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Profound DNA methylomic differences between single- and multi-fraction alpha irradiations of lung fibroblasts

ABSTRACT: Alpha (α)-radiation is a ubiquitous environmental agent with epigenotoxic effects. Human exposure to α-radiation at potentially harmful levels can occur repetitively over the long term via inhalation of naturally occurring radon gas that accumulates in enclosed spaces, or as a result of a single exposure from a nuclear accident. Alterations in epigenetic DNA methylation (DNAm) have been implicated in normal aging and cancer pathogenesis. Nevertheless, the effects of aberrations in the methylome of human lung cells following exposure to single or multiple α-radiation events on these processes remain unexplored. Here, we performed genome-wide DNAm profiling of human embryonic lung fibroblasts from control and irradiated cells using americium-241 α-sources. Cells were α-irradiated in quadruplicates to seven doses using two exposure regimens, a single-fraction (SF) where the total dose was given at once, and a multi-fraction (MF) method, where the total dose was equally distributed over 14 consecutive days. Our results revealed that SF irradiations were prone to a decrease in methylation levels, while MF irradiations mostly increased methylation. The analysis also showed that the gene body (i.e. exons and introns) was the region mostly altered by both the SF hypomethylation and the MF hypermethylation. Additionally, the MF irradiations induced the highest number of differentially methylated regions in genes associated with DNAm biomarkers of aging, carcinogenesis, and cardiovascular disease. The DNAm profile of the oncogenes and tumour suppressor genes suggested that the fibroblasts elicited a defensive response to the MF α-irradiation. Key DNAm events of ionizing radiation exposure including changes in methyl levels in mitochondria dysfunction-related genes were mainly identified in the MF groups. However, these alterations were under-represented, indicating that the mitochondria undergo adaptive mechanisms, aside from methylation, in response to radiation-induced oxidative stress. In this study, we identified a contrasting methylomic profile in the lung fibroblasts α-irradiated to SF compared with MF exposures. These findings demonstrate that the methylome response of the lung cells to α-radiation is highly dependent on both the total dose and the exposure regimen. They also provide novel insights into potential biomarkers of α-radiation which may lead to the development of innovative approaches to detect, prevent and treat α-particle related diseases.

ORGANISM(S): Homo sapiens

PROVIDER: GSE242767 | GEO | 2023/10/09

REPOSITORIES: GEO

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Project description:While modern radiotherapy technologies can precisely deliver higher doses of radiation to tumors; thus, reducing overall radiation exposure to normal tissues, moderate dose and normal tissue toxicity still remains a significant limitation. The present study profiled the global effects on transcript and miR expression in Human Coronary Artery Endothelial Cells (HCAECs) using single-dose irradiation (SD, 10Gy) or multi-fractionated irradiation (MF, 2Gy x 5) regimens. Longitudinal timepoints were collected after a SD or final dose of MF irradiation for analysis using Agilent Human Gene Expression and miRNA microarray platforms. Compared to SD, the exposure to MF resulted in robust transcript and miR expression changes in terms of the number and magnitude. For data analysis, statistically significant mRNAs (2-fold) and miRs (1.5-fold) were processed by Ingenuity Pathway Analysis (IPA) to uncover miRs associated with target transcripts from several cellular pathways post-irradiation. Interestingly, MF radiation induced a cohort of mRNAs and miRs that coordinate the induction of immune response pathway under tight regulation. Additionally, mRNAs and miRs associated with DNA replication, recombination and repair, apoptosis, cardiovascular events and angiogenesis were revealed. Human Coronary Artery Endothelial Cells (HCAECs) were irradiated in a PANTAK high frequency X-ray generator (Precision X-ray Inc., N. Bedford, CT), operated at 300kV and 10MA. The dose rate was 1.6 Gy per min. Cells were plated into T75cm2 flasks (1-1.5 x 10^6 for single dose radiation and 0.6-0.8 x 10^6 for fractionated radiation). After 24h, cells were exposed to a total of 10 Gy radiation administered either as a single-dose radiation (SD), or as multi-fractionated radiation of 2 Gy x 5 (MF). These non-isoeffective doses were selected to simulate clinical hypofractionated and conventionally fractionated radiotherapy regimens. For the MF protocol, cells were exposed to 2 Gy radiation twice a day, at 6h interval. The cells were approximately 90% confluent at the time of harvesting. For both protocols, radiation-induced changes were analyzed at 6h and 24h after a SD and 6h and 24h after the final dose of fractionated irradiation. Separate controls were maintained for SD and MF radiation protocols.

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Profound DNA methylomic differences between single- and multi-fraction alpha irradiations of lung fibroblasts

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