The Evidence for Excess Risk of Cancer and Non-Cancer Disease at Low Doses and Dose Rates.

The question of whether there are excess radiation-associated health risks at low dose is controversial. We present evidence of excess cancer risks in a number of (largely pediatrically or in utero exposed) groups exposed to low doses of radiation (<0.1 Gy). Moreover, the available data on biological mechanisms do not provide support for the idea of a low-dose threshold or hormesis for any of these endpoints. There are emerging data suggesting risks of cardiovascular disease and cataract at low doses, but this is less well established. This large body of evidence does not suggest and, indeed, is not statistically compatible with any very large threshold in dose (>10 mGy), or with possible beneficial effects from exposures. The presented data suggest that exposure to low-dose radiation causes excess cancer risks and quite possibly also excess risks of various non-cancer endpoints.

Dose-rate effects in radiation biology and radiation protection.

Quantification of biological effects (cancer, other diseases, and cell damage) associated with exposure to ionising radiation has been a major issue for the International Commission on Radiological Protection (ICRP) since its foundation in 1928. While there is a wealth of information on the effects on human health for whole-body doses above approximately 100 mGy, the effects associated with doses below 100 mGy are still being investigated and debated intensively. The current radiological protection approach, proposed by ICRP for workers and the public, is largely based on risks obtained from high-dose and high-dose-rate studies, such as the Japanese Life Span Study on atomic bomb survivors. The risk coefficients obtained from these studies can be reduced by the dose and dose-rate effectiveness factor (DDREF) to account for the assumed lower effectiveness of low-dose and low-dose-rate exposures. The 2007 ICRP Recommendations continue to propose a value of 2 for DDREF, while other international organisations suggest either application of different values or abandonment of the factor. This paper summarises the current status of discussions, and highlights issues that are relevant to reassessing the magnitude and application of DDREF.

Evidence for variation in human radiosensitivity and its potential impact on radiological protection.

Radiological protection standards generally assume that all members of the population are equally sensitive to the adverse health effects associated with radiation exposure, recognising the age- and sex-related differences in sensitivity to radiation-induced cancer. It has become very clear over recent years that genetic and lifestyle factors can play important roles in the susceptibility of individuals to a range of diseases; as such, the same may apply to radiation-associated diseases. Evidence is accumulating from studies at many levels of biological organisation - cells, experimental organisms, and humans - that a range of radiosensitivity exists between individuals in the population. Consideration of improvements in radiological protection practices to take account of such differences will require the availability of robust and accurate ways to assess the sensitivity of an individual or population subgroup. In addition, there will need to be careful consideration of the ethical aspects relating to use of individual sensitivity information. These ethical considerations are very likely to be exposure context dependent, and require careful risk-benefit balance consideration before practical application.

The lens of the eye: exposures in the UK medical sector and mechanistic studies of radiation effects.

The recommendation from the International Commission on Radiological Protection that the occupational equivalent dose limit for the lens of the eye should be reduced to 20 mSv year(-1), averaged over 5 years with no year exceeding 50 mSv, has stimulated a discussion on the practicalities of implementation of this revised dose limit, and the most appropriate risk and protection framework to adopt. This brief paper provides an overview of some of the drivers behind the move to a lower recommended dose limit. The issue of implementation in the medical sector in the UK has been addressed through a small-scale survey of doses to the lens of the eye amongst interventional cardiologists and radiologists. In addition, a mechanistic study of early and late post-irradiation changes in the lens of the eye in in-vivo-exposed mice is outlined. Surveys and studies such as those described can contribute to a deeper understanding of fundamental and practical issues, and therefore contribute to a robust evidence base for ensuring adequate protection of the eye while avoiding undesirable restrictions to working practices.

Live cell detection of chromosome 2 deletion and Sfpi1/PU1 loss in radiation-induced mouse acute myeloid leukaemia.

