After irradiation with high-LET particles, on the other hand, locally concentrated energy deposition causes serious chromatin fragmentation and mobilization within cell nucleus micro-volumes along the particle tracks. This situation allows mutual contacts of many short chromatin fragments from one or several neighboring chromosomes and thus easy formation of complex chromatin translocations, irrespectively of the original chromatin architecture and chromatin architecture changes during repair. Chromosomal translocations in cells exposed to high-LET radiation thus occur due to physical rather than biological (repair) processes. We have already mentioned that heterochromatin architecture protects DNA from low-LET radiation as heterochromatin-binding proteins prevent DNA interaction with free radicals, mostly mediating harmful effects of low-LET radiation. With high-LET radiation, however, most damage to DNA is caused by the direct effect of radiation particles or emitted secondary electrons. In this case, heterochromatin represents a more dangerous chromatin architecture, as particles cannot be stopped by any chromatin architecture and heterochromatin provides more DNA targets per a volume unit compared to euchromatin. Hence, in cells exposed to high-LET radiation, translocations in heterochromatin tend to be more complex than in euchromatin (Box 3.8).

#### **Box 3.8 In a Nutshell: DNA Damage and Repair in the Context of Chromatin Architecture**

# **3.4.3 DNA Damage and Repair Processes at the Nanoscale**

Using a variety of tools of super-resolution microscopy and image data computing has revealed that γH2AX foci in cell nuclei exposed to low-LET X-rays are subdivided into several equally sized, functionally relevant clusters. The number of clusters increased with the radiation dose according to the well-known linear-quadratic dependence and decreased at later time periods postirradiation. Calculations of the persistence of homology revealed a highly similar topology of γH2AX and other repair protein clusters, especially when these clusters were closely associated with heterochromatin regions. During the repair period, size and topology of these clusters seem to be maintained as long as they are attached to chromatin at actively repairing DSB sites. These fndings suggest a functional relevance of the focus/cluster topology [57].

For instance, while the γH2AX clusters had a typical diameter of about 400 nm–600 m, the MRE11 clusters were smaller (about 200 nm) and usually completely embedded within γH2AX clusters [58]. The sizes of clusters were independent of repair time and cell type. On the other hand, the topological similarity of clusters followed the dynamics of the repair protein interaction with chromatin; that is, binding to damage sites was accompanied by ordering while detachments caused the relaxation of topological arrangements. In contrast, γH2AX and MRE11 clusters spontaneously occurring in the nonirradiated cells (e.g., due to replication defects) did not show this topological similarity.

Recent studies discovered spatial distribution changes of tri-methylated H3K9 histone (H3K9me3), ALU repeat sequences (ALU), or long interspersed nuclear element (LINE)-like L1 sequences, indicating chromatin reorganization or movement and DNA strand relaxation after radiation exposure, followed by recovery during repair [59]. Altogether, described results suggest a functional relevance of chromatin and repair focus nano-architecture in DSB repair process and their regulation (Box 3.9).

# **Box 3.9 In a Nutshell: DNA Damage and Repair Processes on the Nanoscale**

# **3.5 Consequences of DNA Damage Misrepair or Unrepair**

Lack of repair (unrepair) and misrepair of DNA damage can lead to increased chromosome breaks or rearrangements and mutations usually referred to as a status of genomic or genetic instability (GI). GI is usually associated with loss of cell cycle control, senescence, and cell death and in humans with pathological disorders including premature aging and predisposition to various types of cancer and inherited diseases [60]. On the other hand, GI is also fundamental for evolution and induction of genetic diversity. It is known that genomic integrity is carefully supervised by specifc surveillance mechanisms like DNA damage checkpoint, DNA repair, or mitotic checkpoint. A defciency in the regulation of any of these mechanisms often leads to GI, which can predispose a cell to malignant transformation [61].

# **3.5.1 DNA Lesions and Repair**

In huge DNA molecules in the cell, nucleus genes are present. These genes are responsible for the development and function of the cell and the whole organism, because they code proteins. Due to this fact, unrepaired or misrepaired DNA lesions, which can lead to gene mutations, can promote changes in the structure of the encoded protein or lead to the decrease or complete loss of its expression. The types of DNA lesions occurring were already discussed in Sect. 3.3. Based on the current experimental and theoretical evidence, the most repair-resistant lesions are not the single ones but a combination of them in a short DNA segment of 10–20 bp called clustered damage. Clustered DNA lesions are considered the signature of ionizing radiations especially for particle radiation [45]. Various studies suggest that the probability for a break or other DNA lesion to be incorrectly processed and amended is fairly low when damage is spatially separated but increases drastically when multiple breaks and/or non-break lesions coincide. For an analytical description of DNA repair pathways, the reader can refer to Sect. 3.4. As was already mentioned in Sect. 3.3, the DNA molecule consists of nucleotides (deoxyribose + phosphate group + base), which can be for simplicity named based on the four bases [adenine (A), cytosine (C), guanine (G), thymine (T)]. Thus, the DNA alphabet is a very easy one; it only consists of four letters. These four letters are then combined to give rise to groups of three, which defne the amino acids that are then the new alphabet for the translation to proteins. For more details on DNA-to-RNA transcription and RNA-to-protein translation, see for example [62]. Even if the cells have a very sophisticated DNA damage response and repair system, it may happen that not all the damage is removed. A mutation is when a permanent change in the DNA sequence occurs. Mutations can be divided into somatic or germline mutation in terms of what kind of cell is affected. A germline mutation occurs in a sperm or in an egg and can be passed to offspring. Somatic mutations occur in cells of the body and cannot be passed to next generations. Mutations can also be grouped as point or chromosomal mutations. Point mutations are when a single nucleotide is replaced with another single nucleotide, or deleted, or inserted in a place that it should not be. Point mutations do not always have signifcant consequences on the encoded protein. For example, as is shown in Table 3.4, the mutation can be silent. This means that even if there is a change in the original DNA sequence, the fnal product of the transcription will be the same, because there are several combinations of the DNA alphabet that lead to the same amino acid. In other cases, the mutation can lead to the change of the fnal amino acid (missense mutation) or to the creation of a stop codon (nonsense mutation), which then affects the fnal protein.

# **3.5.2 Mitotic Cell Death, Senescence, Cytoplasmic DNA**

Mitotic cell death, also called mitotic catastrophe (MC), is the process when a cell dies during or right after mitosis [63]. It can be triggered by DNA damage and its mis- and unrepair and therefore through radiation. MC can be both a caspasedependent, regulated and caspase-independent, unregulated pathway of cell death. Some characteristic morphologies can be found in Fig. 3.14a.

Senescence in biology refers to a process by which a cell ages and permanently and irreversibly stops dividing but does not die [63]. The number of senescent cells increases with age, but senescence also plays an important role during development as well as during wound healing and can be triggered by radiation. In culture, senescent cells exhibit a different morphology compared to non-senescent cells, called "fried egg" appearance (see Fig. 3.14b). It was shown that among other features, the radiation dose plays a major role in the induction of either senescence or apoptosis and necrosis. In some cell lines, senescence is the major response to low doses of radiation, whereas higher doses lead to apoptosis or necrosis. In IR-treated tissue, enhanced senescence may lead to pathogenic onsets, such as loss of organ function.

**Fig. 3.14** Morphologies of mitotic catastrophe (**a**) and senescence (**b**). (**a**) Fluorescence image of cancer cells undergoing mitosis. The DNA is labeled with DAPI and mitotic spindles using α-tubulin staining. The cells exhibiting mitotic catastrophe are treated with photodynamic therapy (PDT), Taxol (Tx), or nocodazole (Nc). The control shows normal

mitotic spindles. The treated cells show various types of altered spindles and mitosis. Scale bar: 10 μm. Reproduced with permission (CCBY) from Mascaraque et al. [64]. (**b**) Phase-contrast images of Chang cells. Senescence was induced using 1 mM of deferoxamine. (Reproduced with permission (CCBY) from Kwon et al. [65])

Recent advances in the feld indicate that a further consequence of DNA damage misrepair or unrepair can be the release of cytoplasmic DNA that can also trigger immune responses. In general, it is widely accepted that immune signaling can be activated by the presence of DNA in unusual locations, such as the cytoplasm or the endosomes, as DNA is normally located in the nucleus of eukaryotic cells. Emerging evidence indicates a cross talk between DNA repair machinery and the immune system, and more specifcally it has been discovered that DDR factors like DNA repair proteins can enhance innate immune signaling [66]. Defects in DDR and proper processing of DNA damage can therefore trigger a multitude of cellular phenotypes, including autoinfammatory disease, cellular senescence, and cancer. Genotoxic agents such as radiations or high oxidative stress can act as the primary instigators for immune signaling activation through the release of a wide range of biological and chemical factors often referred to as "danger signals" or damage-associated molecular patterns (DAMPs) [67] (Box 3.10).

# **Box 3.10 In a Nutshell: Consequences of DNA Damage Misrepair and Unrepair**

# **3.6 Cytogenetics and DNA Damage Measurements for Assessment of Radiation Efects**

Cytogenetic techniques can be used to analyze chromosomal aberrations in metaphase and morphological abnormalities of DNA content in interphase nuclei. The applicability of these aberrations in the felds of biological dosimetry, clinical cytogenetics, and environmental monitoring is based on a large number of radiobiological and DNA-repair theories.

# **3.6.1 Micronuclei and Other Nuclear Anomalies**

As described before, when cells are exposed to a variety of genotoxic agents (chemical/physical/radiation/DNAdamaging agents), they cause defects in DNA, chromosomes, and other cellular components. Radiation induces extensive DNA damage such as DSBs that, if misrepaired or unrepaired, ordinarily result in asymmetrical chromosome rearrangements and exchanges, which may lead to formation of small chromatinic bodies also known as micronuclei (MN) (see Fig. 3.15). MN are tiny extranuclear bodies that contain damaged chromosome fragments and/or whole chromosomes that were not incorporated into the nucleus after cell division and are surrounded by a membrane. As a variety of genotoxic agents may damage DNA and the mitotic machinery by multiple mechanisms, leading to MN formation, MN are not IR specifc.

It is now well established that MN are formed from acentric chromatid fragments caused by misrepaired or unrepaired DNA breaks or lagging acentric chromosomes due to mitotic spindle failure at an anaphase. Additionally, the formation of DNA DSBs and MN is sometimes the result of simultaneous excision repair of damages (e.g., 8-oxodeoxyguanosine) and inappropriate bases' (e.g., uracil) incorporation in proximity on opposite complementary DNA strands.

A whole chromosome lagging behind (chromosome malsegregation) during anaphase also results in MN formation. Mal-segregation usually happens due to absence or inappropriate attachment of spindle microtubules to chromosome kinetochore. However, the potential mechanisms behind the formation of MN are hypomethylation repeat sequences in centromeric and pericentromeric DNA, defects in kinetochore proteins or assembly, dysfunctional spindle, defective anaphase checkpoint genes, and malfunctioning in cell cycle control system. Sometimes, mis-segregation events occur when the centromeres of the dicentric chromosomes are pulled towards opposite poles of cells with suffcient forces to detach the chromosome from spindle during anaphase, thus resulting in micronucleus formation from whole chromosome loss.

Furthermore, multiple extrachromosomal acentric double minutes (DMs), cytogenetic hallmarks of genomic amplifcation, can aggregate after DNA damage and generate cytoplasmic MN that are subsequently eliminated from the cell.

Other nuclear anomalies such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) (see Fig. 3.15) are sensi-

**Fig. 3.15** Mechanisms by which genotoxic agents cause micronuclei and other nuclear anomalies. Micronuclei (MN) can originate from lagging acentric chromosomes or chromatid fragments or whole chromosomes at anaphase in mitosis. Nuclear bud (NBUD) formation represents the process of extrusion of the amplifed/surplus DNA, DNA repair-recombinational protein complexes, and possibly excess chro-

mosomes from aneuploidic cells. Nucleoplasmic bridges (NPBs) originate from dicentric chromosomes. This arises because the centromeres of dicentric chromosomes are often pulled in opposite directions and defective separation of sister chromatids occurs during anaphase leading to bridge formation, which can be observed as an NPB in telophase

tive and reliable biomarkers for early genotoxic instability and chromosomal breakages and rearrangements. NPBs originate as an aftereffect of misrepair of DNA strand breaks or failure of complete chromatid separation to opposite poles of the cell during anaphase. It can also originate from telomere end-to-end fusion mechanism, a fundamental indication of and a marker for loss of telomere function, which is caused by (a) excessively short telomeres, (b) dysfunctional telomeres due to loss of telomere-binding proteins without telomere erosion, (c) inappropriate assembly of telomere-capping protein structure, (d) defects in recombinational repair proteins, or (e) lack of telomeres. Another distinctive nuclear anomaly, NBUDs, is one of the precursors of MN and is associated with chromosomal instability events. Most NBUDs originate from interstitial or terminal acentric fragments and represent the expulsion of undesirable amplifed extrachromosomal DNA content, which localizes to specifc sites at the periphery of the nucleus and is eventually eliminated via nuclear budding during the S phase of cell cycle. It is also plausible that NBUDs might occur after elimination of DNA repair-protein complexes in the cytoplasm (Box 3.11).

