RESUMO
INTRODUCTION: DNA double-strand breaks (DSBs) induced by ionizing radiation pose a significant threat to genome integrity, necessitating robust repair mechanisms. This study explores the responses of repair-deficient cells to low dose rate (LDR) radiation. Non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways play pivotal roles in maintaining genomic stability. The hypothesis posits distinct cellular outcomes under LDR exposure compared to acute radiation, impacting DNA repair mechanisms and cell survival. MATERIALS AND METHODS: Chinese hamster ovary (CHO) cells, featuring deficiencies in NHEJ, HR, Fanconi Anemia, and PARP pathways, were systematically studied. Clonogenic assays for acute and LDR gamma-ray exposures, cell growth inhibition analyses, and γ-H2AX foci assays were conducted, encompassing varied dose rates to comprehensively assess cellular responses. RESULTS: NHEJ mutants exhibited an unexpected inverse dose rate effect, challenging conventional expectations. HR mutants displayed unique radiosensitivity patterns, aligning with responses to major DNA-damaging agents. LDR exposure induced cell cycle alterations, growth delays, and giant cell formation, revealing context-dependent sensitivities. γ-H2AX foci assays indicated DSB accumulation during LDR exposure. DISCUSSION: These findings challenge established paradigms, emphasizing the intricate interplay between repair pathways and dose rates. The study offers comprehensive insights into repair-deficient cell responses, urging a reevaluation of conventional dose-response models and providing potential avenues for targeted therapeutic strategies in diverse radiation scenarios.
Assuntos
Reparo do DNA por Junção de Extremidades , Reparo do DNA , Cricetinae , Animais , Células CHO , Cricetulus , Reparo do DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo de DNA por Recombinação , DNARESUMO
Following carbon ion beam irradiation in mammalian cells, such as used in carbon ion radiotherapy (CIRT), it has been suggested that the balance between whether nonhomologous end joining (NHEJ) or homologous recombination (HR) is utilized depends on the DNA double-strand break (DSB) complexity. Here, we quantified DSB distribution and identified the importance of each DSB repair pathway at increasing depths within the carbon ion spread-out Bragg peak (SOBP) beam range. Chinese hamster ovary (CHO) cell lines were irradiated in a single biological system capable of incorporating the full carbon ion SOBP beam range. Cytotoxicity and DSB distribution/repair kinetics were examined at increasing beam depths using cell survival as an endpoint and γ-H2AX as a surrogate marker for DSBs. We observed that proximal SOBP had the highest number of total foci/cell and lowest survival, while distal SOBP had the most dense tracks. Both NHEJ- and HR-deficient CHO cells portrayed an increase in radiosensitivity throughout the full carbon beam range, although NHEJ-deficient cells were the most radiosensitive cell line from beam entrance up to proximal SOBP and demonstrated a dose-dependent decrease in ability to repair DSBs. In contrast, HR-deficient cells had the greatest ratio of survival fraction at entrance depth to the lowest survival fraction within the SOBP and demonstrated a linear energy transfer (LET)-dependent decrease in ability to repair DSBs. Collectively, our results provide insight into treatment planning and potential targets to inhibit, as HR was a more beneficial pathway to inhibit than NHEJ to enhance the cell killing effect of CIRT in targeted tumor cells within the SOBP while maintaining limited unwanted damage to surrounding healthy cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Cricetinae , Animais , Humanos , Cricetulus , Células CHO , DNA , Carbono , Reparo do DNA por Junção de ExtremidadesRESUMO
Cu2+ and Co2+ are metals known to increase DNA damage in the presence of hydrogen peroxide through a Fenton-type reaction. We hypothesized that these metals could increase DNA damage following irradiations of increasing LET values as hydrogen peroxide is a product of the radiolysis of water. The reaction mixtures contain either double- or single-stranded DNA in solution with Cu2+ or Co2+ and were irradiated either with X-ray, carbon-ion or iron-ion beams, or they were treated with hydrogen peroxide or bleomycin at increasing radiation dosages or chemical concentrations. DNA damage was then assessed via gel electrophoresis followed with a band intensity analysis. DNA damage was the greatest when DNA in the solution with either metal was treated with only hydrogen peroxide followed by the DNA damage of DNA in the solution with either metal post irradiation of low-LET (X-Ray) or high-LET (carbon-ion and iron-ion), respectively, and demonstrated the least damage after treatment with bleomycin. Cu2+ portrayed greater DNA damage than Co2+ following all experimental conditions. The metals' effect caused more DNA damage and was observed to be LET-dependent for single-strand break formation but inversely dependent for double-strand break formation. These results suggest that Cu2+ is more efficient than Co2+ at inducing both DNA single-strand and double-strand breaks following all irradiations and chemical treatments.
RESUMO
DNA double-strand breaks (DSBs) are the main factor behind carbon-ion radiation therapy (CIRT)-induced cell death. Nuclear interactions along the beam path between the primary carbon ions and targets result in nuclear fragmentation of carbon ions and recoiled particles. These secondary particles travel further distances past the Bragg peak to the tail region, leading to unwanted biological effects that may result in cytotoxicity in critical organs and secondary induced tumors following CIRT. Here, we confirmed that the density of the DSB distributions increases as the cell survival decreases at the Bragg peak and demonstrated that by visualizing DSBs, the various LET fragmentation ions and recoiled particles produced differences in their biological effects in the post-Bragg peak tail regions. This suggests that the density of the DSBs within the high-LET track structures, rather than only their presence, is important for inducing cell death. These results are essential for CIRT treatment planning to limit the amount of healthy cell damage and reducing both the late effect and the secondary tumor-associated risk.
