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

Abstract

Background: 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-irradiation events on these processes remain unexplored.

Results: 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 DNAm levels, while MF irradiations mostly increased DNAm. The analysis also showed that the gene body (i.e., exons and introns) was the region most 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 tumor suppressor genes suggests that the fibroblasts manifested a defensive response to the MF α-irradiation. Key DNAm events of ionizing radiation exposure, including changes in methylation 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 DNAm, in response to radiation-induced oxidative stress.

Conclusions: 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 contribute to the development of innovative approaches to detect, prevent, and treat α-particle-related diseases.

Keywords: Aging; Alpha particles; Cancer; Lung; Methylome; Mitochondria; Radiation; Radon.

Fig. 1

Differentially methylated regions (DMRs) in lung fibroblasts following irradiation to single-fraction (SF) doses of α-particles. A Total number of DMRs identified using the Bioconductor MEDIPS package. B UpSet plot showing the number of the identified DMRs in the fibroblasts across the different SF α-irradiation doses. C Bar chart displaying the number of hypo and hyperDMRs across all chromosomes. D Percentages of the genomic regions that were found to be differentially enriched in each irradiation group. E Bar chart displaying the number of hypo and hyperDMRs identified in each genomic region. Each irradiation group includes four biological replicates per dose, each independently irradiated and assessed. Abbreviations: hypoDMRs, hypomethylated DMRs; hyperDMRs, hypermethylated DMRs; TSS, transcription start site; TTS, transcription termination site; 3’ UTR, 3’ untranslated region; and 5’ UTR, 5’ untranslated region

Fig. 2

Differentially methylated regions (DMRs) in lung fibroblasts following irradiation to multi-fraction (MF) doses of α-particles. A Total number of DMRs identified using the Bioconductor MEDIPS package. B UpSet plot showing the number of the identified DMRs in the fibroblasts across the different MF α-irradiation doses. C Bar chart displaying the number of hypo and hyperDMRs across all chromosomes. D Percentages of the genomic regions that were found to be differentially enriched in each irradiation group. E Bar chart displaying the number of hypo and hyperDMRs identified in each genomic region. Each dose of ionizing radiation was equally distributed every 24 h over 14 consecutive days. Each irradiation group includes four biological replicates per dose, each independently irradiated and assessed. Abbreviations: hypoDMRs, hypomethylated DMRs; hyperDMRs, hypermethylated DMRs; TSS, transcription start site; TTS, transcription termination site; 3’ UTR, 3’ untranslated region; and 5’ UTR, 5’ untranslated region

Fig. 3

Aging-associated DNA methylation (DNAm) events identified in the α-irradiated lung fibroblasts. These events are based on the DNAm clock from Horvath [33] and on the PhenoAge DNAm clock from Levine et al. [34]. A Number of differentially methylated regions (DMRs) from the α-irradiated fibroblasts whose neighboring genes were associated with aging as established by the two selected DNAm clocks. B Table displaying the enrichment analysis results of the aging-associated genes whose DNAm levels were altered in the α-irradiated fibroblasts, computed using the cumulative distribution function of the hypergeometric equation from [49]. C Top 5 functional classification of the genes affected by the aging-associated DMRs using Ingenuity Pathway Analysis. Abbreviations: SF, single-fraction; MF, multi-fraction; TSS, transcription start site; TTS, transcription termination site; 3’ UTR, 3’ untranslated region; and 5’ UTR, 5’ untranslated region

Fig. 4

Promoters and gene body regions whose methylome was altered by α-irradiation in lung fibroblasts. A Number of promoters that were either hypo or hypermethylated following single-fraction (SF) α-irradiation. B Number of promoters that were either hypo or hypermethylated following multi-fraction (MF) α-irradiation. C Table displaying the top diseases and biological functions associated with the affected promoters in the α-irradiated fibroblasts characterized using QIAGEN Ingenuity Pathway Analysis. D Number of gene body regions that were hypermethylated following MF α-irradiation. (Venn diagrams created using Oliveros [63])

Fig. 5

Nuclear-encoded mitochondrial genes identified in the fibroblasts following irradiations to α-particles. A Table displaying the results from the enrichment analysis of the nuclear-encoded mitochondrial genes whose methylation levels were altered in the α-irradiated fibroblasts, computed using the cumulative distribution function of the hypergeometric equation [49]. B UpSet plot exhibiting the number of nuclear-encoded mitochondrial genes harboring DMRs identified in the fibroblasts across the different α-irradiation doses