#### **Box 3.11 In a Nutshell: Micronuclei and Other Nuclear Anomalies**

Micronucleus assays are frequently used to assess genotoxicity and cytotoxicity of different chemical and physical factors, including IR-induced DNA damage. The cytokinesisblock micronucleus assay can measure MN, NPBs, and NBUDs. A diverse range of reliable micronucleus tests (Fig. 3.16) are executed with different cell types, eventually refecting chromosomal aberrations, ongoing DNA injury, initial stage in the development of genomic instability, and tumorigenesis. In the widely used cytokinesis-blocked MN assay, MN are scored in once-divided binucleated cells, where cytokinesis is blocked with addition of cytochalasin B, an inhibitor of microflament ring assembly necessary for the completion of cytokinesis. In order to get statistically solid results, a huge amount of cells need to be scored. Therefore, automatic analysis of MN boosts the reliability of

**Fig. 3.16** Depending on the cell type, different micronucleus assays can be employed to assess and determine the genotoxicity and cytotoxicity of different chemical and physical factors. Applications of each assay are outlined in their respective boxes. The most popular CBMN assay can be applied to cultured human lymphocytes or cell lines to measure MN and other chromosomal instability biomarkers such as NPBs and NBUD. The mammalian erythrocyte micronucleus assay is performed on immature erythrocytes from bone marrow to determine cytogenetic damage after radiation exposure. The buccal micronucleus cytome assay is done in rapidly dividing buccal epithelial exfoliated cells (oral cavity) to analyze MN and other cytogenetic biomarkers (source of DNA damage, cytotoxicity, etc.). Occasionally, MN assay is performed on nasal mucosa cells or urine-derived cells for detection of chromosomal damage caused by environmental and lifestyle factors, occupational exposures, prognosis of cancer, and certain diseases. Although the objective and method of performance are similar to CBMN or bone marrow MN assays, these tests have not gained much popularity so far

the assays. Concomitantly, it increases the statistical validity after analyzing a large number of cells in one go. Additionally, the existing automatic/semiautomatic micronucleus scoring by microscopic systems, by fow cytometry and imaging fow cytometry, gives high accuracy and sensitivity and leads to rapid analysis (Box 3.12).

#### **Box 3.12 In a Nutshell: The Use of Micronucleus Assay**

# **3.6.2 Chromosomal Aberrations**

Chromosomal mutations, also called chromosomal aberrations (CA), are observed at the frst mitosis after irradiation and are those that incorporate chromosomal changes, such as deletions, inversions, insertions, substitutions, duplications, or translocations of parts of chromosomes. For better understanding, some types of mutations are shown in Fig. 3.17.

The mutations shown can also lead to other aberrations. Three which should be mentioned are dicentric and ring chromosomes as well as acentric fragments as shown in Fig. 3.18. A dicentric chromosome is created when two chromosomes with two centromeres are fused. In metaphase, they are visible as one chromosome with two centromeres. This aberration will most likely die during mitosis. Acentric fragments are either fragments of a single chromosome or fused parts of different chromosomes containing no centromere. A ring chromosome is a chromosome which has two breaks on the opposing ends and is fused to form a ring. Both aberrations cannot be pulled into a daughter cell and most of germ line cell or a cell in early embryogenesis, several genetic disorders can occur [69]. For a more detailed view on this, refer to Chaps. 2 and 7. The frequency of radiation-induced CAs rises with increasing radiation dose to the cells. Different types of CAs depend on the phase of cell cycle at which the nucleus is exposed to irradiation. Chromosome-type aberrations (Table 3.5) occur when pre-synthetic phase (G1) is exposed to irradiation, while chromatid-type aberrations (Table 3.6) appear if irradiation occurs during post-synthetic phase (G2). In chromosome-type aberrations, more than one break is unable to rejoin at the correct ends that often results in abnor-

mal chromosomes. There is much hidden damage present, some of which is transmitted to future cell generations. In

**Fig. 3.18** Human metaphase cell irradiated with 5 Gy gamma rays. Two dicentric chromosomes, three acentric fragments, and a ring chromosome could be found. From https://www.qst.go.jp/site/nirsenglish/1369.html (accessed 05/2022)

**Fig. 3.17** Types of chromosomal mutations. Nonlethal aberrations are observed at the frst mitosis after irradiation. Duplication: one or more copies of a DNA segment/a region of a chromosome are formed. Inversion: A segment of a chromosome breaks off and reinserts in reverse orientation within the same chromosome. Deletion: A part of a

chromosome/one or more nucleotides from a segment of DNA are missing or deleted. Translocation: It involves two chromosomes in which a piece of one chromosome breaks off and rejoins to another chromosome. Insertion: A segment of one chromosome is removed and inserted to another chromosome or the same chromosome

**Table 3.5** Chromosome-type aberrations

**Table 3.6** Chromatid-type aberrations

contrast, radiation can induce chromatid aberrations during late S and G2 phases, when sister chromatids are being duplicated and the DNA DSBs may result in chromatid breaks (deletions), interchanges, or triradials. Mostly, sister chromatids or non-sister chromatids of homologous chromosomes are affected by all the breaks and rejoins. The chromosomal aberrations serve as a biological dosimeter—an indicator of radiation exposure. Furthermore, radiationinduced CAs delineate an early marker of late effects, including cell killing and transformation.

A series of methods and techniques (Fig. 3.19) have been developing to assess stable or unstable type of CAs in order to evaluate the potential of a test compound (chemical/mutagen/ radiation exposure). Human peripheral blood lymphocytes offer unique possibilities to study somatic cell division (in vitro) and thus have been utilized for detection of CAs (Box 3.13).

**Fig. 3.19** Techniques to assess constitutional or acquired chromosomal abnormalities using standard banding techniques (left) or advanced molecular cytogenetic techniques (right). Standard cytogenetic techniques are traditionally performed by karyotyping of stained metaphase chromosomes or by fow cytometry. Chromosome banding is used to produce alternating light and dark regions, also referred to as "cytogenetic bands," along a chromosome with the use of special stains (abbreviations are listed below). Chromosome banding patterns are essential in pairing and ordering all the chromosomes, known as karyotyping. Flow cytometry-based procedures have been developed to assess numerical (ploidy) and structural (telomere length) chromosomal aberrations in mitotic cells largely based on DNA content. To overcome the limitations of the banding analysis, advanced cytogenetic techniques are introduced. In techniques based on ISH, fuorescently labeled "painting" probes are used to localize nucleic acid sequences. FISH identifes chromosomal rearrangements and mapping-specifc genes on individual mitotic chromosomes. GISH determines the origin

# **Box 3.13 In a Nutshell: Chromosome-Type and Chromatid-Type Abberations**

of genomes or chromatins in hybrids. RISH reveals cellular patterns of mRNA expression in cells. CGH-based techniques provide an overview of chromosome ploidy level (gain and loss) throughout the whole genome. CGH with the use of microarrays—aCGH—detects aneuploidies, deletions, duplications, and amplifcations based on DNA content. Southern blotting and PCR-based molecular cytogenetic techniques have good potential to detect chromosomal abnormalities from trace amounts of specifc regions of DNA/RNA. *G-banding* Giemsa banding, *Q-banding* quinacrine fuorescence banding, *R-banding* reverse banding, *C-banding* centromere banding, *ISH* in situ hybridization, *FISH* fuorescence in situ hybridization, *GISH* genomic in situ hybridization, *RISH* RNA in situ hybridization, *CGH* comparative genomic hybridization, *aCGH* array comparative genomic hybridization, *QF-PCR* quantitative fuorescence polymerase chain reaction, *qPCR* quantitative polymerase chain reaction, *MAPH* multiplex amplifable probe hybridization, *MLPA* multiplex ligation-dependent probe amplifcation

From the mentioned chromosomal mutations, the translocations are especially dangerous as, in contrast to many other types of chromosomal aberrations, they can be tolerated by the cells. They usually neither cause loss of genetic material nor mitotic cell death and are thus transmitted to the next cell generations. At the same time, translocations are highly oncogenic or affect cell physiology in other ways. Translocations may be simple; reciprocal; i.e., if chromatin fragments are exchanged between two chromosomes; or even complex [70]. Translocations mostly arise due to erroneous DNA end joining by classical NHEJ or mutagenic alternative repair pathways. Although homologous recombination is generally considered a highly precise repair mechanism, recombination between repetitive sequences especially in heterochromatin may also lead to chromatin exchanges [48]. In addition, HR can trigger chromosomal translocations when its intermediates are resolved by crossover between allelic or nonhomologous chromosomes [70]. Although translocations are not associated with extensive losses of the genetic material, they can generate fusion genes (and proteins) with aberrant, often oncogenic, functions. An example could be the reciprocal translocation t(9;22) (q34;q11) between genes BCR and ABL [71], which is responsible for the development of the well-known chronic myeloid leukemia (for terminology and categorization of translocation types, the reader is referred to specialized books on medical genetics or cytogenetics, e.g., Griffths et al. [70]). In addition to formation of fusion genes, translocations may activate proto-oncogenes by repositioning them along or between the DNA molecules into a close proximity of a strong promoter of some other gene. If the reading frame of the translocated gene is shifted, its function may be lost. However, the gene activity can be changed also epigenetically, if a gene is moved into an incorrect chromatin environment. This is often a cause of the tumor suppressor silencing, after a tumor suppressor is translocated close to a genetically inactive heterochromatin domain. In the context of radiobiology, it is important to emphasize that cell exposures to different radiation types lead to different types of translocations. Cells irradiated with photonic radiation with low LET mostly contain interchromosomal translocations where one chromatin fragment is translocated to another chromosome or two fragments are reciprocally exchanged between two chromosomes. These lesions are usually simple, but the proportion of complex translocations increases with the radiation dose. Cells exposed to a particle high-LET radiation, on the other hand, mostly suffer from complex chromosomal translocations arising as the consequence of extensive chromatin fragmentation by highly localized energy deposition along the particle tracks [72]. For the same reason, high-LET radiation preferential generates intrachromosomal translocations affecting a single chromosome at multiple sites. To explain this phenomenon, it should be emphasized that chromosomes in the interphase cells occur in the form of chromosomal territories with only a limited extent of mutual intermingling along their borders, as explained in Sect. 3.5. Hence, the areas of chromosome territory borders where translocations between the neighboring chromosomes can be formed represent only a small proportion of the nuclear volume along the radiation particle track [53, 54]. With increasing doses and more particles transversing a single nucleus, however, extensive rearrangements of the genome affecting high numbers of chromosomes can be detected (Box 3.14).

#### **Box 3.14 In a Nutshell: Chromosomal Translocations**

# **3.6.3 Premature Chromosome Condensation**

Chromosome condensation, the landmark event at the onset of prophase, is the dramatic reorganization of the isolated patches of long thin chromatin strands at the nuclear periphery into compact short chromosomes that can be visualized at metaphase during mitosis or meiosis in eukaryotic cells. Maturation-promoting factor (also called mitosis-promoting factor or M phase-promoting factor, abbreviated MPF), the p34cdc2/cyclin B complex, serves as a master cell cycle regulator for the M-phase transition and chromatin condensation by phosphorylated condensins (Fig. 3.20). MPF activity mainly depends on the cellular concentration of cyclin B, which usually oscillates through cell cycle. During cell division, chromatin condenses and individualizes to discrete chromosomes, which are further segregated by mitotic spindle fbers. Once divided, chromatin decondenses to reestablish its interphase structure component facilitating DNA replication and protein-making processes.

**Fig. 3.20** The presence and action of MPF protein in the cell control premature chromosome condensation induction. Cyclin B oscillates through the cell cycle being undetectable during interphase, very low in G1, gradually increasing from S, reaching maximum in G2, and decreasing abruptly at G2/M transition. This corresponds to the MPF activity during cell cycle. *MPF* maturation/mitosis-promoting factor

**Fig. 3.21** Premature chromosome condensations (PCCs) at various stages of the cell cycle: darkly stained metaphase chromosomes belong to mitotic CHO cells, whereas the lighter stained to the interphase CHO

Chromosome condensation may also occur prematurely in interphase test cells when they are fused to mitotic cells or chemically using specifc phosphatase inhibitors. The most common approach is the use of Chinese hamster ovary (CHO) cells as mitotic inducer cells. Following cell fusion, the MPF present in a mitotic cell interacts with the interphase nucleus causing dissolution of its nuclear membrane and premature chromosome condensation of interphase chromosomes. This phenomenon is known as *premature chromosome condensation (PCC)*. The morphology of prematurely condensed chromosomes (PCCs) depends on the stage of the interphase cell in the cell cycle (i.e., G0, G1, S, and G2) (Fig. 3.21). PCCs in G0-phase cells exhibit single chromatids, highly condensed and distinct. During the G1 phase, G1-PCCs are despiralized single chromatid chromosomes, while chromosomes condensed during the S phase (S-PCCs) have a "pulverized" appearance because of less condensed chromatin at the sites of replication [73]. Condensation during the G2 phase (G2-PCCs) yields distinct

cells. (**a**) G0-PCCs, (**b**) G1-PCCs, (**c**) S-PCCs (reproduced with permission (CCBY) from Pantelias et al. [73]), (**d**) G2-PCCs. *CHO* Chinese hamster ovary

elongated double-chromatid chromosomes. Consequently, cell fusion-mediated or chemical induction of PCCs has been proven a powerful cytogenetic tool in radiobiology to study the conversion of radiation-induced DNA lesions into chromosomal aberrations at various cell cycle stages since it enables visualization and quantifcation of radiation-induced numerical and structural chromosomal alterations directly in interphase cells.

PCC can be induced either by fusion of human lymphocytes with mitotic cells (fusion-mediated PCC) or with the use of specifc chemicals (chemical-induced PCC).

In the case of fusion-mediated PCC, the condensation was at frst achieved with the use of fusogenic viruses (such as Sendai virus or its equivalent). However, an important disadvantage of this method is that the fusion effciency depends on various notable factors [74]. These diffculties were overcome by using cell-fusing chemical agents (e.g., polyethylene glycol—PEG). PEG overcomes these diffculties and can be widely used for radiation cytogenetic studies.

Chemical-induced PCC exploits specifc inhibitors for serine/threonine protein phosphatase, which can activate endogenous intracellular MPF, which is much simpler and easier than fusion-induced PCC. Chemicals that can be used for the achievement of drug-induced PCC are okadaic acid, calyculin A, 2-aminopurine, staurosporine, wortmannin, and sodium vanadate. A limitation of this method is that no PCC can be induced in G0 resting-phase cells (Box 3.15).

