Cellular effects of ionizing radiation : Relevant for understanding cancer risk after medical and environmental radiation exposures

Radiation-induced cancers are stochastic and delayed effects of exposure to ionizing radiation. The dose-response relationship for radiation-induced cancers at both low dose/low dose rates and high doses (doses encountered during radiotherapy) remains unclear. Uncertainties observed in epidemiological studies at low doses and dose rates hamper cancer risk estimation at this dose level. Assessing dose-response relationships for radiotherapy-induced cancers is also complicated due to the inherent difficulty in assessing the doses absorbed by tissues at the site of tumours. In addition, the modulatory effect of chemotherapy on the incidence of radiotherapy-induced cancer risk has been debated. Although included as a modifying factor of the incidence of radiotherapy-induced cancers, results from epidemiological studies do not provide sufficient evidence to support this claim. This thesis summarizes studies conducted to improve the understanding of the association of cancer incidence and radiation dose at clinically relevant (high) doses, low doses and low dose rates, as well as the modulatory role of platinum-based chemotherapy on radiation-induced carcinogenesis.  In Paper I, we investigated the competitive relationship between cell killing and the accumulation of DNA damage and genomic instability using two normal cell types (VH10 fibroblasts and AHH-1 lymphoblasts). Dose fractionation schemes were designed based on the cell growth characteristics of each cell type. Cells were irradiated at 0.25, 0.5, 1.0, or 2 Gy per fraction, representing the various dose levels within a radiation field, to simulate the heterogeneous dose distribution across normal tissue during radiotherapy. Following fractionated radiation exposure, the effects on cell growth, cell survival, radiosensitivity, and the accumulation of residual DNA damage and genomic instability were analyzed as a function of dose per fraction and the total absorbed dose. The accumulation of DNA damage and markers of genomic instability associated with DNA damage depended on cell type-specific factors. In Paper II, we investigated the modulatory effects of combining cisplatin and radiation on the accumulation of micronuclei (a biomarker of DNA damage and carcinogenesis) in peripheral blood lymphocytes of patients receiving treatment for gynaecological cancers. We also determined the modulatory effects of the combination of both agents on cell death and cell proliferation, by scoring the frequency of apoptotic and binucleated cells. We compared the frequency of these markers between patients receiving treatment with radiotherapy alone and a combination of cisplatin and radiotherapy. There was a decline in the frequency of micronuclei in patients receiving a combination of cisplatin and radiotherapy. We conducted in vitro experiments in Paper III using AHH-1 and VH10 cells. We investigated the effects of the concurrent combination of cisplatin treatment and multifractionated radiation exposure at 1 Gy per fraction on cell growth, cell survival, cell death, changes in radiosensitivity, accumulation of DNA damage, and other markers of genomic instability as well as the expression of cancer stem cell markers. We also investigated the interaction between cisplatin and radiation exposure in our schedule. The concurrent combination of cisplatin and radiation did not increase the accumulation of markers of genomic instability. In Paper IV, we investigated the short and long-term effects of radiation exposure at low doses and low dose rates on global gene expression, cell growth and cell survival of VH10 fibroblasts to identify unique dose rate signatures that could be useful biomarkers in determining if the application of DDREF is accurate. Except for the differential expression of DMXL2, the long-term effects of LDLDR exposure on global gene expression, cell growth and cell survival of VH10 fibroblasts were negligible. These results suggest that the accumulation of DNA damage and other markers of genomic instability is regulated by cell type-specific factors at these dose levels

Cellular effects of ionizing radiation : Relevant for understanding cancer risk after medical and environmental radiation exposures

