DNA damage by reactive oxygen species (ROS) and its biological consequences
Reactive oxygen species (ROS) are generated in cells not only under the influence of xenobiotic agents (peroxides, photosensitizers, other oxidants) and radiation (UV, X-rays), but also endogenously, e.g. as by-products of the oxygen metabolism (Fig. 1). The ROS are genotoxic, i.e. they induce DNA modifications and subsequently mutations. Therefore, the cellular generation of ROS constitutes a serious threat to the integrity of the cellular genome, despite of the existence of efficient defense mechanisms (antioxidants, specific DNA repair), and it is supposed to be causally involved in the generation of cancer and other age-correlated diseases (Fig. 2). Our research aims at a better understanding of the genotoxicity of ROS and its role for the induction of cancer and other diseases.
Fig. 1: Mechanisms of DNA damage by reactive oxygen species (ROS). ROS are generated in cells both endogenously (oxygen metabolism) and exogenously (drugs, radiation).
Fig. 2: The basal "steady-state" levels of oxidative DNA modifications contribute to the generation of cancer and other diseases. The continuous generation of oxidative DNA modifications by ROS from the oxygen metabolism and the simultaneous repair by specific mechanisms result in basal "steady-state" levels of the modifications, which in the figure are shown as red ink an a box. The DNA modifications contribute to the spontaneous mutation frequency of the cells during replication, which can activate tumor genes and thereby initiate carcinogenesis. Other mutations, e.g. in the mitochondrial DNA, might affect the energy supply of the cells and thus cause degenerative diseases.
Specific Research Topics
1. Characterization of the gentoxic effects of drugs and other agents
In this area of research, we analyse the type and extent of the cellular oxidative DNA damage that is induced by various drugs (photosensitizers, radiomimetic drugs, oxidants) and correlate the DNA damage with those consequences that are relevant for carcinogenesis, such as induction of mutations and DNA repair. For this end, the the pattern of DNA modifications, the repair and the induction of mutations are determined in cultured mammalian cells. The influence of the chromatin structure on the damage generation and mutagenesis is of specific interest. (Examples of published results: O. Will et al., Mutat. Res. 435, 89 (1999); D. Ballmaier and B. Epe, Toxicology 221, 166 (2006))
2. Relevance of oxidative DNA damage for the spontaneous cancer incidence
The balance between generation of oxidative DNA modifications (most probably by endogenously generated reactive oxygen species) and their removal by specific repair enzymes results in steady-state levels of these lesions that can be detected in apparently all types of cells under physiological conditions (see Fig. 2). Since several of the oxidative DNA modifications are known to be mutagenic, increased steady-state levels should be associated with higher mutation rates and therefore more frequent tumor development. In our projects we want to test this hypothesis and thus assess the role of ROS for the initiation of carcinogenesis. In addition, we want to identify both cellular constituents and exogenous compounds that influence the steady-state levels of oxidative modifications in cultured cells and in vivo. This should allow to find and characterize new protective drugs. (Examples of published results: C. Trapp et al., Oncogene 26, 4044 (2007); C. Trapp et al, Cancer Res. 67, 5156 (2007))
3. Repair mechanisms of oxidative DNA base modifications
Oxidative DNA basemodifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) are repaired predominantly by base excision repair. However, our results indicated an unexpected relevance of other proteins, in particular Cockayne Syndrome B protein (CSB) and poly(ADP-ribosyl)polymerase 1 (Parp1). The underlying mechanisms remain to be established. In addition, the influence of changes of the chromatin structure on the repair mechanisms and kinetics are currently of specific interest . (Examples of published results: M. Osterod et al., Oncogene 21, 8232 (2002); C. Flohr et al., Nucleic Acids Res. 31, 5332 (2003))
4. Mechanisms and consequences of oxidative DNA damage induced by ultraviolet radiation and visible light
Sunlight gives rise to DNA damage by two mechanisms. On the one hand, DNA directly absorbs radiation in the UVC and UVB range of the spectrum (up to ~320 nm). The absorption gives rise to characteristic photoproducts, especially the formation of pyrimidine dimers. Their mutagenic properties have been well established. On the other hand, some so far unidentified cellular constituents (probably porphyrins or flavins) act as endogenous photosensitizers that react directly with DNA or give rise to the formation of ROS. These reactions result in oxidative DNA damage which is also mutagenic. The contribution of the indirect (photosensitizer mediated) mechanisms to the cancer risk induced by direct sun light is, as yet, not known and is therefore being investigated in this research field. It is anticipated that the indirect mechanisms will not be as effective as direct DNA excitation, but that they will make an important contribution to the genotoxicity of sunlight in the longer wavelength range where DNA has little or no absorption. (Examples of published results: C. Kielbassa et al., Carcinogenesis 18, 811 (1997); S. Hoffmann-Dörr et al., Mutat. Res. 572, 142 (2005)
5. Design of photosensitizers and targeting systems for photodynamic therapy
Photodynamic therapy, i.e. the irradiation of tumor tissue with laser light (plus fiber optics) after systemic application of a photosensitizer, is a promising new treatment for certain types of cancer. Suitable photosensitizers should absorb efficiently in the red range of the spectrum, generate singlet oxygen in high yields after excitation, not be toxic in the absence of light and accumulate in tumor cells. In the project, various novel types of photosensitizers (e.g. squaraines) are synthetised by a collaborating group (D. Ramaiah, India) and tested for their photobiological properties. In addition, it is planned to attach the photosensitizers to carrier systems that allow specific targeting to tumor cells. Example of published results: D. Ramaiah et al., Photochem. Photobiol. 79, 99 (2004)