Oxidized nucleotides as a predictor of radiation sensitivity

Abstract: The direct and indirect effects of IR can lead to DNA damage and activation of DNA repair. The indirect effects of ionizing radiation mediated by reactive oxygen species (ROS) which produced through radiolysis of water and if not taken care of by the antioxidant system, can also give rise to oxidative stress. ROS can affect the DNA or RNA directly or indirectly. Directly by causing damage to the DNA/RNA bases present in their structures. Indirectly by causing modifications of the dNTPs and rNTPs which later become incorporated into DNA or RNA. The presence of modified base in RNA seems to be less important as in a cell several thousand RNA transcripts of a gene are available. However, under severe oxidative stress, biological effects of oxidized rNTP or damaged RNA can become important. In the dNTP, one such modification is 8-oxo-dGTP, which can be incorporated in front of an A or a C and lead to mutation during replication. In our previous studies we have shown a correlation between serum/urinary 8-oxo-dG levels and individual radiosensitivity in breast and head and neck cancer patients receiving radiotherapy. We have also shown that a protein called MTH1 hydrolyses 8-oxo-dGTP to 8-oxo-dGMP. 8-Oxo-dGMP becomes dephosphorylated to 8-oxo-dG which can then excrete from the cells to the extracellular milieu. In our recent publication, included in the thesis, we aimed to investigate whether the oxidative stress marker, 8-oxo-dG, is a predictor of tumor response. We used modified ELISA, originally developed at Stockholm University, with a two-step filtration to analyze 8-oxo-dG in serum. The relationship between 8-oxo-dG levels and tumour response was studied in esophageal and gastric cancer patients who received radiotherapy and chemotherapy. In the radiotherapy and the merged radiotherapy and chemotherapy groups, the background levels of serum 8-oxo-dG were significantly lower in responder than in non-responder patients and the increments after treatment were greater. In comparison with patients whose serum 8-oxo-dG levels decrease after treatment, patients with increasing levels had a longer median “progression-free survival”. The results suggest that serum levels of 8-oxo-dG or oxidative stress response in general may potentially be used to predict the sensitivity and outcome of radiotherapy and chemotherapy of upper gastrointestinal tumours. Since the patient cohort is small more investigation is needed to validate the results.In our ongoing project we investigate cytoplasmic extracts from organs of irradiated mice; liver and brain. In this project we are trying to establish working protocols to measure the nucleotide pool imbalance and modifications arising from IR-induced ROS. We look at the possibilities of finding additional nucleotide pool modifications that can be used as stress biomarkers e.g. modified adenosines and other markers. We are establishing an HPLC method for detecting these modifications. Our results so far are that we can quantify the rNTPs, however the dNTPs are more difficult to detect in the cytoplasm isolated from organs. Our results also indicate a trend that inosine levels increase while adenosine decreases by irradiation of mice.  For our future studies inosine and adenosine are interesting to investigate. Also the proteins involved in the DNA damage and repair and oxidative stress pathways will be investigated in the liver and brain of irradiated mice.