William F. Morgan

Review and evaluation of updated research on the health effects associated with low-dose ionising radiation

While radiation health risks at low doses have traditionally been estimated from high-dose studies, we have reviewed recent literature and concluded that the mechanisms of action for many biological endpoints may be different at low doses from those observed at high doses; that acute doses <100 mSv may be too small to allow epidemiological detection of excess cancers given the background of naturally occurring cancers; that low-dose radiation research should use holistic approaches such as systems-based methods to develop models that define the shape of the dose-response relationship; and that these results should be combined with the latest epidemiology to produce a comprehensive understanding of radiation effects that addresses both damage, likely with a linear effect, and response, possibly with non-linear consequences. Continued research is needed to understand how radiobiology and epidemiology advances should be used to effectively model radiation worker risks.

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.

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

Purpose:u2003This review examines the evidence for the hypothesis that epigenetics are involved in the initiation and perpetuation of radiation-induced genomic instability (RIGI). Conclusion:u2003In 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.

Non-targeted effects induced by ionizing radiation: Mechanisms and potential impact on radiation induced health effects

Not-targeted effects represent a paradigm shift from the DNA centric view that ionizing radiation only elicits biological effects and subsequent health consequences as a result of an energy deposition event in the cell nucleus. While this is likely true at higher radiation doses (>1 Gy), at low doses (<100 mGy) non-targeted effects associated with radiation exposure might play a significant role. Here definitions of non-targeted effects are presented, the potential mechanisms for the communication of signals and signaling networks from irradiated cells/tissues are proposed, and the various effects of this intra- and intercellular signaling are described. We conclude with speculation on how these observations might lead to and impact long-term human health outcomes.

Metabolomic Response of Human Skin Tissue to Low Dose Ionizing Radiation

Understanding how human organs respond to ionizing radiation (IR) at a systems biology level and identifying biomarkers for IR exposure at low doses can help provide a scientific basis for establishing radiation protection standards. Little is known regarding the physiological responses to low dose IR at the metabolite level, which represents the end-point of biochemical processes inside cells. Using a full thickness human skin tissue model and GC-MS-based metabolomic analysis, we examined the metabolic perturbations at three time points (3, 24 and 48 h) after exposure to 3, 10 and 200 cGy of X-rays. PLS-DA score plots revealed dose- and time-dependent clustering between sham and irradiated groups. Importantly, delayed metabolic responses were observed at low dose IR. When compared with the high dose at 200 cGy, a comparable number of significantly changed metabolites were detected 48 h after exposure to low doses (3 and 10 cGy) of irradiation. Biochemical pathway analysis showed perturbations to DNA/RNA damage and repair, lipid and energy metabolisms, even at low doses of IR.

Quantitative proteomic analysis of mitochondrial proteins reveals prosurvival mechanisms in the perpetuation of radiation-induced genomic instability.

Radiation-induced genomic instability is a well-studied phenomenon that is measured as mitotically heritable genetic alterations observed in the progeny of an irradiated cell. The mechanisms that perpetuate this instability are unclear; however, a role for chronic oxidative stress has consistently been demonstrated. In the chromosomally unstable LS12 cell line, oxidative stress and genomic instability were correlated with mitochondrial dysfunction. To clarify this mitochondrial dysfunction and gain insight into the mechanisms underlying radiation-induced genomic instability we have evaluated the mitochondrial subproteome and performed quantitative mass spectrometry analysis of LS12 cells. Of 98 quantified mitochondrial proteins, 17 met criteria for fold changes and reproducibility; and 11 were statistically significant in comparison with the stable parental GM10115 cell line. Previous observations implicated defects in the electron transport chain (ETC) in the LS12 cell mitochondrial dysfunction. Proteomic analysis supports these observations, demonstrating significantly reduced levels of mitochondrial cytochrome c, the intermediary between complexes III and IV of the ETC. Results also suggest that LS12 cells compensate for ETC dysfunction and oxidative stress through increased levels of tricarboxylic acid cycle enzymes and upregulation of proteins that protect against oxidative stress and apoptosis. More than one cellular defect is likely to contribute to the genomic instability phenotype, and evaluation of gene and microRNA expression suggests that epigenetics play a role in the phenotype. These data suggest that LS12 cells have adapted mechanisms that allow survival under suboptimal conditions of oxidative stress and compromised mitochondrial function to perpetuate genomic instability.

Microdistribution and Long-term Retention of 239Pu (NO3)4 in the Respiratory Tracts of an Acutely Exposed Plutonium Worker and Experimental Beagle Dogs

The long-term retention of inhaled soluble forms of plutonium raises concerns as to the potential health effects in persons working in nuclear energy or the nuclear weapons program. The distributions of long-term retained inhaled plutonium-nitrate [(239)Pu (NO(3))(4)] deposited in the lungs of an accidentally exposed nuclear worker (Human Case 0269) and in the lungs of experimentally exposed beagle dogs with varying initial lung depositions were determined via autoradiographs of selected histologic lung, lymph node, trachea, and nasal turbinate tissue sections. These studies showed that both the human and dogs had a nonuniform distribution of plutonium throughout the lung tissue. Fibrotic scar tissue effectively encapsulated a portion of the plutonium and prevented its clearance from the body or translocation to other tissues and diminished dose to organ parenchyma. Alpha radiation activity from deposited plutonium in Human Case 0269 was observed primarily along the subpleural regions while no alpha activity was seen in the tracheobronchial lymph nodes of this individual. However, relatively high activity levels in the tracheobronchial lymph nodes of the beagles indicated the lymphatic system was effective in clearing deposited plutonium from the lung tissues. In both the human case and beagle dogs, the appearance of retained plutonium within the respiratory tract was inconsistent with current biokinetic models of clearance for soluble forms of plutonium. Bound plutonium can have a marked effect on the dose to the lungs and subsequent radiation exposure has the potential to increase cancer risk.

