Kinetics of propagation of bystander effects in human cells cultures exposed to low fluences of high LET radiations

International audienceWe and others have previously shown, in confluent cell cultures exposed to low fluences of a particles, that the proportion of cells that upregulate stress-inducible proteins is much higher that the number of cells irradiated. This phenomenon, called bystander effect, is now well accepted and is thought to impact the health risks of exposure to ionizing radiation. Here, we investigate the kinetics of propagation of signaling events that lead to induction of DNA damage in bystander cells in confluent normal human AG1522 fibroblasts exposed to a mean dose of 0.2 cGy from 3.2 MeV a particles (LET ; 124 keV/lm) or 1 GeV/n iron ions (LET ; 151 keV/lm). We evaluated the formation of 53BP1 foci (p53 binding protein 1), which localizes at sites of DNA double strand breaks, as a function of time after irradiation. The fraction of cells whose nuclei were traversed by an irradiating particle was derived from Poisson statistics and estimates of cell geometry, particle fluence and energy loss. At a mean dose of 0.2 cGy, only 1.4% and 1.2% of the cells are traversed through the nucleus by a particle or iron ion tracks, respectively. The number of 53BP1 foci in control cells was ; 0.61 foci per cell. In a particle-irradiated cell cultures, the mean number of foci per cell was 0.73 (p,0.001) at 15 min; it reached 0.90 by 3h (p,0.001) after irradiation, following which a decrease was observed. The same trend was detected when the fraction of cells with foci was considered: it reached 61% at 3h which is higher than the expected 47.4% of the cells (46% in control + 1.4% traversed). The increase in foci formation over the expected value was eliminated when the cells were incubated with a specific inhibitor of ATM (Ataxia Telangiectasia Mutated protein). Analyses of bystander effects in iron ion-irradiated cell cultures and the effect of partial oxygen tension on the kinetic of 53BP1 foci formation in low fluences alpha particle-irradiated cell populations are in progress

Non-targeted Stressful Effects in Normal Human Fibroblast Cultures Exposed to Low Fluences of High Charge, High-Energy (HZE) Particles: Kinetics of Biologic Responses and Significance of Secondary Radiations.

International audienceThe induction of nontargeted stressful effects in cell populations exposed to low fluences of high-charge (Z) and high-energy (E) particles is relevant to estimates of the health risks of space radiation. We investigated the up-regulation of stress markers in confluent normal human fibroblast cultures exposed to 1,000 MeV/u iron ions [linear energy transfer (LET) ∼151 keV/μm] or 600 MeV/u silicon ions (LET ∼50 keV/μm) at mean absorbed doses as low as 0.2 cGy, wherein 1-3% of the cells were targeted through the nucleus by a primary particle. Within 24 h postirradiation, significant increases in the levels of phospho-TP53 (serine 15), p21(Waf1) (CDKN1A), HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation were detected, which suggested participation in the stress response of cells not targeted by primary particles. This was supported by in situ studies that indicated greater increases in 53BP1 foci formation, a marker of DNA damage. than expected from the number of primary particle traversals. The effect was expressed as early as 15 min after exposure, peaked at 1 h and decreased by 24 h. A similar tendency occurred after exposure of the cell cultures to 0.2 cGy of 3.7 MeV α particles (LET ∼109 keV/μm) that targets ∼1.6% of nuclei, but not after 0.2 cGy from 290 MeV/u carbon ions (LET ∼13 keV/μm) by which, on average, ∼13% of the nuclei were hit, which highlights the importance of radiation quality in the induced effect. Simulations with the FLUKA multi-particle transport code revealed that fragmentation products, other than electrons, in cell cultures exposed to HZE particles comprise <1% of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10-20 μm. Thus, the latter are unlikely to significantly contribute to stressful effects in cells not targeted by primary HZE particles

RENEB accident simulation exercise

Purpose: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event. Materials and methods: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners. Results: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes). Conclusions: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested

Effet de proximité induit par ions lourds d'origine cosmique : cinétique des réponses biologiques, mécanismes et importance des radiations secondaires