The CBA/H mouse model of radiation-induced acute myeloid leukaemia (rAML) has been studied for decades to bring to light the molecular mechanisms associated with multistage carcinogenesis. A specific interstitial deletion of chromosome 2 found in a high proportion of rAML is recognised as the initiating event. The deletion leads to the loss of Sfpi, a gene essential for haematopoietic development. Its product, the transcription factor PU.1 acts as a tumour suppressor in this model. Although the deletion can be detected early following ionising radiation exposure by cytogenetic techniques, precise characterisation of the haematopoietic cells carrying the deletion and the study of their fate in vivo cannot be achieved. Here, using a genetically engineered C57BL/6 mouse model expressing the GFP fluorescent molecule under the control of the Sfpi1 promoter, which we have bred onto the rAML-susceptible CBA/H strain, we demonstrate that GFP expression did not interfere with X-ray induced leukaemia incidence and that GFP fluorescence in live leukaemic cells is a surrogate marker of radiation-induced chromosome 2 deletions with or without point mutations on the remaining allele of the Sfpi1 gene. This study presents the first experimental evidence for the detection of this leukaemia initiating event in live leukemic cells.

Frequency of acute myeloid leukaemia-associated mouse chromosome 2 deletions in X-ray exposed immature haematopoietic progenitors and stem cells.

Exposure to ionising radiation can lead to an increased risk of cancer, particularly leukaemia. In radiation-induced acute myeloid leukaemia (rAML), a partial hemizygous deletion of mouse chromosome 2 is a common feature in several susceptible strains. The deletion is an early event detectable 24h after exposure in bone marrow cells using cytogenetic techniques. Expanding clones of bone marrow cells with chromosome 2 deletions can be detected less than a year after exposure to ionising radiation in around half of the irradiated mice. Ultimately, 15-25% of exposed animals develop AML. It is generally assumed that leukaemia originates in an early progenitor cell or haematopoietic stem cell, but it is unknown whether the original chromosome damage occurs at a similar frequency in committed progenitors and stem cells. In this study, we monitored the frequency of chromosome 2 deletions in immature bone marrow cells (Lin(-)) and haematopoietic stem cells/multipotent progenitor cells (LSK) by several techniques, fluorescent in situ hybridisation (FISH) and through use of a reporter gene model, flow cytometry and colony forming units in spleen (CFU-S) following ex vivo or in vivo exposure. We showed that partial chromosome 2 deletions are present in the LSK subpopulation, but cannot be detected in Lin(-) cells and CFU-S12 cells. Furthermore, we transplanted irradiated Lin(-) or LSK cells into host animals to determine whether specific irradiated cell populations acquire an increased proliferative advantage compared to unirradiated cells. Interestingly, the irradiated LSK subpopulation containing cells carrying chromosome 2 deletions does not appear to repopulate as well as the unirradiated population, suggesting that the chromosomal deletion does not provide an advantage for growth and in vivo repopulation, at least at early stages following occurrence.

Sfpi1/PU.1 mutations in mouse radiation-induced acute myeloid leukaemias affect mRNA and protein abundance and associate with disrupted transcription.

Radiation-induced acute myeloid leukaemias (AMLs) in mice are characterised by deletions and point mutations in the Sfpi1/PU.1 transcription factor. Six AML cell lines were used to examine the impact of three previously described R235 point mutations. AML cells carry myeloid and stem cell markers and the R235 mutations differentially affect mRNA and protein abundance. Expression of Sfpi1/PU.1 target genes was deregulated in a broadly similar fashion irrespective of R235 mutation including Flt3, which is frequently subject to activating mutations in human myeloid leukaemias. While R235 mutations differentially affect protein abundance they resulted in similar disruption of Sfpi1/PU.1 functions.

Radiation cataractogenesis: a review of recent studies.

The lens of the eye is recognized as one of the most radiosensitive tissues in the human body, and it is known that cataracts can be induced by acute doses of less than 2 Gy of low-LET ionizing radiation and less than 5 Gy of protracted radiation. Although much work has been carried out in this area, the exact mechanisms of radiation cataractogenesis are still not fully understood. In particular, the question of the threshold dose for cataract development is not resolved. Cataracts have been classified as a deterministic effect of radiation exposure with a threshold of approximately 2 Gy. Here we review the combined results of recent mechanistic and human studies regarding induction of cataracts by ionizing radiation. These studies indicate that the threshold for cataract development is certainly less than was previously estimated, of the order of 0.5 Gy, or that radiation cataractogenesis may in fact be more accurately described by a linear, no-threshold model.

A possible role for telomerase RNA and telomere length in global mitotic recombination.

