Bystander Response Following High-Dose X-irradiation; Time-dependent Nature of GammaH2AX Foci and Cell Death Consequences.

Background: The paradigm shifts in target theory could be defined as the radiation-triggered bystander response in which the radiation deleterious effects occurred in the adjacent cells. Objective: This study aims to assess bystander response in terms of DNA damage and their possible cell death consequences following high-dose radiotherapy. Temporal characteristics of gH2AX foci as a manifestation of DNA damage were also evaluated. Material and Methods: In this experimental study, bystander response was investigated in human carcinoma cells of HeLa and HN5, neighboring those that received high doses. Medium transfer was performed from 10 Gy-irradiated donors to 1.5 Gy-irradiated recipients. GammaH2AX foci, clonogenic and apoptosis assays were investigated. The gH2AX foci time-point study was implemented 1, 4, and 24 h after the medium exchange. Results: DNA damage was enhanced in HeLa and HN5 bystander cells with the ratio of 1.27 and 1.72, respectively, which terminated in more than two-fold clonogenic survival decrease, along with gradual apoptosis increase. GammH2AX foci temporal characterization revealed maximum foci scoring at the 1 h time-point in HeLa, and also 4 h in HN5, which remained even 24 h after the medium sharing in higher level than the control group. Conclusion: The time-dependent nature of bystander-induced gH2AX foci as a DNA damage surrogate marker was highlighted with the persistent foci at 24 h. considering an outcome of bystander-induced DNA damage, predominant role of clonogenic cell death was also elicited compared to apoptosis. Moreover, the role of high-dose bystander response observed in the current work clarified bystander potential implications in radiotherapy.

Evaluation of Capability and Relationship of Different Radiobiological Endpoints for Radiosensitivity Prediction in Human Tumor Cell Lines Compared with Clonogenic Survival.

Background: Establishing a predictive assay of radiosensitivity (as an appropriate, practical and cost-effective method) has been challenging. Objective: The purpose of this study is to evaluate the capability and relationship of various endpoints, including GammaH2AX, micronuclei; and apoptosis in determining the human tumor cell lines radiosensitivities compared with clonogenic survival. Material and Methods: In an experimental in-vitro study, the response of carcinoma cell lines of HN5 and HeLa to 2 Gy of 6 MV photon beam was investigated via various assays. Results: Survival fraction at 2 Gy (SF2) of HeLa and HN5 was indicated as 0.42 ± 0.06 and 0.5 ± 0.03 respectively, proposing more radioresistance of HN5. This finding was confirmed with "2 Gy apoptosis enhancement ratio" which was 1.77 and 1.42 in HeLa and HN5. The increased levels of DNA DSBs were observed after irradiation; significant in HeLa with enhancement rate of 19.24. The micronuclei formation followed an ascending trend post irradiation; but with the least difference between two cells. Although the relationship between micronuclei and clonogenic survival was moderate (R2 = 0.35), a good correlation was observed between apoptosis and clonogenic survival (R2 = 0.71). Conclusion: The results of studied endpoints agreed with the SF2, highlighting their capabilities in radiosensitivity prediction. In terms of the enhancement ratio, gammaH2AX foci scoring could be a valid indicator of radiosensitivity but not the exact surrogate marker of survival because no correlation was observed. Moreover, considering the chief determents comprising lack of time and money, the apoptotic induction might be an appropriate indicator with the best correlation coefficient.

Decreased Fetal Movements: A Sign of Placental SARS-CoV-2 Infection with Perinatal Brain Injury.

Neonatal COVID-19 is rare and mainly results from postnatal transmission. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, can infect the placenta and compromise its function. We present two cases of decreased fetal movements and abnormal fetal heart rhythm 5 days after mild maternal COVID-19, requiring emergency caesarean section at 29 + 3 and 32 + 1 weeks of gestation, and leading to brain injury. Placental examination revealed extensive and multifocal chronic intervillositis, with intense cytoplasmic positivity for SARS-CoV-2 spike antibody and SARS-CoV-2 detection by RT-qPCR. Vertical transmission was confirmed in one case, and both neonates developed extensive cystic peri-ventricular leukomalacia.

Enhanced response of radioresistant carcinoma cell line to heterogeneous dose distribution of grid; the role of high-dose bystander effect.

PURPOSE: The classical dogma that restricted the radiation effect to the directly irradiated cells has been challenged by the bystander effect. This off-target phenomenon which was manifested in adjacent cells via signaling of fully exposed cells might be involved in high-dose Grid therapy as well. Here, an in-vitro study was performed to examine the possible extent of carcinoma cells response to the inhomogeneous dose distribution of Grid irradiation in the context of the bystander effect. MATERIALS AND METHODS: Bystander effect was investigated in human carcinoma cell lines of HeLa and HN5 adjacent to those received high-dose Grid irradiation using 'medium transfer' and 'cell-to-cell contact' strategies. Based on the Grid peak-to-valley dose profile, medium transfer was exerted from 10 Gy uniformly exposed donors to 1.5 Gy uniformly irradiated recipients. Cell-contact bystander was evaluated after nonuniform dose distribution of 10 Gy Grid irradiation using cloning cylinders. GammaH2AX foci, micronucleus and clonogenic assays besides gene expression analysis were performed. RESULTS: Various parameters (ɑ/ß, D37, D50) extracted from survival curve which fitted to the Linear Quadratic model, verified more radioresistance of HN5. Survival fraction at 2 Gy (SF2) indicated as 0.42 ± 0.06 in HeLa and 0.5 ± 0.03 in HN5. The level of survival decrease, DNA damages and micronucleus of cells located in the Grid shielded areas (1.5 Gy cell-to-cell contact bystander cells) were significantly more than the values obtained from cells which were irradiated by merely uniform dose of 1.5 Gy. The gH2AX foci and micronuclei frequencies were enhanced in cell-contact bystander approximately more than 1.8 times. Relative expression of DNA damage repair pathway genes (Xrcc6 and H2afx) in bystander cells increased significantly. The most cell survival reduction (11.6 times) was revealed in the Grid bystander cells of radioresistant cell line (HN5). No statistically significant difference between 10 Gy uniform beam and Grid non-uniform beam was observed. CONCLUSIONS: Various endpoints confirmed an augmented response of cells in the valley dose region of the Grid block significantly (compared with the cells irradiated by identical dose of uniform beam), suggesting the role of high-dose bystander effect which was more pronounced in resistant carcinoma cell lines. These findings could provide a partial explanation for the Grid beneficial response seen in a number of pre-clinical and clinical studies.