Dopamine oxidation products inhibit Na+, K+-ATPase activity in crude synaptosomal-mitochondrial fraction from rat brain

The diverse damaging effects of dopamine (DA) oxidation products on brain subcellular components including mitochondrial electron transport chain have been implicated in dopaminergic neuronal death in Parkinson's disease. It has been shown in this study that DA (50-200 μM) causes dose-dependent inhibition of Na₊, K₊-ATPase activity of rat brain crude synaptosomal-mitochondrial fraction during in vitro incubation up to 2 h. The enzyme inactivation is prevented by catalase and the metal-chelator (diethylenetriamine penta-acetic acid) but not by superoxide dismutase or hydroxyl-radical scavengers like mannitol and dimethylsulphoxide (DMSO). Further, reduced glutathione and cysteine, markedly prevent DA-mediated inactivation of Na₊, K₊-ATPase. Under similar conditions of incubation, DA (200 μM) leads to the formation of quinoprotein adducts (protein-cysteinyl catechol) with synaptosomal-mitochondrial proteins and the phenomenon is also prevented by glutathione (5 mM) or cysteine (5 mM).

The available data imply that the inactivation of Na₊, K₊-ATPase in this system involves both H₂O₂ and metal ions. The reactive quinones by forming adducts with protein thiols also probably contribute to the process, since reduced glutathione and cysteine which scavenge quinones from the system protect Na₊, K₊-ATPase from DA-mediated damage. The inactivation of neuronal Na₊, K₊-ATPase by DA may give rise to various toxic sequelae with potential implications for dopaminergic cell death in Parkinson's disease.     

Bioactive phospholipid oxidation products

Oxidation of phospholipids results in chain-shortened fragments and oxygenated derivatives of polyunsaturated sn-2 fatty acyl residues, generating a myriad of phospholipid products. Certain oxidation products of phosphatidylcholine bind to and activate the human receptor for PAF, and these PAF-like lipids are potent, selective inflammatory mediators. Formation of PAF-like lipids is nonenzymatic and so their accumulation is unregulated. PAF-like lipids are produced in vivo in response to oxidative stresses and are responsible for attendant acute inflammatory responses. PAF-like lipids almost exclusively contain an ether-linked alkyl residue at the sn-1 position of the phosphatidylcholine backbone and molecular identification of these is facilitated by phospholipase A₁ treatment to remove the bulk of the inactive phospholipids. The identity of biologically active species generated by oxidative fragmentation and oxidation can be elucidated by understanding relevant reactions leading to the formation of PAF-like lipids, and then their structure can be established by tandem mass spectrometry and chemical synthesis.     

The formation of phosphatidylcholine oxidation products by stimulated phagocytes

Phagocytic cells produce a variety of oxidants as part of the immune defence, which react readily both with proteins and lipids, and could contribute to the oxidation of low density lipoprotein in atherosclerosis. We have investigated the oxidation of phospholipid vesicles by neutrophils and mononuclear cells, to provide a model of lipid oxidation in the absence of competing protein. Phorbol 12-myristate 13-acetate-stimulated neutrophils were incubated with phospholipid vesicles containing dipalmitoyl phosphatidylcholine, palmitoyl-arachidonoyl phosphatidylcholine (PAPC) and stearoyl-oleoyl phosphatidylcholine, before extraction of the lipids for analysis by HPLC coupled to electrospray mass spectrometry. The formation of monohydroperoxides (814 m/z) and bishydroperoxides (846 m/z) of PAPC was observed. However, the major oxidized product occurred at 828 m/z, and was identified as 1-palmitoyl-2-(5,6-epoxyisoprostane E₂)-sn-glycero-3-phosphocholine. These products were also formed in incubations where the neutrophils were replaced by mononuclear cells, and the amounts produced per million cells were similar. These results show that following oxidative attack by phagocytes stimulated by PMA, intact phospholipid oxidation products can be detected. The identification of an epoxyisoprostane phospholipid as the major product of phagocyte-induced phospholipid oxidation is novel, and in view of its inflammatory properties has implications for phagocyte involvement in atherogenesis.     

Secondary products of lipid oxidation

In the last decade, a multitude of secondary products have been identified from the radical and photosensitized oxidations of polyunsaturated lipids. These secondary products consist of oxygenated monomeric materials including epoxy-hydroperoxides, oxo-hydroperoxides, hydroperoxy epidioxides, dihydroperoxides, hydroperoxy bis-epidioxides, and hydroperoxy bicycloendoperoxides. More recently, higher molecular weight dimeric compounds have been identified from autoxidized methyl linoleate and linolenate. Decomposition of these oxidation products form a wide range of carbonyl compounds, hydrocarbons, furans, and other materials that contribute to the flavor deterioration of foods and that are implicated in biological oxidation. The interaction of some of these degradation products with DNA may be involved in cell-damaging reactions.     

Intracellular oxidation of hydroethidine: Compartmentalization and cytotoxicity of oxidation products

Hydroethidine (HE) is a blue fluorescent dye that is intracellularly converted into red-emitting products on two-electron oxidation. One of these products, namely 2-hydroxyethidium, is formed as the result of HE superoxide anion-specific oxidation, and so HE is widely used for the detection of superoxide in cells and tissues. In our experiments we exploited three cell lines of different origin: K562 (human leukemia cells), A431 (human epidermoid carcinoma cells), and SCE2304 (human mesenchymal stem cells derived from endometrium). Using fluorescent microscopy and flow cytometry analysis, we showed that HE intracellular oxidation products accumulate mostly in the cell mitochondria. This accumulation provokes gradual depolarization of mitochondrial membrane, affects oxygen consumption rate in HE-treated cells, and causes cellular apoptosis in the case of high HE concentrations and/or long cell incubations with HE, as well as a high rate of HE oxidation in cells exposed to some stimuli.     

The oxidation products of Haematoxylin and their role in biological staining

Synopsis Haematoxylin and its oxidation products were studied by t.l.c., and i.r., n.m.r., u.v. and visible spectroscopy. Oxidation (ripening) of Haematoxylin with oxygen, from the atmosphere or a cylinder, gave Haematein as the only major product; whereas with non-gaseous oxidants such as hydrogen peroxide, potassium permanganate and sodium iodate, further oxidation readily occurred. Such over-ripening always produced the same major product: a yellowish quinone-carboxylic acid, which we term Oxyhaematein. Oxyhaematein did not form a complex with aluminium ions under staining conditions but did act as an acid dye. Thus the deleterious effects of the overripening of Haematoxylin and the reported yellow-orange acidophil background staining sometimes seen with metal-Haematoxylin stains may both be explained. However, an additional, or alternative, explanation is available for this orange staining: acidic solutions of Haematein were found to react with acidophils of tissue sections to give an orange-brown product, not extracted by the processing fluids.     

Cholesteryl ester oxidation products in atherosclerosis

Lipid oxidation products are formed at sites of increased oxidant stress and have been shown to accumulate in atherosclerotic lesions. Although recent studies have focused on the formation and metabolism of oxidized lipids, very little is known about their biological activities and possible (patho)physiological functions. Oxidation of cholesteryl esters containing unsaturated fatty acids leads to the formation of hydroperoxides that are either reduced to alcohols or degrade into biologically active "core-aldehydes". In this review, the mechanisms of formation and metabolic fate of oxidized cholesteryl esters, their occurrence, as well as possible biological activities are discussed. Based on the current knowledge, cholesteryl ester oxidation leads to the formation of biologically active substances, which could actively contribute to the progression of atherosclerotic lesions and their resulting complications.