Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader.

Free radicals and other so-called 'reactive species' are constantly produced in the brain in vivo. Some arise by 'accidents of chemistry', an example of which may be the leakage of electrons from the mitochondrial electron transport chain to generate superoxide radical (O2*-). Others are generated for useful purposes, such as the role of nitric oxide in neurotransmission and the production of O2*- by activated microglia. Because of its high ATP demand, the brain consumes O2 rapidly, and is thus susceptible to interference with mitochondrial function, which can in turn lead to increased O2*- formation. The brain contains multiple antioxidant defences, of which the mitochondrial manganese-containing superoxide dismutase and reduced glutathione seem especially important. Iron is a powerful promoter of free radical damage, able to catalyse generation of highly reactive hydroxyl, alkoxyl and peroxyl radicals from hydrogen peroxide and lipid peroxides, respectively. Although most iron in the brain is stored in ferritin, 'catalytic' iron is readily mobilised from injured brain tissue. Increased levels of oxidative damage to DNA, lipids and proteins have been detected by a range of assays in post-mortem tissues from patients with Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis, and at least some of these changes may occur early in disease progression. The accumulation and precipitation of proteins that occur in these diseases may be aggravated by oxidative damage, and may in turn cause more oxidative damage by interfering with the function of the proteasome. Indeed, it has been shown that proteasomal inhibition increases levels of oxidative damage not only to proteins but also to other biomolecules. Hence, there are many attempts to develop antioxidants that can cross the blood-brain barrier and decrease oxidative damage. Natural antioxidants such as vitamin E (tocopherol), carotenoids and flavonoids do not readily enter the brain in the adult, and the lazaroid antioxidant tirilazad (U-74006F) appears to localise in the blood-brain barrier. Other antioxidants under development include modified spin traps and low molecular mass scavengers of O2*-. One possible source of lead compounds is the use of traditional remedies claimed to improve brain function. Little is known about the impact of dietary antioxidants upon the development and progression of neurodegenerative diseases, especially Alzheimer's disease. Several agents already in therapeutic use might exert some of their effects by antioxidant action, including selegiline (deprenyl), apomorphine and nitecapone.

Role of free radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment.

Polyunsaturated fatty acid (PUFA) levels (an index of the amount of substrate available for lipid peroxidation) were measured in several brain regions from patients who died with Parkinson's disease and age-matched control human postmortem brains. PUFA levels were reduced in parkinsonian substantia nigra compared to other brain regions and to control tissue. However, basal malondialdehyde (MDA; an intermediate in the lipid peroxidation process) levels were increased in parkinsonian nigra compared with other parkinsonian brain regions and control tissue. Expressing basal MDA levels in terms of PUFA content, the difference between parkinsonian and control substantia nigra was even more pronounced. Stimulating MDA production by incubating tissue with FeSO4 plus ascorbic acid, FeSO4 plus H2O2, or air alone produced lower MDA levels in the parkinsonian substantia nigra, probably reflecting the lower PUFA content. These results may indicate that an increased level of lipid peroxidation continues to occur in the parkinsonian nigra up to the time of death, perhaps because of continued exposure to excess free radicals derived from some endogenous or exogenous neurotoxic species.

Basal Lipid Peroxidation in Substantia Nigra Is Increased in Parkinson's Disease

Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson's disease.

Metabolism of the neurotoxic tertiary amine, MPTP, by brain monoamine oxidase.

The mechanism of cytotoxicity of 6-hydroxydopamine, 2,4,5-trihydroxyphenylalanine, dopa, dopamine, norepinephrine, and epinephrine was explored by asking whether cytotoxicity was a reflection of the potential for autoxidation of each polyphenol or of the sulfhydryl reactivity of its quinone products. The cytotoxicity of the polyphenols, as measured by inhibition of [3H]thymidine incorporation into DNA by C1300 neuroblastoma cells in tissue culture, correlated with the rate of autoxidation, as measured spectrophotometrically or by oxygen electrode studies. Polarographic determinations of the oxidation potentials of the polyphenols were also predictive of cytotoxicity; the most cytotoxic compounds had the most negative half-wave potentials and thus were the most readily oxidized. By contrast, the sulfhydryl reactivity of the quinone oxidation products of the polyphenols, as measured by inhibition of purified calf thymus DNA polymerase α, exhibited an inverse relationship to the cytotoxicity of the polyphenols; the most toxic compounds, 6-hydroxydopamine and 2,4,5-trihydroxyphenylalanine, were oxidized to the least reactive quinone products. An alternative mechanism of toxicity was observed with N -acetyldopamine, which was oxidized to 4-(2- N -acetylaminoethyl)-1,2-benzoquinone, a potent sulfhydryl reagent. N -Acetyldopamine was more toxic than predicted by its half-wave potential or its rate of autoxidation. Furthermore, while norepinephrine completely neutralized 6-hydroxydopamine and 2,4,5-trihydroxyphenylalanine as cytotoxic agents, the toxicity of N -acetyldopamine was minimally affected. Thus we conclude that 6-hydroxydopamine and 2,4,5-trihydroxyphenylalanine kill cells through the production of H2O2, O2[unknown], and OH·, while for dopamine and dopa the reaction of quinone oxidation products with nucleophiles probably also contributes to their cytotoxicity.

Autoxidation versus covalent binding of quinones as the mechanism of toxicity of dopamine, 6-hydroxydopamine, and related compounds toward C1300 neuroblastoma cells in vitro

Sylvia M. Tiffany

Papers

Autoxidation versus covalent binding of quinones as the mechanism of toxicity of dopamine, 6-hydroxydopamine, and related compounds toward C1300 neuroblastoma cells in vitro

The Toxicity of Melanin Precursors