Publications

It has been suggested in the literature that neuroleptic drugs may be able to exert antioxidant and/or pro-oxidant actions in vivo. The feasibility of this was tested by measuring the ability of chlorpromazine, prochlorperazine, metoclopramide, methotrimeprazine and haloperidol to scavenge biologically relevant oxygen-derived species in vitro. None of the drugs reacted with superoxide radical at a significant rate. Chlorpromazine, prochlorperazine, metoclopramide and methotrimeprazine were very powerful scavengers of hydroxyl radicals, reacting at almost a diffusion-controlled rate. Chlorpromazine showed some ability to inhibit iron ion-dependent hydroxyl radical formation. Chlorpromazine, methotrimeprazine, promethazine and prochlorperazine were powerful inhibitors of iron ion-dependent liposomal lipid peroxidation, scavengers of organic peroxyl radicals and inhibitors of haem protein/hydrogen peroxide-dependent peroxidation of arachidonic acid. Chlorpromazine, prochlorperazine, metoclopramide, methotrimeprazine and haloperidol were powerful scavengers of hypochlorous acid. Haloperidol showed no ability to inhibit lipid peroxidation or to scavenge peroxyl radicals, and reproducibly increased lipid peroxidation catalysed by haem proteins, in both the presence and absence of hydrogen peroxide. The relevance of these in vitro observations to events in vivo is discussed.

The reaction of ozone (O 3 ) with human blood plasma was studied to help understand possible events that could occur in the respiratory tract. Uric acid (quantitatively the most important scavenger) and ascorbic acid were oxidized quickly, protein‐SH groups were lost more slowly, and there was no loss of bilirubin or α‐tocopherol. There was little formation of lipid hydroperoxides and no detectable formation of 4‐hydroxynonenal, hexanal or nonanal, or changes in lipoprotein electrophoretic mobility. Uric acid in human upper airway secretions may play a significant role in removing inhaled O 3 . Oxidative damage to lipids must not be assumed to be the key mechanism of respiratory tract O 3 toxicity.

Peroxynitrite (ONOO−) is a powerful oxidant and cytotoxic species formed by the rapid reaction between nitrogen monoxide (nitric oxide, . NO) and superoxide (O . 2 −). At neutral pH ONOO− is partly protonated and this protonated form, peroxynitrous acid (ONOOH), decomposes rapidly to nitrate, forming (an) intermediate(s) with reactivity similar to . OH and . NO2. Peroxynitrite can hydroxylate and nitrate aromatic rings, and aromatic nitration of phenols such as tyrosine by ONOOH is proposed to proceed via a radical mechanism, with intermediate formation of . NO2. Modification of tyrosine by . NO2 also involves nitration via a radical mechanism. Aromatic nitration of phenols by ONOO− has been shown to be enhanced by superoxide dismutase or Fe3+-EDTA, which were proposed to catalyze heterolytic cleavage of ONOOH to form a nitrating species similar to the nitronium ion NO+ 2. We investigated possible mechanisms of tyrosine modification by various reactive nitrogen species, including ONOO−, 3-morpholinosydnonimine (SIN-1), and . NO2. Reaction of tyrosine with ONOO− leads to formation of 3-nitrotyrosine and dityrosine, indicating intermediate formation of tyrosyl radicals. The pH dependence of formation of both 3-nitrotyrosine and dityrosine by ONOO− suggests that intermediate formation of ONOOH is required. Qualitatively similar results were obtained when ONOOH was generated continuously by H2O2 and NaNO2 at mildy acidic pH or with SIN-1, a compound which at neutral pH releases both . NO and O . 2 −, presumably producing ONOO−. However, relatively low yields of nitrotyrosine were obtained with SIN-1, possibly because of competing reactions of tyrosyl radicals with . NO or O . 2 −. Possible involvement of . NO2 in tyrosine modification by ONOO− was studied using hydroxyl radical scavengers, which can increase the radical yield during decomposition of ONOOH and thereby enhance generation of . NO2. Hydroxyl radical scavengers did not affect tyrosine modification by . NO2 directly and slightly inhibited tyrosine modification by authentic ONOO−. However, when ONOO− was produced at a slower rate, either by SIN-I or by H2O2/NaNO2 at acidic pH, hydroxyl radical scavengers were found to significantly enhance tyrosine nitration. Our results suggest that ONOO− or ONOO−-generating systems induce nitration of tyrosine (or tyrosine residues in proteins) via intermediate formation of tyrosyl radicals and . NO2.

Considerable data implicate oxidative damage in influenza pathogenesis. We examined temporal changes in oxidative damage using accurate biomarkers in an adult cohort with acute influenza infection and their relationships with clinical parameters. Clinical information and blood samples were collected during their acute illness and 3 months later. A fatigue questionnaire was administered 3 months following influenza infection. Thirty-five patients (mean age, 34 years) with polymerase chain reaction-confirmed influenza A infection were included; all patients returned for follow-up assessments. Adjusted levels of plasma F2-isoprostanes, total hydroxyeicosatetraenoic products (HETEs), 7β-hydroxycholesterol and 7-ketocholesterol, serum gamma-glutamyltransferase, and high-sensitivity C-reactive protein (hsCRP) were increased during the acute illness compared with age-matched controls. Despite clinical recovery, levels of these biomarkers remained higher at month 3 compared with controls. A proportion of patients had persistent symptoms such as fatigue (23%), myalgia (14%), and arthralgia (11%) at month 3. Patients with significant fatigue had higher baseline levels of plasma F2-isoprostanes, F4-neuroprostanes, and total HETEs compared to those without fatigue. By contrast, patients with persistent arthralgia and myalgia had higher baseline levels of serum hsCRP compared to those without these symptoms. Our observations lead to the hypothesis that oxidative damage participates in the pathogenesis of influenza infection and postinfectious fatigue.

Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H₂0₂), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H₂0₂ production. H₂0₂ gene ration from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37°C under 95% air - 5% C0₂ was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferrioxamine, but partial inhibition by EDTA and diethylenetriaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37°C led to an upward drift of pH, even under an atmosphere of 95% air - 5% C0₂. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H₂0₂ generated by ascorbate and phenolic compounds, but there was still substantial H₂0₂ generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H₂0₂ generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H₂0₂ generation is discussed.

Plasma from patients with iron overload resulting from idiopathic hemochromatosis contains nontransferrin-bound iron, measurable by the bleomycin assay. During venesection therapy, the concentration of bleomycin iron declines in a way highly correlated with plasma ferritin concentrations. Even when patients had been venesected to give very low total plasma iron concentrations and high transferrin iron-binding capacity, bleomycin-detectable iron was still present at low concentrations. Bleomycin-detectable iron can stimulate damaging free radical reactions, and its persistence in plasma even after prolonged venesection might contribute to the tissue damage that results from iron overload.