The Effects of Combined Exposure to Simulated Microgravity, Ionizing Radiation, and Cortisol on the In Vitro Wound Healing Process.

Human spaceflight is associated with several health-related issues as a result of long-term exposure to microgravity, ionizing radiation, and higher levels of psychological stress. Frequent reported skin problems in space include rashes, itches, and a delayed wound healing. Access to space is restricted by financial and logistical issues; as a consequence, experimental sample sizes are often small, which limits the generalization of the results. Earth-based simulation models can be used to investigate cellular responses as a result of exposure to certain spaceflight stressors. Here, we describe the development of an in vitro model of the simulated spaceflight environment, which we used to investigate the combined effect of simulated microgravity using the random positioning machine (RPM), ionizing radiation, and stress hormones on the wound-healing capacity of human dermal fibroblasts. Fibroblasts were exposed to cortisol, after which they were irradiated with different radiation qualities (including X-rays, protons, carbon ions, and iron ions) followed by exposure to simulated microgravity using a random positioning machine (RPM). Data related to the inflammatory, proliferation, and remodeling phase of wound healing has been collected. Results show that spaceflight stressors can interfere with the wound healing process at any phase. Moreover, several interactions between the different spaceflight stressors were found. This highlights the complexity that needs to be taken into account when studying the effect of spaceflight stressors on certain biological processes and for the aim of countermeasures development.

Modulation of VEGF Expression and Oxidative Stress Response by Iodine Deficiency in Irradiated Cancerous and Non-Cancerous Breast Cells.

Breast cancer remains a major concern and its physiopathology is influenced by iodine deficiency (ID) and radiation exposure. Since radiation and ID can separately induce oxidative stress (OS) and microvascular responses in breast, their combination could additively increase these responses. Therefore, ID was induced in MCF7 and MCF12A breast cell lines by medium change. Cells were then X-irradiated with doses of 0.05, 0.1, or 3 Gy. In MCF12A cells, both ID and radiation (0.1 and 3 Gy) increased OS and vascular endothelial growth factor (VEGF) expression, with an additive effect when the highest dose was combined with ID. However, in MCF7 cells no additive effect was observed. VEGF mRNA up-regulation was reactive oxygen species (ROS)-dependent, involving radiation-induced mitochondrial ROS. Results on total VEGF mRNA hold true for the pro-angiogenic isoform VEGF165 mRNA, but the treatments did not modulate the anti-angiogenic isoform VEGF165b. Radiation-induced antioxidant response was differentially regulated upon ID in both cell lines. Thus, radiation response is modulated according to iodine status and cell type and can lead to additive effects on ROS and VEGF. As these are often involved in cancer initiation and progression, we believe that iodine status should be taken into account in radiation prevention policies.

Folic Acid Fortification Prevents Morphological and Behavioral Consequences of X-Ray Exposure During Neurulation.

Previous studies suggested a causal link between pre-natal exposure to ionizing radiation and birth defects such as microphthalmos and exencephaly. In mice, these defects arise primarily after high-dose X-irradiation during early neurulation. However, the impact of sublethal (low) X-ray doses during this early developmental time window on adult behavior and morphology of central nervous system structures is not known. In addition, the efficacy of folic acid (FA) in preventing radiation-induced birth defects and persistent radiation-induced anomalies has remained unexplored. To assess the efficacy of FA in preventing radiation-induced defects, pregnant C57BL6/J mice were X-irradiated at embryonic day (E)7.5 and were fed FA-fortified food. FA partially prevented radiation-induced (1.0 Gy) anophthalmos, exencephaly and gastroschisis at E18, and reduced the number of pre-natal deaths, fetal weight loss and defects in the cervical vertebrae resulting from irradiation. Furthermore, FA food fortification counteracted radiation-induced impairments in vision and olfaction, which were evidenced after exposure to doses ≥0.1 Gy. These findings coincided with the observation of a reduction in thickness of the retinal ganglion cell and nerve fiber layer, and a decreased axial length of the eye following exposure to 0.5 Gy. Finally, MRI studies revealed a volumetric decrease of the hippocampus, striatum, thalamus, midbrain and pons following 0.5 Gy irradiation, which could be partially ameliorated after FA food fortification. Altogether, our study is the first to offer detailed insights into the long-term consequences of X-ray exposure during neurulation, and supports the use of FA as a radioprotectant and antiteratogen to counter the detrimental effects of X-ray exposure during this crucial period of gestation.

A detailed characterization of congenital defects and mortality following moderate X-ray doses during neurulation.

