Janet E. Baulch

Epigenetic changes and nontargeted radiation effects—Is there a link?

It is now well accepted that the effects of ionizing radiation (IR) exposure can be noticed far beyond the borders of the directly irradiated tissue. IR can affect neighboring cells in the proximity, giving rise to a bystander effect. IR effects can also span several generations and influence the progeny of exposed parents, leading to transgeneration effects. Bystander and transgeneration IR effects are linked to the phenomenon of the IR‐induced genome instability that manifests itself as chromosome aberrations, gene mutations, late cell death, and aneuploidy. While the occurrence of the abovementioned phenomena is well documented, the exact mechanisms that lead to their development have still to be delineated. Evidence suggests that the IR‐induced genome instability, bystander, and transgeneration effects may be epigenetically mediated. The epigenetic changes encompass DNA methylation, histone modification, and RNA‐associated silencing. Recent studies demonstrated that IR exposure alters epigenetic parameters in the directly exposed tissues and in the distant bystander tissues. Transgeneration radiation effects were also proposed to be of an epigenetic nature. We will discuss the role of the epigenetic mechanisms in radiation responses, bystander effects, and transgeneration effects. Environ. Mol. Mutagen., 2008.

Radiation-induced epigenetic alterations after low and high LET irradiations

Epigenetics, including DNA methylation and microRNA (miRNA) expression, could be the missing link in understanding radiation-induced genomic instability (RIGI). This study tests the hypothesis that irradiation induces epigenetic aberrations, which could eventually lead to RIGI, and that the epigenetic aberrations induced by low linear energy transfer (LET) irradiation are different than those induced by high LET irradiations. GM10115 cells were irradiated with low LET X-rays and high LET iron (Fe) ions and evaluated for DNA damage, cell survival and chromosomal instability. The cells were also evaluated for specific locus methylation of nuclear factor-kappa B (NFκB), tumor suppressor in lung cancer 1 (TSLC1) and cadherin 1 (CDH1) gene promoter regions, long interspersed nuclear element 1 (LINE-1) and Alu repeat element methylation, CpG and non-CpG global methylation and miRNA expression levels. Irradiated cells showed increased micronucleus induction and cell killing immediately following exposure, but were chromosomally stable at delayed times post-irradiation. At this same delayed time, alterations in repeat element and global DNA methylation and miRNA expression were observed. Analyses of DNA methylation predominantly showed hypomethylation, however hypermethylation was also observed. We demonstrate that miRNA expression levels can be altered after X-ray irradiation and that these miRNA are involved in chromatin remodeling and DNA methylation. A higher incidence of epigenetic changes was observed after exposure to X-rays than Fe ions even though Fe ions elicited more chromosomal damage and cell killing. This distinction is apparent at miRNA analyses at which only three miRNA involved in two major pathways were altered after high LET irradiations while six miRNA involved in five major pathways were altered after low LET irradiations. This study also shows that the irradiated cells acquire epigenetic changes suggesting that epigenetic aberrations may arise in the cell without initiating chromosomal instability.

What happens to your brain on the way to Mars

Animal models reveal an unexpected sensitivity of mature neurons in the brain to the charged particles found in space. As NASA prepares for the first manned spaceflight to Mars, questions have surfaced concerning the potential for increased risks associated with exposure to the spectrum of highly energetic nuclei that comprise galactic cosmic rays. Animal models have revealed an unexpected sensitivity of mature neurons in the brain to charged particles found in space. Astronaut autonomy during long-term space travel is particularly critical as is the need to properly manage planned and unanticipated events, activities that could be compromised by accumulating particle traversals through the brain. Using mice subjected to space-relevant fluences of charged particles, we show significant cortical- and hippocampal-based performance decrements 6 weeks after acute exposure. Animals manifesting cognitive decrements exhibited marked and persistent radiation-induced reductions in dendritic complexity and spine density along medial prefrontal cortical neurons known to mediate neurotransmission specifically interrogated by our behavioral tasks. Significant increases in postsynaptic density protein 95 (PSD-95) revealed major radiation-induced alterations in synaptic integrity. Impaired behavioral performance of individual animals correlated significantly with reduced spine density and trended with increased synaptic puncta, thereby providing quantitative measures of risk for developing cognitive decrements. Our data indicate an unexpected and unique susceptibility of the central nervous system to space radiation exposure, and argue that the underlying radiation sensitivity of delicate neuronal structure may well predispose astronauts to unintended mission-critical performance decrements and/or longer-term neurocognitive sequelae.

