Application of radiation omics in the development of adverse outcome pathway networks: an example of radiation-induced cardiovascular disease.

BACKGROUND: Epidemiological studies have indicated that exposure of the heart to doses of ionizing radiation as low as 0.5 Gy increases the risk of cardiac morbidity and mortality with a latency period of decades. The damaging effects of radiation to myocardial and endothelial structures and functions have been confirmed radiobiologically at high dose, but much less are known at low dose. Integration of radiation biology and epidemiology data is a recommended approach to improve the radiation risk assessment process. The adverse outcome pathway (AOP) framework offers a comprehensive tool to compile and translate mechanistic information into pathological endpoints which may be relevant for risk assessment at the different levels of a biological system. Omics technologies enable the generation of large volumes of biological data at various levels of complexity, from molecular pathways to functional organisms. Given the quality and quantity of available data across levels of biology, omics data can be attractive sources of information for use within the AOP framework. It is anticipated that radiation omics studies could improve our understanding of the molecular mechanisms behind the adverse effects of radiation on the cardiovascular system. In this review, we explored the available omics studies on radiation-induced cardiovascular disease (CVD) and their applicability to the proposed AOP for CVD. RESULTS: The results of 80 omics studies published on radiation-induced CVD over the past 20 years have been discussed in the context of the AOP of CVD proposed by Chauhan et al. Most of the available omics data on radiation-induced CVD are from proteomics, transcriptomics, and metabolomics, whereas few datasets were available from epigenomics and multi-omics. The omics data presented here show great promise in providing information for several key events (KEs) of the proposed AOP of CVD, particularly oxidative stress, alterations of energy metabolism, extracellular matrix (ECM), and vascular remodeling. CONCLUSIONS: The omics data presented here shows promise to inform the various levels of the proposed AOP of CVD. However, the data highlight the urgent need of designing omics studies to address the knowledge gap concerning different radiation scenarios, time after exposure, and experimental models. This review presents the evidence to build a qualitative omics-informed AOP and provides views on the potential benefits and challenges in using omics data to assess risk-related outcomes.

Expert consultation is vital for adverse outcome pathway development: a case example of cardiovascular effects of ionizing radiation.

BACKGROUND: The circulatory system distributes nutrients, signaling molecules, and immune cells to vital organs and soft tissues. Epidemiological, animal, and in vitro cellular mechanistic studies have highlighted that exposure to ionizing radiation (IR) can induce molecular changes in cellular and subcellular milieus leading to long-term health impacts, particularly on the circulatory system. Although the mechanisms for the pathologies are not fully elucidated, endothelial dysfunction is proven to be a critical event via radiation-induced oxidative stress mediators. To delineate connectivities of events specifically to cardiovascular disease (CVD) initiation and progression, the adverse outcome pathway (AOP) approach was used with consultation from field experts. AOPs are a means to organize information around a disease of interest to a regulatory question. An AOP begins with a molecular initiating event and ends in an adverse outcome via sequential linkages of key event relationships that are supported by evidence in the form of the modified Bradford-Hill criteria. Detailed guidelines on building AOPs are provided by the Organisation for Economic Cooperation and Development (OECD) AOP program. Here, we report on the questions and discussions needed to develop an AOP for CVD resulting from IR exposure. A recent workshop jointly organized by the MELODI (Multidisciplinary European Low Dose Initiative) and the ALLIANCE (European Radioecology Alliance) associations brought together experts from the OECD to present the AOP approach and tools with examples from the toxicology field. As part of this workshop, four working groups were formed to discuss the identification of adverse outcomes relevant to radiation exposures and development of potential AOPs, one of which was focused on IR-induced cardiovascular effects. Each working group comprised subject matter experts and radiation researchers interested in the specific disease area and included an AOP coach. CONCLUSION: The CVD working group identified the critical questions of interest for AOP development, including the exposure scenario that would inform the evidence, the mechanisms of toxicity, the initiating event, intermediate key events/relationships, and the type of data currently available. This commentary describes the four-day discussion of the CVD working group, its outcomes, and demonstrates how collaboration and expert consultation is vital to informing AOP construction.

Low-dose radiobiology program at Canadian nuclear laboratories: past, present, and future.