Telomeres are specialised structures at the ends of mammalian chromosomes with many unique properties. Recombinational events at telomeres are more frequent than in the remainder of the genome by several orders of magnitude. This study examined the influence of telomerase status and telomere length on genome-wide recombination assessed by genomic sister chromatid exchange (G-SCE). Telomerase deficiency per se appears to increase G-SCE frequencies in splenocytes but as telomeres shorten through subsequent generations of mTerc(-/-) mice this increase is progressively lost. Telomerase status and telomere length also influences the induction of G-SCE by UV light. Even when mitotic recombination is affected by PARP deficiency, mTerc and telomere length interact to further affect G-SCE frequencies. Taken together the data presented here demonstrate that telomerase status and telomere length can affect recombination frequencies genome-wide.

TPD52 and NFKB1 gene expression levels correlate with G2 chromosomal radiosensitivity in lymphocytes of women with and at risk of hereditary breast cancer.

PURPOSE: To evaluate a transcriptomic approach to identify healthy women at increased risk of breast cancer due to G2-radiosensitivity and look at transcripts that are differentially expressed between individuals. MATERIALS AND METHODS: We perform the first study to assess the association of G2 radiosensitivity with basal gene expression in cultured T-lymphocytes from 11 women with breast cancer and 12 healthy female relatives using Affymetrix GeneChips. RESULTS: Transcripts associated with radiosensitivity and breast cancer risk were predominantly involved in innate immunity and inflammation, such as interleukins and chemokines. Genes differentially expressed in radiosensitive individuals were more similarly expressed in close family members than in un-related individuals, suggesting heritability of the trait. The expression of tumour protein D52 (TPD52), a gene implicated in cell proliferation, apoptosis, and vesicle trafficking was the most strongly correlated with G2 score while nuclear factor (kappa)-B (NFKB1) was highly inversely correlated with G2 score. NFKB1 is known to be activated by irradiation and its inhibition has been previously shown to increase radiosensitivity. CONCLUSIONS: Gene expression analysis of lymphocytes may provide a quantitative measure of radiation response potential and is a promising marker of breast cancer susceptibility.

Wavelength dependence of cellular responses in human melanocytes and melanoma cells following exposure to ultraviolet radiation.

PURPOSE: To examine the wavelength dependence of cellular responses in human melanocytes and human melanoma cells exposed to ultraviolet radiation (UVR). MATERIALS AND METHODS: Primary human melanocytes and G361 human melanoma cells were exposed to ultraviolet-C (UVC), ultraviolet-B (UVB), or ultraviolet-A (UVA) radiation. Dose-response relationships for clonal cell survival were assessed, and flow cytometry was used to monitor cell cycle distributions for up to one week post-irradiation. Chromosomal aberrations were scored in exposed and unexposed melanoma cells. RESULTS: G361 melanoma cells were more sensitive than melanocytes to killing by UVB and UVC radiation. This difference in sensitivity between cell types was much less marked following UVA irradiation. The melanoma cells showed a sustained, dose-dependent G2/M block following exposure with all wavelengths; in addition, transit through S phase was slowed following UVA irradiation. There was no apparent block to G1 cells entering S phase at any wavelength. Melanocytes, on the other hand, showed a marked G1 arrest, particularly following UVA irradiation. Cytogenetic results showed a dose-dependent increase in chromatid-type aberrations, mostly gaps, breaks and exchanges, in exposed melanoma cells. CONCLUSION: These results show that G361 malignant melanoma cells have lost the ability to regulate the cell cycle at the G1/S checkpoint and are more sensitive than melanocytes to cell killing by UVC and UVB but not UVA radiation. Similarly, exposure of these melanoma cells to UVC and UVB, and to a much lesser extent UVA, induced chromatid aberrations. UVA nevertheless induced strong cell cycle delays in both cell types, indicating that UVA exposure can significantly affect genome metabolism.

In utero and neonatal sensitivity of ApcMin/+ mice to radiation-induced intestinal neoplasia.

