Redox processes and oxidative stress in cell and tissue damage

University dissertation from Stockholm : Karolinska Institutet, Dept of Laboratory Medicine

Abstract: Oxidative stress is described as an imbalance between oxidants and antioxidants, with a shift towards the oxidants. When the antioxidative systems are insufficient or the production of radicals is increased oxidative damage can occur, damaging DNA, lipids, and proteins. Oxidative stress has been implicated in several pathologies. This study has been focused on oxidative damage caused to the cysteine residues on proteins. The main function of the thioredoxin (Trx) and the glutaredoxin (Grx) systems is to reduce protein thiols and they therefore play an important role in the protection against oxidative stress. Alzheimer’s disease is a progressive disease with a high prevalence in an aging population. Oxidative stress has been implicated in the disease, as demonstrated by elevated levels of oxidized proteins, lipids, carbohydrates and nucleic acids. In our study we were able to show that Trx and Grx were secreted to CSF, and that the levels of the proteins correlated with the previously validated markers tau and p-tau in patients with different stages of AD. The secretion was not caused by cell death as the levels of lactate dehydrogenase did not change between the different stages. Furthermore, decreased axonal staining of Grx1 and Grx2, as well as decreased mitochondrial staining of Trx2 was observed in AD hippocampus. Parkinson’s disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra. The neurotransmitter dopamine has been implicated in the pathology, as rupture of the dopamine vesicles leads to increased cytosolic dopamine levels. Free in the cytosol, dopamine gets oxidized with a release of radicals in the process. We can show a direct interaction between the Trx system and the dopamine-quinone. Furthermore, a protective effect of TrxR against dopamine toxicity was observed in both the cell line SH-SY5Y and in the nematode C. elegans. In addition, decreased levels of Trx1 and TrxR1 were observed in substantia nigra from PD patients. Ischemia causes a conversion of xanthine dehydrogenase into xanthine oxidase, upon reperfusion xanthine oxidase releases radicals, leading to an increased oxidative stress in the tissues. Ischemia/reperfusion was induced in human livers, and samples were taken before induction of ischemia, after ischemia and after reperfusion. We demonstrated that the major reperfusion damage was to the sinusoidal endothelial lining, where a retraction of the lining occurred after ischemia, but after 20 minutes of reperfusion the cell lining had partially recovered. No significant changes in mRNA levels of redox proteins could be observed, but likely redistribution of Trx occurred in the hepatocytes. Cell lines are commonly used in medical research, but a consensus in how to culture the cells is lacking. In order to study the effect of media selection, three different cell lines were cultured in four commonly used cell culturing media. We could observe increased proliferation of cells grown in the high glucose containing DMEM. Increased expression for mesenchymal marker Vimentin in A549 cells cultured in DMEM, and decreased expression of epithelial marker CK18, which indicates a possible change in phenotype due to the selection of media. Furthermore, increased enzymatic activity of TrxR in cells cultured in MEM compared to the other media was observed, and decreased selenite toxicity in cells cultured in DMEM compared to culture in RPMI or F12. This study highlights the importance of consistency in the choice of cell culturing media for the outcome of any cell experiment. In conclusion, the thioredoxin family of proteins has been shown to be implicated in several pathological conditions where oxidative stress is believed to be important.

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