Radiation-induced genomic instability: Are epigenetic mechanisms the missing link?

Purpose: This review examines the evidence for the hypothesis that epigenetics are involved in the initiation and perpetuation of radiation-induced genomic instability (RIGI). Conclusion: In addition to the extensively studied targeted effects of radiation, it is now apparent that non-targeted delayed effects such as RIGI are also important post-irradiation outcomes. In RIGI, unirradiated progeny cells display phenotypic changes at delayed times after radiation of the parental cell. RIGI is thought to be important in the process of carcinogenesis; however, the mechanism by which this occurs remains to be elucidated. In the genomically unstable clones developed by Morgan and colleagues, radiation-induced mutations, double-strand breaks, or changes in messenger RNA (mRNA) levels alone could not account for the initiation or perpetuation of RIGI. Since changes in the DNA sequence could not fully explain the mechanism of RIGI, inherited epigenetic changes may be involved. Epigenetics are known to play an important role in many cellular processes and epigenetic aberrations can lead to carcinogenesis. Recent studies in the field of radiation biology suggest that the changes in methylation patterns may be involved in RIGI. Together these clues have led us to hypothesise that epigenetics may be the missing link in understanding the mechanism behind RIGI.

Lack of evidence for low-LET radiation induced bystander response in normal human fibroblasts and colon carcinoma cells

Purpose: To investigate radiation-induced bystander responses and to determine the role of gap junction intercellular communication and the radiation environment in propagating this response. Materials and methods: We used medium transfer and targeted irradiation to examine radiation-induced bystander effects in primary human fibroblast (AG01522) and human colon carcinoma (RKO36) cells. We examined the effect of variables such as gap junction intercellular communication, linear energy transfer (LET), and the role of the radiation environment in non-targeted responses. Endpoints included clonogenic survival, micronucleus formation and foci formation at histone 2AX over doses ranging from 10–100 cGy. Results: The results showed no evidence of a low-LET radiation-induced bystander response for the endpoints of clonogenic survival and induction of DNA damage. Nor did we see evidence of a high-LET, Fe ion radiation (1 GeV/n) induced bystander effect. However, direct comparison for 3.2 MeV α-particle exposures showed a statistically significant medium transfer bystander effect for this high-LET radiation. Conclusions: From our results, it is evident that there are many confounding factors influencing bystander responses as reported in the literature. Our observations reflect the inherent variability in biological systems and the difficulties in extrapolating from in vitro models to radiation risks in humans.

The Effect of Radiation Quality on Genomic DNA Methylation Profiles in Irradiated Human Cell Lines

Abstract It has been acknowledged for many years that radiation exposure induces delayed, non-targeted effects in the progeny of the irradiated cell. Evidence is beginning to demonstrate that among these delayed effects of radiation are epigenetic aberrations, including altered DNA methylation. To test the hypothesis that differences in radiation quality affect radiation-induced DNA methylation profiles, normal AG01522 and RKO colon carcinoma cells were exposed to low-LET X rays and protons or high-LET iron ions. DNA methylation was then evaluated at delayed times using assays for p16 and MGMT promoter, LINE-1 and alu repeat element, and global methylation. The results of these experiments demonstrated radiation-induced changes in repeat element and global DNA methylation patterns at ∼20 population doublings postirradiation. Further, radiation-induced changes in repeat element and global DNA methylation were more similar between proton- and iron-ion-irradiated cells than X-irradiated cells, suggesting that radiation quality rather than LET alone affects the radiation-induced epigenetic profile. Since alterations in DNA methylation have also emerged as one of the most consistent molecular alterations in cancer, these data also suggest the possibility that radiation-induced carcinogenic risk might be affected by radiation quality.