BACKGROUND: Both epidemiological and animal studies have previously indicated a link between in utero radiation exposure and birth defects such as microphthalmos, anophthalmos, and exencephaly. However, detailed knowledge on embryonic radiosensitivity during different stages of neurulation is limited, especially in terms of neural tube defect and eye defect development. METHODS: To assess the most radiosensitive stage during neurulation, pregnant C57BL6/J mice were X-irradiated (0.5 Gy or 1.0 Gy) at embryonic days (E)7, E7.5, E8, E8.5, or E9. Next, the fetuses were scored macroscopically for various defects and prenatal resorptions/deaths were counted. In addition, cranial skeletal development was ascertained using the alcian-alizarin method. Furthermore, postnatal/young adult survival was followed until 5 weeks (W5) of age, after X-irradiation at E7.5 (0.1 Gy, 0.5 Gy, or 1.0 Gy). In addition, body and brain weights were registered at adult age (W10) following X-ray exposure at E7.5 (0.1 Gy, 0.5 Gy). RESULTS: Several malformations, including microphthalmos and exencephaly, were most evident after irradiation at E7.5, with significance starting respectively at 0.5 Gy and 1.0 Gy. Prenatal mortality and weight were significantly affected in all irradiated groups. Long-term follow-up of E7.5 irradiated animals revealed a reduction in survival at 5 weeks of age after high dose exposure (1.0 Gy), while lower doses (0.5 Gy, 0.1 Gy) did not affect brain and body weight at postnatal week 10. CONCLUSIONS: With this study, we gained more insight in radiosensitivity throughout neurulation, and offered a better defined model to further study radiation-induced malformations and the underlying mechanisms.

Radiation sensitivity of the gastrula-stage embryo: Chromosome aberrations and mutation induction in lacZ transgenic mice: The roles of DNA double-strand break repair systems.

At the gastrula phase of development, just after the onset of implantation, the embryo proper is characterized by extremely rapid cell proliferation. The importance of DNA repair is illustrated by embryonic lethality at this stage after ablation of the genes involved. Insight into mutation induction is called for by the fact that women often do not realize they are pregnant, shortly after implantation, a circumstance which may have important consequences when women are subjected to medical imaging using ionizing radiation. We screened gastrula embryos for DNA synthesis, nuclear morphology, growth, and chromosome aberrations (CA) shortly after irradiation with doses up to 2.5Gy. In order to obtain an insight into the importance of DNA repair for CA induction, we included mutants for the non-homologous end joining (NHEJ) and homologous recombination repair (HRR) pathways, as well as Parp1-/- and p53+/- embryos. With the pUR288 shuttle vector assay, we determined the radiation sensitivity for point mutations and small deletions detected in young adults. We found increased numbers of abnormal nuclei 5h after irradiation; an indication of disturbed development was also observed around this time. Chromosome aberrations 7h after irradiation arose in all genotypes and were mainly of the chromatid type, in agreement with a cell cycle dominated by S-phase. Increased frequencies of CA were found for NHEJ and HR mutants. Gastrula embryos are unusual in that they are low in exchange induction, even after compromised HR. Gastrula embryos were radiation sensitive in the pUR288 shuttle vector assay, giving the highest mutation induction ever reported for this genetic toxicology model. On theoretical grounds, a delayed radiation response must be involved. The compromised developmental profile after doses up to 2.5Gy likely is caused by both apoptosis and later cell death due to large deletions. Our data indicate a distinct radiation-sensitive profile of gastrula embryos, including some stage-specific aspects that are not as yet understood.

Dose- and time-dependent gene expression alterations in prostate and colon cancer cells after in vitro exposure to carbon ion and X-irradiation.

Hadrontherapy is an advanced form of radiotherapy that uses beams of charged particles (such as protons and carbon ions). Compared with conventional radiotherapy, the main advantages of carbon ion therapy are the precise absorbed dose localization, along with an increased relative biological effectiveness (RBE). This high ballistic accuracy of particle beams deposits the maximal dose to the tumor, while damage to the surrounding healthy tissue is limited. Currently, hadrontherapy is being used for the treatment of specific types of cancer. Previous in vitro studies have shown that, under certain circumstances, exposure to charged particles may inhibit cell motility and migration. In the present study, we investigated the expression of four motility-related genes in prostate (PC3) and colon (Caco-2) cancer cell lines after exposure to different radiation types. Cells were irradiated with various absorbed doses (0, 0.5 and 2 Gy) of accelerated (13)C-ions at the GANIL facility (Caen, France) or with X-rays. Clonogenic assays were performed to determine the RBE. RT-qPCR analysis showed dose- and time-dependent changes in the expression of CCDC88A, FN1, MYH9 and ROCK1 in both cell lines. However, whereas in PC3 cells the response to carbon ion irradiation was enhanced compared with X-irradiation, the effect was the opposite in Caco-2 cells, indicating cell-type-specific responses to the different radiation types.

