Effects of reactive oxygen species on the cellular vacuolar apparatus

Detta är en avhandling från Linköping : Linköpings universitet

Sammanfattning: Reactive and potentially harmful oxygen species occur, and are continuously formed, in all cells of a living organism. Increased production of these metabolites are seen in a number of pathological states, for instance in inflammatory foci or during reoxygenation of ischemic tissue. Reactive oxygen species are considered to be associated with aging, the development of cancer and atherosclerosis.The main aim of this study was to investigate the effects of reactive oxygen species on the cellular vacuolar apparatus, in particlar lysosomes.It was found that neither superoxide radicals nor hydrogen peroxide alone had any damaging effect on lysosomes. With the presence of iron and, when hydrogen peroxide is concerned, a reducing agent damage to lysosomes was detected in the form of impaired proton gradient, leakage of lysosomal enzyme and lipid peroxidation, suggesting involvement of hydroxyl radicals.By refining and modifying an existing cytochemical method it was also possible to detect iron at both light- and electron microscopicallevel with preserved ultrastructure. Iron was mainly found in secondary lysosomes.It was also found that hydrogen peroxide-toxicity to cultured cells is iron dependent and could be much reduced by deferoxamine, an iron-chelator. This drug also prevented the hydrogen peroxide-mediated loss of the lysosomal proton gradient, suggesting intralysosomal Fentonreactions with generation of hydroxyl radicals.Acridine orange is an acidotropic photosensitizer which mainly accumulates in lysosomes. By exposing acridine orange-loaded cultured cells to blue light reactive oxygen, found to be singlet oxygen, was generated intralysosomally. This lead to loss of the proton gradient over the lysosomal membrane and a decrease in lysosomal cathepsin activity. Inhibition of lysosomal proteases lead to a reduction in acridine orange-mediated cell death suggesting a role for these enzymes in cell death. Electron microscopy revealed bleb-formation and increased autophagocytosis in photosensitized cells.

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