Abstract
The neutrophil oxidative burst during inflammation is a main source of oxidants in vivo. As a result of neutrophil activation, the inflammatory oxidants produced include H2O2 and chlorinated oxidants (HOCl and chloramines). These oxidants have been linked to endothelial dysfunction, a key component of various inflammatory diseases such as cancer, atherosclerosis and obesity.
The relevance of chloramines to inflammatory disease is an area of great interest. Some chloramines have been shown to traverse membranes and are cytotoxic, reacting with a number of cell components, with particular preference for Cys and Met residues. Understanding which cellular targets are preferentially oxidised by chloramines could provide important clues as to how endothelial dysfunction might be mediated in the context of inflammation. The aim of this thesis was to explore the cellular thiol targets HOCl and cell-permeable chloramines (glycine chloramine, GlyCl; and monochloramine, NH2Cl) in an endothelial cell model. Specifically, the focus was to investigate each oxidant‘s effects on cells grown in culture, with treatment exposure kept to a minimum in order to specifically identify early protein thiol modifications which could be involved in the determination of cell fate and/or antioxidant protection.
Peroxiredoxins (Prxs, a family of thiol proteins known to detoxify cells against H2O2) in both endothelial cells and erythrocytes were shown to react with chloramines and HOCl, but accumulated in a different redox state than that seen with H2O2. Instead of becoming inactivated due to hyperoxidation, as observed with H2O2, Prxs were reversibly oxidised to disulfides and thus remained catalytically active following chloramine or HOCl treatment, suggesting that these enzymes may be important in detoxifying cells against chlorinated compounds. In addition, this study provided some evidence that Prx redox status could be involved in (or at least related to) cell viability in an endothelial model.
This study has also identified novel proteins which specifically underwent thiol redox modifications in cells briefly exposed to cell-permeable chlorinated oxidants. The two-dimensional SDS-PAGE approach used illustrated that many proteins underwent thiol oxidation following treatment, and a subset of these proteins were selected and identified by mass spectrometry. Many of the proteins identified by this method are known to participate in antioxidant protection of cells and/or in apoptotic cell signalling, which makes them worthy of further exploration.
Finally, haem oxygenase-1 (HO-1), another important antioxidant enzyme with strong links to inflammatory disease, was shown to be highly induced in cells exposed to low doses of NH2Cl. Surprisingly, nuclear translocation of nuclear factor erythroid-2-related factor 2, one of the major transcription factors known to induce HO-1 expression, could not be detected. The highly sensitive HO-1 expression in response to chloramine treatment, in addition to its known roles in protecting cells against inflammatory tissue injury, mean that HO-1 is likely involved in protecting cells against oxidative injury during inflammation.
This study has explored a number of thiol proteins which undergo redox modification in response to exposure to chlorinated oxidants. The identification and characterisation of these targets helps to understand the inflammatory condition and the roles specific to the various chlorinated oxidants. Further understanding of the processes involved in inflammatory disease will assist in directing future studies toward the development of therapeutics for preventing inflammatory tissue damage.