Studies on the adaptive response in mouse female germ cells X-irradiated in vitro at two different stages of maturation.

BACKGROUND: Radioadaptation is a phenomenon whereby cells exposed to a low dose of ionizing radiation are more resistant to a much higher dose delivered some time thereafter. This phenomenon could result from the activation of damage repair and/or antioxidant defense systems by the low dose. MATERIALS AND METHODS: The existence of a cytogenetic adaptive response in female germ cells was investigated using a recently developed in vitro system. Mouse ovarian follicles were cultured from an early preantral stage up to ovulation. The follicles were X-irradiated with either 2 or 4 Gy ("challenge dose") preceded or not by 50 mGy ("conditioning dose", 5 h earlier), on days 0 or 12 of the culture. Ovulated oocytes were collected on day 13, fixed and analyzed for the presence of chromosome aberrations. RESULTS: Irradiation with 2 or 4 Gy on days 0 or 12 did not influence ovulation but had dose-dependent effects on the germinal vesicle breakdown of the oocytes. It also caused dose-dependent chromosome damage, with a greater sensitivity of oocytes to this effect when irradiation occurred on day 12 than on day 0. Prior irradiation of oocytes with the dose of 50 mGy led to a reduction in the yield of chromosome aberrations when irradiation occurred on day 12 but not on day 0. CONCLUSION: These results suggest that pre-irradiation of mouse pre-ovulatory oocytes with a low conditioning dose could confer on them some protection against radiation-induced chromosomal damage by a subsequent challenge dose of a few Gy.

Histone H1 and cdk1 kinase activities in early embryos of four mouse strains after X-irradiation.

The cdk1/cyclin B1 complex is a universal regulator known to be responsible for driving the cell-cycle from the G2- to the M-phase. To investigate the effects of irradiation on the activity of this complex in preimplantation embryos, we irradiated one- and two-cell mouse embryos with X-rays, and measured the fluctuations of histone H1 and cdk1 kinase activity. Four mouse strains with different radiation sensitivities were chosen: the BALB/c and the Heiligenberger (radiation-sensitive) and the C57BL and the CF1 (radiation-resistant) strains. Embryos irradiated in the first cell-cycle arrested in the G2-phase. However, the dynamics of this radiation-induced G2-block were different between the mouse strains tested. Indeed, in the C57BL and the CF1 strains, X-irradiation with 2.5 Gy induced a very short G2 block before the one-cell embryos could then proceed to mitosis. On the contrary, X-irradiation in BALB/c induced a G2-arrest that lasted about 20 h, with the percentage of embryos blocked in G2 depending on the dose, whilst in the Heiligenberger strain, all irradiated embryos developed a G2-block, which was dependent in duration on the radiation dose. In all mouse strains, the histone H1 kinase activity remained low during the G2 arrest, while it showed values comparable to that of control embryos during mitosis. X-irradiation is known to induce a change in the phosphorylation state of the cdk1 protein kinase in adult somatic cells. In embryos from the BALB/c and C57BL strains, the histone H1 kinase activities were confirmed by the cdk1 phosphorylation pattern: the inactive and phosphorylated form of cdk1 was observed in G2 arrested 1-cell embryos, while the active and dephosphorylated form of cdk1 was present in dividing control and irradiated 1-cell embryos. X-irradiation at the 2-cell stage only induced a short G2-arrest in all tested mouse strains. In conclusion, cell-cycle effects in early embryos under normal conditions and after irradiation are strictly paralleled by changes in the activity of the central cell-cycle driving enzyme complex.

Intracellular signal transduction in mouse oocytes and irradiated early embryos.