Radiation-induced cancers are stochastic and delayed effects of exposure to ionizing radiation. The dose-response relationship for radiation-induced cancers at both low dose/low dose rates and high doses (doses encountered during radiotherapy) remains unclear. Uncertainties observed in epidemiological studies at low doses and dose rates hamper cancer risk estimation at this dose level. Assessing dose-response relationships for radiotherapy-induced cancers is also complicated due to the inherent difficulty in assessing the doses absorbed by tissues at the site of tumours. In addition, the modulatory effect of chemotherapy on the incidence of radiotherapy-induced cancer risk has been debated. Although included as a modifying factor of the incidence of radiotherapy-induced cancers, results from epidemiological studies do not provide sufficient evidence to support this claim. This thesis summarizes studies conducted to improve the understanding of the association of cancer incidence and radiation dose at clinically relevant (high) doses, low doses and low dose rates, as well as the modulatory role of platinum-based chemotherapy on radiation-induced carcinogenesis.  In Paper I, we investigated the competitive relationship between cell killing and the accumulation of DNA damage and genomic instability using two normal cell types (VH10 fibroblasts and AHH-1 lymphoblasts). Dose fractionation schemes were designed based on the cell growth characteristics of each cell type. Cells were irradiated at 0.25, 0.5, 1.0, or 2 Gy per fraction, representing the various dose levels within a radiation field, to simulate the heterogeneous dose distribution across normal tissue during radiotherapy. Following fractionated radiation exposure, the effects on cell growth, cell survival, radiosensitivity, and the accumulation of residual DNA damage and genomic instability were analyzed as a function of dose per fraction and the total absorbed dose. The accumulation of DNA damage and markers of genomic instability associated with DNA damage depended on cell type-specific factors. In Paper II, we investigated the modulatory effects of combining cisplatin and radiation on the accumulation of micronuclei (a biomarker of DNA damage and carcinogenesis) in peripheral blood lymphocytes of patients receiving treatment for gynaecological cancers. We also determined the modulatory effects of the combination of both agents on cell death and cell proliferation, by scoring the frequency of apoptotic and binucleated cells. We compared the frequency of these markers between patients receiving treatment with radiotherapy alone and a combination of cisplatin and radiotherapy. There was a decline in the frequency of micronuclei in patients receiving a combination of cisplatin and radiotherapy. We conducted in vitro experiments in Paper III using AHH-1 and VH10 cells. We investigated the effects of the concurrent combination of cisplatin treatment and multifractionated radiation exposure at 1 Gy per fraction on cell growth, cell survival, cell death, changes in radiosensitivity, accumulation of DNA damage, and other markers of genomic instability as well as the expression of cancer stem cell markers. We also investigated the interaction between cisplatin and radiation exposure in our schedule. The concurrent combination of cisplatin and radiation did not increase the accumulation of markers of genomic instability. In Paper IV, we investigated the short and long-term effects of radiation exposure at low doses and low dose rates on global gene expression, cell growth and cell survival of VH10 fibroblasts to identify unique dose rate signatures that could be useful biomarkers in determining if the application of DDREF is accurate. Except for the differential expression of DMXL2, the long-term effects of LDLDR exposure on global gene expression, cell growth and cell survival of VH10 fibroblasts were negligible. These results suggest that the accumulation of DNA damage and other markers of genomic instability is regulated by cell type-specific factors at these dose levels

Impact of fractionated cisplatin and radiation treatment on cell growth and accumulation of DNA damage in two normal cell types differing in origin

Abstract Evidence on the impact of chemotherapy on radiotherapy-induced second malignant neoplasms is controversial. We estimated how cisplatin modulates the in vitro response of two normal cell types to fractionated radiation. AHH-1 lymphoblasts and VH10 fibroblasts were irradiated at 1 Gy/fraction 5 and 3 times per week during 12 and 19 days, respectively, and simultaneously treated with 0.1, 0.2, 0.4, 0.8, 1.7 and 3.3 µM of cisplatin twice a week. Cell growth during treatment was monitored. Cell growth/cell death and endpoints related to accumulation of DNA damage and, thus, carcinogenesis, were studied up to 21 days post treatment in cells exposed to radiation and the lowest cisplatin doses. Radiation alone significantly reduced cell growth. The impact of cisplatin alone below 3.3 µM was minimal. Except the lowest dose of cisplatin in VH10 cells, cisplatin reduced the inhibitory effect of radiation on cell growth. Delayed cell death was highest in the combination groups while the accumulation of DNA damage did not reveal a clear pattern. In conclusion, fractionated, concomitant exposure to radiation and cisplatin reduces the inhibitory effect of radiation on cell proliferation of normal cells and does not potentiate delayed effects resulting from accumulation of DNA damage