PURPOSE: To assess the sensitivity of ApcMin/+ mice (adenomatous polyposis coli Apc, multiple intestinal neoplasia, Min) to the development of intestinal adenomas after x-irradiation in utero, as neonates, or as young adults. MATERIALS AND METHODS: CHB6 ApcMin/+ mice were exposed to an acute dose of 2 Gy x-rays either in utero on day 7 or 14 post-conception, as 2-day or 10-day neonates or as 35-day young adults. Tumour identification and counting was performed 200-214 days later. RESULTS: Irradiation as 10-day-old neonates resulted in a significantly greater overall tumour incidence (average of about 130 tumours per animal) than irradiation as 35-day-old young adults (about 70 tumours). Irradiation as 2-day-old neonates resulted in an intermediate incidence (about 85 tumours). In contrast, the greatest tumour incidence observed after in utero irradiation of ApcMin/+ mice, of about 44 tumours per animal after 2 Gy irradiation at 14 days post-conception, was significantly lower than the incidence in irradiated adults. Tumour incidences after irradiation as 7-day embryos was not significantly raised above numbers in unirradiated controls (about 30 tumours). These tumour numbers include cystic crypts, largely radiation-induced, which were classed as early stage microadenomas on the basis of loss of wild-type Apc+ and expression of beta-catenin. CONCLUSIONS: The sensitivity of ApcMin/+ mice to the induction of intestinal tumours by radiation was shown to be in the order: 10 d neonates>2 d neonates>35 d young adults>14 d fetus>7 d embryo.

Assessing radiation-associated mutational risk to the germline: repetitive DNA sequences as mutational targets and biomarkers.

This review assesses recent data on mutational risk to the germline after radiation exposure obtained by molecular analysis of tandemly repeated DNA loci (TRDLs): minisatellites in humans and expanded simple tandem repeats in mice. Some studies, particularly those including exposure to internal emitters, indicate that TRDL mutation can be used as a marker of human radiation exposure; most human studies, however, are negative. Although mouse studies have suggested that TRDL mutation analysis may be more widely applicable in biomonitoring, there are important differences between the structure of mouse and human TRDLs. Mutational mechanisms probably differ between the two species, and so care should be taken in predicting effects in humans from mouse data. In mice and humans, TRDL mutations are largely untargeted with only limited evidence of dose dependence. Transgenerational mutation has been observed in mice but not in humans, but the mechanisms driving such mutation transmission are unknown. Some minisatellite variants are associated with human diseases and may affect gene transcription, but causal relationships have not yet been established. It is concluded that at present the TRDL mutation data do not warrant a dramatic revision of germline or cancer risk estimates for radiation.

Evidence for complex multigenic inheritance of radiation AML susceptibility in mice revealed using a surrogate phenotypic assay.

The mapping of genes which affect individual cancer risk is an important but complex challenge. A surrogate assay of susceptibility to radiation-induced acute myeloid leukaemia (AML) in the mouse based on chromosomal radiosensitivity has been developed and validated. This assay was applied to the mapping of radiation-induced AML risk modifier loci by association with microsatellite markers. A region on chromosome (chr) 18 with strong association is identified and confirmed by backcross analysis. Additional loci on chrs 8 and 13 show significant association. A key candidate gene Rbbp8 on chr18 is identified. Rbbp8 is shown to be upregulated in response to X-irradiation in the AML sensitive CBA strain but not AML resistant C57BL/6 strain. This study demonstrates the strength of utilizing surrogate endpoints of cancer susceptibility in the mapping of mouse loci and identifies additional loci that may affect radiation cancer risk.

Long but dysfunctional telomeres correlate with chromosomal radiosensitivity in a mouse AML cell line.

PURPOSE: To compare the chromosomal radiosensitivity of C3H mouse acute myeloid leukaemia (AML) cell lines 7926 and 8709 and to investigate the mechanistic basis of the radiosensitivity observed in 7926. MATERIALS AND METHODS: Yields of chromosome aberrations following X-irradiation were determined in Giemsa-stained metaphases. Cell cycle phase distributions were determined by BrdU incorporation and microscopy, apoptosis was assessed by caspase assays. Telomerase activity (TRAP assay), telomere length (Q-FISH and Southern blotting) and telomere function (Robertsonian-like fusion formation) were also examined. The expression levels of telomerase components, telomerase regulators and DNA PKcs were determined on Northern blots. RESULTS: A total of 4.5-7.6-fold elevated chromosome aberration yields were found in 7926 by comparison with 8709 3-24h after 0.5 and 1 Gy X-ray exposure. This difference could not be accounted for by differences in chromatid break-rejoining rates, cell cycle phase distribution or the induction of apoptosis. Telomeres and telomerase were dysfunctional in 7926. However, average telomere length was approximately two-fold greater than in 8709. CONCLUSION: Defective telomere function in 7926 correlates with chromosomal radiosensitivity. This implicates telomere function in addition to telomere length as a determinant of chromosomal radiosensitivity.

