In a radiological examination, low-energy X-radiation is used (<100 keV). For other radiological procedures, the energy used is several MeV. ICRP in publication 103 has currently considered that photons irrespective of their energy have the same radiation weighting factor. Nevertheless, there are topological differences at the nanoscale of X-ray energy deposition as a function of its energy spectrum, meaning that the different interactions with living matter could vary in biological efficacy. To study these differences, we characterized our irradiation conditions in terms of energy spectra of secondary electrons at the cell nucleus level, using Monte Carlo simulations. We evaluated signaling of DNA damage by monitoring a large number of gammaH2A.X foci after exposure of G0/G1-phase synchronized human primary endothelial cells at a dose from 0,25 to 5 Gy at 40 kV, 220 kV and 4 MV X-rays. Number and spatial distribution of gammaH2A.X foci were explorad. In parallel, we investogated cell behavior through cell damage, gammaH2A.X foci, cell behavior, missegragation death and ability of a mother cell to produce two daughter cells. We also studied the missgregation rate after cell division. We report a higher number of DNA double-strand breaks signaled by gammaH2A.X for 40 kVp and/or 220 kVp compared to 4 MVp for the highest tested doses of 2 and 5 Gy. We observe no difference between the biological endpoint studies with 40 kVp and 220 kVp X-ray spectra. This lack of difference could be explained by the relative similarity of the calculated energy spectra of secondary electrons at the cell monolayer. The energy spectrum of secondary electrons seems to be more closely related to the level of DNA damage measured by gamma H2A.X the the initial spectrum of photon energy or voltage settings. Our results indicate that as energy spectrum of secondary electrons increases, the DNA damage signaled by gammaH2A.X decreases and this effect is observable beyond 220 kVp.