Jeremy P.E. Spencer

Conjugates of catecholamines with cysteine and GSH in Parkinson's disease: possible mechanisms of formation involving reactive oxygen species

Abstract: Oxidation of l‐3,4‐dihydroxyphenylalanine (l‐DOPA) and dopamine (DA) to generate semiquinones/quinones, oxygen radicals, and other reactive oxygen species may play a role in neuronal cell death in Parkinsons disease (PD). In particular, semiquinones/quinones can form conjugates with thiol compounds such as GSH and cysteine. Exposure of l‐DOPA, DA, and other catecholamines to a system generating O2•− radical led to O2•−‐dependent depletion of added GSH (or cysteine), accompanied by the formation of thiol‐DA or ‐DOPA adducts as detected by HPLC. Superoxide could additionally cause destruction of these adducts. Iron or copper ions could also promote conjugate formation between GSH or cysteine and DA and l‐DOPA, especially if H2O2 was present. We applied HPLC to measure glutathionyl and cysteinyl conjugates of l‐DOPA, DA, and 3,4‐dihydroxyphenylacetic acid (DOPAC) in postmortem brain samples from PD patients and normal control subjects. Conjugates were detected in most brain areas examined, but levels were highest in the substantia nigra and putamen. In most regions, adduct levels were lower in PD, but there were significant increases in cysteinyl adducts of l‐DOPA, DA, and DOPAC in PD substantia nigra, suggesting that acceleration of l‐DOPA/DA oxidation occurs in PD, although we cannot say if this is a primary feature of the disease or if it is related to therapy with l‐DOPA. In vitro, conjugate formation could be inhibited by the dithiol dihydrolipoate but not by its oxidised form, lipoic acid.

An evaluation of the antioxidant and antiviral action of extracts of rosemary and Provençal herbs.

Extracts of herbs and spices are increasingly of interest in the food industry because they retard oxidative degradation of lipids. There is also increasing interest in the antiviral activity of plant products. A liquid, deodorized rosemary extract and an oily extract of a mixture of Provençal herbs were tested for antioxidant and antiviral action in vitro. The rosemary extract (Herbor 025) and the extract of Provençal herbs (Spice Cocktail) inhibited peroxidation of phospholipid liposomes with 50% inhibition concentration values of 0.0009% (v/v) and 0.0035% (v/v), respectively. Herbor 025 and the spice cocktail (at 0.2%, v/v) reacted with trichloromethylperoxyl radical with calculated rates of 2.7 x 10(4) s-1 and 1.5 x 10(3) s-1, respectively. The main active components in the herbal preparations, carnosol and carnosic acid, at 0.05% (v/v) react with rate constants of (1-3) x 10(6) M-1 sec-1 and 2.7 x 10(7) M-1 sec-1, respectively. Both extracts show good antioxidant activity in the Rancimat test, especially in lard. Herbor 025 and the spice cocktail inhibited human immunodeficiency virus (HIV) infection at very low concentrations which were also cytotoxic. However, purified carnosol exhibited definite anti-HIV activity at a concentration (8 microM) which was not cytotoxic. Both preparations promoted some DNA damage in the copper-phenanthroline and the bleomycin-iron systems. The two herbal preparations possess antioxidant properties that may make them useful in the food matrix.

Inhibition of peroxynitrite dependent DNA base modification and tyrosine nitration by the extra virgin olive oil-derived antioxidant hydroxytyrosol

Hydroxytyrosol is one of the o-diphenolic compounds in extra virgin olive oil and has been suggested to be a potent antioxidant. The superoxide radical (O2*-) and nitric oxide (NO*) can react very rapidly to form peroxynitrite (ONOO ), a reactive tissue damaging species thought to be involved in the pathology of several chronic diseases. Hydroxytyrosol was highly protective against the peroxynitrite-dependent nitration of tyrosine and DNA damage by peroxynitrite in vitro. Given that extra virgin olive oil is consumed daily by many humans, hydroxytyrosol derived from this diet could conceivably provide a defense against damage by oxidants in vivo. The biological activity of hydroxytyrosol in vivo will depend on its intake, uptake and access to cellular compartments.