Widespread evidence indicates that exposure of cell cultures to α particles results in significant biological changes in both the irradiated and non-irradiated bystander cells in the population. The induction of non-targeted biological responses in cell cultures exposed to low fluences of high charge (Z) and high energy (E) particles is relevant to estimates of the health risks of space radiation and to radiotherapy. Here, we investigated the mechanisms underlying the induction of stressful effects in confluent normal human fibroblast cultures exposed to low fluences of 1000 MeV/u iron ions (linear energy transfer (LET) ~151 keV/µm), 600 MeV/u silicon ions (LET ~50 keV/µm) or 290 MeV/u carbon ions (LET ~13 keV/µm). We compared the results with those obtained in cell cultures exposed, in parallel, to low fluences of 0.92 MeV/u α particles (LET ~109 keV/µm).Induction of DNA damage, changes in gene expression, protein carbonylation and lipid peroxidation during 24 h after exposure of confluent cultures to mean doses as low as 0.2 cGy of iron or silicon ions strongly supported the propagation of stressful effects from irradiated to bystander cells. At a mean dose of 0.2 cGy, only ~1 and 3 % of the cells would be targeted through the nucleus by an iron or silicon ion, respectively. Within 24 h post-irradiation, immunoblot analyses revealed significant increases in the levels of phospho-TP53 (serine 15), p21Waf1 (also known as CDKN1A), HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation. The magnitude of the responses suggested participation of non-targeted cells in the response. Furthermore, when the irradiated cell populations were subcultured in fresh medium shortly after irradiation, greater than expected increases in the levels of these markers were also observed during 24 h. Together, the results imply a rapidly propagated and persistent bystander effect. In situ analyses in confluent cultures showed 53BP1 foci formation, a marker of DNA damage, in more cells than expected based on the fraction of cells traversed through the nucleus by an iron or silicon ion. The effect was expressed as early as 15 min after exposure, peaked at 1 h and decreased by 24 h. A similar tendency occurred after exposure to a mean absorbed dose of 0.2 cGy of 3.7 MeV α particles, but not after 0.2 cGy of 290 MeV/u carbon ions.Analyses in dishes that incorporate a CR-39 solid state nuclear track detector bottom identified the cells irradiated with iron or silicon ions and further supported the participation of bystander cells in the stress response. Mechanistic studies indicated that gap junction intercellular communication, DNA repair, and oxidative metabolism participate in the propagation of the induced effects.We also considered the possible contribution of secondary particles produced along the primary particle tracks to the biological responses. Simulations with the FLUKA multi-particle transport code revealed that fragmentation products, other than electrons, in cells cultures exposed to HZE particles comprise <1 % of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10-20 µm Thus, the latter are unlikely to significantly contribute to the stressful effects in cells not targeted by primary HZE particles.De nombreuses études ont montré que l'exposition de cultures cellulaires à des particules α conduit à des changements biologiques importants autant dans les cellules irradiées que dans les cellules bystander non-irradiées. L'étude des réponses biologiques non-ciblées dans des cultures cellulaires exposées à de faibles fluences d’ions lourds permet d’estimer les risques pour la santé du rayonnement spatial et de la radiothérapie. Nous avons caractérisé les mécanismes sous-jacents de l'induction d'effets stressants dans des cultures confluentes de fibroblastes normaux humains exposés à de faibles fluences d’ions fer de 1000 MeV/u (transfert d'énergie linéique (TEL) ~151 keV/µm), d’ions silicium de 600 MeV/u (TEL ~50 keV/µm) ou d’ions carbone de 290 MeV/u (TEL ~13 keV/µm). Nous avons comparé ces résultats avec ceux obtenus dans des cultures cellulaires exposées, en parallèle, à de faibles fluences de particules α de 0,92 MeV/u (TEL ~109 keV/µm). L'induction de dommages à l'ADN, les changements dans l'expression des gènes, la carbonylation des protéines et la peroxydation lipidique durant les 24 h suivant l'exposition de cultures confluentes à de faibles doses (0,2 cGy et plus) d’ions fer ou d'ions silicium ont très largement contribué à la propagation d’effets stressants des cellules irradiées aux cellules bystander non-irradiées. Pour une dose moyenne de 0,2 cGy, seules ~1 et 3 % des cellules seraient irradiées dans le noyau par un ion, respectivement, fer ou silicium. Les immunoblots ont révélés des augmentations significatives des niveaux de phospho-TP53 (sérine 15), p21Waf1 (CDKN1A), HDM2, phospho-ERK1/2, de carbonylation des protéines et de peroxydation lipidique dans les 24 h suivant l’exposition. L'ampleur de ces réponses suggère la participation de cellules non ciblées dans les effets observés. De plus, lorsque les populations cellulaires irradiées ont été ré-ensemencées dans un milieu de culture frais peu après l'irradiation, les niveaux de ces marqueurs ont aussi augmentés durant 24 h. Ensemble, ces résultats montrent un effet rapidement propagé et persistant. Des analyses in situ réalisées dans des cultures cellulaires confluentes ont montré que la formation de foyers de la protéine 53BP1, marqueur de dommages à l'ADN, touchait un nombre de cellules plus important que celui auguré par la fraction de cellules traversées dans le noyau par un ion fer ou silicium. Cet effet est exprimé dès 15 min suivant l'exposition, atteint son maximum 1 h après l’exposition puis diminue jusqu’à 24 h. Une tendance similaire s'est produite après exposition à une dose moyenne absorbée de 0,2 cGy de particules α de 3,7 MeV, mais non après 0,2 cGy d’ions carbone de 290 MeV/u.Des analyses utilisant des puits de cultures intégrant une fine épaisseur de CR-39, détecteur solide de traces nucléaires, et permettant ainsi l’identification des cellules irradiées aux ions fer ou silicium, confirment la participation de cellules bystander dans la réponse au stress. Des études mécanistiques ont, de plus, indiqué que les jonctions gap permettant la communication intercellulaire, certaines voies de la réparation de l’ADN, ainsi que le métabolisme oxydatif participent à la propagation des effets non ciblés induit par des radiations de haut TEL. Nous avons également examiné la contribution possible des particules secondaires produites le long des traces d’ions primaires dans les réponses biologiques. Les simulations réalisées avec le code de transport de particules FLUKA ont révélé que la dose due aux produits de fragmentation, autres que les électrons, est inférieure à 1 % de la dose absorbée dans les cultures cellulaires exposées à des ions lourds. De plus, la dose radiale des ions lourds secondaires est limitée à ~10-20 µm autour de l’ion primaire. Ainsi, ces derniers sont peu susceptibles de contribuer de manière significative à la réponse biologique observée dans des cellules non ciblées par des ions lourds primaire