#### **Box 3.15 In a Nutshell: Premature Chromosome Condensation**

Because of its unique properties, PCC is used for visualizing and scoring chromosomal damage induced by radiation or other clastogenic agents, measuring the induction yield and repair kinetics of chromosome damage in cells at various cell cycle stages immediately after irradiation. It can also be used for the study of condensation dynamics and conformational changes that occur during the cell cycle. The data obtained using the PCC assay can correlate radiation-induced DNA damage and CAs observable at metaphase [75].

Mitotic cell fusion-induced PCC in human lymphocytes (G0-PCC) allows early detection of cytogenetic damage in interphase, the stage of human lymphocytes in peripheral blood, and is the most suitable technique especially for biodosimetry applications in radiation emergency accidents as well as for triage biodosimetry [76]. A later ring PCC (rPCC) assay is an alternative biodosimetry method to the "gold standard" cytogenetic approach (dicentric analysis in metaphase) for high-dose exposure to radiation and can be applied in a simulated mass casualty accident either after chemical induction of PCC [77] or by means of cell fusion providing a much faster assessment of dose [78].

# **3.6.4 Chromothripsis-Like Alterations**

During the last decade, it has been reported that high-LET radiation induces chromothripsis-like complex chromosomal alterations, resembling the phenomenon of chromothripsis appearing in tumors [79]. The term chromothripsis arises from the Greek dialect (chromo for chromosome and thripsis for shattering into pieces), and it was initially described in 2011 by Stephens et al. [80]. Rather than a progressive accumulation of sequential alterations induced in the genome, chromothripsis is a process where chromosome segments undergo tremendous but localized shattering and random rearrangements in a single catastrophic event. Inaccurate rejoining of the induced chromosome fragments results in a new genomic arrangement and the formation of complex chromosomal aberrations that may trigger carcinogenesis (Fig. 3.22).

The mechanisms responsible for chromothripsis are still under debate. However, studies have shown several situations that could be catastrophic for the cell and result in chromothripsis. One possible mechanism proposed is that DNA damage such as DSBs and chromosomal aberrations may cause aberrant mitosis and formation of MN including one or more chromosomes that may undergo localized shattering and chromothripsis. Chromosome shuttering and chromothripsis may emerge in MN when the main nucleus enters mitosis while DNA is still being replicated within micronuclei. Additionally, PCC induces a mechanical stress in the asynchronous micronucleated cells leading to chromosome shattering [73]. Random genomic rearrangements in micronuclei can then be integrated into the cell's genome, triggering amplifcation of oncogenes and cancer development [81]. Other additional mechanisms have also been proposed, such as dicentric chromosome formation, telomere erosion, and abortive apoptosis [82].

Regarding radiation-induced chromothripsis-like chromosomal alterations, it was tested recently whether clustered DNA lesions and chromatin decompaction induced by high-LET irradiation can subsequently evolve in localized chromosome shattering in chromosome domains along the particle tracks. This is a critical risk for chromothripsis to occur, and the results obtained provided experimental evidence that high-LET particle radiation is effective in inducing chromothripsis-like aberrations, which can be used as a fngerprint of high-LET exposure [83]. These discoveries are valuable in the felds of radiation oncology and space radiation protection, since chromothripsis-like aberrations can be responsible for adverse effects and increase the hazard for secondary induced cancer.

**Fig. 3.23** Radiation-induced DNA damage foci. 53BP1 (left, cyan) and γΗ2ΑΧ (middle, magenta) foci in HeLa cells irradiated with 1.2 Gy alpha particles and spatially fxed at 60 min postirradiation.

Colocalization of γΗ2ΑΧ and 53BP1 foci is shown (right). Yellow line indicates the cell nucleus

# **3.6.5 Ionizing Radiation-Induced Foci (IRIF)**

This chapter is dedicated to the importance of ionizing radiation-induced foci (IRIF) (Fig. 3.23) in DNA damage measurements. Traditional biomarkers of radiation exposures are chromosomal aberrations and micronuclei. In contrast to quantifcation of these biomarkers, which emerge due to repair errors in some cells only, IRIF of certain proteins and posttranslational modifcations are formed in all cells on all DSB damages, almost immediately after irradiation. Hence, these IRIF can be considered specifc biomarkers of DSB lesions [84]. This allows easier and faster victim triage. Moreover, naturally occurring amplifcation of the DSB damage signal, associated with extensive focal accumulation of γH2AX and numerous repair proteins at DSB sites (for detailed description on DNA repair, see Sect. 3.4), offers the unprecedented sensitivity of radiation dose estimation via the pure counting of IRIF on immunofuorescence microscopy images [84]. The radiation dose absorbed by the cells can be estimated by simple counting of such IRIF or, more automatically, by measuring the integrated intensity of the IRIF signal for high numbers of individual cells by fow cytometry [85]. Under the optimal conditions, especially the time range around 30 min after irradiation, the reported minimal detectable values lie in the range of mGy [86].

Furthermore, DNA damage induction and repair processes can be studied in individual cells using the IRIF assay. In practice, this is important in situations where individual cells can be differentially affected by irradiation, such as in the cases of a partial-body exposure. The ability to study individual cells is critically important also for radiobiological research as individual cells, even if irradiated homogeneously, appear in different phases of the cell cycle, belong to specifc (cancer) cell clones, may be to a various extent affected by the bystander effect, etc.

On the other hand, the biochemical nature of IRIF means that their formation potentially depends on various factors, which may introduce some variability to DSB quantifcation. It remains a subject of discussion whether all DSBs necessarily require IRIF formation for successful repair. Additionally, some foci may persist at DSB sites even after the break rejoining. A real obstacle could follow from the fact that IRIF occur, to some extent, in nonirradiated cells. However, recent results have proved that the spontaneously forming foci differ in size and topology from the radiationinduced ones. So, staining patterns corresponding, for instance, to replication-stressed or apoptotic cells can be distinguished from IRIF related to DNA repair [87]. Importantly, this phenomenon is more prominent only in cancer cells, which are not relevant for biodosimetry. In any case, "the second γH2AX assay intercomparison exercise" carried out in the framework of the European biodosimetry network (RENEB) confrmed a high fdelity of irradiated victims' triage (dose categorization, rather than dosimetry) based on IRIF detection of the postradiation modifcation of histone variant H2AX, called γH2AX [84].

γH2AX is formed by the phosphorylation of histone H2AX at ser139 [57]. This process is mediated by ATM, ATR, and DNA-PK kinases, appears in minutes after DNA breakage, and spreads over ~2 Mbps of DSB-surrounding chromatin. Due to this extent of chromatin modifcation, γH2AX can be microscopically visualized as compact IRIF at DSB sites of 400–600 nm size as described in Sect. 3.5.

The number of γH2AX foci at a particular time postirradiation corresponds to a dynamic equilibrium between the IRIF formation and disassembly as shown in Fig. 3.24. This is the reason why the maximum γH2AX numbers per cell are detected with a short delay after irradiation and the numbers of counted γH2AX are slightly lower compared to physically detected DNA breaks (PFGE, comet assay) [49].

For most cell types, the peak number of γH2AX is detected in the time window between 30 min and 1 h postirradiation on average, and some shift to later postirradiation times may appear in cancer cells as they often suffer from DSB repair defects. If the integrated γH2AX signal is measured by fow cytometry, the maximal values are measured later than with focus counting, at about 1 h postirradiation, as the size of γH2AX foci grows longer than their number [49]. After reaching the peak value, the number of γH2AX foci rapidly reduces (Fig. 3.24) and, at 24 h postirradiation, only few DSBs that are repaired only with diffculty persist in cells irradiated with medium doses (in order of Gy) of low-LET radiation. However, a substantial proportion of DSBs may still be detected at this late period of time or even after several days postirradiation in

**Fig. 3.24** DNA repair kinetics. (**a**) Formation and disassembly of γH2AX foci in human cancer cells irradiated with 1 Gy or 2 Gy X-rays. (**b**) Representative microscopic images for γH2AX foci 1 h and 2 h

after X-ray irradiation. (Reproduced with permission (CCBY) from Mariotti et al. [88])

cells exposed to high-LET radiation or high doses of low-LET radiation. From the perspective of biodosimetry, this means that the highest precision of the absorbed dose estimation can be achieved in a few-hour window immediately after irradiation. This requirement can be fulflled during planned medical care, where, in addition, the monitoring of γH2AX foci formation and disassembly (DSB repair kinetics) may be used to identify patients hypersensitive to radiotherapy or radioresistant tumors. However, in the case of unpredicted accidents with mass screening, IRIF-based biodosimetry must rely on the persistent foci due to the necessary reaction time. This requires suitable mathematical models for the absorbed dose estimation and restricts the method applicability to the acute photon dose range of ~0.5 to ~8.5 Gy and days after exposure (i.e., 1 day after 1 Gy and 14 days after 8.5 Gy) [89]. For military countermeasures, it should also be kept in mind that some chemical warfare agents, such as mustard gas, also generate γH2AX foci. Furthermore, background levels may vary due to non-irradiation-induced IRIF, which are also counted and vary individually, so this assay is best suited for triage rather for accurate dosimetry.

In addition to the analysis of γH2AX IRIF numbers, the spatial distribution of γH2AX foci can be determined by microscopy. This is an important advantage of microscopy over fow cytometry as low-LET and high-LET exposures can be distinguished according to nuclear topology of γH2AX foci [90] as described in Sect. 3.5. On the other hand, fow cytometry offers more room for automation than microscopy and can analyze much higher cell numbers, making it the more suitable method for routine biodosimetry in most circumstances (Box 3.16).

# **Box 3.16 In a Nutshell: γH2AX as Radiation Damage Marker**

γH2AX attracts numerous proteins with specifc signaling and/or repair functions to DSB sites. These proteins, in turn, form IRIF with protein-specifc time occurrence and extent of colocalization with γH2AX. Hence, IRIF formed by numerous repair proteins can be used to quantify DSBs and estimate the absorbed dose in the same way as it was described above for γH2AX. Alternatively, repair protein and γH2AX foci can be detected simultaneously to enhance the fdelity of DSB evaluation. Furthermore, the protein composition and structure of IRIF protein complexes (e.g., their specifc persistent homology at the nanoscale), and differences of these parameters in specifc chromatin domains and after exposure to different types of ionizing radiation, help to understand the mechanisms of DNA repair.

Some proteins like 53BP1 form IRIF morphologically comparable to γH2AX foci. Others, which are required in only a few copies (Ku70 and Ku80), are too tiny and can be visualized only with electron microscopy or super-resolution optical microscopy [91]. Other proteins [such as MRE11, NBS1, or ATM (Fig. 3.25)] create small, but large enough, IRIF to be recognized by standard immunofuorescence microscopy. However, these proteins are, in addition to their IRIF location, also dispersed over the cell nucleus. As IRIF and free aggregates of these proteins may be similar in size, and cannot be discriminated by antibody staining, it is often diffcult to reliably distinguish these IRIF from the background [52]. Depending on the function of a particular protein in the repair process, IRIF appear immediately (e.g., MRE11, NBS1, 53BP1) or only later after irradiation (BRCA1, BRCA2, RAD51, etc.). This timing may correspond with repair pathway specifcity of a given protein. Some proteins, such as 53BP1 [57], are involved in the regulation of both major DSB repair pathways (NHEJ and HR), while other proteins are selective either for NHEJ or for HR (BRCA1, BRCA2, RAD51).

IRIF of repair pathway nonselective proteins, such as 53BP1, occur in all cells and colocalize with most γH2AX foci [57]. 53BP1 is thus a good DSB marker for biodosimetry, in addition to γH2AX. Moreover, 53BP1 foci have similar size and shape as γH2AX foci so that 53BP1 and γH2AX foci extensively colocalize (Fig. 3.23). This fact improves DSB detection in cells where both types of foci are labeled simultaneously. Co-labeling of γH2AX and 53BP1 foci may be especially useful when cells were exposed to low radiation doses generating only few DSBs or if cancer cells with a strong background signal are analyzed. A signifcant improvement of DSB number estimation due to γH2AX and 53BP1 co-detection is experienced also in cells exposed to high-LET radiation, where DSBs are extensively clustered and can be thus discriminated only with limitation. However, super-resolution microscopy methods, such as singlemolecule localization microscopy (SMLM) or STED microscopy, are necessary for more precise analysis of IRIF foci or even their internal composition and arrangement [48, 57].

It should be noted that not all γH2AX foci necessarily colocalize with 53BP1 (or other repair proteins) at early time periods postirradiation. This includes also the period of 30 min postirradiation when the maximum γH2AX focus

**Table 3.7** DNA repair proteins and occurrence

numbers are detected. On the other hand, at late time periods after irradiation, 53BP1 foci may persist in cells without being accompanied by γH2AX. These non-colocalizing 53BP1 foci probably label and protect incompletely repaired chromatin [93].

Moreover, as IRIF form also at sites of single-stranded DNA breaks (SSB) or oxidative base damages, co-labeling of γH2AX with suitable markers of these lesions (e.g., XRCC1 or OGG1) [94] can provide information on the complexity of individual DNA damage sites. This information may be correlated to various factors, such as the LET of the incidental radiation or chromatin density and genetic activity at DSB sites [16]. Table 3.7 shows a summary of the IRIF markers mentioned in this section and their occurrence (Box 3.17).

