Background: Exposure to low dose rate (LDR) radiation may accelerate aging processes. Previously, we identified numerous LDR-induced pathways involved in oxidative stress (OS) and antioxidant systems, suggesting that these pathways protect against premature senescence (PS). This study aimed to investigate if there are differences between young replicative senescent (RS) and PS cells considering DNA repair kinetics, OS, and DNA damage localized in the telomeres. Methods: We established PS cells by culturing and passaging young primary fibroblasts exposed to LDR. Then, RS cells were established by culturing and passaging young fibroblasts until they stopped proliferating. Senescence was characterized by analyzing telomere length and senescence-associated β-galactosidase (SA-β-gal) staining. DNA damage and repair were evaluated with γH2AX foci formation; telomere identification was carried out using the fluorescence in situ hybridization (FISH) probe; and oxidative stress was assessed by measuring 8-oxo-dG in the medium. Results: The data indicate the following: young cells have a better ability to cope with LDR-induced oxidative stress; RS and PS have higher steady-state levels of DNA damage; RS have slower DNA repair kinetics; and PS/RS have elevated levels of telomeric DNA damage. Conclusion: Our main conclusion is that PS and RS differ regarding DNA repair kinetics and SA-β-gal levels.

It is well-known that the cytotoxicity and mutagenic effects of high dose rate (HDR) ionizing radiation (IR) are increased by increasing the dose but less is known about the effects of chronic low dose rate (LDR). In vitro, we have shown that in addition to the immediate interaction of IR with DNA (the direct and indirect effects), low doses and chronic LDR exposure induce endogenous oxidative stress. During elevated oxidative stress, reactive oxygen species (ROS) react with DNA modifying its structure. Here, BL6 mice were exposed to IR at LDR and HDR and were then sacrificed 3 hours and 3 weeks after exposure to examine early and late effects of IR. The levels of micronuclei, MN, were determined in bone marrow cells. Our data indicate that the effects of 200 mGy on MN-induction are transient, but 500 and 1000 mGy (both HDR and LDR) lead to increased levels of MN up to 3 weeks after the exposure.

Reactive oxygen species (ROS) at a normal level are important molecules involved in several cellular processes including immune response and cell signalling. Overproduction of ROS may lead to elevated oxidative stress and consequently to age-related diseases. Most of the studies related to oxidative stress in humans have been done on blood samples. However, blood sampling might be painful, requires special qualified personnel, and has to be performed at medical centers. An alternative to blood is saliva. Saliva sampling is noninvasive and can be performed by the donor. Biomarker determination in saliva is becoming an important part of laboratory diagnosis, but method development is needed before it can be used in the clinics. In the present investigation, 16 donors performed extensive physical exercise by cycling and keeping their heart rate at 80% of maximum for 20 minutes. The physical activity was repeated 3 times: before tomato juice intake, after daily intake of 100 ml tomato juice during 3 weeks, and finally 3 weeks after finishing tomato juice intake (washout period). The level of the stress biomarker, salivary 8-oxo-dG, was determined before and after the physical activity. The results indicate that (a) 20 min extensive physical activity increases the level of 8-oxo-dG in saliva significantly (p=0.0078) and (b) daily intake of 100 ml tomato juice may inhibit (p=0.052) overproduction of salivary 8-oxo-dG by 20 min physical activity. We conclude that the 20 min extensive physical activity increases the level of salivary 8-oxo-dG in healthy donors and 100 ml daily intake of tomato juice may inhibit the increase of 8-oxo-dG in saliva.

The quality of the ionizing radiation (IR) can be described in terms of its nature, photons or particles, and their corresponding energies. The energy is classified in terms of High or Low linear energy transfer that will produce a different distribution of DNA damage and other molecules in the cell either by direct action or indirect action. Indirect action leads to the production of reactive oxygen species (ROS) modifying nucleotides in DNA or free dNTPs. 8-oxo-dGTP is formed through ROS endogenously when there is an imbalance between the antioxidants defence systems and the production of ROS levels in favour of ROS, leading to an oxidative stress condition. Organisms, organs, and cell types show different degrees of radiosensitivity, and this thesis aimed to investigate the underlying mechanisms of IR induced oxidative stress and its relation with radiosensitivity.

In previous studies, we identified proteins involved in radiation response with a focus on low dose radiation response. Cell models were established in which the expression of some protein/s was downregulated by knocking down/out using CRISPR/Cas9 or shRNA technology. The knockdown or knockout cells were exposed to different doses at low dose rates (LDR) or high dose rate (HDR) to investigate the role of these genes/proteins for survival (radiosensitivity), mutation induction, stress response, differentiation, etc. and they were subjected to further studies in this thesis.

Publication I, cell lines with hMTH1, and MYH knockdown were established and exposed to 0.5 and 1 Gy administered at different dose rates. We found that LDR induces significantly increased levels of extracellular 8-oxo-dG compared to HDR. We also found that hMTH1 and MYH play together an important role in the protection of cells against ROS-induced mutagenicity.