Impact of the redox environment on propagation of radiation bystander effects: The modulating effect of oxidative metabolism and oxygen partial pressure

International audienceRedox modulated pathways play important roles in out-of-field effects of ionizing radiation. We investigated how the redox environment impacts the magnitude of propagation of stressful effects from irradiated to bystander cells. Normal human fibroblasts that have incorporated [3H]-thymidine were intimately co-cultured with bystander cells in a strategy that allowed isolation of bystander cells with high purity. The antioxidant glutathione peroxidase (GPX) was maintained either at wild-type conditions or overexpressed in the bystanders. Following 24 h of coculture, levels of stress-responsive p21Waf1, p-Hdm2, and connexin43 proteins were increased in bystander cells expressing wild-type GPX relative to respective controls. These levels were significantly attenuated when GPX was ectopically overexpressed, demonstrating by direct approach the involvement of a regulator of intracellular redox homeostasis. Evidence of participation of pro-oxidant compounds was generated by exposing confluent cell cultures to low fluences of 3.7 MeV α particles in presence or absence of t-butyl hydroperoxide. By 3 h post-exposure to fluences wherein only ∼2% of cells are traversed through the nucleus by a particle track, increases in chromosomal damage were greater than expected in absence of the drug (p < 0.001) and further enhanced in its presence (p < 0.05). While maintenance and irradiation of cell cultures at low oxygen pressure (pO2 3.8 mm Hg) to mimic in vivo still supported the participation of bystander cells in responses assessed by chromosomal damage and stress-responsive protein levels (p < 0.001), the effects were attenuated compared to ambient pO2 (155 mm Hg) (p < 0.05). Together, the results show that bystander effects are attenuated at below ambient pO2 and when metabolic oxidative stress is reduced but increased when the basal redox environment tilts towards oxidizing conditions. They are consistent with bystander effects being independent of radiation dose rate

The modulating effect of Oxidative Metabolism and Oxygen Partial Pressure on Propagation of Radiation Bystander Effects