#### **Box 3.17 In a Nutshell: Ionizing Radiation Induced Foci**

# **3.7 Oxidative Stress: Redox Control and Mitochondrial DNA Damage**

# **3.7.1 Oxidative Stress and Consequences for Cell Macromolecules**

Exposure to IR induces oxidative damage to cellular molecules such as proteins, lipids, and DNA as a result of oxidative stress (OS), a consequence of the indirect effects of IR (see Chap. 2 and Sect. 3.2), as shown in Fig. 3.26. OS refers to a state of imbalance between oxidants and antioxidants, in favor of oxidants, due to either antioxidant depletion or oxidant accumulation. Oxidants include reactive oxygen (ROS) and nitrogen (RNS) species that comprise free radicals, which are characterized by oneself or more unpaired electrons in the outer shell, and non-radical reactive species. A list of radicals and non-radicals can be found in Table 3.8. Some of these species, e.g., superoxide and hydroxyl radical, are short-lived due to their high reactivity towards other molecules, while others, like hydrogen peroxide, are more stable. Among the ROS, the hydroxyl radical is particularly toxic and involved in the mediation of IR-induced lesions to cell biomolecules. By analogy to OS, nitrosative stress is mentioned when referring to RNS.

Oxidants are produced from exogenous, such as air pollutants, xenobiotics, and IR, and endogenous sources as normal cellular metabolism by-products. Examples are the mitochondrial electron transport chain (ETC), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, and peroxidases. Low to moderate ROS levels are crucial in physiological function of cell to avoid oxidative stress involved in aging and several neurodegenerative diseases, diabetes, cancer, atherosclerosis, etc. ROS are also signaling molecules involved in the IR non-targeted effects (see Chap. 2).

OS occurs in pathologic conditions, when the cellular antioxidant defenses are overwhelmed by free radicals and oxidants. Their great oxidative ability leads to oxidative damages to cellular biomolecules (DNA, proteins, and lipids) resulting in multiple damage affecting cell membrane, cellular signaling, and genome integrity. The accepted radiation biology paradigm considered DNA for a long time as the critical IR target and the primary cause for the harmful effects of IR, due to its content of genetic information, with nucleic acid damage being extensively characterized, without consideration that damaged lipids and proteins may also have detrimental effects on cellular function.

Further targets of radiation-generated ROS are lipids, major constituents of the cell membrane, because of their molecular structure containing abundant reactive double bonds [96]. Upon ROS reaction with polyunsaturated fatty acids (PUFA), chain reactions occur, leading to lipid peroxidation (LP) and generation of toxic decomposition products such as malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), and isoprostanes (IsoPs), which are quantifable markers of LP reactions. Biological LP consequences include changes in the permeability and fuidity of the membrane lipid bilayer, ion gradient disruption across membrane, and alterations in membrane-associated protein activity [96].

Potential oxidative damage to proteins is multiple, cysteine, methionine, and tyrosine residues. Chemical modifcations include oxidation, carbonylation, and nitration and lead to posttranslational modifcations inducing conformational changes affecting protein structure and function, i.e., loss of enzyme activity.

While the physical and chemical reactions initiated by radiation occur in less than a millisecond, the resulting biological effects may take hours, days, months, or years to be expressed and may differ among individuals due to varying intrinsic radiosensitivity. In particular, since the oxidative damage extent depends on the antioxidant availability, increased expression of antioxidant defense systems has been linked to decreased radiosensitivity [97].

OS also has a central role within the infammatory process. ROS such as superoxide can rapidly combine with NO to form other RNS, such as peroxynitrite, and is 3–4 times faster than the dismutation of superoxide by the SOD. The RNS, in turn, induces nitrosative stress, which adds to the pro-infammatory burden of ROS. Injured cells release chemoattractant molecules, and NO increases vascular permeability and vasodilation that trigger local infammation. Neutrophils are the frst infammatory cells to arrive at the site of injury, and the increased expression of intercellular adhesion molecule 1 (ICAM-1) and platelet endothelial cell adhesion molecule 1 (PECAM-1) on disrupted endothelial surfaces contributes to neutrophil extravasation. When leukocytes come into contact with collagen fragments and fbronectin, they release pro-infammatory cytokines like tumor necrosis factor alpha (TNF-α), IL-1, and IL-6 that increase ROS production and lead to even greater local infammation that can perpetuate inducing chronic radiation injury, which in some cases develop into fbrosis [98] (Box 3.18).

#### **Box 3.18 In a Nutshell: Oxidative Stress**

# **3.7.2 Redox Control: Antioxidant Defenses**

In order to cope with ROS and RNS, living organisms have evolved essential antioxidant defense mechanisms (Fig. 3.27). These are classifed as enzymatic and nonenzymatic systems or as high-molecular-weight and lowmolecular-weight compounds. The frst line of antioxidant defenses includes the highly abundant glutathione (GSH), catalase, glutathione peroxidase (GPx), and superoxide dismutase (SOD). GSH acts directly as an oxidant scavenger or indirectly as a cofactor of several enzymes such as the GPx. SOD exists in three isoforms using different metals as cofactors: SOD1, which is predominantly cytoplasmic; SOD2, which is mitochondrial; and SOD3, which is extracellular. SOD1 and SOD3 contain copper (Cu) and zinc (Zn), whereas SOD2 has manganese (Mn) in its active site. They catalyze the dismutation of °O2– to H2O2 afterwards converted to water by catalase, GPx, or peroxiredoxin (Prx). GPx transforms reduced GSH to its oxidized form (GSSG). GSH pool regenerates by de novo synthesis and glutathione reductase using NADPH as a reducing equivalent. GPx is also involved in hydroperoxide detoxifcation. Prx is involved in hydroperoxides and peroxynitrite detoxifcation, using thioredoxin (Trx) as a source of reducing equivalents. The most reactive and highly toxic °OH is produced from H2O2 in the presence of reduced transition metal, a reaction known as the Fenton reaction. Apart from GSH, nonenzymatic antioxidants include endogenous compounds which are produced in organism (uric acid, lipoic acid, l-arginine …) and exogenous compounds which are supplemented through the diet, i.e., carotenoids, ascorbic acid (vitamin C), vitamin E and derivatives (tocopherols and tocotrienols), polyphenols (curcumin, resveratrol, quercetin …), and others.

Glutathione is the major low-molecular-weight thiol in mammals. It plays a key role in cell resistance against oxidative and nitrosative damage by providing reducing equivalents to enzymes involved in the metabolism of ROS, by eliminating potentially toxic oxidation products, and by reducing oxidized or nitrosated protein thiols. In its reduced form (GSH), glutathione is the principal intracellular antioxidant. The conversion of the oxidized form (GSSG) into GSH is done by glutathione reductase (GR) in the presence of NADPH, which is generated by glucose-6-phosphate dehydrogenase in the pentose phosphate pathway (Fig. 3.27). Hence, any damages to these enzymes can compromise GSH functions. The processes of glutathione synthesis, transport, utilization, and metabolism are tightly controlled to maintain intracellular glutathione homeostasis and redox balance. Glutathione is exclusively synthesized in the cytosol and about 85% of it remains there, mainly in the reduced form. The ratio of GSH:GSSG in the cytosol is conservatively estimated at about 10,000:1–50,000:1, and the concentration of the cytosolic GSH is as high as 10 mM, while GSSG in the cytosol is as low as nanomolar concentration [99]. Directly and indirectly, GSH effectively scavenges free radicals and other reactive species (e.g., hydroxyl radical, lipid peroxyl radical, peroxynitrite, and H2O2) through enzymatic reactions, such as those catalyzed by GPxs, glutathione-Stransferases (GST), formaldehyde dehydrogenase, maleylacetoacetate isomerase, and glyoxalase I (Fig. 3.27). GSH also helps to recover other important antioxidants as vitamin C.

OS was shown to promote the activation of redox-sensitive transcription factors such as the nuclear factor erythroid 2-related factor 2 (NRF2) and the nuclear factor kappa B (NF-κB). The NRF2 transcription factor plays a central role in the maintenance of cellular redox homeostasis via the coordinated transcriptional upregulation of numerous antioxidant proteins (Fig. 3.28). These include more than 500

**Fig. 3.28** NRF2 protection against oxidative stress and excessive infammatory responses involved in IR injury. NRF2 induces antioxidant response genes, like SOD, CAT, GPX, and GST that enhance ROS elimination. In addition, expression of enzymes such as GR and GS increases GSH cellular content and antioxidant capacity of the cell. Reduction in ROS levels decreases the expression of NFKβ, the main contributor to the infammatory response. Moreover, NRF2 enhances the expression of HO-1 and its activity in the production of CO that reduces NFKβ activity, pro-infammatory cytokine secretion (IL-6,

genes that are crucial to metabolize electrophilic attack and protect against OS and infammatory damage. Kelch-like ECH-associated protein 1 (KEAP1) is a key cytoplasmic repressor of NRF2. KEAP1 interaction with NRF2 leads to NRF2 proteasomal degradation. In the presence of OS or inducers, key "sensor" cysteine thiol groups on KEAP1 are modifed, disrupting the degradation process and allowing

TNFα, and IL-1β), and pro-infammatory enzyme activity (COX-2 and iNOS). *ARE* antioxidant-responsive element, *NRF2* NF-E2-related factor 2, *SOD* superoxide dismutase, *CAT* catalase, *GPx* glutathione peroxidase, *GST* glutathione S-transferase, *GS* glutathione synthetase, *GR* glutathione reductase, *GSH* glutathione, *ROS* reactive oxygen species, *NFKβ* nuclear factor kappa β, *IL-6 and 10* interleukin 6 and 10, *IL-1β* interleukin 1 beta, *TNFα* tumor necrosis factor alpha, *COX-2* cyclooxygenase 2, *iNOS* inducible nitric oxide synthase, *HO-1* heme oxygenase 1

NRF2 to directly translocate into the nucleus. NRF2 then upregulates the expression of enzymes involved in the synthesis and recycling of GSH, such as the catalytic and modulator subunits of glutamate–cysteine ligase (GCLC and GCLM), GR, GPx, SOD, and several GST. Moreover, several proteins within the redoxin family, such as Trx, TrxRs, Prxs, and sulfredoxins, are also upregulated by NRF2 [100]

**Fig. 3.29** Mitochondria as the key player in radiation-induced oxidative stress-mediated apoptosis. Various stimuli like radiation or improper functioning of the oxidative phosphorylation induce oxidative stress via ROS production. This causes the mitochondria to dysfunction and subsequently leads to cell death by apoptosis. *NAD+* nicotinamide adenine dinucleotide, *NADH* nicotinamide adenine dinucleotide hydrogen, *H+* hydrogen, *FAD* favin adenine dinucleotide, *FADH2* favin

adenine dinucleotide hydrogen, *ATP* adenosine triphosphate, *ADP* adenosine diphosphate, *Mn-SOD* manganese superoxide dismutase, *GPx* glutathione peroxidase, *H2O2* hydrogen peroxide, *CuZn-SOD* copper zinc superoxide dismutase, *ROS* reactive oxygen species, *Bcl-2* B-cell lymphoma 2, *Bax* Bcl2-associated X, *APAF1* apoptotic proteaseactivating factor 1

as shown in Fig. 3.29. NRF2 stimulates the mitochondrial biogenesis program through activation of nuclear respiratory factor 1 and indirectly prevents/attenuates infammation, because NRF2 activation results in the expression of previously mentioned antioxidant enzymes, which detoxify ROS, and in turn this reduces the expression of NLRP3 infammasome and NFKβ (the main regulator of pro-infammatory response). Moreover, NRF2 upregulates heme oxygenase activity (HO-1) and increases CO production, which in turn reduces NFKβ activity. In response to this, pro-infammatory cytokine (IL6 and TNFa) production is reduced, and at the same time the production of anti-infammatory cytokines (such as IL10) increases. As a consequence of these changes, NRF2 facilitates cells to survive oxidative stress and the infammatory response that aggravates their cytotoxic effects (Fig. 3.29).

# **3.7.3 The Role of Mitochondria in Oxidative Stress**

Mts are double-membrane multifunctional organelles associated with biosynthesis, metabolism, cell survival, signaling of ROS, etc. In the late 1960s, it was found that radiation could signifcantly modify the structural form of mts and also the mitochondrial DNA. Human mtDNA is a 16,569 base pair (bp) double-strand circular DNA molecule containing 37 genes, encoding 13 polypeptides for the mt electron transport chain, 2 ribosomal RNA, and 22 transfer RNA for mt protein synthesis. Somatic cells have an average of 100– 500 mts with 1–15 mtDNA molecules per mitochondrion.

Although nuclear DNA (nDNA) is the main IR target, mts are constantly removing excess ROS created during energy production and mtDNA is much more vulnerable to IR effects than nDNA. mtDNA is generally repaired less effciently than nDNA [101], although it uses the same repair mechanisms such as BER, MMR, and HR but not NER and classical NHEJ. Furthermore, the histones for better exposure protection are lacking. Together, this leads to a mutation rate which is 10–1000 times higher than nDNA [102]. Both direct IR exposure and irradiated cell-conditioned medium induce mtDNA damage and alter directed protein synthesis. As a consequence, IR exposure can cause the loss of mt membrane potential, leading to mt undergoing either fssion, division of one mitochondrion, or fusion, combination of several mitochondria, autophagy (mitophagy), apoptosis, modifcation in the mtDNA copy number per cell (mtD-NAcn), and cause DNA damage and mutations, like point mutations or deletions. A common deletion mutation of 4977 base pair deletion in mtDNA genes coding for subunits of the mitochondrial ATPase, NADH dehydrogenase complex I, and cytochrome c oxidase is known as a marker for oxidative damage [101].

Changes in mtDNAcn or mutations in mtDNA both caused by high intra-mtROS control mt-dependent methylation potential of nDNA by decreasing methyltransferase activity and thus causing global DNA hypomethylation or changes in the expression of specifc genes [103]. Global DNA methylation levels depend on human mtDNA variants and are also tissue specifc and, therefore, may be connected with the differences in susceptibility to the pathogenic processes resulting from IR exposure and OS in different tissues [103].