Health risks associated with the exposure of humans to low-dose ionizing radiation are currently estimated using the Linear-No-Threshold model. Over the last few decades, however, this model has been widely criticized for inconsistency with a large body of experimental evidence. Substantial efforts have been made to delineate biological mechanisms and health-related outcomes of low-dose radiation. These include a large DOE-funded Low Dose program operated in the 2000s, as well as the EU funded programs, previously NOTE and DOREMI and currently MELODI. Although not as widely known, the Atomic Energy of Canada Limited (AECL) in Chalk River, operated a low-dose radiobiology program since as early as 1948. The Canadian Nuclear Laboratories (CNL), the successor to AECL since 2015, has expanded this program into new areas making it the world's most robust, centrally coordinated and long-lived research efforts to delineate the biological effects of low-dose radiation. The purpose of this review is to provide a high-level overview of the low-dose radiobiology program maintained at CNL while capturing the historical perspectives. Past studies carried out at CNL have substantially influenced the area of low-dose radiobiology, exemplified by highly cited papers showing delays in spontaneous tumorigenesis in low-dose irradiated mice. The current low-dose research program at CNL is not only addressing a wide range of mechanistic questions about the biological effects of low doses - from genetic to epigenetic to immunological questions - but also moving toward novel areas, such as the dosimetry and health consequences of space radiation and the use of low-dose radiation in cancer therapy and regenerative medicine.

Tissue-specific splicing of a ubiquitously expressed transcription factor is essential for muscle differentiation.

Alternate splicing contributes extensively to cellular complexity by generating protein isoforms with divergent functions. However, the role of alternate isoforms in development remains poorly understood. Mef2 transcription factors are essential transducers of cell signaling that modulate differentiation of many cell types. Among Mef2 family members, Mef2D is unique, as it undergoes tissue-specific splicing to generate a muscle-specific isoform. Since the ubiquitously expressed (Mef2Dα1) and muscle-specific (Mef2Dα2) isoforms of Mef2D are both expressed in muscle, we examined the relative contribution of each Mef2D isoform to differentiation. Using both in vitro and in vivo models, we demonstrate that Mef2D isoforms act antagonistically to modulate differentiation. While chromatin immunoprecipitation (ChIP) sequencing analysis shows that the Mef2D isoforms bind an overlapping set of genes, only Mef2Dα2 activates late muscle transcription. Mechanistically, the differential ability of Mef2D isoforms to activate transcription depends on their susceptibility to phosphorylation by protein kinase A (PKA). Phosphorylation of Mef2Dα1 by PKA provokes its association with corepressors. Conversely, exon switching allows Mef2Dα2 to escape this inhibitory phosphorylation, permitting recruitment of Ash2L for transactivation of muscle genes. Thus, our results reveal a novel mechanism in which a tissue-specific alternate splicing event has evolved that permits a ubiquitously expressed transcription factor to escape inhibitory signaling for temporal regulation of gene expression.

MLL5, a trithorax homolog, indirectly regulates H3K4 methylation, represses cyclin A2 expression, and promotes myogenic differentiation.

Most cells in adult tissues are nondividing. In skeletal muscle, differentiated myofibers have exited the cell cycle permanently, whereas satellite stem cells withdraw transiently, returning to active proliferation to repair damaged myofibers. We have examined the epigenetic mechanisms operating in conditional quiescence by analyzing the function of a predicted chromatin regulator mixed lineage leukemia 5 (MLL5) in a culture model of reversible arrest. MLL5 is induced in quiescent myoblasts and regulates both the cell cycle and differentiation via a hierarchy of chromatin and transcriptional regulators. Knocking down MLL5 delays entry of quiescent myoblasts into S phase, but hastens S-phase completion. Cyclin A2 (CycA) mRNA is no longer restricted to S phase, but is induced throughout G(0)/G(1), with activation of the cell cycle regulated element (CCRE) in the CycA promoter. Overexpressed MLL5 physically associates with the CCRE and impairs its activity. MLL5 also regulates CycA indirectly: Cux, an activator of CycA promoter and S phase is induced in RNAi cells, and Brm/Brg1, CCRE-binding repressors that promote differentiation are repressed. In knockdown cells, H3K4 methylation at the CCRE is reduced, reflecting quantitative global changes in methylation. MLL5 appears to lack intrinsic histone methyl transferase activity, but regulates expression of histone-modifying enzymes LSD1 and SET7/9, suggesting an indirect mechanism. Finally, expression of muscle regulators Pax7, Myf5, and myogenin is impaired in MLL5 knockdown cells, which are profoundly differentiation defective. Collectively, our results suggest that MLL5 plays an integral role in novel chromatin regulatory mechanisms that suppress inappropriate expression of S-phase-promoting genes and maintain expression of determination genes in quiescent cells.