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Phototherapy using narrowband ultraviolet-B (NB-UVB) has been shown to be more effective than conventional broadband UVB (BB-UVB) in treating a variety of skin diseases. To assess the difference in carcinogenic potential between NB-UVB and BB-UVB, we investigated the cytotoxicity via colony formation assay, genotoxicity via sister chromatid exchange (SCE) assay, mutagenicity via hypoxanthine phosphoribosyltransferase (HPRT) mutation assay, as well as cyclobutane pyrimidine dimer (CPD) formation and reactive oxygen species (ROS) generation in Chinese hamster ovary (CHO) and their NER mutant cells. The radiation dose required to reduce survival to 10% (D10 value) demonstrated BB-UVB was 10 times more cytotoxic than NB-UVB, and revealed that NB-UVB also induces DNA damage repaired by nucleotide excision repair. We also found that BB-UVB more efficiently induced SCEs and HPRT mutations per absorbed energy dosage (J/m2) than NB-UVB. However, SCE and HPRT mutation frequencies were observed to rise in noncytotoxic dosages of NB-UVB exposure. BB-UVB and NB-UVB both produced a significant increase in CPD formation and ROS formation (p < 0.05); however, higher dosages were required for NB-UVB. These results suggest that NB-UVB is less cytotoxic and genotoxic than BB-UVB, but can still produce genotoxic effects even at noncytotoxic doses.
Assuntos
Dano ao DNA/efeitos da radiação , Mutagênese/efeitos da radiação , Mutagênicos/toxicidade , Pele/efeitos da radiação , Animais , Células CHO , Cricetinae , Cricetulus , Dano ao DNA/genética , Humanos , Mutagênese/genética , Mutação/efeitos da radiação , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/efeitos da radiação , Pele/metabolismo , Raios UltravioletaRESUMO
Blue light is commonly used for the treatment of Neonatal Jaundice and as a photodynamic therapy for cancer. In comparison to ultraviolet light, blue light has a lower toxicity due to the differences in photon energies. However, blue light can still be mutagenic to cells. The proposed mechanism suggests blue light exposure induces reactive oxygen species inducing oxidative stress. In this study, we examined how blue light exposure caused genotoxic effects utilizing Chinese hamster ovary (CHO) cells and UV135 cells when exposed to fluorescent blue light. Cytotoxic effects of blue light exposure were quantified through cellular oxidative stress analysis, cell survival assay, and in cell cycle arrest experiments. Genotoxicity was studied in sister chromatid exchange (SCE) only, and endoreduplication formation. Following blue light exposure, an increase of cell cycle arrest, oxidative stress, and cytotoxicity was observed. Blue light treatment also produced an increased amount of SCE, and more importantly, induced endoreduplicated chromosomes. In conclusion, exposure to blue light resulted in significant genotoxicity of the treated cells.
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Endorreduplicação/efeitos da radiação , Luz/efeitos adversos , Estresse Oxidativo/efeitos da radiação , Animais , Células CHO , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Cricetulus , Dano ao DNA/efeitos da radiação , Mutagênicos/efeitos adversos , Troca de Cromátide Irmã/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
The sharp high dose Bragg peak of a carbon-ion beam helps it to deliver the highest dosage to the malignant cells while leaving the normal cells relatively unharmed. However, the precise range in which it distributes dosages that significantly induce cell death or genotoxicity surrounding its Bragg peak remains unclear. To evaluate biological effects of carbon-ion radiation through entrance to post Bragg peak in a single biological system, CHO and xrs5 cells were cultured in T-175 cell culture flasks and irradiated with 290 MeV/n monoenergetic carbon-ions with initial dosages upon entrance to the flask of 1, 2, or 3 Gy for cell survival assays or 1 Gy for cytokinesis block micronuclei assays. Under all initial dosages, the biological Bragg peak and the highest micronuclei formation was observed at the depth of 14.5 cm. Moreover, as the initial dosage increased the range displaying a significant decrease in survival fraction increased as well (P < 0.0001). Intriguingly from 1 Gy to 3 Gy, we observed a significant increase in reappearance of colony formation depth (P < 0.05), possibly indicating the nuclear fragmentation lethality potential of the carbon-ion. By means of our single system approach, we can achieve a more comprehensive understanding of biological effects surrounding of carbon-ions Bragg peak.
Assuntos
Radioterapia com Íons Pesados , Neoplasias/radioterapia , Animais , Células CHO , Sobrevivência Celular/efeitos da radiação , Cricetulus , Relação Dose-Resposta à Radiação , Radioterapia com Íons Pesados/métodosRESUMO
DMSO, glycerol, and ascorbic acid (AA) are used in pharmaceuticals and known to display radioprotective effects. The present study investigates radioprotective properties of novel glyceryl glucoside, ascorbic acid 2-glucoside, glyceryl ascorbate, and palmitoyl ascorbic acid 2-glucoside (PA). Gamma-rays or high-LET carbon-ions were irradiated in the presence of tested chemicals. Lambda DNA damage, cell survival, and micronuclei formation of CHO cells were analyzed to evaluate radioprotective properties. Radiation-induced Lambda DNA damage was reduced with chemical pre-treatment in a concentration-dependent manner. This confirmed tested chemicals were radical scavengers. For gamma-irradiation, enhanced cell survival and reduction of micronuclei formation were observed for all chemicals. For carbon-ion irradiation, DMSO, glycerol, and PA displayed radioprotection for cell survival. Based on cell survival curves, protection levels by PA were confirmed and comparable between gamma-rays and high-LET carbon-ions. Micronuclei formation was only decreased with AA and a high concentration of glycerol treatment, and not decreased with PA treatment. This suggests that mechanisms of protection against high-LET carbon-ions by PA can differ from normal radical scavenging effects that protect DNA from damage.