OS also appears to target the mitochondrial DNA polymerase-γ activity required for replication and repair of mtDNA, thereby reducing the overall repair capacity. Therefore, subsequent to radiation exposure, mtDNA might be damaged, with an ensuing decrease in respiratory chain activity and decrease of mitochondrial function, giving rise to an increased ROS production. Moreover, mutations in mtDNA could lead to an increase in accessibility of reduced components of the ETCs to O2, which may result in an increase in prooxidant formation. The functional disablement can be weighed by the limitations of the complexes I and III of the mitochondria, reduction of succinate-induced respiratory competence, augmented ROS levels, and increased mitochondrial protein oxidation. The net consequence is persistent metabolic OS that continues to cause de novo oxidative damage to critical biological structures. Such mitochondrial dysfunction can lead to stress signals, which lead to reduced electron transport chain (ETC), and oxidative phosphorylation can cause imbalance in the mitochondrial ROS production, decrease in the mitochondrial membrane potential, and lesser cellular ATP or energy. Although mts are the main producer of ROS, mts themselves can be susceptible to the pathological outcomes once targeted by ROS. By triggering the mitochondrial stress and downstream signaling, the increased levels of free radicals linked to the mtDNA oxidative damage lead to apoptosis.

One of the crucial steps in the process of apoptosis is the permeability transition pore opening (mPTP), followed by drop in the mitochondrial membrane potential. Opening of the pore increases the permeability of the mitochondrial membrane to molecules, leading to mitochondrial swelling and necrosis. NO produced at the basal level (e.g., 5 μM) could S-nitrosylate cyclophilin D (CypD), a critical mPTP regulatory component. This prevents the association of CypD with mPTP that is required for opening the pore and confers a protection to the cell under a stress. On the other hand, NO produced at a high concentration (e.g., 500 μM) could produce peroxynitrite in the presence of large amounts of ROS. Peroxynitrite could oxidize mPTP leading to its opening, which would lead to the opening of mPTP, loss of ATP production, and necrosis. The damaged mitochondria generated excessive ROS like hydrogen peroxide and superoxide anion, which provokes the mitochondrion-driven ROS propagation. ROS themselves accelerate the production of mitochondrial ROS. This process is also called as ROSinstigated ROS release (RIRR) by initiating as intermitochondria signaling network [104] (Fig. 3.28). Oxidative insult by radiation to the mt alters the mitochondrial membrane potential and causes the leakage of cytochrome c from the inner membrane compartment, which elicits a sequence of signal transduction progression, the outcome of which is apoptotic cell death. Once the mitochondria are severely stressed, the pro-apoptotic factors like Bax create pores on the mitochondrial membrane, which lets the release of cytochrome c in the cell cytoplasm. It interacts with Apaf-1 to form a complex called apoptosome (Apaf-1, cytochrome c, and ATP). Caspase-9 then gets activated and commences the action of other caspases like caspase-3, -6, and -7. These lead to DNA fragmentation and cell degradation, thereby pushing the cells towards apoptosis. This kind of cell death is known as mitochondrial mediated cell death or intrinsic pathway of apoptosis (Fig. 3.29). However, in this case, apoptosis plays a role in abashing cells that induce excessive ROS.

# **3.7.4 Oxidative Stress Biomarkers**

Biomarkers of OS can be classifed as molecules that are modifed by interactions with ROS or molecules of the antioxidant system that change in response to increased OS. ROS levels can also be monitored using fuorescent probes of commercial kits, which specifcally detect intracellular ROS such as H2O2, NO, or °O2 <sup>−</sup>. However, assays that monitor ROS levels are unlikely to be useful for biomonitoring purposes due to the short half-life of ROS and the fact that the response is not specifc to radiation exposure.

#### **<IP_ADDRESS> Antioxidant Defenses**

S-Glutathionylation is the posttranslational modifcation of protein cysteine residues by the addition of glutathione. This modifcation can prevent proteolysis caused by the excessive oxidation of protein cysteine residues under oxidative or nitrosative stress conditions. Measuring S-glutathionylation of the proteins as biomarkers (Fig. 3.30) is hampered by diffculty in accessing the tissue in which these modifcations occur. Nevertheless, S-glutathionylation of hemoglobin has been proposed as a biomarker of OS strengthened by fnding that it occurs in the circulating erythrocytes in parallel with S-glutathionylation of molecules in the vasculature or myocardium [105].

The participation of GSH in antioxidant reactions, either chemically or enzymatically via GPx, results in its own oxidation to GSSG. Decrease in intracellular GSH/GSSG ratio is one of the most used biomarkers of OS. In these conditions, GSSG is preferentially secreted out of the cell, and therefore, blood levels of GSH and GSSG may refect changes in glutathione status in other less accessible tissues. 6 h after a single dose of irradiation (equivalent to 5 Gy), GSH/GSSG ratio decreases in blood. The decrease in GSH/GSSG is mainly due to an increase in the concentration of GSSG, because GSH levels do not change signifcantly [106].

# **<IP_ADDRESS> Total Antioxidant Capacity (TAC) and Other Antioxidant Biomarkers**

Antioxidants protect the body from the harmful effects of free radical damage. Thus, measurement of antioxidant levels in target tissues or biofuids has been widely used to assess the extent of oxidant exposure and, in turn, OS. TAC is the measure of the free radical amount scavenged by a test solution, being used to evaluate the antioxidant capacity of biological samples (tissues or biofuids). The TAC system involves enzymes (SOD, CAT, GPxs, and other enzymes), endogenous antioxidants, and dietary antioxidants (mentioned before), which are generally decreased when OS increases. TAC can be easily measured in cells, tissue lysates, and biological fuids by commercial colorimetric kits and represents a global approach (integrated parameter considered as the cumulative effect of all antioxidants of the biological sample) if no specifc antioxidant molecule is to be investigated. One of the critical points is that the results obtained with different methods are not always comparable, depending on the different technologies used for their assessment. Moreover, as mentioned by Dr. Sies (who coined the concept of oxidative stress): "neither the term 'total' nor the term 'capacity' are applicable to the in vivo assays using an arbitrarily selected oxidant generator assaying a sample removed from its biological context, which is characterized by enzymatic maintenance of steady state" [107]. For that reason, we agree with him "that investigators should measure individually parameters associated with oxidative stress (GSH, urate, ascorbate, tocopherol, etc.) and antioxidant enzymes activities (in tissues samples and lymphocytes (in the case of blood samples) if their want to have an idea of the exposure of the entire organism to oxidative stress" [108].

# **<IP_ADDRESS> Oxidation Products of DNA, Lipids, and Proteins**

The "comet assay" and newer techniques [e.g., gas chromatography, high-pressure liquid chromatography (HPLC), immunoassays] can distinguish gross DNA damage produced by IR and damage from oxidation (for a detailed description, see Chap. 7). For low doses of radiation, the total number of induced DNA alterations is probably small when compared with the total number of equivalent alterations from endogenous sources. At DNA level, guanine is the most susceptible base to OS, and its oxidation at the C8 of the imidazole ring of deoxyguanosine generates 7,8-dihydro-8-oxo-2′deoxyguanosine (8-oxodG), which is the most predominant and stable DNA oxidative lesion in the genome (Fig. 3.30). A failure to repair oxidized bases creates a risk of mutation during DNA replication. For example, 8-oxodG mispairs with deoxyadenosine (dA) rather than deoxycytosine (dC) resulting in a C-A point mutation, thus increasing the risk of carcinogenesis. Besides the impact of confounding factors like age, sex, and smoking habits, with the help of correction factors, 8-oxodG levels are good and sensitive biological indicators of OS, which can be quantifed in serum or urine samples, using HPLC coupled with mass spectrometry [109]. 8-OxodG can be removed by NER or BER with the action of 8-oxodG DNA glycosylase 1 (OGG1), a base excision DNA repair enzyme that cleaves the N-glycosidic bond between the base and the deoxyribose, generating an apurinic/apyrimidinic site (AP) and triggering the BER mechanism. DNA strand breaks and AP sites are effective substrates to activate DNA damage sensor PARP1. Overactivation of PARP1 is associated with apoptosis-inducing factor (AIF)-mediated and caspase-independent cell death. OGG1 seems to guard genome integrity through lesion repair or cell death depending on the magnitude of guanine oxidation. OGG1 may also be measured as an OS marker.

As previously mentioned, lipid peroxidation products include MDA, 4-HNE, or IsoPs and can be used as oxidative stress biomarkers (Fig. 3.30). The latter are prostaglandin-like molecules formed by the nonenzymatic peroxidation of arachidonic acid (AA). MDA may be formed as a result of enzymatic and free radical peroxidation of PUFAs containing at least three double bonds and is also formed during prostaglandin synthesis. MDA can also react with DNA bases to form deoxyguanosine, deoxyadenosine, and deoxycytidine adducts, and these DNA-MDA adducts have mutagenic effects. Phospholipids containing linoleic acid and AA are considered the main source for 4-HNE production. Many different analytical methods are available for the measurement of MDA, 4-HNE, or IsoPs in biological samples and are reviewed by Tsikas [96].

It has been estimated that proteins scavenge a majority (50–75%) of generated reactive species. To function as biomarkers, protein oxidation products must be stable, accumulate in detectable concentrations, and correlate with OS exposition. Protein carbonylation is an irreversible protein modifcation, associated with alterations in functional and structural integrity of proteins, contributing to cellular dysfunction and tissue damages. Due to relatively early formation during OS, higher stability in comparison to other oxidation products, and simple analysis methods, protein carbonyls are one of the most OS biomarkers. Protein carbonyls can be easily quantifed in plasma, serum, tissue samples, and also saliva by enzyme-linked immunosorbent assay (ELISA) [110].

The reaction between °NO and °O2 <sup>−</sup> forms peroxynitrite, which can nitrate tyrosine residues in proteins. This process is in competition with the enzymatic dismutation of °O2 <sup>−</sup> and the diffusion of °NO across cells and tissues. Peroxynitritemediated damage has been implicated in a wide range of disease pathologies, and 3-nitrotyrosine (3-NT) and nitrated proteins have been established as a footprint of nitro/oxidative biomarker of progression and severity in conditions. The measurement of 3-NT can be performed in plasma, serum, as well as tissue samples by special mass spectrometry. Commercially available ELISAs are usually used in clinical studies due to standardization and easy sample preparation. In turn, several limitations have been highlighted in the literature, such as low sensitivity and minor specifcity. This and other protein oxidation biomarkers in human diseases are extensively reviewed by Kehm et al. [110]. The advancement of proteomics will allow us to assess changes in proteins (including the assessment of carbonylated, S-glutathionylation, S-nitrated, and/or N-nitrated derivatives) that serve as biomarkers of exposure to IR. An overview of the oxidation products of DNA, lipids, and proteins formed can be found in Fig. 3.30.

#### J. Reindl et al.

# **3.8 Cell Cycle Efects**

The cell cycle is a fundamental process through which the cell grows and accurately duplicates the genetic material before it divides to give rise to two daughter cells. The cell cycle is divided into two phases: interphase in which the cell spends most of its time, followed by mitosis during which the cell divides into two daughter cells. The interphase has three distinct phases. The frst phase is the G1 phase in which the cell grows and prepares itself for DNA synthesis. Second is the S phase, when the cell actually duplicate its DNA. The third phase is the G2 phase, where it prepares itself for mitosis. The duration of G1 varies considerably from cell to cell, while S, G2, and mitosis show less variation. Quiescence is a reversible state of a cell in which it does not divide but retains the ability to reenter cell cycle. This state is also called G0 phase.

The transition from one cell cycle phase to another is controlled by a variety of proteins, cyclins, and cyclindependent kinases. If the system identifes any inaccuracies, the transition from one phase to the next will be delayed and the cells arrested in the so-called cell cycle checkpoints [111]. Cells, which enter mitosis with unrepaired DNA damage, will most likely fail to divide properly resulting in cell death. In order to provide time for DNA damage repair or, if repair is not the best solution for inducing cell death, e.g. apoptosis, before DNA synthesis (S phase) and in particular mitosis is initiated, radiation induces arrest in checkpoints at the end of the G1 and G2 phases. Since the process that kills the cells after radiation damage is related to cell division, cells in G0 or cells which are differentiated or in senescence and have lost the ability to proliferate are very resistant to radiation [112].

# **3.8.1 Cycle-Dependent Kinases and Cyclins**

Cell division is a highly regulated progression allowing cells to divide and to generate daughter cells. The regulation is necessary for the recognition and restoration of genetic injury along with the prevention of uninhibited cell division. It is regulated by cyclins and cyclin-dependent kinases (CDKs). CDKs are serine or threonine kinases, which unite with a separate subunit of functional cyclins, which presents domains essential for enzymatic activity. CDKs are known to have a crucial function not only in cell division but also in amending the transcription responses. Hence, the deregulation of CDKs is a characteristic of cancers and utilized for anticancer therapy purposes. On the other hand, cyclins establish the activity of CDKs as their levels keep changing during the cell cycle. Depending on their participation and function during the cell cycle, cyclins are divided into four categories: G1 cyclins, i.e., D cyclins; G1/S cyclin, i.e., cyclin E; S-phase cyclins, i.e., cyclins E and A; and M-phase cyclins, i.e., B cyclins. Researchers have discovered around 20 CDK-associated proteins, which makes the cell cycle a complex process that involves the combination of CDKs (Cdk1, Cdk2, Cdk3, Cdk5, Cdk4, Cdk6, Cdk7, Cdk8, etc.) and cyclins (A1, A2, B1, B2, B3, C, D1, D2, D3, E1, E2, F, etc.) in distinct phases of the cell cycle endowing extra governance to the cell cycle apparatus (Table 3.9 and Fig. 3.31). Cyclins impart the specifcity for substrates and normal cell cycle regulation, which includes the subunit binding, localization, activation/deactivation, etc. to the Cdk/cyclin complexes [113].