Protection against oxidative damage and cell death by the natural antioxidant ergothioneine.

The natural antioxidant ergothioneine (EGT) was tested for its ability to inhibit cell death caused by hydrogen peroxide (H2O2) and to inhibit DNA oxidation by peroxynitrite (ONOO-) in human neuronal hybridoma cell line (N-18-RE-105). High concentrations of EGT (5 mM) were tolerated by the N-18-RE-105 cells. N-acetylcysteine (NAC) was not well tolerated by the cells at concentrations greater than 3 mM (cell viability averaged 50%). Increasing concentrations of EGT increases cell viability in the presence of NAC. EGT at concentrations up to 2 mM weakly improved cell viability in the presence of H2O2. NAC at concentrations up to 2 mM weakly decreased, but not significantly, the viability of the cells. At a higher concentration of 5 mM, NAC weakly protected the neuronal cells against the H2O2-induced cell death. The protection was significantly enhanced by preincubation with EGT. Ergothioneine inhibited ONOO(-)-induced oxidative damage in isolated calf thymus DNA and DNA in N-18-RE-105 cells. The concentration of EGT in human and mammalian tissue has been estimated to be 1-2 mM, which suggests that EGT may serve as a non-toxic thiol buffering antioxidant in vivo and may find applications in pharmaceutical preparations where oxidative stability is desired.

Characterization of food antioxidants, illustrated using commercial garlic and ginger preparations

Abstract Methods for the characterization of antioxidants are presented and illustrated by their application to commercial garlic and ginger preparations, since it has been widely speculated that garlic and ginger might be beneficial to human health because they exert ‘antioxidant activity’. The sample of commercial ginger powder, tested at concentrations up to 5 mg/ml, inhibited the peroxidation of phospholipid liposomes, but a sample of one commercial garlic preparation was less effective. Both preparations could scavenge peroxyl radicals, but the garlic preparation was again less effective. The ginger and garlic preparations were powerful scavengers of hydroxyl radicals (OH) and were able to react with hypochlorous acid (HOCl) at a rate sufficient to protect catalase and alpha-l-antiproteinase against inactivation. However, they could also interact with iron chelates to facilitate OH generation from H 2 O 2 (‘pro-oxidant’ activity). Ginger (but not the garlic preparation) also exerted pro-oxidant action in the bleomycin assay, accelerating damage to DNA in the presence of a bleomycin-ferric iron complex. Our results illustrate the use of antioxidant characterization methods.

DNA damage in human respiratory tract epithelial cells: damage by gas phase cigarette smoke apparently involves attack by reactive nitrogen species in addition to oxygen radicals

Treatment of human respiratory tract tracheobronchial epithelial cells with gas‐phase cigarette smoke led to dose‐dependent DNA strand breakage that was highly correlated with multiple chemical modifications of all four DNA bases. The pattern of base damage suggests attack by hydroxyl radicals (OH:). However, by far the most important base damage in quantitative terms was formation of xanthine and hypoxanthine, presumably resulting from deamination of guanine and adenine respectively. Hence, DNA damage by cigarette smoke may involve reactive nitrogen species as well as reactive oxygen species.

DNA strand breakage and base modification induced by hydrogen peroxide treatment of human respiratory tract epithelial cells

Treatment of human respiratory tract epithelial cells with H2O2 led to concentration‐dependent DNA strand breakage that was highly‐correlated with multiple chemical modifications of all four DNA bases, suggesting that damage is due to hydroxyl radical, OH.. However, the major base damage occurred to adenine. Hence, conclusions made about the occurrence and the extent of oxidative DNA damage on the basis only of changes in 8‐hydroxyguanine should be approached with caution.