Cell Type-Specific Patterns in the Accumulation of DNA Damage Following Multifractional Radiation Exposure

Predicting the risk of second malignant neoplasms is complicated by uncertainties regarding the shape of the dose–response relationship at high doses. Limited understanding of the competitive relationship between cell killing and the accumulation of DNA lesions at high doses, as well as the effects of other modulatory factors unique to radiation exposure during radiotherapy, such as dose heterogeneity across normal tissue and dose fractionation, contribute to these uncertainties. The aim of this study was to analyze the impact of fractionated irradiations on two cell systems, focusing on the endpoints relevant for cancer induction. To simulate the heterogeneous dose distribution across normal tissue during radiotherapy, exponentially growing VH10 fibroblasts and AHH-1 lymphoblasts were irradiated with 9 and 12 fractions (VH10) and 10 fractions (AHH-1) at 0.25, 0.5, 1, or 2 Gy per fraction. The effects on cell growth, cell survival, radiosensitivity and the accumulation of residual DNA damage lesions were analyzed as functions of dose per fraction and the total absorbed dose. Residual γH2AX foci and other DNA damage markers (micronuclei, nuclear buds, and giant nuclei) were accumulated at high doses in both cell types, but in a cell type-dependent manner. The competitive relationship between cell killing and the accumulation of carcinogenic DNA damage following multifractional radiation exposure is cell type-specific

Image_1_Short- and long-term effects of radiation exposure at low dose and low dose rate in normal human VH10 fibroblasts.pdf

IntroductionExperimental studies complement epidemiological data on the biological effects of low doses and dose rates of ionizing radiation and help in determining the dose and dose rate effectiveness factor.MethodsHuman VH10 skin fibroblasts exposed to 25, 50, and 100 mGy of 137Cs gamma radiation at 1.6, 8, 12 mGy/h, and at a high dose rate of 23.4 Gy/h, were analyzed for radiation-induced short- and long-term effects. Two sample cohorts, i.e., discovery (n = 30) and validation (n = 12), were subjected to RNA sequencing. The pool of the results from those six experiments with shared conditions (1.6 mGy/h; 24 h), together with an earlier time point (0 h), constituted a third cohort (n = 12).ResultsThe 100 mGy-exposed cells at all abovementioned dose rates, harvested at 0/24 h and 21 days after exposure, showed no strong gene expression changes. DMXL2, involved in the regulation of the NOTCH signaling pathway, presented a consistent upregulation among both the discovery and validation cohorts, and was validated by qPCR. Gene set enrichment analysis revealed that the NOTCH pathway was upregulated in the pooled cohort (p = 0.76, normalized enrichment score (NES) = 0.86). Apart from upregulated apical junction and downregulated DNA repair, few pathways were consistently changed across exposed cohorts. Concurringly, cell viability assays, performed 1, 3, and 6 days post irradiation, and colony forming assay, seeded just after exposure, did not reveal any statistically significant early effects on cell growth or survival patterns. Tendencies of increased viability (day 6) and reduced colony size (day 21) were observed at 12 mGy/h and 23.4 Gy/min. Furthermore, no long-term changes were observed in cell growth curves generated up to 70 days after exposure.DiscussionIn conclusion, low doses of gamma radiation given at low dose rates had no strong cytotoxic effects on radioresistant VH10 cells.</p