**Table 3.9** Cyclins, CDKs, and their function throughout cell cycle

**Fig. 3.31** Overview of cell cycle: functions of different phases, cyclins and CDKS, and CDIs

# **3.8.2 Activation of CDKs by Binding to Cyclins**

CDKs have a very limited activity without the presence of a cyclin. To be an active kinase, it should be bound to its cyclin partner and its activity can be further altered by phosphorylation and association of additional proteins like p27. Every CDK/cyclin complex possesses a distinct function that is restricted to a specifc cell cycle phase (Table 3.9). Cdk4 and/or Cdk6 are activated by D-type cyclins in the beginning of the G1 phase, and it commences phosphorylation of the retinoblastoma protein (Rb) family (Rb, p107, and p130). This releases the E2F transcription factor and causes the activation and transcription of the E2F-responsive genes that are necessary for the cell cycle progression. The cyclin A and E types are the early E2F-responsive genes. During the later G1 phase, cyclin E binds to Cdk2 to activate it and executes the phosphorylation of Rb (pocket proteins), provoking the further activation of the E2F intervened transcription. This assists in the crossing over of the cell cycle checkpoints at the periphery of the G1/S phase, and to S-phase commencement. Cdk2 unites with cyclin A and aids the progression of the S phase. During the inception of the S phase, A-type cyclins are synthesized, which phosphorylates proteins associated with DNA replication. Going further, at the time of G2/M transition, the activity of Cdk1/ cyclin A is necessary for the induction of the prophase. Lastly, Cdk1/cyclin B complexes dynamically contribute to the completion of the mitosis process. Cdk1 activity fuctuates throughout cell cycle succession and is profcient of governing varied cell cycle adaptations (G1/S, S, and G2/M phases) by connecting with diverse cell cycle phase-associated specifc cyclins, and several processes like action of CDK-activating kinase (CAK) and inhibitory phosphorylation on CDK. Regulating the cyclin levels and action of CDK inhibitors during the cell cycle assures that CDKs are active in the precise stage of the cell cycle. Cells exploit many processes such as transcriptional control of cyclin genes and breakdown of cyclins; the transcriptional control of the cyclin subunits is one way that ensures appropriate temporal expression of the cyclins and degradation of cyclins, to confne cyclins to the proper cell cycle phase and to keep them at the accurate concentration [114]. Ubiquitinmediated protein degradation is one of the most crucial regulatory controls that confne the cyclins to the proper cell cycle phase. However, SCF (Skp1, Cullin, and F-box proteins) and APC/C (anaphase-promoting complex or cydosome) are two ubiquitin proteins involved in the degradation of cyclins. During the G1-S-phase transition, SCF controls degrading G1 cyclins (cyclin D), while APC/C degrades the cyclins of the S phase and mitosis, thus advancing the exit from mitosis. To control the CDK activity, the regulation of cyclin levels is not the only mechanism. Other mechanisms like activation and inhibition of phosphorylation actions on the CDK subunit and existence of inhibitors are critical in controlling cyclin-CDK activity [114].

# **3.8.3 Inhibitors of Cyclin-Dependent Kinases**

CDK inhibitors are a family of proteins that can bind directly to the cyclin-CDK complex and hinder its activity. In the transition of the G1-S phases, these proteins play a very crucial role. CKIs implicated in controlling the S phase and mitotic CKIs are indispensable to avoid early commencement of the S- and M-phase CDKs. However, in human cancers, genes coding these CKIs are often mutated leading to aberrant cell cycle regulation. During normal or extreme conditions (DNA damage, telomere dysfunction, and stress), the functions and activities of the CDK/cyclin complexes are governed and controlled by two families of CKIs. The INK4 family comprises the p16INK4a, p15INK4b, p18INK4c, and p19INK4d which can specifcally bind to Cdk4 and Cdk6 and hinder the activity of the D-type cyclin. The other Cip/Kip family (p21Cip1/ Waf1/Sdi1, p27Kip1, p57Kip2) obstructs Cdk2/cyclin E, Cdk2/cyclin A, Cdk1/cyclin A, as well as Cdk1/cyclin B activity. The p21 protein hinders the formation of cyclin/CDK protein complexes that are required for the progression from the G1 phase to the S phase of the cell cycle (Box 3.19).

#### **Box 3.19 In a Nutshell: Cell Cycle and Radiation Response**

# **3.8.4 Cell Cycle Phase and Radiosensitivity**

To study the variation of radiosensitivity with position in the cell cycle, it is necessary to synchronize the cells to get a population of cells that are all in the same cell cycle phase.

For cells in culture, there are three main techniques.

removed, the cells will go through the cell cycle synchronously for some time before they become more and more asynchronous. The most used drug is hydroxyl urea, which arrests cells at the border between G1 and S. The advantage of this method is that it can also be used in vivo.

3. Mitotic selection was introduced by Terasima and Tolmach [115] and is the most used synchronization method in cell culture in vitro. As cells enter mitosis, they round up and become less attached to the fask bottom. By then shaking the fask, the mitotic cells will detach and can be collected with the medium. The cells can then be irradiated at different time points as they go through cell cycle.

The frst age-response curve by Terasima and Tolmach [115] using 3 Gy irradiation of HeLa cells is shown in Fig. 3.32, left panel, together with a curve showing the fraction of labeled cells after pulsed incorporation of [3H]-thymidine during S phase. Cell survival was measured as the ability to form a macroscopic colony. The data indicate four times higher survival if the dose is delivered during early G1 compared to at the start of S phase. Furthermore, there is an increase in cell survival with age during S phase; that is, the radioresistance increases as more and more of the DNA is synthesized. HeLa cells are HPV infected and do not have functional p53, which normally would give the cells time for repair before entering S phase. The cells irradiated early in G1 will have time for repair, which is refected in a high survival, while the cells in late G1 are more sensitive, because they may enter S phase with unrepaired DNA damages. Cell lines with short G1 are sensitive throughout G1. Terasima and Tolmach also irradiated the synchronized cells with various radiation doses and thereby recorded complete dose-response curves for HeLa cells irradiated in different phases of the cell cycle (see Fig. 3.32, right panel). These curves confrmed the variation in radiosensitivity through the cell cycle, as was demonstrated by the age-response curves. In addition, they also showed that cells irradiated while in mitosis are far more radiosensitive than cells irradiated in any part of interphase.

Measurements of the radiosensitivity of cells in G2 are technically diffcult, and it has become customary to suppose that cells are radiosensitive if irradiated in G2. However, the radiosensitivity of cells in G2 has been shown to be dose dependent to a quite different degree than in any other phase of the cell cycle. Cells are hyper-radiosensitive for small radiation doses because the mechanism for early radiationinduced G2 arrest by ATM is not activated by radiation doses in the range below about 0.3–0.5 Gy (Box 3.20).

#### **Box 3.20 In a Nutshell: Radioresistance and Radiosensitivity and Cell Cycle**

**Fig. 3.32** Age-response of cells after radiation. Left: Age-response curves for HeLa-S3 cells (open circles: synchronized cells, triangles: asynchronous cells) irradiated with 3 Gy X-rays (= 300 rad) at different time points after selection in mitosis and the fraction of cells with incorporated [3H]-thymidine in DNA after a 20-min pulse (black circles,

right *y*-axis). Right: Dose-response curves for HeLa-S3 cells synchronized by mitotic selection and X-irradiated at different times after selection. 0 h: mitosis, 5 h: early G1 phase, 14 h: S phase, 19 h: late S/ G2 phase. [Reproduced with permission from Terasima and Tolmach [115]]

# **3.9 Telomeres and Senescence**

# **3.9.1 Telomeres and Their Role**

Telomeres are nucleoprotein structures located at the end of each linear chromosome in the cell nucleus. They are composed by tandem repeats of the G-rich hexanucleotide TTAGGG and are typically 10–15 kb long [116]. These structures are organized into heterochromatin domains, and they play a signifcant role in maintaining genome stability. There are at least two very important functions of telomeres in eukaryotes. The frst one is the protection of the linear DNA molecules from the DNA repair mechanisms, which may recognize these sites as double-strand breaks. Secondly, they defne the maximum number of cell cycles that a cell may undergo [116]. At each cell cycle division, telomeres shorten by 50–200 bp due to the DNA end-replication problem [117]. This problem results from the ineffcient copying of the last base pairs of the linear DNA molecule by DNA polymerase. After several cell divisions, the length of telomeres reaches a critical threshold, which means that the cell can no longer divide. The cell has then reached its Hayfick limit, and it proceeds to senescence. Telomere shortening is thus a very-well-known hallmark of cellular senescence and aging. A good example of the telomere shortening is the defciency of the adaptive immune system in older individuals caused by T cells reaching their Hayfick limit [118].

The telomere attrition can be opposed by an RNAdependent DNA polymerase known as telomerase. This enzyme can elongate the telomeres by adding 5′-TTAGGG-3′ repeats to the chromosomes 3′ terminal ends. Telomerase is connected with cells' immortality; thus, it is present in germline and malignant cells. There is only little or no telomerase in most somatic cells [118]. This information is summarized in Fig. 3.33. An inverse correlation between the telomere length and the radiation-induced cytogenetic damage was found for lymphocytes, fbroblasts, epithelial cells, and many cancer cell lines. It was shown that telomere shortening leads to chromosome fusion, chromosome bridges, or higher frequencies of micronuclei. Thus, telomere shortening is closely linked to the cell radiosensitivity. Therefore, targeting the telomeres could be a very good radiosensitizing method in our fght with cancer during radiotherapy [116].

# **3.9.2 Senescence and Its Role**

As described in Sect. 3.7, cellular senescence is a cell state triggered by extrinsic (cellular stressors) and intrinsic (physiological processes) factors. It is characterized by a prolonged and generally irreversible cell cycle arrest, associated with secretory features, macromolecular damage, and altered metabolism, with its function to remove potentially harmful cells from the proliferative pool [120]. Senescent cells are

**Fig. 3.33** Telomeres, their shortening, the senescence state, and immortal cells. An adult cell chromosome with telomeres and the enzyme telomerase, which plays a crucial role in telomere end lengthening (left). Telomere characteristics in an adult cell's chromosome,

after multiple replications, at cell senescence, and when the cell is immortal (left to right, blue box). (Adapted with permission from Aunan et al. [119])

detected at any life stage from embryogenesis (contributes to tissue development) to adulthood (to prevent the proliferation of damaged cells). Yet, senescent cells can also potentiate various aspects of tumorigenesis, including proliferation, metastasis, and immunosuppression by secreting a collection of pro-infammatory factors collectively termed as senescence-associated secretory phenotype (SASP) [121]. It is important to clarify that senescence is a distinct form of cell cycle arrest and distinct from quiescence, where cells can reenter the cell cycle when favorable growth conditions are restored; terminal differentiation, where cells exhibit functional and morphological changes resulting in loss of original cellular identity; and cell death, where cells are being eliminated and are thus nonfunctional. The existence of multiple senescence programs and the nonspecifcity of current senescence markers make it diffcult to fully unveil the complex mechanism behind senescence (current understanding presented in Fig. 3.34). It is therefore recommended to apply a multi-marker approach when investigating cellular senescence [120]. Yet, it is currently accepted that two main signaling pathways initiate and maintain the cell cycle arrest: p53–p21–retinoblastoma protein (RB) and p16INK4A– RB. As a consequence, depending on the senescence program, senescent cells express a multitude of hallmarks such as morphological alterations, senescence-associated betagalactosidase (SA-β-gal), and SASP among others [122].

• Senescence in developmental processes, i.e., in embryogenesis and organogenesis, is induced by paracrine signaling and is mediated by the expression of the cell cycle inhibitor p21. Although SA-β-gal is highly expressed, developmental senescence is not associated with DNA damage, does not secrete the typical range of SASP cytokines, and is independent of p53 and p16INK4a. Senescence in wound healing prevents excessive fbrosis by secreting PDGFA-enriched SASP to stimulate appropriate skin repair. Senescence causes, or at least contributes to, organismal aging through the shortening of telomeres followed by the induction of p16INK4a and resulting in an accumulation of senescent cells over time. Studies by Baker et al. [123], frst in BubR1-mutant mice (Cdkn2ap16 knockout mice) and then later in naturally aged mice, demonstrated that in the absence of p16INK4a, it is possible to inhibit the production of senescent cells and improve health span [123]. Also, SASP triggers multiple intercellular communication paths that also promote aging. Finally, the elimination of senescent cells improved several age-associated conditions. Senescence in cancer has shown a dual role as tumor suppressor and tumor promoter. Senescence is a key mechanism of tumor suppression via the inhibition of proliferation of cancer cells or by stimulating immune surveillance. Yet, cells induced to senescence by oncogenes or chemotherapy exhibit stem-

**Fig. 3.34** Overview of cellular senescence processes. *ROS* reactive oxygen species, *ATM* ataxia-telangiectasia mutated, *ATR* ATM and Rad3-related protein, *Cdk2/4/6* cyclin-dependent kinase 2/4/6, *RB* reti-

noblastoma tumor suppressor gene, *SASP* senescence-associated secretory phenotype, *SA-β-gal* senescence-associated beta-galactosidase

like properties that promote cancer. Several stressors can induce cellular senescence and radiation in one of them. Thus, IR may cause cell cycle arrest resulting in a prematurely induced senescence phenotype (including SA-βgal, p16INK4a, p21, and SASP), which is p53 dependent [121]. Unfortunately, the accumulation of these senescent cells can have a negative impact by promoting tumorigenesis. Thus, eliminating senescent cells from tumors and surrounding healthy tissues may be a successful and benefcial adjuvant strategy (Box 3.21).

#### **Box 3.21 In a Nutshell: Telomeres and Senescence**

# **3.10 Cell Death Mechanisms**

In response to IR, multiple, molecularly distinct forms of cell death may be initiated. Although the decision points of their initiation are not completely clear, it is known that the level of the DNA damage but also the individual signaling status of different cell death pathways in different cell types, e.g., hematological vs. epithelial cells, infuence the decision regarding the cell death route.