Publication II, the role of NRF2 was investigated for the radiosensitivity of glioblastoma cancer stem cells (CSCs). The neutrosphere cells from the U87MG cell line were irradiated with three different radiation qualities. The results show that cells exposed to LDR produce significantly higher levels of extracellular 8-oxo-dG compared to HDR and carbon ion irradiated cells. Lower proliferation, self-renewal, and neurosphere formation were observed in both LDR and HDR irradiated NRF2-knockdown cells as compared with the wild type. The results show that NRF2 plays an important role in the radiosensitivity of neurosphere cells isolated from the U87MG cell line.

Publication III, we examined the relation between 8-oxo-dG levels and the outcome of radiotherapy and chemotherapy in gastrointestinal cancer patients. The results showed that patients with improved treatment outcomes (responders), had lower levels of the stress marker extracellular 8-oxo-dG before the start of the treatment and the levels were increased 2 weeks after completing the treatment.

Publication IV, mice were whole-body irradiated with different doses administered at LDR and HDR. Three hours or three weeks after exposure, the immune cell populations in the spleens were phenotyped. The effects of dose, dose rate, and time after exposure and interaction between them were investigated to check which of the factors had the main effect on the change of immune cell populations. The results indicate that there was a pro-inflammatory short-term effect at high doses for both HDR and LDR. The results also indicate a pro-inflammatory effect of low doses of radiation three weeks after exposure.

The presence of glioma stem cells (GSCs), which are enriched in neurospheres, may be connected to the radioresistance of glioblastoma (GBM) due to their enhanced antioxidant defense and elevated DNA repair capacity. The aim was to evaluate the responses to different radiation qualities and to reduce radioresistance of U87MG cells, a GBM cell line. U87MG cells were cultured in a 3D model and irradiated with low (24 mGy/h) and high (0.39 Gy/min) dose rates of low LET gamma and high LET carbon ions (1-2 Gy/min). Thereafter, expression of proteins related to oxidative stress response, extracellular 8-oxo-dG, and neurospheres were determined. LD50 for carbon ions was significantly lower compared to LD50 of high and low dose rate gamma radiation. A significantly higher level of 8-oxo-dG was detected in the media of cells exposed to a low dose rate as compared to a high dose rate of gamma or carbon ions. A downregulation of oxidative stress proteins was also observed (NRF2, hMTH1, and SOD1). The NRF2 gene was knocked down by CRISPR/Cas9 in neurosphere cells, resulting in less self-renewal, more differentiated cells, and less proliferation capacity after irradiation with low and high dose rate gamma rays. Overall, U87MG glioma neurospheres presented differential responses to distinct radiation qualities and NRF2 plays an important role in cellular sensitivity to radiation.

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.

The level of oxidative stress is important in the initiation and progression of various age-related diseases, such as cancer. The level of oxidative stress may also play a significant role in cancer patients' response to treatment. We aimed to investigate whether serum 8-oxo-dG as a marker of oxidative stress is a predictor of tumour response. We used modified ELISA with a two-step filtration to analyse 8-oxo-dG in serum. The relationship between 8-oxo-dG levels, tumour response, and toxicity was studied in 19 oesophageal cancer patients who received radiotherapy and 16 gastric cancer patients who received chemotherapy. In the radiotherapy and the merged radio-and chemotherapy groups, the baseline levels of 8-oxo-dG were significantly lower in responder patients than in nonresponder 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. Our results, although preliminary, suggest that serum levels of 8-oxo-dG may potentially be used to predict the sensitivity and outcome of radiotherapy and chemotherapy of upper gastrointestinal tumours. Patients with 8-oxo-dG levels that are low prior to treatment and subsequently increase after treatment may be more likely to benefit from the therapy.

Our previous results showed that in addition to the immediate interaction of ionising radiation with DNA (direct and indirect effect), low-dose and chronic low-dose rate of irradiation induce endogenous oxidative stress. During oxidative stress, free radicals react with DNA, nucleoside triphosphates (dNTPs), proteins and lipids, and modify their structures. The MYH and MTH1 genes play important roles in preventing mutations induced by 8-hydroxy-guanine, which is an oxidised product of guanine. In this study, we used short-hairpin RNA to permanently knockdown MYH and MTH1 proteins in human lymphoblastoid TK6 cells. Knockdown and wild-type cells were chronically exposed to low dose rates of gamma-radiation (between 1.4 and 30 mGy/h). The cells were also subjected to acute doses delivered at a high-dose rate. Growth rate, extracellular 8-hydroxy-2'-deoxyguanosine, clonogenic cell survival and mutant frequencies were analysed in all cell types. A reduced level of cell growth and survival as well as increased mutant frequencies were observed in cells lacking both MYH and MTH1 proteins as compared to cells lacking only MYH and wild-type cells. To sum up, our results suggest that low-dose rates elevate oxidative stress. MTH1 together with MYH plays an important role in protection against mutations induced by modified dNTPs during chronic oxidative stress. In addition, we found no dose-rate effect at the level of mutations in the wild-type TK6 and MYH-KD cells. Our data interestingly indicate a dose-rate threshold for mutation induction in MTH1/MYH double knockdown cells.