International audienceIntroduction:Redox modulated pathways play important roles in out-of-field effects of ionizing radiation. We investigated how the redox environment impacts the propagation of stressful effects from irradiated to bystander cells.Materials and Methods:- Normal human fibroblasts that have incorporated [3H]-thymidine were intimately co-cultured with bystander cells during 24 h in a strategy that allowed isolation of bystander cells with high purity. The antioxidant glutathione peroxidase (GPX) was maintained either at wild-type conditions or overexpressed in the bystanders.- Confluent cell cultures pre-treated with t-butyl-hydroperoxide (t-BOOH), an oxidizing agent, or maintained in low oxygen pressure environment approaching in vivo conditions were exposed alongside respective controls to low fluences of 3.7 MeV α particles. Results:- Following 24 h of coculture with [3H]-thymidine-labelled cells, levels of stress-responsive p21Waf1, p-Hdm2, and connexin43 proteins were increased in bystander cells expressing wild-type GPX relative to respective controls. These levels were significantly attenuated when GPX was ectopically overexpressed in the bystanders, demonstrating by direct approach the involvement of oxidative metabolism.- By exposing confluent cell cultures to 3.7 MeV α particles wherein only ~2 % of cells are traversed through the nucleus by a particle track, increases in chromosomal damage 3 h post-exposure were greater than expected (p<0.001) and further enhanced in presence of t-BOOH (p<0.05). While maintaining and irradiating cell cultures at low oxygen pressure (3.3 or 6.7 mmHg) still supported the participation of bystander cells in responses assessed by chromosomal damage and stress-responsive protein levels (p<0.001), the effects were attenuated compared to ambient pO2 (141 mm Hg) (p<0.05).Conclusions: Together, the results show that bystander effects are attenuated at below ambient pO2 and when metabolic oxidative stress is reduced but increased when the basal redox environment tilts towards oxidizing conditions. They are consistent with bystander effects being independent of radiation dose rate

Low fluences of High Charge, High Energy (HZE) particles: kinetics of biologic responses and significance of secondary radiations

International audienceThe induction of non-targeted stressful effects in cell populations exposed to low fluences of high charge (Z) and high energy (E) particles is relevant to estimates of the outcome of hadron therapy and the long term health risks of space radiation. To gain greater knowledge of HZE-particle-induced non-targeted effects, we investigated the regulation of stress markers in confluent normal human fibroblast cultures exposed to 1000 MeV/u iron ions (linear energy transfer (LET) ~151 keV/μm) or 600 MeV/u silicon ions (LET ~50 keV/μm) at mean absorbed doses as low as 0.2 cGy, wherein 1-3 % of the cells were targeted through the nucleus by a primary particle. Within 24 h post-irradiation, significant increases in the levels of phospho-TP53 (S15), p21Waf1, HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation were detected, which suggested participation in the stress response of cells not targeted by primary particles. This was supported by in situ studies that indicated greater increases in 53BP1 foci formation, a marker of DNA damage, than expected from the number of primary particle traversals. The effect was expressed as early as 15 min after exposure, peaked at 1 h, and decreased by 24 h. A similar tendency occurred after exposure of the cell cultures to 0.2 cGy of 3.7 MeV α particles (LET ~109 keV/μm) that targets ~1.6 % of nuclei, but not after 0.2 cGy from 290 MeV/u carbon ions (LET ~13 keV/μm) by which, on average, ~13 % of the nuclei were hit, which highlights the importance of radiation quality in the induced effect. Together, the above data suggested participation of bystander cells in the observed responses. To investigate whether secondary particles are a factor in expression of the apparent HZE-particle-induced bystander effects, the tracks of the secondaries were simulated with the multi-particle transport code FLUKA and absorbed doses received by the monolayer of cells adjacent to the targeted cells were assessed. The simulations revealed that fragmentation products, other than electrons, in cell cultures exposed to HZE particles comprise <1 % of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10-20 µm. Thus, the latter are unlikely to significantly contribute to stressful effects in cells not targeted by primary HZE particles. In contrast, electrons contributed up to 40 % of the total dose. Benchmarking the FLUKA code with the MCNPX code is expected to generate rather consistent results for the neutron dose

Espace privé/Espace public, in Théâtre/Public, n°179, Gennevilliers, 4e trimestre 2005

Direction d'un dossier de revue en collaboration avec Bénédicte Boisson