## 3 Results
### BNCT Causes More Complex DNA Damage Than X-rays  
Cells irradiated with the neutron thermal flux in the presence of the \({}^{10}\)B carrier BPA yielded a significantly higher proportion of complex-type CAs than following X-rays (Figures 3 and 4). Specifically, for X-rays at 0.5 Gy, the occurrence of such aberration type was almost negligible by either technique whereas at the same dose in BNCT-treated samples, analysis by WCP and m-FISH showed frequencies of around 0.08 and 0.25 complex rearrangements per cell, respectively (Figure 3). In the case of m-FISH, the latter value increased to 1.9 \(\pm\) 0.1 at 2 Gy for BPA-treated samples compared to a complex CA frequency of just 0.08 \(\pm\) 0.03 for MCF-10 irradiated with X-rays. In particular, the results found for 2 Gy demonstrate well the appropriateness of m-FISH-based karyotype-wide analysis in unveiling the \"true\" occurrence of radiation-induced DNA complex damage as 2.5-fold more complex-type CAs were found after labeling by m-FISH than after WCP in BNCT-treated samples (hatched vs. plain bars in Figure 3). That the spectrum of measured CAs shifts towards a greater complexity following BNCT than after X-ray irradiation is immediately visible in Figure 4, which shows the relative weight of complex CAs on the overall yield of CAs. An example of the complexity in the spectrum of aberrations that m-FISH can detect is shown in Figure 5, which compares two karyotypes from control and 2 Gy BNCT. These results strongly suggest an effect of the BNCT-associated fusion reaction's high-LET products [5; 28]. As expected, no complex aberrations were found in unirradiated controls. Figure 2: Comparison between the frequency of total aberrations per cell yielded by BNCT (hatched bars) and X-ray irradiation and measured using WCP or m-FISH for the two doses used. Unirradiated controls show similarly low levels of spontaneous CAs. Figure 4: Incidence of complex-type damage following BNCT (hatched bars) or X-rays as obtained using WCP and m-FISH: the two doses that used complex CAs are more frequently observed after BNCT than following X-rays. Figure 5: Reconstructed karyotypes by m-FISH from PCC spreads: (**a**) karyotype from an unirradiated cell; (**b**) karyotype from a PCC following 2 Gy BNCT. The irradiated cell shows an aberrated karyotype with a significant excess of complex CAs plus other rearrangements, namely, three complex CAs involving chromosomes 10 and 4, another involving chromosomes 9 and 18 (both with 3 breaks), and another involving chromosomes 3, 6, and 8 and X. Additionally, a non-reciprocal translocation between chromosomes 13 and 17, and several fragments from chromosomes 1, 2, 5, 16 and 20 were found; (**c**) karyotype from a PCC exposed to 2 Gy of X-rays showing a simple exchange (incomplete translocation between chromosomes 3 and 11) and an acentric fragment from one of the two copies of chromosome 1. Figure 3: Occurrence of complex aberrations per cell obtained using WCP and m-FISH for the two doses used following BNCT (hatched bars) or X-rays. BNCT Greatly Enhances the Degree of Complexity Associated with Complex Aberrations Detected by m-FISH  
m-FISH analysis allowed us to measure the number of chromosomes and that of breaks involved per complex aberrations normalized by the total number scored. These figures were taken as representing the degree of complexity associated with the treatment with BNCT or X-rays [5]. As clearly shown by Figures 6 and 7, a very marked level of complexity accompanies the complex exchanges found in the BNCT-exposed samples, concurrent with the notion of high LET-induced chromosomal damage, while the much fewer complex CAs observed after X-ray irradiation (Figures 3 and 4) exhibited a minimal number of chromosomes or breaks involved even at the highest X-ray dose, that is 0.19 \(\pm\) 0.04 and 0.24 \(\pm\) 0.04, respectively.