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Iron plays a potential role in oxidative stress-mediated injuries and pathologies e.g. rheumatoid arthritis (RA). Four decades ago it was suggested that iron may have a crucial role in the progression of inflammation in RA. Indeed, free radicals generated by iron can cause damage to lipids, proteins, carbohydrates, and DNA. It is this destructive process that is believed to occur in rheumatoid joints. However, none had differentiated between the role of iron in both acute and chronic phases of the disease and the origin of this 'labile' iron. Since RA cells are chronically exposed to oxidative stress, we have therefore chosen Jurkat cells to be our cell model. We used the parental (J16) cell line was used to mimic the acute phase of oxidative stress and the H2O2-resistant (HJ16) cells to mimic the chronic phase. By using hydrogen peroxide (H2O2) as the oxidising agent, we aim to study the role of iron in acute and chronic phase of oxidative stress and to know its origin. In the present study, we found that both antioxidants and H2O2-induced labile iron are modulated when cells are chronically exposed to H2O2. HJ16 cells contain higher total intracellular glutathione levels and glutathione peroxidase activity than J16 cells while the superoxide dismutase and catalase activity are similar. Haem oxygenase-1 (HO-1) was not detectable nor was it induced in these cell lines; HO-2 on the other hand was expressed but not induced. Although they had the same ‘basal’ LIP and L-Ft levels, J16 cells contain more than 7-fold higher H-Ft levels than in HJ16 cells. It was also found that H2O2-induced labile iron is directly correlated with necrotic cell death. These results are consistent with the conclusion that both antioxidant defence mechanism and labile iron status are modulated in cells chronically exposed to H2O2. We have also shown that the ‘basal’ and ‘H2O2-induced’ NFκB activation was higher in the HJ16 cells. We have also provided a link between labile iron release, lysosomal membrane damage and the ensuing necrotic cell death following H2O2 treatment.

Cadmium (Cd), is a non-essential metal with no known physiological function. It is known to alter redox state by disrupting the mitochondrial electron transport chain, as well as inactivating protein and non-protein thiols. It is thus believed that oxidative stress may comprise an important part of the mechanism of Cd toxicity. Accordingly, the initial cellular response to acute Cd exposure is defensive, where various anti-oxidant defence systems are triggered. One of the induced systems is the haem oxygenase-1 (HO-1). Its activation is mediated by the transcription factor Nrf2, which is the general regulator of cellular defence against oxidative stress. The protective effects of HO-1 are mediated, in part, through the generation of potent anti-oxidant bilirubin (BR) and its metabolites, which exploit the intrinsic antioxidant properties of these species at a cellular level. The oxidative metabolism of BR is an important route of detoxification in addition to glucuronidation. However, the major enzyme(s) involved in this oxidative degradation are not known. This thesis presents evidence for a major role of the hepatic cytochrome P450 2a5 (Cyp2a5) in BR degradation during Cd intoxication, where the BR levels are elevated following induction of HO-1. Treatment of DBA/2J male mice with CdCl2 induced both the Cyp2a5 and HO-1, and increased the microsomal BR degradation activity. By way of contrast, the total cytochrome P450 (CYP) content and the expression of Cyp1a2 were down-regulated by the treatment. The induction of the HO-1 and Cyp2a5 was significant at the mRNA, protein and enzyme activity levels. In each case, the up-regulation of the HO-1 preceded that of the Cyp2a5 with a 5-10 hr interval. In addition, BR totally inhibited the microsomal coumarin hydroxylase activity (a Cyp2a5-catalysed reaction) with an IC50 approximately equal to the substrate concentration. The MROD activity, catalysed mainly by the Cyp1a2, was inhibited up to 36% by BR. The microsomal BR degradation was inhibited by coumarin and by a monoclonal antibody against the Cyp2a5 by about 90%. In addition, 7-methoxyresorufin, a substrate for Cyp1a2, inhibited BR degradation activity by approximately 20%. A study using Nrf2 null mutant mice suggests that Cd-mediated induction of Cyp2a5 is dependent on the transcription factor Nrf2. Additionally, acute exposure to Cd activated localisation of Nrf2 from the cytoplasm to the nucleus. Furthermore, electrophoretic mobility shift assay (EMSA) analysis suggests that Cd induced sequence-specific binding of various species of the StRE-binding proteins on the 5’-flanking region of the Cyp2a5 gene. Collectively, these observations strongly suggest that BR may act as a substrate for the hepatic Cyp2a5, a major catalyst for BR degradation under conditions of substantial elevation of BR levels following induction of HO-1 by Cd. Secondly, the concurrent up-regulation of the HO-1 and Cyp2a5 during Cd-mediated injury implicates a coordinated regulation of two enzyme systems in the maintenance of balancing BR production and elimination. Finally, StRE-binding proteins, in particular Nrf2, may be involved in the regulation of the Cyp2a5 gene, which leads to the oxidation of BR. However, the respective roles of these factors in the regulation of the Cyp2a5 gene, as well as the coordinated regulation of ho-1 and Cyp2a5 genes remain an open question, requiring further investigations.