts hydrogen from NADH to generate NAD., which is not spin trapped by DMPO, but reacts with molecular oxygen to produce the superoxide radical detected by EPR. In the absence of DMPO, the oxygen consumption is measured using the Clark-type electrode. Production of ascorbate radical anion is also enhanced in a system of phenolphthalein, ascorbic acid, hydrogen peroxide, and lactoperoxidase. Ascorbate inhibits oxygen consumption when phenolphthalein is metabolized in the presence of either glutathione or NADH by reducing radical intermediates to their parent molecules and forming the relatively stable ascorbate anion radical. The detection of enhanced free radical production in these three systems, a consequence of futile metabolism (or redox cycling), suggests that phenolphthalein may be a significant source of oxidative stress in physiological systems. Parallel EPR and oxygen consumption studies with phenolphthalein glucuronide give analogous results, but with lesser enhancement of free radical production.

Free radical intermediates during peroxidase oxidation of 2-t-butyl-4-methoxyphenol, 2,6-di-t-butyl-4-methylphenol, and related phenol compounds.

2-t-butyl-4-methoxyphenol (BHA) and 2,6-di-t-butyl-4-methylphenol (BHT) are widely used antioxidant food additives that are generally recognized as safe by the Food and Drug Administration. Previously reported studies have suggested that the ip LD50 of BHA may be as much as 2 orders of magnitude lower than its oral LD50. Metabolic activation of BHA to reactive intermediates possibly may be responsible for this result and may be related to other reported toxic effects. BHT has been reported to cause haemorrhagic lung damage and possible hepatocarcinogenicity in test animals. The present studies report investigations by electron spin resonance spectroscopy of free radical metabolites of BHA, BHT and related compounds. The primary, unstable phenoxy free radical of BHA has been generated by oxidation with horseradish peroxidase and hydrogen peroxide and detected by ESR spectroscopy. A scheme has been proposed for the peroxidatic oxidation of BHA. The ESR spectrum of the di-BHA dimer, one product of BHA oxidation, has been observed, analyzed, and reported. ESR studies have been extended to other phenol derivatives structurally related to BHA and suspected to be substrates for peroxidase. Similarly it has been found that BHT and structurally related phenols are substrates for peroxidation by horseradish peroxidase and hydrogen peroxide. In agreement with previous chemical and biochemical studies, it has been found that ortho-disubstituted phenols are oxidized to more stable phenoxy radicals than are ortho-monosubstituted phenols. The ESR hyperfine coupling constants for the phenoxy radicals studied are in agreement with those for similar radicals produced by chemical oxidation. Attention has been drawn to the biochemical and toxicological implications of these and related studies of BHA and BHT peroxidation.

The metabolism of 17 beta-estradiol by lactoperoxidase: a possible source of oxidative stress in breast cancer.

Electron spin resonance (ESR) spectroscopy and oxygen consumption measurements using a Clark-type oxygen electrode have been used to study the metabolism of the estrogen 17 beta-estradiol by lactoperoxidase. Evidence for a one-electron oxidation of estradiol to its reactive phenoxyl radical intermediate is presented. The phenoxyl radical metabolite abstracts hydrogen from reduced glutathione generating the glutathione thiyl radical, which is spin trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and subsequently detected by ESR spectroscopy. In the absence of DMPO, molecular oxygen is consumed by a sequence of reactions initiated by the glutathione thiyl radical. Similarly, the estradiol phenoxyl radical abstracts hydrogen from reduced beta-nicotinamide-adenine dinucleotide (NADH) to generate the NAD. radical. The NAD. radical is not spin trapped by DMPO, but instead reduces molecular oxygen to the superoxide radical, which is then spin-trapped by DMPO. The superoxide generated may either spontaneously dismutate to form hydrogen peroxide or react with another NADH to form NAD., thus propagating a chain reaction leading to oxygen consumption and hydrogen peroxide accumulation. Ascorbate inhibits oxygen consumption when estradiol is metabolized in the presence of either glutathione or NADH by reducing radical intermediates back to their parent molecules and forming the relatively stable ascorbate radical. These results demonstrate that the futile metabolism of micromolar quantities of estradiol catalyzes the oxidation of much greater concentrations of biochemical reducing cofactors, such as glutathione and NADH, with hydrogen peroxide produced as a consequence. The accumulation of intracellular hydrogen peroxide could explain the hydroxyl radical-induced DNA base lesions recently reported for female breast cancer tissue.

The fate of the oxidizing tyrosyl radical in the presence of glutathione and ascorbate. Implications for the radical sink hypothesis.

Cellular systems contain as much as millimolar concentrations of both ascorbate and GSH, although the GSH concentration is often 10-fold that of ascorbate. It has been proposed that GSH and superoxide dismutase (SOD) act in a concerted effort to eliminate biologically generated radicals. The tyrosyl radical (Tyr.) generated by horseradish peroxidase in the presence of hydrogen peroxide can react with GSH to form the glutathione thiyl radical (GS.). GS. can react with the glutathione anion (GS-) to form the disulfide radical anion (GSSG-). This highly reactive disulfide radical anion will reduce molecular oxygen, forming superoxide and glutathione disulfide (GSSG). In a concerted effort, SOD will catalyze the dismutation of superoxide, resulting in the elimination of the radical. The physiological relevance of this GSH/SOD concerted effort is questionable. In a tyrosyl radical-generating system containing ascorbate (100 microM) and GSH (8 mM), the ascorbate nearly eliminated oxygen consumption and diminished GS. formation. In the presence of ascorbate, the tyrosyl radical will oxidize ascorbate to form the ascorbate radical. When measuring the ascorbate radical directly using fast-flow electron spin resonance, only minor changes in the ascorbate radical electron spin resonance signal intensity occurred in the presence of GSH. These results indicate that in the presence of physiological concentrations of ascorbate and GSH, GSH is not involved in the detoxification pathway of oxidizing free radicals formed by peroxidases.