Impaired cell proliferation in mice that persists across at least two generations after paternal irradiation

Irradiation of male F0 mice 6 to 7 weeks prior to mating causes significant changes in the proliferation of F1 and F2 embryonic cells. These changes are revealed as a competitive cell proliferation disadvantage in chimera assays when the affected embryo is paired with a normal embryo in an aggregation chimera. This effect has been observed previously to be transmitted to F1 embryos for absorbed doses from 0.01 to 1.0 Gy; 0.01 Gy is about 100-fold lower than detectable using conventional germline mutation assays. However, until now there has been no reported cross-generation heritability. We now report that this competitive cell proliferation disadvantage persists without degradation in the F2 generation of embryos when F0 males received 1.0 Gy from gamma irradiation 6 and 7 weeks prior to conception of F1 males.

WR-1065, the active metabolite of amifostine, mitigates radiation-induced delayed genomic instability.

Compounds that can protect cells from the effects of radiation are important for clinical use, in the event of an accidental or terrorist-generated radiation event, and for astronauts traveling in space. One of the major concerns regarding the use of radio-protective agents is that they may protect cells initially, but predispose surviving cells to increased genomic instability later. In this study we used WR-1065, the active metabolite of amifostine, to determine how protection from direct effects of high- and low-LET radiation exposure influences genomic stability. When added 30 min before irradiation and in high concentrations, WR-1065 protected cells from immediate radiation-induced effects as well as from delayed genomic instability. Lower, nontoxic concentrations of WR-1065 did not protect cells from death; however, it was effective in significantly decreasing delayed genomic instability in the progeny of irradiated cells. The observed increase in manganese superoxide dismutase protein levels and activity may provide an explanation for this effect. These results confirm that WR-1065 is protective against both low- and high-LET radiation-induced genomic instability in surviving cells.

Elimination of microglia improves cognitive function following cranial irradiation

Cranial irradiation for the treatment of brain cancer elicits progressive and severe cognitive dysfunction that is associated with significant neuropathology. Radiation injury in the CNS has been linked to persistent microglial activation, and we find upregulation of pro-inflammatory genes even 6 weeks after irradiation. We hypothesize that depletion of microglia in the irradiated brain would have a neuroprotective effect. Adult mice received acute head only irradiation (9 Gy) and were administered a dietary inhibitor (PLX5622) of colony stimulating factor-1 receptor (CSF1R) to deplete microglia post-irradiation. Cohorts of mice maintained on a normal and PLX5662 diet were analyzed for cognitive changes using a battery of behavioral tasks 4–6 weeks later. PLX5622 treatment caused a rapid and near complete elimination of microglia in the brain within 3 days of treatment. Irradiation of animals given a normal diet caused characteristic behavioral deficits designed to test medial pre-frontal cortex (mPFC) and hippocampal learning and memory and caused increased microglial activation. Animals receiving the PLX5622 diet exhibited no radiation-induced cognitive deficits, and exhibited near complete loss of IBA-1 and CD68 positive microglia in the mPFC and hippocampus. Our data demonstrate that elimination of microglia through CSF1R inhibition can ameliorate radiation-induced cognitive deficits in mice.

High LET 56Fe ion irradiation induces tissue-specific changes in DNA methylation in the mouse

DNA methylation is an epigenetic mechanism that drives phenotype and that can be altered by environmental exposures including radiation. The majority of human radiation exposures occur in a relatively low dose range; however, the biological response to low dose radiation is poorly understood. Based on previous observations, we hypothesized that in vivo changes in DNA methylation would be observed in mice following exposure to doses of high linear energy transfer (LET) 56Fe ion radiation between 10 and 100 cGy. We evaluated the DNA methylation status of genes for which expression can be regulated by methylation and that play significant roles in radiation responses or carcinogenic processes including apoptosis, metastasis, cell cycle regulation, and DNA repair (DAPK1, EVL, 14.3.3, p16, MGMT, and IGFBP3). We also evaluated DNA methylation of repeat elements in the genome that are typically highly methylated. No changes in liver DNA methylation were observed. Although no change in DNA methylation was observed for the repeat elements in the lungs of these same mice, significant changes were observed for the genes of interest as a direct effect and a delayed effect of irradiation 1, 7, 30, and 120 days post exposure. At delayed times, differences in methylation profiles among genes were observed. DNA methylation profiles also significantly differed based on dose, with the lowest dose frequently affecting the largest change. The results of this study are the first to demonstrate in vivo high LET radiation‐induced changes in DNA methylation that are tissue and locus specific, and dose and time dependent. Environ. Mol. Mutagen. 55:266–277, 2014.