Cytogenetic effects of densely ionising radiation in human lymphocytes: impact of cell cycle delays

The classical cytogenetic assay to estimate the dose to which an individual has been exposed relies on the measurement of chromosome aberrations in lymphocytes at the first post-irradiation mitosis 48 h after in vitro stimulation. However, evidence is accumulating that this protocol results in an underestimation of the cytogenetic effects of high LET radiation due to a selective delay of damaged cells. To address this issue, human lymphocytes were irradiated with C-ions (25-mm extended Bragg peak, LET: 60-85 keV/ micro m) and aberrations were measured in cells reaching the first mitosis after 48, 60, 72 and 84 h and in G2-phase cells collected after 48 h by calyculin A induced premature chromosome condensation (PCC). The results were compared with recently published data on the effects of X-rays and 200 MeV/u Fe-ions (LET: 440 keV/ micro m) on lymphocytes of the same donor (Ritter et al., 2002a). The experiments show clearly that the aberration yield rises in first-generation metaphase (M1) with culture time and that this effect increases with LET. Obviously, severely damaged cells suffer a prolonged arrest in G2. The mitotic delay has a profound effect on the RBE: RBE values estimated from the PCC data were about two times higher than those obtained by conventional metaphase analysis at 48 h. Altogether, these observations argue against the use of single sampling times to quantify high LET induced chromosomal damage in metaphase cells.     

Women's pupillary responses to sexually significant others during the hormonal cycle

Women's sexual preferences can change over the hormonal cycle, as several studies, based on responses to questionnaires, diaries, and ratings of photographs, have indicated increased sexual interests around the time of ovulation. However, fewer studies have measured changes in attention or interest to sexually significant stimuli in terms of physiological responses that are not under voluntary control and measure sexual interest indirectly (i.e., without mention of sexual feelings or activities). In the present study, we indexed changes in sexual interest in terms of changes in the eye pupil's size. Pupillary diameter is known to have a proportional relation to the observer's level of interest and attention to a visual stimulus as well as to physical pleasure. Fourteen women (7 being "pill" users) viewed photos on a computer screen while their pupil diameters were recorded using an infrared eye-tracking device. Three measures were taken for each participant during three time windows that estimated the ovulatory, luteal, and menstrual phase of the cycle. We found an increase in mean pupil diameter for sexually significant stimuli during the fertile phase and this pupillary change was also specific to pictures of the participants' actual sexual partners. Moreover, this effect was only seen for women who did not use oral contraceptives. These findings confirm that women's attention for sexually significant stimuli is higher during their fertile phase of the menstrual cycle, and that changes in sexual interest are implicitly measurable using pupillometry.     

Differentiation and delayed cell death in embryonal stem cells exposed to low doses of ionising radiation

Embryonal stem cells have been used to study the effects of environmentally relevant doses of radiation on cell death and differentation. The ES cells were found to have a greater than 60% chance of surviving the traversal of a single alpha-particle, the lowest possible dose of high linear energy transfer radiation a cell may receive. The ES cells appeared to possess the cell cycle checkpoints believed to prevent the transmission of the radiation damage. However, delayed effects were observed in the progeny. An increased incidence of apoptosis and haempoietic differentiation capacity was found to persist in the ES cell population over many cell divisions. Since both cell death and differentiation are known to play a key role in tissue kinetics, an ES cell model will provide a valuable and versatile cell system for studying the role of cell death and differentiation in the pathology of radiogenic diseases.     

Requirements for apoptotic cell contact in regulation of macrophage responses

An important consequence of macrophage engulfment of apoptotic cells is suppression of inflammatory responses, which was first defined by assay of TNF-alpha release stimulated by LPS. These effects are apparently mediated in part by paracrine effects of TGF-beta released by the subset of stimulated macrophages that ingest apoptotic cells, which suppresses neighboring cells. However, the apoptotic cell-derived signal that stimulates TGF-beta release, and the nature of any additional signals required for the anti-inflammatory response remain poorly defined. In this study, we investigate the requirements for apoptotic cell engagement of macrophage surface receptors in these responses. We show that the apoptotic cell receptors CD36 and alphavbeta3 contribute to apoptotic cell phagocytosis by mouse macrophages, but are not essential for anti-inflammatory responses, suggesting that the mechanisms of response and phagocytosis are separate. In further defining requirements for response, we confirm the importance of TGF-beta in suppression by apoptotic cells, and identify an additional level of control of these effects. We show that LPS-stimulated mouse macrophage TNF-alpha release is only suppressed if macrophages have first contacted apoptotic cells, and hence, bystander macrophages are refractory to TGF-beta released by phagocytosing macrophages. We conclude that the profound suppression of LPS-driven TNF-alpha release by macrophage populations requires hitherto obscure contact-dependent licensing of macrophage responsiveness to TGF-beta by apoptotic cells.     