High and Low Doses of Ionizing Radiation Induce Different Secretome Profiles in a Human Skin Model

It is postulated that secreted soluble factors are important contributors of bystander effect and adaptive responses observed in low dose ionizing radiation. Using multidimensional liquid chromatography-mass spectrometry based proteomics, we quantified the changes of skin tissue secretome – the proteins secreted from a full thickness, reconstituted 3-dimensional skin tissue model 48 hr after exposure to 3, 10 and 200 cGy of X-rays. Overall, 135 proteins showed statistical significant difference between the sham (0 cGy) and any of the irradiated groups (3, 10 or 200 cGy) on the basis of Dunnett adjusted t-test; among these, 97 proteins showed a trend of downregulation and 9 proteins showed a trend of upregulation with increasing radiation dose. In addition, there were 21 and 8 proteins observed to have irregular trends with the 10 cGy irradiated group either having the highest or the lowest level among all three radiated doses. Moreover, two proteins, carboxypeptidase E and ubiquitin carboxyl-terminal hydrolase isozyme L1 were sensitive to ionizing radiation, but relatively independent of radiation dose. Conversely, proteasome activator complex subunit 2 protein appeared to be sensitive to the dose of radiation, as rapid upregulation of this protein was observed when radiation doses were increased from 3, to 10 or 200 cGy. These results suggest that different mechanisms of action exist at the secretome level for low and high doses of ionizing radiation.

Genetic and Epigenetic Changes in Chromosomally Stable and Unstable Progeny of Irradiated Cells

Radiation induced genomic instability is a well-studied phenomenon, the underlying mechanisms of which are poorly understood. Persistent oxidative stress, mitochondrial dysfunction, elevated cytokine levels and epigenetic changes are among the mechanisms invoked in the perpetuation of the phenotype. To determine whether epigenetic aberrations affect genomic instability we measured DNA methylation, mRNA and microRNA (miR) levels in well characterized chromosomally stable and unstable clonally expanded single cell survivors of irradiation. While no changes in DNA methylation were observed for the gene promoters evaluated, increased LINE-1 methylation was observed for two unstable clones (LS12 and CS9) and decreased Alu element methylation was observed for the other two unstable clones (115 and Fe5.0–8). These relationships also manifested for mRNA and miR expression. mRNA identified for the LS12 and CS9 clones were most similar to each other (261 mRNA), while the 115 and Fe5.0–8 clones were more similar to each other, and surprisingly also similar to the two stable clones, 114 and 118 (286 mRNA among these four clones). Pathway analysis showed enrichment for pathways involved in mitochondrial function and cellular redox, themes routinely invoked in genomic instability. The commonalities between the two subgroups of clones were also observed for miR. The number of miR for which anti-correlated mRNA were identified suggests that these miR exert functional effects in each clone. The results demonstrate significant genetic and epigenetic changes in unstable cells, but similar changes are almost as equally common in chromosomally stable cells. Possible conclusions might be that the chromosomally stable clones have some other form of instability, or that some of the observed changes represent a sort of radiation signature and that other changes are related to genomic instability. Irrespective, these findings again suggest that a spectrum of changes both drive genomic instability and permit unstable cells to persist and proliferate.

Carcinogenic and inflammatory effects of plutonium-nitrate retention in an exposed nuclear worker and beagle dogs.

Abstract Purpose: Plutonium-nitrate has a moderately rapid translocation rate from the lung to blood stream. Previous studies have shown an unexpected retention of soluble plutonium in the beagles and human case studied here. The inflammatory responses that may be associated with long-term exposure to ionizing radiation were characterized. These pathways include tissue injury, apoptosis, and gene expression modifications. Other protein modifications related to carcinogenesis and inflammation and the various factors that may play a role in orchestrating complex interactions which influence tissue integrity following irradiation were investigated. Materials and methods: We have examined numerous lung samples from a plutonium-exposed worker, a human control, and a variety of plutonium-exposed beagle dogs using immunohistochemistry and quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Results: The exposed human showed interstitial fibrosis in peripheral regions of the lung, but no pulmonary tumors. Beagles with similar doses were diagnosed with tumors in bronchiolo-alveolar, peripheral and sub-pleural alveolar regions of the lung. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed an elevation of apoptosis in tracheal mucosa, tumor cells, and nuclear debris in the alveoli and lymph nodes of the beagles but not in the human case. In both the beagles and human there were statistically significant modifications in the expression of Fas ligand (FASLG), B-cell lymphoma 2 (BCL2), and Caspase 3 (CASP3). Conclusions: The data suggests that FASLG, BCL2, CASP3 and apoptosis play a role in the inflammatory responses following prolonged plutonium exposure. Utilizing these unique tissues revealed which pathways are triggered following the internal deposition and long-term retention of plutonium-nitrate in a human and a large animal model.