In order to determine the effect of X-irradiation on intracellular signal transduction in mouse oocytes and embryos, JNK, ERK and p38 kinase activities were measured by the state of phosphorylation of their respective substrates (c-Jun, Elk-1 and ATF-2, respectively) in two mouse strains differing in radiation sensitivity, namely C57BL and BALB/c. In a first step, control oocytes and embryos were compared for their respective kinase activities at various stages of oocyte maturation (germinal vesicle and metaphases of 1st and 2nd meiosis stages) and early embryonic development (1-, 2-, 4-, 8- and 16-cell, morula and blastula stages). Levels of p38, ERK or JNK kinase activities were shown to vary with the stage of oocyte maturation and embryo development. In a second step, 1- and 2-cell embryos were X-irradiated with 2.5 Gy during the S-phase of the 1st or the 2nd cell-cycle, respectively. There were no significant differences in p38, ERK and JNK kinase activities between control and irradiated embryos, whatever the stage or mouse strain was considered. In conclusion, p38, ERK and JNK kinase activities were shown to vary during oocyte maturation and early embryonic development. Apparently, X-irradiation did not affect these kinase activities at the 1- and 2-cell stages in either mouse strains regardless of their difference in radiation sensitivity.

Telomere shortening is associated with malformation in p53-deficient mice after irradiation during specific stages of development.

The natural ends of linear chromosomes, the telomeres, recruit specific proteins in the formation of protective caps that preserve the integrity of the genome. Unprotected chromosomes induce DNA damage checkpoint cascades and ultimately lead to senescence both in mouse and man in a p53 dependent manner and initial telomere length setting therefore determines the proliferative capacity of each cell. Yet, only little information is available on telomere biology during embryonic development. We have previously shown that the p53 gene plays a crucial role in the development of malformations (exencephaly, gastroschisis, polydactyly, cleft palate and dwarfism) in control and irradiated mouse embryos. Here, we investigated telomere biology and the outcome of radiation exposure in wild type (p53+/+) and p53-mutant (p53+/-- and--/--) C57BL mouse foetuses irradiated at three different developmental stages. We show that telomeres are significantly shorter in malformed foetuses as compared to normal counterparts. In addition, our results indicate that the observed telomere attrition is primarily associated with p53-deficiency but is also modulated by irradiation, more specifically during the gastrulation and organogenesis stages. In conclusion, we formulate a hypothesis in which telomere shortening is linked to the absence of p53 in mouse foetuses and that when, in the presence of shorter telomeres, these foetuses are irradiated, the chance for the occurrence of developmental defects increases substantially.

Developmental abnormalities induced by X-irradiation in p53 deficient mice.

In order to assess the influence of p53 inactivation on radiation-induced developmental effects, male mice heterozygous for the wild-type p53 allele (mimicking the human Li-Fraumeni syndrome) were crossed with C57BL females, and their heterozygous p53+/- progeny were mated with each other to obtain p53+/-, p53-/- and p53+/+ embryos. Pregnant females were X-irradiated with 0.5 Gy on days 1 (pre-implantation period), 8 or 11 (organogenesis period) of gestation. Dissection of the pregnant females occurred on day 19 of gestation. The p53 genotype of the foetuses was determined by PCR from small pieces of soft tissues. Exencephaly was the only external malformation found in the control group. It affected essentially p53-/- female foetuses. A number of p53+/- and p53+/- control foetuses also showed dwarfism, or underdevelopment. In the group irradiated on day 1, the frequency of abnormal foetuses was, paradoxically, lower than that found in the control group. As in that group, exencephaly and dwarfism constituted the only anomalies that were found. Exencephaly affected only homozygous p53-/- females, while dwarfism concerned either p53-/- or p53+/- foetuses, with a majority of females. Irradiation on day 8 of gestation induced a significant increase in the frequency of abnormal foetuses, compared to the control group. Various malformations were observed in addition to exencephaly, including gastroschisis, polydactyly, cephalic oedema and cleft palate. All malformed foetuses were either homozygous p53-/- or heterozygous p53+/- while most affected foetuses were females, as was the case for dwarf individuals. Irradiation on day 11 did not cause an increase in the frequency of abnormal foetuses, in comparison with the controls. However, a large spectrum of external malformations was again noticed, as in the group irradiated on day 8. All affected foetuses were homozygous p53-/- and there were slightly more abnormal females than males (3 out of 5). No dwarfs were found in this group. Overall, these results confirm the importance of the p53 tumour-suppressor protein for normal embryonic development. They clearly show that homozygous p53-/- (or heterozygous p53+/- to a lesser extent) foetuses are more at risk for radiation-induction of external malformations during the organogenesis period, and that the risk of developing such malformations is much higher for females than for males. In contrast to results published very recently by others, we found that malformed foetuses resulting from an X-irradiation with a low-dose during the highly sensitive period of gastrulation are able to survive to birth.