Genetic analysis of ionising radiation sensitive mutants of cultured mammalian cell lines

The genetic diversity of a range of ionising radiation sensitive mutants of cultured mammalian cell lines has been examined. Hybrids were constructed from suitably marked diploid cells by cell fusion and selected using resistance to HAT and ouabain. Hybrids were examined for ploidy and gamma-ray sensitivity. The data suggest that at least 8 and possibly 9 complementation groups exist which confer sensitivity to ionising radiation. Mutants in at least 3 distinct complementation groups have a reduced ability to rejoin DNA double-strand breaks.     

Regulation of the apoptotic response to radiation damage in B cell development

B lymphocyte precursor cells are ultrasensitive to DNA damage induced by irradiation and drugs and die by apoptosis at very low levels of exposure. Previous studies have shown that this high level sensitivity is p53-dependent, associated with very low level expression of Bcl-2 protein and can be reversed by expression of a bcl-2 transgene. We show here that transition from the pro-B to pre-B and then mature B cell stages of murine lymphopoiesis is accompanied by changes in proliferating cells in sensitivity to X-irradiation induced apoptosis and that this is paralleled by variation in the ratio of anti-(Bcl-2/Bcl-chiL) to pro-(Bax) apoptotic proteins. These are however not fixed or invariant features of developmental stage as they can be modulated by interactions via adhesive interactions with stromal cells, stromal proteins and growth factors. We interpret these data in the context of the stringent developmental regulation of clonal lymphopoiesis and the contingency programming of cells that have extensive proliferative potential with a very low threshold for apoptosis following DNA damage.     

Assessment of genome damage in occupational exposure to ionising radiation and ultrasound

The mutagenic effects of low doses of radiation on occupationally exposed subjects were studied on lymphocyte culture using two methods: analysis of structural chromosome aberrations and micronucleus assay. The results obtained in subjects exposed to ionising radiation alone were compared to those exposed to both ionising radiation and ultrasound. A correlation between the total number of chromosome aberrations and distribution of micronuclei in the genome of somatic cells show higher deviation in the group exposed to X-ray and ultrasound than in the group exposed to X-rays alone. The degree of genome damage in occupational exposure to X-rays and ultrasound were discussed.     

Individual response of humans to ionising radiation: governing factors and importance for radiological protection

Tissue reactions and stochastic effects after exposure to ionising radiation are variable between individuals but the factors and mechanisms governing individual responses are not well understood. Individual responses can be measured at different levels of biological organization and using different endpoints following varying doses of radiation, including: cancers, non-cancer diseases and mortality in the whole organism; normal tissue reactions after exposures; and, cellular endpoints such as chromosomal damage and molecular alterations. There is no doubt that many factors influence the responses of people to radiation to different degrees. In addition to the obvious general factors of radiation quality, dose, dose rate and the tissue (sub)volume irradiated, recognized and potential determining factors include age, sex, life style (e.g., smoking, diet, possibly body mass index), environmental factors, genetics and epigenetics, stochastic distribution of cellular events, and systemic comorbidities such as diabetes or viral infections. Genetic factors are commonly thought to be a substantial contributor to individual response to radiation. Apart from a small number of rare monogenic diseases such as ataxia telangiectasia, the inheritance of an abnormally responsive phenotype among a population of healthy individuals does not follow a classical Mendelian inheritance pattern. Rather it is considered to be a multi-factorial, complex trait.     

