**2.2 Double strand break repair in irradiated human lymphocytes**

To evaluate the repair of DSBs in PBL irradiated with -rays or low-energy protons, we analyzed -H2AX kinetics through foci formation and disappearance. The presence of nuclear foci was monitored by *in situ* immunofluorescence at different time points after IR. Figure 2 shows the different -H2AX foci pattern at 2h after IR with high- and low-LET radiation, reflecting the sparsely and densely nature of IR.

Fig. 2. Visualization by *in situ* immunofluorescence of -H2AX foci in human PBL irradiated with -rays or low-energy protons. The pattern of -H2AX localization within the nucleus is strictly dependent on the quality of radiation. Low-LET radiation, such as -rays, hit the cells throughout all directions, and DSBs are sparsely distributed; on the contrary, high-LET radiation such as protons, give raise to clustered DNA damage along tracks.

In irradiated PBL the kinetics of DSB repair was different according to the quality of radiation. In particular, the fraction of foci-positive cells was higher in -irradiated than in proton-irradiated lymphocytes at all times, except at 24h after IR. Early after irradiation (30 min and 2h) -H2AX foci were present in 80% and 43% of PBL, irradiated respectively with -rays and protons (Fig. 3A). This difference is mainly due to the quality of radiation: while sparsely IR as -rays lose their energy throughout all directions thus hitting all nuclei, densely IR as protons, hits the fraction of cells along their track. The preferential production of complex aberrations is related to the unique energy deposition patterns produced by densely ionizing radiation, causing highly localized multiple DNA damage. At 6h after IR the percentage of foci-positive cells decreased, revealing the repair capacity of DSBs in both kind of irradiated lymphocytes, although the repair kinetics was faster in -irradiated PBL. At 24h after IR the percentage of -H2AX foci positive cells tended to reach the value of nonirradiated PBL, either in - and in proton-irradiated PBL.

The mean number of -H2AX foci per nucleus was higher in PBL irradiated with -rays than with protons, at all times after IR (Fig. 3B). In our experiments, most of PBL displayed 10–20 or more -H2AX foci/nucleus 30 min after irradiation, giving a maximum yield of 4 foci/Gy, a number similar to that reported for human PBL irradiated with X-rays (about 10 foci/Gy) (Sak et al., 2007; Schertan et al., 2008), but much lower than that determined in human fibroblasts (32.2 foci/Gy) (Hamada et al., 2006). It has been reported that the number of -H2AX foci is well consistent with the number of theoretically calculated DSB/Gy of sparsely ionizing radiation (i.e. about 40) (Ward, 1991), if one DSB is contained per focus.

The DNA-Damage Response to Ionizing Radiation in Human Lymphocytes 9

on human cells grown in microgravity during space flights or in modeled microgravity (MMG) on Earth, report changes among genes involved in apoptosis induction, cell adhesion, cytoskeletal features and cell differentiation, even if large differences in culture conditions, cell types and methods to simulate microgravity were adopted in those experiments (Hammond et al., 2000; Lewis et al., 2001, Torigoe et al., 2001, Infanger et al., 2007). While the genotoxic effects of ionizing radiation have been intensely studied, the consequence of the reduced gravity together with radiation is still unclear. Therefore, it is of special importance to verify whether DDR is affected by the combined effects of IR and microgravity, in view of the prolonged permanence of man in future space missions. To analyze the possibility that a reduced gravitational force impairs the DDR pathway, increasing the risk of the exposure to conditions occurring during spaceflight, we studied the DDR to ionizing radiation in human PBL incubated in MMG and in parallel static conditions. Microgravity was simulated by culturing PBL in the Rotating Wall Vessel bioreactor (Synthecon, Cellon, Fig. 4) placed inside a humidified incubator, vertically

The Rotating Wall Vessel was developed at the NASA Johnson Space Center (Houston, TX) to simulate, as accurately as possible, culture conditions predicted to occur during experiments in space. In the rotating system, the gravity is balanced by equal and opposite mechanical forces (centrifugal, Coriolis and shear components), and the gravitational vector is reduced to about 10−2 g. In these conditions, single cells are nearly always in suspension, rotating quasi-stationary with the fluid, in a low-shear culture environment (Unsworth 1998, Maccarone et al., 2003). Ground based (1 g) PBL cultures, both irradiated and non-irradiated,

were kept at the same cell density in flasks inside a humidified incubator for 24 h.

**3.1 The DNA-damage response of human peripheral lymphocytes cultured in** 

The DNA-damage response was investigated in human PBL irradiated *in vitro* with different doses of gamma rays and incubated for 24 h in 1 g or in modeled microgravity (MMG). While cell survival was only slight affected by MMG, the *HPRT* mutant frequency significantly increased in PBL incubated in MMG after irradiation compared with those

rotating at 23 rpm.

Fig. 4. Rotating Wall Vessel Bioreactor (Synthecon).

**microgravity after γ-irradiation** 

Fig. 3. Kinetics of -H2AX foci in PBL irradiated with -rays and low energy protons during the time after irradiation. A) Fraction of cells positive for -H2AX foci and B) mean number of -H2AX foci per nucleus.

The lower number of foci detected in peripheral lymphocytes could depend on the large amount of heterochromatin of resting cells, from which -H2AX foci are mostly excluded (Cowell et al., 2007) as well as on the small nuclear volume, where overlapping foci are difficult to detect separately. Thus, in accordance with the observations of Scherthan et al., (2008) we hypothesize that -H2AX foci detected very early after irradiation contained more than one DSB; later on, the number of foci decreased and probably each foci contained only one DSB. Furthermore , we found a size increase of -H2AX foci in cells irradiated with protons, as compared with gamma irradiations, probably as a consequence of DSBs clusters induced by high-LET radiation. Our results are in accordance with those in melanoma cells exposed to low- and high-LET radiation (Ibañez et al., 2009).