The cellular factors that infuence include cell type, position in cell cycle when irradiated, DNA repair capacity, as well as functionality of TP53 and similar DNA-damaging sensors [124]. The dose and radiation quality also contribute to the cellular IR response to cell death, and in the tissue, the oxygen levels may impact the cell death route taken [124]. In this section, an overview of four cell death mechanisms are given: (I) mitotic cell death/mitotic catastrophe, (II) apoptosis, (III) necrosis, and (IV) autophagy (Fig. 3.35), some of which are also interconnected in the cell. Furthermore, the underlying molecular mechanisms and importance of these forms of cell death following IR are also described alongside methods of assessment.

# **3.10.1 Mitotic Cell Death/Mitotic Catastrophe**

Mitotic catastrophe (MC) is an important type of IR-induced cell death mechanism, which is triggered when cells enter into the mitotic phase without appropriately completing the S and G2 cell cycle phases [125]. Hence, MC controls cells that are often incapable of successfully completing mitosis. MC works by activating mitotic arrest, and later it may lead to a controlled or a regulated cell death mechanism or senescence. Therefore, MC is a controled cell death that usually follows the intrinsic apoptotic pathway route [124] (Fig. 3.37). MC is also promoted when the proteins that regulate the G2 phase like the p21CDKN1A, checkpoint kinases 1 and 2 (CHK1/2), ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia, and Rad3-related protein (ATR) are inhibited. MC basically commences with the irregular condensation of the chromatin around the nucleoli, which looks similar to early chromosome condensation. Cells may die in the same cell cycle or in the successive cell cycle progression or division after IR. The anomalous mitosis in such cases leads to unusual segregation of the chromosomes and cell division. As a consequence, this causes formation of giant cells which exhibit the uncharacteristic nuclear morphology and numerous micronuclei and nuclei. Also, it is noteworthy that MC induced by IR is accompanied with excess duplication of chromosomes and hyper-amplifcation, which results in a mitosis that is multipolar and later development of micronuclei. DNA damage and faws in the DNA repair processes lead to centrosome hyper-amplifcation. Cyclin-dependent kinase 2 (CDK2) and cyclin A or E initiate the amplifcation of the centrosomes at the boundary of G1/S phase. This is often observed in cells that lack a functional TP53; however, in cells with a functional TP53 and p21CDKN1A, which is known as an inhibitor of CDK2, cellular senescence is promoted.

The outcome of MC in the form of cell death can be elicited in the mitotic phase or in the successive interphase. Some cells activate apoptotic pathways in the metaphase that results in delayed apoptosis, i.e., it can take up to 6 days after IR. Cells that get away with the mitotic arrest of the mitotic

**Fig. 3.35** Overview of cell death and cell death-protective mechanisms in response to radiation. Radiation-induced cell death is infuenced by different factors, such as radiation factors, cell intrinsic

cell death are frequently observed to have an unsuccessful cytokinesis consequentially exhibiting tetraploid anomalous nuclei developing into giant cells. Giant cells that possess a functional TP53 will eventually undergo apoptosis following the mitochondrial pathway of apoptosis in the subsequent G1 phase. However, cells with mutant TP53 or defcient TP53 function go on with a few number of cell cycles and attain a growing amount of chromosomal anomalies before they fnally succumb to either delayed apoptosis or necrotic form of cell death [125]. As the cells that undergo MC are usually the ones who have lost the potential to carry out any further replication, MC is frequently referred to as a genuine type of cell death. One of the most common properties exhibited by cancer cells is that of defects in cell cycle checkpoints. This lets the cells enduring IR-induced damage to hastily inscribe in the mitotic process even with the misrepaired DNA that eventually leads to MC. More than a few cell division attempts can take place before adequate genetic injuries mount up to activate mitotic death, emphasizing why solid tumors frequently display deferred reactions to IR [124]. MC is triggered after IR exhibits diverse mechanisms of action (Table 3.10) [126].

factors, and cellular microenvironment factors (left). Cell death pathways are listed to the right. The mechanisms and importance of these principal cell death forms are described in detail in the text

**Table 3.10** Examples of IR-induced MC in different tumor cell lines

#### **<IP_ADDRESS> Mode of Action of Mitotic Catastrophe**

During MC, the mitotic damage is recognized and guides the cell into one of the three potential antiproliferative fates (Fig. 3.36).  Another cell death pathway that cells can take is by mitotic slippage. Here, cells go out from mitosis and cell death is triggered in the next G1 cell cycle transition. Lastly, cells with a MC character can also undergo senescence after exiting mitosis.

MC may not at all time be accompanied by mitotic arrest. Nevertheless, the mechanism of action that dictates cell fate of subsequent MC continues to remain unclear [127]. When

**Fig. 3.36** Cell death pathways operative in mitotic catastrophe. Different signaling events triggered in response to a nonfunctional mitosis are shown. Upon DNA damage, cells which lack functional p53

can go out from mitosis without commencing cytokines or initiate cell death even in mitosis. Apoptosis and necrosis signaling in the context of mitotic catastrophe are depicted

mitotic arrest is extended, the amount of cyclin B is decreased albeit the spindle assembly checkpoint (SAC) is functional. As a result, if cyclin B levels drop below the verge that determines mitotic exit, slippage occurs (Box 3.22).

#### **Box 3.22 In a Nutshell: Ionizing Radiation Induced Cell Death**

# **3.10.2 Apoptosis**

Apoptosis (originally from Greek language translated "falling off") is also known as "cellular suicide." It is a cell death process which may be executed under normal physiology, e.g., organism development, but also in the context of disease. Apoptosis is a highly controlled pathway with distinct molecular features. Thus, some of the rapidly proliferating cells undergo apoptosis, which is an essential part of neurogenesis and tissue development in humans as well as in other mammalians. During apoptosis, cells are disposed in a complex but well-ordered fashion which involves energy-requiring molecularly defned effector mechanisms [128]. To simplify, apoptosis allows the cells to self-destruct with limited tissue damage when they are exposed to different triggers/signals which can be endogenous, e.g., formed DNA damages, telomere shortening, or encountered from the outside of the cell, e.g., cytotoxic or DNA-damaging agents, IR exposure, loss of growth factors, cytokine or glucocorticoid hormone level alterations, or hypoxia [128].

**Fig. 3.37** The intrinsic and extrinsic route to apoptosis. Intrinsic stress signals (e.g., DNA damage, hypoxia, metabolic stress) or lethal stimuli (e.g., IR exposure) can induce intrinsic mitochondrial apoptosis (middle). Cleaved or truncated Bid (tBid) can also connect the extrinsic pathway to the intrinsic route. In the extrinsic pathway, ligands for death receptors (left) can trigger caspase activation, but the pathway can also be activated when some dependence receptors are inactivated (right). Abbreviations: *FasL* Fas ligand, *TRAIL* TNF-related apoptosisinducing ligand, *TNF* tumor necrosis factor, *Fas* Fas cell surface death receptor, *TRAILR* TNF-related apoptosis-inducing ligand receptor, *TNFR* tumor necrosis factor receptor, *TRADD* TNFR1-associated death

Apoptosis results in the production of apoptotic bodies, which are cell fragments, e.g., collapsed cytoskeleton, disassembled nuclear envelope, and fragments of nuclear DNA. An apoptotic cell is also marked by certain "fnd-me" and "eat-me" signals at the cell surface, which allow the dying cell to be recognized and rapidly engulfed by different macrophage subtypes in the near or distant tissue, thereby avoiding infammation. A well-recognized potential "eatme" signal is the expression of phosphatidylserine (PS) on the outer side of plasma membrane, which in turn is being used for assessing early apoptotic cells [129].

In the 1990s, studies which resulted in authors being awarded a Nobel Prize revealed that core machinery compo-

domain protein, *FADD* Fas-associated protein with death domain, *caspase* cysteine-aspartic proteases, *BID* BH3-interacting domain death agonist, *tBID* truncated BID, *Bcl-2* B-cell lymphoma 2 (an apoptotic inhibitor), *BCL2L1* Bcl-2-like 1, *MOMP* mitochondrial outer membrane permeabilization, *BH3* Bcl-2 homology 3, *DIABLO* direct inhibitor of apoptosis-binding protein with low pI, *APAF-1* apoptotic peptidase-activating factor 1, *Bax* Bcl2-associated X (an apoptotic regulator), *Bak* Bcl-2 homologous antagonist/killer, *XIAP* X-linked inhibitor of apoptosis protein, *SMAC* second mitochondria-derived activator of caspase, *UNC5B* Unc-5 netrin receptor B

nents of some apoptotic pathways are highly conserved from nematodes to humans [130]. Subsequently, research on the molecular mechanisms regulating apoptosis has established two major routes of this cell death type, namely intrinsic and extrinsic apoptosis, respectively (Fig. 3.37).

# **<IP_ADDRESS> Intrinsic Pathway to Apoptotic Execution**

Multiple perturbations may trigger intrinsic apoptotic cell death, e.g., growth factor withdrawal, cytokine alterations, endoplasmic reticulum stress, replication stress, formation of reactive oxygen species (ROS), microtubular alterations or mitotic defects, and IR-induced DNA damage. In the context of DNA damage, mitochondrial release of apoptogenic proteins is central. This commences in part via mitochondrial outer membrane permeabilization (MOMP) that allows cytochrome C and other proteins to be released to cytosol. Once there, cytochrome forms the apoptosome complex together with apoptotic peptidase-activating factor 1 (APAF-1), where pro-caspase-9 is cleaved to active caspase-9 (CASP9). Subsequently, CASP9 cleaves the effector caspases (e.g., caspase-3, caspase-6, and caspase-7), which then causes degradation of cell signaling and structural proteins resulting in an apoptotic morphology. The BCL-2 proteins are regulators of MOMP. These can either promote, e.g., BCL-2 associated X apoptosis regulator (BAX) or BCL-2 antagonist/ killer (BAK) or block MOMP, e.g., BCL-2 or BCL-XL members [131]. Another set of BCL-2 members, which only have a BH3 domain, can also promote MOMP, but they act via alleviation of BCL-2 or BCL-XL function or via promotion of BAX/BAK activity. Examples thereof are BCL2 associated agonist of cell death (BAD), BH3-interacting domain death agonist (BID), BCL2-interacting mediator of cell death (BIM), NOXA, and TP53-upregulated modulator of apoptosis (PUMA).

In DNA damage-induced apoptosis, TP53 and BAX/BAK proteins are important. The BCL-2 family members also sense other cellular clues to elicit intrinsic apoptosis including alterations in growth factor receptor/PI3K signaling or microtubule disruption, both of which may have impact in the context of IR-induced cell death. In addition, the mitogenactivated protein kinases 8 and 9 (MAPK8 and MAPK9), more commonly referred to as c-jun N-terminal kinase 1/2 (JNK1/JNK2), are known to regulate the BCL-2 rheostat by phosphorylation of BCL-2 and BAD, via induction of NOXA and PUMA by TP53 transcriptional regulation as well as by association of BIM to microtubuli [132].

# **<IP_ADDRESS> Extrinsic Pathway to Apoptotic Execution**

The extrinsic pathway starts by the activation of membrane receptors, so-called death receptors (DRs), e.g., FAS/CD95 cell surface death receptor and TNF receptor superfamily member 1A (TNFRSF1A)/TNFR1, and is driven by initiator caspases, e.g., caspase-8 (CASP8) and caspase-10 (CASP10). The extrinsic pathway is also used by various immune cells to trigger apoptotic cell death in tumor cells including TRAIL [133]. In addition, the infammatory cytokine TNF-α produced by activated macrophages, which binds to the TNFR1 and TNFR2 receptors in most human cells, can elicit apoptotic response. Moreover, cytotoxic lymphocytes carry the FasL, which binds and activates the FAS receptor on the surface of the target cell that is followed by death-inducing signaling complex (DISC) formation. Subsequently, adapter proteins bind to the intracellular region of aggregated DISC complex, causing the accumulation of procaspase-8 molecules, which via proteolytic cleavage initiate a proteolytic cascade leading to effector caspase activation. There is also an amplifcation step where further release of mitochondrialocalized pro-apoptotic factors takes place to amplify the initial CASP-3 activation (Box 3.23).

#### **Box 3.23 In a Nutshell: Apoptosis**

# **<IP_ADDRESS> Activation of Apoptosis by Ionizing Radiation**

IR-induced DNA damages, e.g., unrepaired DNA SSBs or DSBs, primarily trigger apoptosis via the intrinsic pathway [134]; however, at certain IR doses and in certain cell types, the extrinsic apoptotic pathway may also be executed. IR can also initiate mitochondria-mediated signaling in response to ceramide production/formation at the plasma membrane. Moreover, IR can trigger the production of O2 <sup>−</sup> and ROS (like H2O2 or OH− radicals), which via release of Ca2+ and cytochrome c from mitochondria can cause apoptosis [135].

One important signaling regulator of apoptosis in response to IR is TP53 [136] (Fig. 3.38). Thus, TP53 is phosphorylated in response to DDR signaling, accumulates in the nucleus, and binds to promoters of target genes, e.g., BAX, PUMA, NOXA, p53AIP1, and APAF-1. This results in an alteration in their transcription and hence expression levels, which is followed by mitochondria-mediated apoptosis.

The extrinsic pathway may also play a role in IR-induced apoptosis in which TP53 may upregulate the expression of the FAS receptor and its ligands, which subsequently causes downstream transactivation of initiator CASP8 and apoptosis.

IR may moreover activate the ceramide pathway at the plasma membrane, wherein formation of ROS inficts lipid

**Fig. 3.38** TP53-mediated intrinsic route to apoptosis. The mechanisms of TP53-induced apoptosis through the Bcl-2-regulated pathways in cells undergoing stress are shown. DNA damage triggers stress signaling, which in turn causes stabilization of the TP53 protein in the nucleus. Subsequently, TP53 as a nuclear transcription factor increases the expression of BH3-only proteins such as PUMA and NOXA and downregulation of BCL-2 or BCL-XL expression. The BH3-only proteins bind and inhibit the anti-apoptotic or pro-survival BCL-2 family proteins, so as to unleash the cell death effectors (BAX/BAK) which are often held as hallmarks of apoptosis in affected cells. Oligomerization of BAX/BAK causes MOMP, with subsequent release of cytochrome c,

oxidative damage in the membrane (Fig. 3.39). Subsequently, acid sphingomyelinase is activated, and second messenger ceramide is released as a result of sphingomyelin hydrolysis. IR-induced DNA damage may also trigger mitochondrial ceramide synthase resulting in the accumulation of ceramide which subsequently can induce apoptosis [137].