Rb at the interface between cell cycle and apoptotic decisions

The retinoblastoma (RB) gene was the first tumor suppressor to be identified, and it continues to be the subject of intense scientific interest. Not only is the RB gene mutated in the rare eye tumor and some other cancers, the Rb protein is functionally inactivated in virtually all human cancers, suggesting that it plays a general role in cellular homeostasis. Rb initially was envisaged as a simple 'on-off' regulator of the cell cycle, and this function was thought to account for its role as a tumor suppressor. Subsequently, however, closer scrutiny revealed unexpected and complex properties of Rb that together contribute to the unique role of Rb in cell biology. For example, Rb appears to be dispensable for normal cell cycling, but it has a special role in triggering permanent cell cycle exit associated with differentiation and senescence. Further, although the role of Rb as tumor suppressor is firmly established, it also has the ability to block apoptosis, which is generally thought to be a property of oncogenes. Our lab has been interested in understanding the dual and seemingly incongruous roles of Rb in cell cycle control and apoptosis. For many of these studies, we have chosen the melanocyte lineage as a model cell system because of the established role for Rb in melanocyte differentiation and survival, and the frequent deregulation of the Rb pathway in melanoma.     

Eurados review of retrospective dosimetry techniques for internal exposures to ionising radiation and their applications

Radiation and Environmental Biophysics - This work presents an overview of the applications of retrospective dosimetry techniques in case of incorporation of radionuclides. The fact that internal...     

Dose limits for occupational exposure to ionising radiation and genotoxic carcinogens: a German perspective

This paper summarises the view of the German Commission on Radiological Protection (“Strahlenschutzkommission”, SSK) on the rationale behind the currently valid dose limits and dose constraints for workers recommended by the International Commission on Radiological Protection (ICRP). The paper includes a discussion of the reasoning behind current dose limits followed by a discussion of the detriment used by ICRP as a measure for stochastic health effects. Studies on radiation-induced cancer are reviewed because this endpoint represents the most important contribution to detriment. Recent findings on radiation-induced circulatory disease that are currently not included in detriment calculation are also reviewed. It appeared that for detriment calculations the contribution of circulatory diseases plays only a secondary role, although the uncertainties involved in their risk estimates are considerable. These discussions are complemented by a review of the procedures currently in use in Germany, or in discussion elsewhere, to define limits for genotoxic carcinogens. To put these concepts in perspective, actual occupational radiation exposures are exemplified with data from Germany, for the year 2012, and regulations in Germany are compared to the recommendations issued by ICRP. Conclusions include, among others, considerations on radiation protection concepts currently in use and recommendations of the SSK on the limitation of annual effective dose and effective dose cumulated over a whole working life.     

Mast cells dysregulate apoptotic and cell cycle genes in mucosal squamous cell carcinoma

Background  

Mucosal squamous cell carcinoma of the head and neck is a disease of high mortality and morbidity. Interactions between the squamous cell carcinoma and the host's local immunity, and how the latter contributes to the biological behavior of the tumor are unclear. In vivo studies have demonstrated sequential mast cell infiltration and degranulation during squamous cell carcinogenesis. The degree of mast cell activation correlates closely with distinct phases of hyperkeratosis, dysplasia, carcinoma in-situ and invasive carcinoma. However, the role of mast cells in carcinogenesis is unclear.     

Contribution of growth and cell cycle checkpoints to radiation survival in Drosophila

Cell cycle checkpoints contribute to survival after exposure to ionizing radiation (IR) by arresting the cell cycle and permitting repair. As such, yeast and mammalian cells lacking checkpoints are more sensitive to killing by IR. We reported previously that Drosophila larvae mutant for grp (encoding a homolog of Chk1) survive IR as well as wild type despite being deficient in cell cycle checkpoints. This discrepancy could be due to differences either among species or between unicellular and multicellular systems. Here, we provide evidence that Grapes is needed for survival of Drosophila S2 cells after exposure to similar doses of IR, suggesting that multicellular organisms may utilize checkpoint-independent mechanisms to survive irradiation. The dispensability of checkpoints in multicellular organisms could be due to replacement of damaged cells by regeneration through increased nutritional uptake and compensatory proliferation. In support of this idea, we find that inhibition of nutritional uptake (by starvation or onset of pupariation) or inhibition of growth factor signaling and downstream targets (by mutations in cdk4, chico, or dmyc) reduced the radiation survival of larvae. Further, some of these treatments are more detrimental for grp mutants, suggesting that the need for compensatory proliferation is greater for checkpoint mutants. The difference in survival of grp and wild-type larvae allowed us to screen for small molecules that act as genotype-specific radiation sensitizers in a multicellular context. A pilot screen of a small molecule library from the National Cancer Institute yielded known and approved radio-sensitizing anticancer drugs. Since radiation is a common treatment option for human cancers, we propose that Drosophila may be used as an in vivo screening tool for genotype-specific drugs that enhance the effect of radiation therapy.