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 O2
¿-
, 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 ¿/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 Gadd45¿ 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:BRADFORD/oai:bradscholars.brad.ac.uk:10454/4326 |
| Date | January 2009 |
| Creators | Shalbaf, Mohammad |
| Contributors | Schallreuter, Karin U. |
| Publisher | University of Bradford, Centre of Skin Sciences, Clinical and Experimental Dermatology, Department of Biomedical Sciences. |
| Source Sets | Bradford Scholars |
| Language | English |
| Detected Language | English |
| Type | Thesis, doctoral, PhD |
| Rights | <a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/"><img alt="Creative Commons License" style="border-width:0" src="http://i.creativecommons.org/l/by-nc-nd/3.0/88x31.png" /></a><br />The University of Bradford theses are licenced under a <a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/">Creative Commons Licence</a>. |
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