**2. Cellular effects of ionizing radiation in human lymphocytes**

#### **2.1 Surviving fraction,** *HPRT* **mutant frequency and molecular characterization of mutations in irradiated human lymphocytes**

To contribute to the understanding of the DDR pathway following radiation-induced damage, we studied the effects of IR on human peripheral blood lymphocytes (PBL) irradiated *in vitro* with different doses of -rays and low-energy protons (0.88 MeV; LET: 28keV/m). Irradiated PBL were assayed for cell viability, for mutant frequency at the hypoxanthine-guanine phosphoribosyl transferase (*HPRT*) gene, and for molecular characterization of mutations. The *HPRT* gene, which in humans covers 44 kb and encodes a non-essential protein, allows a wide variety of mutations, from point mutation to total gene deletion, to be detected by using the *HPRT* mutation assay. Deletion of DNA segments is the predominant form of radiation damage in cells that survive irradiation and the mechanisms for producing deletion mutations appear to be very complex and dependent on target cell, gene studied, dose, dose-rate and radiation quality (Schwartz et al., 2000). Large deletions are thought to derive from two DNA double strand breaks close enough to interact each other. Thus, deletion frequency should be dependent on radiation dose and dose-rate. All PBL samples, irradiated either with -rays or protons, showed a dose-dependent cell survival decrease and a *HPRT* mutant frequency increase. In Table 1 we report the data of survival and *HPRT* mutant frequency in human PBL irradiated with different doses of rays and low-energy protons.

Molecular analyses of *HPRT* mutants were carried out in clones derived from PBL exposed to -rays (1-4 Gy) and to low-energy protons (0.5-2Gy), and in non-irradiated clones of the same donors. Among the mutant clones obtained from -irradiated PBL, point mutations were the only kind of mutation in 1Gy irradiated clones, whereas deletions were the prevalent mutations among clones irradiated at 4Gy. In contrast, no partial or total deletions of the *HPRT* gene were detected in mutant clones isolated after proton irradiation. Figure 1

The DNA-Damage Response to Ionizing Radiation in Human Lymphocytes 7

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

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

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 non-

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.

radiation such as protons, give raise to clustered DNA damage along tracks.

irradiated PBL, either in - and in proton-irradiated PBL.

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

radiation, reflecting the sparsely and densely nature of IR.

shows the percentages of mutation types calculated over the total number of mutations derived from human PBL irradiated with both radiation qualities. The difference of the mutational spectrum between -rays and protons probably depends on the nature of IR. Complex gene rearrangements and deletions are assumed to be a specific signature of exposure to high-LET radiation in mammalian cells. Nevertheless, the absence of these kind of mutations in PBL irradiated with protons could be due to their lower survival in comparison with -irradiated PBL, as a consequence of the more cytotoxic than mutagenic lesions induced.


Table 1. Surviving fraction (SF) and *HPRT* mutant frequency (± standard error, S.E.) in human PBL irradiated with -rays and low-energy protons.

Fig. 1. Characterization of *HPRT* mutant clones derived from PBL irradiated with -rays and protons or non-irradiated (0Gy).

shows the percentages of mutation types calculated over the total number of mutations derived from human PBL irradiated with both radiation qualities. The difference of the mutational spectrum between -rays and protons probably depends on the nature of IR. Complex gene rearrangements and deletions are assumed to be a specific signature of exposure to high-LET radiation in mammalian cells. Nevertheless, the absence of these kind of mutations in PBL irradiated with protons could be due to their lower survival in comparison with -irradiated PBL, as a consequence of the more cytotoxic than mutagenic

> 0.5 Gy 99.5 ± 0 2.8 ± 0 1.0 Gy 83.85 ± 11.36 10.5 ± 3.8 2.0 Gy 44.7 ± 7.36 29.2 ± 3.7 3.0 Gy 13.7 ± 4.56 50.6 ± 17.9 4.0 Gy 6.2 ± 2.4 24.7 ± 11.1

> 0.5 Gy 60.84 ± 7.82 5.33 ± 2.18 1.0 Gy 39.87 ± 6.28 9.25 ± 2.8

Table 1. Surviving fraction (SF) and *HPRT* mutant frequency (± standard error, S.E.) in

Fig. 1. Characterization of *HPRT* mutant clones derived from PBL irradiated with -rays and

38.42 ± 11.35 35.5 ± 0 29.9 ± 11.35

human PBL irradiated with -rays and low-energy protons.

SF (%) ± S.E. HPRT MF (x10-6) ± S.E.

16.72 ± 4.86 11.7 ± 1 5.06 ± 0

lesions induced.


Protons

1.5 Gy 2.0 Gy 2.5 Gy

protons or non-irradiated (0Gy).