Telomeric sequences, radiation sensitivity and genomic instability.

PURPOSE: To review the basic features of telomeres with particular emphasis on their potential importance in radiation biology. Recent findings suggest that telomere length can influence radiation sensitivity in mouse and that several human radiosensitive disorders also show abnormalities in telomere dynamics. Numerous studies indicate that telomeric sequences may play a role in determining the stability of certain genomic regions both spontaneously and following irradiation. Furthermore, a number of transmissible genomic instability systems have been described in which it appears that telomere metabolism may be contributing to the delayed effects observed. Features of telomeres and telomere biology relevant to these topics are reviewed. CONCLUSIONS: The evidence that telomeres and the molecular pathways of telomere maintenance can play a role in determining the outcome of radiation exposure is now substantial. Thus, the field of telomere biology deserves continued attention from radiobiologists.

In utero haemopoietic sensitivity to alpha, beta or X-irradiation in CBA/H mice.

PURPOSE: To assess in utero sensitivity to x-rays, alpha-emissions from plutonium-239 and beta-emissions from tritium in terms of induction of chromosomal aberrations in bone marrow cells. MATERIALS AND METHODS: CBA/H mice were exposed to a single dose of X-rays (0.5Gy) on either day 7 or day 14 of pregnancy or given (239)Pu (100 kBq kg(-1)) by intraperitoneal injection on either day 6 or day 13. Tritium was administered to mice throughout pregnancy as either tritiated water, ad libitum in drinking water (total intake averaged 130 MBq), or as homogenized tritiated cress, administered by gastric intubation (total 60 MBq). Irradiated and unexposed control mice and their offspring were sacrificed at 2-8 weeks after birth. Direct metaphase preparations from femoral bone marrow cells from mothers and offspring were used for G-band analysis. RESULTS: The incidence of stable aberrations was significantly and similarly increased in neonatal and maternal marrow samples after exposure to X-rays, (239)Pu or (3)H. The estimated average bone absorbed doses from (239)Pu in pregnant females were similar to the X-ray dose of 0.5 Gy, suggesting a low RBE for alpha-irradiation in adults. The similar levels of damage observed in neonates after X-irradiation and 239Pu exposure are indicative of greater in utero sensitivity to alpha-irradiation since the overall estimated in utero alpha-particle doses to haemopoietic tissue were much lower. In utero doses from (3)H and corresponding maternal doses were around 0.5Gy, showing no evidence of greater in utero sensitivity, no significant difference between the effects of the two forms of tritium, and were consistent with an RBE value of 1-2. CONCLUSIONS: Comparison of stable aberration yields in haemopoietic cells suggests a greater sensitivity to alpha-particles from (239)Pu than X-rays or beta-particles from (3)H for irradiation in utero but a low RBE value in adults.

Lack of detectable transmissible chromosomal instability after in vivo or in vitro exposure of mouse bone marrow cells to 224Ra alpha particles.

Several studies over recent years have highlighted the possibility that radiation can induce transmissible genomic instability. Most of these involve in vitro irradiation and usually in vitro culture. Here it is reported that the short-half-life bone-seeking alpha-particle emitter (224)Ra did not induce excess transmissible chromosomal instability in CBA/H mouse bone marrow cells in a 100-day period after in vivo or in vitro exposure. Similarly, no excess transmissible chromosomal instability could be detected after in vivo whole-body X irradiation. It was noted, however, that short-term culture of murine bone marrow cells elevated yields of aberrations, as did transplantation of untreated marrow into radiation-ablated hosts. These findings emphasize the sensitivity of murine hemopoietic tissue to experimental manipulation and reinforce the importance of appropriate concurrent control experiments in any investigation of transmissible genomic instability.