Several types of stress, either environmental or organic, have genotoxic effects . Ionizing radiation may promote oxidative stress, induce DNA strand breaks and affect cell components, even a few hours after irradiation [16, 17]. In medical imaging, radiation effects are considered stochastic, and mainly of two kinds, malignancy or heritable disease . These effects are dose-dependent and follow a linear no-threshold model . However, there is no direct evidence of cancer risk from cardiac imaging, but only projections from the epidemiological studies . On the opposite end of this, studies at the “bench” level try to provide different types of evidence to help elucidate this issue.
The comet assay (single-cell gel electrophoresis) is one of the methods of choice for the evaluation and measurement of DNA damage. It is a simple, fast, precise, low-cost technique, in which cells are incorporated into an agarose matrix and then have their membranes lysed for the generation of nucleoid structures. Thereafter, DNA is untwined and undergoes electrophoresis. If there are bond breaks, the highly negative molecules move towards the anode [21, 22]. After staining and through visualization in a fluorescence microscope, a comet shape appears, with the nucleus in the head of the comet and the tail consisting of DNA strands or fragments which migrated to the anode. The relative intensity of the tail increases according to the intensity of damage caused by any agent, either ionizing radiation or chemical agents, for example .
In this study, even before exposure to ionizing radiation, 24% of the cells had evidence of some DNA damage, what recalls the variety of other factors that may lead to damage, such as smoking, diabetes, and indeed all currently recognized cardiovascular risk factors [24,25,26]. Importantly, even though there was an increase of the damage index and of classes 1–3 of damage, most cells remained in class 0. Shirazi et al. , also using the comet assay, showed that patients who received Tc-99m sestamibi or thallium-201 injections for MPI had evidence of DNA damage, compared to controls; however, repeated evaluations in the same patients (before/after radiotracer injection) were not available, and therefore a clear inference on the effect of ionizing radiation cannot be made. In the study by Varol et al. , among 27 children who underwent Tc-99m DMSA scintigraphy, DNA damage increased after the test, returning to normal levels after a week. Rief et al.  showed, by immunofluorescence, that strand breaks appeared after Tc-99m sestamibi injection for MPI and disappeared after 24 h. In the current study, a decrease of DNA damage with time could not be demonstrated, as patients were not re-evaluated later.
Additionally, even though DNA damage may occur, there are counteractive, self-protective mechanisms that contribute to reduce radiation effects. In fact, in response to DNA damage, cells activate repair genes . Cheng et al.  have demonstrated that, after exposure to different types of ionizing radiation, the lymphocyte expression of mRNA of several repair genes was increased compared to controls. Won et al.  observed the activation of DNA repair pathways in patients who underwent MPI, by evaluating the phosphorylation of histone 2AX, protein p53 or serine/threonine protein kinase (ATM) in peripheral blood T lymphocytes by flow cytometry and immunohistochemistry, as well as the mRNA expression of repair genes such as BCL2 associated X, damage specific DNA binding protein 2, or Tp53 (a tumor-suppressing gene). Therefore, the biological consequences of DNA damage may be reduced by these mechanisms, helping minimize concerns about the effects of ionizing radiation used in MPI. Finally, new imaging protocols, using stress-only strategies, or new imaging hardware and software, which allow the use of very small radiotracer doses, may lead to further reductions in radiation-induced DNA damage from MPI.
As the collection of the second blood sample was relatively “early” regarding the half-life of Tc-99m sestamibi, the extent of DNA damage induced by the tracer might have been underestimated. This timing was due to the presence of the patients in the Nuclear Medicine laboratory, which typically lasts for up to 90 min. Nonetheless, as Rief et al. have described after performing multiple blood sample analyses, strand breaks can be detected as early as 5 min after radiotracer injection, without major difference when compared to the 1-h sample . Therefore, we believe that our data may be representative of near-maximal radiation effects on the DNA. Additionally, as pointed by Azqueta et al. , DNA repair mechanisms can also occur very quickly, and experiments assessing DNA damage should take care to avoid repair of strand breaks; so, in this context, a shorter timing may also be desirable. Furthermore, due to the cross-sectional nature of this study, a return of damage levels to baseline could not be assessed, as other blood samples were not collected later. Finally, the study was performed with relatively high tracer doses, and therefore the amount of DNA damage might have been overestimated and may currently be substantially less with hardware improvement and new test protocols.