Immunochemical investigation of UV-induced DNA damage

• Main Author: Cooke, Marcus S.
• Published: University of Leicester 1997
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696359

In this thesis, immunochemical and chromatographic techniques were employed to assess the relative contribution of direct/indirect mechanisms to DNA damage in a number of model systems by measuring lesions associated with each mechanism. Efforts to produce polyclonal and monoclonal antibodies to oxidative DNA damage suggested that profiles of antigenicity and immunogenicity arise dependent on the method of reactive oxygen species (ROS) production. Furthermore, an antiserum, raised against DNA damaged by an established ROS-generating system, was shown to recognise direct UV damage and was characterised with a previously developed antiserum to UV-damaged DNA. ELISA demonstrated the main epitopes recognised to be dimerised adjacent thymines, suggested to be cyclobutadithymine, in conjunction with a flanking sequence, specific to each antiserum. Employing a dimer-containing DNA standard, calibrated by GC-MS, the limits of detection for each antiserum were found to be 0.9 and 1.9 fmol, compared with 20-50 fmol for GC-MS. Such results confirm these antisera to be a sensitive approach for determining levels of UV damage in DNA, supported by their successful demonstration of cyclobutadithymine formation in naked DNA following UVC-irradiation. No discernible binding was seen to UVA-irradiated DNA, which was confirmed immunohistochemically in fixed and embedded sections of human skin. The qualitative induction of dimers following UVB-, and ROS lesions, following UVA-irradiation of cultured human keratinocytes, was shown using an antiserum to cyclobutadithymine and a novel affinity technique for oxidised purines. Application of an antibody to 8-oxodG and a novel antiserum to ROS-modified DNA, allowed quantitative ELISA measurements of dimeric and oxidative DNA changes to be made, following irradiation of keratinocytes. It was concluded that oxidative DNA damage, along with cyclobutadithymine, represents an appreciable contribution to total damage following UVC irradiation and that oxidative processes, with little contribution by direct damage, represent the major mechanism responsible for UVA-induced DNA damage.