## 3 Results and Discussion
As said before, additional information about the changes occurring in \(\mu\)-FT-IR spectra of cells exposed to different doses and fixed at different times can be obtained by using the previously described ratiometric approach (see Table 1 for a complete list of the evaluated ratios with the indication of the biomolecular origin). In Figures 4-7, the ratios measured between the areas of some of the different contributions present in the MCF-10A cell FT-IR spectra following different doses of protons and fixed at different times are reported (see panels I and II in Figure S1 for details). As previously observed [24], a limited number of significant changes are present for t\({}_{0}\) samples, and significant changes are detected for the samples fixed after 48 and 72 h, that is, after an appreciable time in which the various cellular repair processes have acted. This is in agreement with the results reported in Table S1, in which a large part of significant wavenumber shifts is present for t\({}_{48}\) and t\({}_{72}\) samples. The DNA1, DNA2, and DM ratios (Figure 4) indicate significant modifications in DNA for t\({}_{48}\) and t\({}_{72}\) samples, but some changes are also present in all cases. Even though it is not possible to demonstrate a well-defined trend, these modifications are indicative of the occurrence of changes in the nucleic acid structure related to DSBs [22; 24; 52], and they agree with the results obtained for other cell samples exposed to ionizing radiation that indicate the occurrence of damage in the primary, secondary, and tertiary structures of  
Figure 4: Comparison of the DNA1, DNA2, and DM absorbance area ratios for MCF-10A human breast epithelial cells for different proton doses and different fixation times. See Table 1 for details about the reported ratios (Mean \(\pm\) SD), which are normalized to the corresponding ratio value of the non-exposed sample. Asterisks indicate significant differences (\(p<0.05\)). nucleic acid [51]. The observed changes are also in agreement with Raman spectroscopy measurements of exposed MCF10A cells [27]. It is worthwhile to note that particular attention is required in analyzing DNA-related structures in infrared spectra, considering the dehydration process in our samples [53; 54]. The PD1, PD2, and PR1 ratios (Figure 5), which are assumed to be indicative of the relative contents of protein and DNA and of protein rearrangement, respectively, also show significant changes only for t\({}_{48}\) and t\({}_{72}\) samples. A similar trend is also evidenced in Figure 6 by the LS (related to the ratio between CH\({}_{3}\) and CH\({}_{2}\) asymmetric stretching band areas and lipid saturation) and PP1 and PP2 (indicative of protein phosphorylation) ratios, and in Figure 7 by the PL2 and PL3 ratios (related to relative contents of proteins and lipids). As far as LS ratio changes are concerned, they can be associated with cell apoptosis processes [24; 47; 53; 55; 56; 57; 53] and with the results of biochemical assays reported by [PERSON] et al. [27]. The changes in the PP1 and PP2 ratios confirm the modifications occurring in DNA [45; 55]. PL1, always related to the ratio between protein and lipid contents, shows significant changes for all fixation times with a  
Figure 5: Comparison of the PD1, PD2, and PR1 absorbance area ratios for MCF-10A human breast epithelial cells for different proton doses and different fixation times. See Table 1 for details on the reported ratios (Mean \(\pm\) SD), which are normalized to the corresponding ratio value of the non-irradiated sample. Asterisks indicate significant differences (\(p<\) 0.05). general increasing trend with dose. These diffuse changes in PL1 and the more contained ones in PL2 and PL3 ratios can corroborate the presence of changes in membrane fluidity already indicated by the shifts in the position of CH\({}_{2}\) symmetric stretching [24; 32; 41]. Figure 6: Comparison of the LS, PP1, and PP2 absorbance area ratios for MCF-10A human breast epithelial cells for different proton doses and different fixation times.