Nowdays there is a plethora of evidence for H₂O₂-mediated oxidative stress in the epidermis as well as in the system in patients with vitiligo (for review see (Schallreuter, Bahadoran et al. 2008). Xanthine dehydrogenase/xanthine oxidase (XDH/XO) catalyses the oxidative hydroxylation of hypoxanthine to xanthine followed by xanthine to uric acid, the last two steps in purine degradation pathway. Under oxidative conditions, XDH is converted to XO. The reactions catalysed by this enzyme generate H₂O₂ and O₂̇⁻, yielding in the presence of ROS accumulation, allantoin from uric acid. Therefore XO has been considered a major biologic source of oxygen-derived free radicals in many organs. The presence of XO in the human epidermis has not been shown so far. In this study several techniques were utilised to nail the presence and activity of XO in epidermal melanocytes and keratinocytes. The enzyme is regulated by H₂O₂ in a concentration dependent manner, where concentrations of 10-6M upregulate activity. Importantly, the results showed that the activity of XO is little affected by H₂O₂ in the mM range. H₂O₂-mediated oxidation of tryptophan and methionine residues in the sequence of XO yields only subtle alterations in the enzyme active site. These findings are in agreement with enzyme kinetics in the presence of 10-3M H₂O₂. Since uric acid is the end product of XO activity and this can be oxidised to allantoin by H₂O₂, we wanted to know whether allantoin is formed in the epidermis of patients with vitiligo. In order to address this issue, we utilised HPLC/mass spectrometry analysis. Analysis of epidermal cell extracts from suction blister tissue identified the presence of allantoin in patients with acute vitiligo, while this product was absent in healthy controls. In conclusion, our results provide evidence for functioning epidermal XO in the human epidermis which 4 can be a major source for the production of H₂O₂ contributing to oxidative stress in vitiligo. In addition, this thesis also demonstrates for the first time the presence of XO in melanosomes, and we showed that both 7BH4 and 7-biopterin inhibit XO activity in a concentration dependent manner. Moreover, XO has the potential to bind to 6/7BH4 and 6/7-biopterin from the pterin/tyrosinase inhibitor complex. This discovery adds another receptor independent mechanism for regulation of tyrosinase within the melanocyte similar to α/ß-MSH as shown earlier (Moore, Wood et al. 1999; Spencer, Chavan et al. 2005). Since the entire epidermis of patients with vitiligo is under H₂O₂-mediated oxidative stress, oxidative DNA damage would be highly expected. This thesis shows for the first time that epidermal 8-oxoG levels as well as plasma level of this oxidised DNA base are significantly increased in patients compared to healthy controls. We have shown that epidermal cells from patients with vitiligo respond to oxidative DNA damage via the overexpression of p21 and Gadd45α leading to a functioning increased short-patch base-excision repair (BER), while increased apoptosis can be ruled out due to lower caspase 3 and cytochrome c response compared to healthy controls. Our results show that patients develop effective DNA repair machinery via hOgg1, APE1 and DNA polymeraseß. Taking into consideration that these patients do not have an increased prevalence for solar-induced skin cancers, our data suggest that BER is a major player in the hierarchy to combat H₂O₂-mediated oxidative stress preventing ROS-induced tumourigenesis in the epidermis of these patients.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:553957 |
| Date | January 2009 |
| Creators | Shalbaf, Mohammad |
| Contributors | Schallreuter, Karin U. |
| Publisher | University of Bradford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10454/4326 |
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