## 4 The Importance of Biomarkers in Monitoring RT Toxicity
### Apoptosis Biomarkers  
Cell death may occur by various mechanisms such as apoptosis, necrosis, mitotic catastrophe, ferrotptosis, senescence, and autophagy [92; 93; 94]. Apoptosis as a programmed cell death usually occurs in multicellular organisms under normal conditions to maintain homeostasis by eliminating damaged cells. It is mediated by intrinsic and extrinsic mechanisms that activate those members of the proteases family that lead to the initiation and amplification of proteolytic cascades: the _intrinsic_ and _extrinsic_ ones. Extrinsic apoptosis is triggered by external signal proteins that bind to specific receptors of the cell surface and lead to the activation of the apoptotic process. Intrinsic apoptosis involves the release of mitochondrial proteins into the cytosol, which activates a proteolytic cascade. In other cases,the two pathways work harmoniously together to activate a member of the pro-apoptotic _Bel2_ proteins for killing the cell. It is of note that when the apoptosis rate among cells deviates from the norm, either excessively or insufficiently, and when there is an inaccurate timing and location of apoptotic death, tumorigenesis and autoimmune mechanisms may be triggered [95]. Since apoptosis is characterized as asynchronous in cells and the persistence of apoptotic cells is relatively short, the in vivo detection of apoptosis biomarkers is complicated. Some of the most studied cells in relation to RT-induced apoptosis are endothelial cells, lymphocytes, and bone marrow progenitor cells [96].  
An acknowledged biomarker related to apoptosis is caspase-3, which actively participates in apoptosis as a primary executioner of this process. Caspase-3 initiates DNA fragmentation through the proteolytic inactivation of DEF45 (DNA fragmentation factor-45)/ICAD (inhibitor of caspase-activated DNase), which releases, in turn, the active complex DEF40/CAD (caspase-activated DNase), the endonuclease associated with the inhibitor [97]. Pathways leading to the caspase-3 activation are shown to be either dependent or independent of the cytochrome c release and the caspase-9 synergy [98]. [PERSON] et al., 2017 reported a dose-dependent increase in active caspase-3 expression levels in human blood lymphocytes irradiated with 1, 2, and 4 Gy gamma radiation, which was investigated after different times of in vitro incubation (24, 48, and 72 h). Accordingly, caspase-3 might be used for detecting differences in the radiation sensitivity of patients before undergoing RT [99], as also reported in several other studies [100; 101; 102]. A well-documented study by [PERSON] et al., 2005 with irradiated MCF-7 breast cancer cells indicates that caspase-3 plays a critical role in RT-induced apoptosis [103]. It has also been reported that caspase-3 is necessary for the radiation-induced apoptosis of human B-lymphocytes and lymphoblastoid cells [104; 105]. The study of [PERSON] et al., 2011 making use of the radiosensitive CD4\({}^{\text{+}}\)CD25\({}^{\text{+}}\) T regulatory cells (Treg) from patients with hepatocellular carcinoma, reveals high expression of radiation-induced active caspase-3 [106]. Similar results were independently obtained with \({}^{137}\)Cs-irradiation of T cells by [PERSON] et al., 2020, who observed an increase in caspase-3 levels due to irradiation [107].  
Annecin V has also been shown as a useful biomarker of radiation-induced apoptosis in human T-lymphocytes and as an indicator of radiation toxicity. This anticoagulant protein may detect apoptotic cells exhibiting on their surface negatively charged phospholipids like phosphatidylinositolserine. Several studies emphasized annexin V as a promising candidate biomarker for evaluating radiation toxicity in oncologic patients [108; 109].