Ceramide may also activate the RAC1/mitogen-activated protein kinase kinase kinase-1 (MAP3K1) pathway by which MAPK8 and the effector CASP-1, -3, and -6 are induced and which also stimulate the DR pathway. MAPK8/JNK1 is known to be triggered in response to IR as well as other apoptotic stimuli, and depending on the duration of activity, it may induce apoptotic signaling. In summary, the rate of apoptotic events after IR may be executed via different routes

formation of the apoptosome complex, and activation of CASP9 and subsequently effector caspases, which causes apoptotic features of the dying cells. Abbreviations: *ROS* reactive oxygen species, *MOMP* mitochondrial outer membrane permeabilization, *BH3* Bcl-2 homology 3, *PUMA* p53 upregulated modulator of apoptosis, *BAD* Bcl-2-associated agonist of cell death, *CHOP* CCAAT/enhancer-binding protein homologous protein, *Bcl-2* B-cell lymphoma 2 (an apoptotic inhibitor), *Bcl-xL* B-cell lymphoma-extra-large, *Bax* Bcl2-associated X (an apoptotic regulator), *Bak* Bcl2 antagonist killer 1, *APAF-1* apoptotic peptidaseactivating factor 1, *caspase* cascade of aspartate-specifc cysteine proteases

and is infuenced by cell type, cell cycle phase, dosage number, as well as radiation quality (Box 3.24).

#### **Box 3.24 In a Nutshell: Ionizing Radiation Induced Apoptosis**

**Fig. 3.39** Overview of ceramide signaling and connection to the apoptotic machinery. IR-induced lipid oxidative damage causes sphingomyelinase activation at the plasma membrane, followed by hydrolysis of sphingomyelin and release of ceramide. High dose of IR-induced DNA DSBs can also trigger the mitochondrial ceramide synthase for *de novo* synthesis of ceramide. Inhibition of SERCA and calcium depletion in ER promote ER stress. Expression of downstream pro-apoptotic factor, e.g., CHOP, increases. The UPR activator proteins, ATF6, IRE1, and PERK, alter ER stress. The PERK pathway via ATF4-dependent NRF2 expression triggers the CHOP-mediated apoptotic pathway. CHOP can also be induced by spliced ATF-6 (in Golgi), which regulates the Bcl-2 protein family. CAPPs can alter the BCL-2 protein family, which deter-

# **<IP_ADDRESS> Methods to Detect Apoptotic Cell Death**

The apoptotic cell features, i.e., cell morphology, and the activation of different apoptotic signaling routes giving rise to distinguishable phenotypes have been extensively studied with multiple methods at hand. The detection of apoptosis includes methods (Fig. 3.40) related to membrane alterations, e.g., PS exposure monitored by annexin V association [129]; DNA fragmentation assessment; cytotoxicity and cell proliferation assays; analyses of mitochondrial effects, i.e., cell permeabilization; loss of mitochondrial potential; BCL-2 family protein complex formation; association of the apoptosome or DISC complex in cytosol; and pro-caspase cleavage later via different antibody-based, enzymatic assays or by fow cytometry [138]. Moreover, less frequently used technologies such as

mines the commitment of cells to apoptosis. Abbreviations: *Cer* ceramide, *CerS1–6* a family of six ceramide synthases, *SMase* sphingomyelinase, *SERCA* sarco-endoplasmic reticulum calcium transport ATPase, *ER* endoplasmic reticulum, *ATF6* activating transcription factor 6, *IRE1* inositol-requiring enzyme 1, *PERK* protein kinase R-like ER kinase, *NRF2* nuclear factor erythroid 2-related factor-2, *ATF4* activating transcription factor 4, *CHOP* CCAAT/enhancer-binding protein homologous protein, *Mt* mitochondria, *CAPPs* ceramide-activated protein phosphatase, *Bcl-2* B-cell lymphoma 2 (an apoptotic inhibitor), *Bcl-xL* B-cell lymphoma-extra-large, *Bax* Bcl-2-associated X (an apoptotic regulator), *RNS* reactive nitrogen species, *ATP* adenosine triphosphate

light-scattering fow cytometry and time-lapse microscopy perfusion platform can be performed to avoid underestimating the extent and timing of apoptosis, temporal aspects of death, cell surface area assessment, cellular adhesion analysis, and genotoxicity-specifc chromatin changes.

# **3.10.3 Necrosis**

Necrosis (from the Greek "nekros" designating "to kill") has for long been seen upon as an uncontrolled, irreversible mode of cell death, while recent work suggests that necrosis is a tightly genetically regulated pathway yet triggering infammatory and/or reparative reactions in the tissue [139]. Necrotic cell death can be classifed into accidental cell death

**Fig. 3.40** Methods to detect cell death, in particular apoptotic cell death. The schematic diagram outlines various biological assays used to determine apoptotic cell death. Some of these assays can also be used to assess other types of cell death. These assays are based on the morphological criteria and distinguishing features of apoptotic pathways, e.g., staining for PS exposure on the outer plasma membrane (by annexin V assay) and caspase-3 activation or PARP cleavage (by, e.g., western blotting). Cell viability assays such as membrane integrity assays and reproductive assays are performed to monitor live cells in culture and measure an enzymatic activity as a marker of viable cells by using different classes of colorimetric reagents and substrates generating a fuorescent signal. Results from these assays do not always indicate apoptosis, but more about cell death in general. DNA labeling assay, functional assays, and morphological mechanism-based assays detect and quantify the cellular events, some of which are specifcally associated with apoptotic cell death, such as formation of apoptotic antibodies, expression of apoptotic inhibitors, caspase activation in either intrinsic or extrinsic pathways, and DNA fragmentation. The principles for each assay are given in the respective yellow boxes. Abbreviations: *MTT* (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide), *LDH* lactate dehydrogenase, *BrdU* bromodeoxyuridine, *PARP* polyadenosine diphosphate-ribose polymerase, *PS* phosphatidylserine

(ACD) and regulated necrotic cell death (RNCD). RNCD can be further classifed into necroptosis, pyroptosis, ferroptosis, NETosis, and methuosis given their molecular routes [139] (Table 3.11).

# **<IP_ADDRESS> The Role of Necrosis in IR Cellular Responses**

Necroptosis, pyroptosis, methuosis, and ferroptosis are all triggered in response to IR [124, 140]. In the context of RT of cancer, necrosis can be induced either directly following DNA damage or indirectly by ROS formation that reacts with lipids generating lipid peroxides. IR has also been linked to lipid peroxidation and ferroptosis, and necroptosis together with ferroptosis was postulated to occur via ATM signaling.

Both ACD and RNCD trigger immunogenic cell death (ICD). In turn, ICD can stimulate an adaptive immune response after antigen is exposed by cells after RT or chemotherapeutics [141]. In case of immunogenic cell death, damage-associated molecular patterns (DAMPs) are delivered and identifed by pathogen recognition receptors (PRRs) exhibited by intrinsic components of the immune system, conducting to the stimulation of an immune response [141]. ACD is an uncontrolled type of cell death which is activated by, e.g., physical damage, hypoxia, infammatory toxins, and high doses of IR. The cells respond by morphological alterations, such as cytoplasmic swelling of the cell organelles, i.e., oncosis [142], which is a result of disturbance of ionic pumps causing Ca+ infux, plasma membrane disruption followed by the leakage of intracellular organelles with accidental deteriorated DNA, and absence of clear chromatin condensation [142]. RNCD comprises upregulation of diverse pro-infammatory proteins and molecules such as nuclear factor-κB, leading to the rupture of the cell membrane causing leakage of the cellular debris, e.g., ATP, DNA, nuclear proteins, heat-shock proteins, and uric acid, into surrounding zones, provoking a cascade of infammation and tissue injury. Thus, the release of proteins/molecules promotes infammasome activation and production of proinfammatory cytokine interleukin-1 beta (IL1). The methods used to detect necrosis are lactate dehydrogenase (LDH) activity measurement and cell-impermeable DNA-binding dye. These techniques are based on the morphological characteristics proving the cellular release and membrane porosity (Table 3.12).

**Table 3.12** Examples of some oncogenes in cancer from Weinberg [143] and Gillies et al. [144]

# **<IP_ADDRESS> Necroptosis/Regulated Necrosis**

Necroptosis, also known as a regulated necrosis, which works in a caspase-independent fashion, exhibits a necrotic morphology with membrane disruption and leakage of organelles (reviewed by Weinlich et al. (2017)). Different stimuli can elicit necroptosis: DRs, e.g., members of the TNFR superfamily, pattern recognition receptors (PRRs), Toll-like receptors (TLRs), T-cell receptors (TCRs), multiple chemotherapeutic drugs, and hypoxia. The process of necroptosis commences by the stimulation of receptorinteracting protein kinases (RIPKs) (Fig. 3.41).

RIPKs are stimulated to go into macromolecular complexes from the membrane receptors with the necrosome with RIPK1 and RIPK3 being the main components. RIPK3 subsequently stimulates mixed-lineage kinase domain-like protein (MLKL) through phosphorylation causing its oligomerization and relocalization, resulting in cell membrane permeabilization and subsequent cell death.

Different techniques can be used to identify necroptosis, e.g., fow cytometry, western blotting, and immunohistochemistry. Through these techniques, the expression levels of MLKL, RIPK3, and RIPK1 are evaluated as well as cell by electron microscopy (Table 3.11).

# **<IP_ADDRESS> Pyroptosis and Ferroptosis: Triggers and Molecular Mechanisms**

Pyroptosis, which is stimulated by IR as well as intracellular pathogenic factors in immune cells, follows a series of caspase-dependent events and is pro-infammatory (reviewed by Yu et al. [146]). Thus, the NOD-like receptors (NLRs) of irradiated/infected macrophages/monocytes recognize cytoplasmic pathogen-associated molecular patterns (PAMPs) as well as DAMPs and trigger infammasome complex production, which activates CASP1. CASP1 in turn activates gasdermin D, which mediates the plasma membrane rupture (Fig. 3.42) as well as the infammatory cytokines interleukin 1β (IL-1β) and IL-18, which further regulate infammation. Pyroptosis also involves cell swelling followed by disintegration of the plasma membrane and leakage of the proinfammatory contents, e.g., DAMPs, IL-1β, and IL-18, contributing to elimination of the immunologic challenges locally or systemically. Pyroptosis can be detected by LDH assay, fuorescence microscopy, western blot analysis (for identifcation of gasdermin D, IL-1β), and measurement of the cell intake of propidium iodide (Table 3.11).

Ferroptosis is a form of caspase-independent regulated necrosis and is distinguished by excessive iron-dependent lipid peroxidation. It presents a necrotic morphology with altered mitochondria, i.e., small mitochondria, fewer cristae, rupture of outer membrane, and an electron-dense ultrastructure. Execution of ferroptosis is decided by the equilibrium between ROS production due to iron increase and antioxidant protection mechanisms that impede lipid peroxidation.

**Fig. 3.41** Summary of regulated necrotic cell death. (**a**) Necroptosis elicited by DR, TLR, and viruses stimulates RIPK3 and then MLKL, which is required for membrane disruption. (**b**) Pyroptosis induced by GSDMD following its cleavage by CASP1 and CASP11. The main elicitors: PAMPs and DAMPs, or cytosolic LPS. (**c**) Ferroptosis is dependent on the balance between ROS production due to iron accumulation and antioxidant defense mechanisms that inhibit lipid peroxidation. The ACSL4–LPCAT3–ALOX15 pathway mediates lipid peroxidation, while system xc- (comprising SLC7A11, GPX4, and NFE2L2) impeded this process. (**d**) NETosis is triggered by NET leakage, which is mediated by ROS generation and histone citrullination. (**e**) Methuosis is associated with macropinocytosis. Nascent micropinosomes fused forming large vacuoles that contain late endosomal markers (LAMP1 and Rab7). These do not recycle or unify with lysosomes

Thus, ferroptosis is activated after lipid peroxidation in a process catalyzed by iron, either in a Fenton-like manner or through lipoxygenases (Fig. 3.41). Accordingly, the oxidation of polyunsaturated fatty acids (PUFAs), like arachidonic acid (AA), is necessary for lipotoxicity in ferroptosis, which takes place via a catalytic pathway comprising acyl-CoA synthetase long-chain family member 4 (ACSL4), lysophosphatidylcholine acyltransferase 3 (LPCAT3), and arachidonate lipoxygenases (ALOXs, specifcally ALOX15) [147]. In

causing cell death. Reproduced with permission (CCBY) from Tang et al. [145]. *DR* death receptor, *TLR* Toll-like receptor, *RIPK3* receptorinteracting protein kinases 3, *MLKL* mixed-lineage kinase domain-like protein, *GSDMD* gasdermin D, *CASP1* caspase 1, *CASP11* caspase 11, *PAMPs* pathogen-associated molecular patterns, *DAMPs* damageassociated molecular patterns, or cytosolic, *LPS* lipopolysaccharide, *ACSL4* acyl-CoA synthetase long-chain family member 4, *LPCAT3* lysophosphatidylcholine acyltransferase 3, *ALOX15* arachidonate lipoxygenases (ALOXs, specifcally ALOX15), *SLC7A11* the catalytic subunit solute carrier family 7 member 11, *GPX4* glutathione peroxidase 4, *NFE2L2* nuclear factor erythroid 2-like 2, *NET* NETosis extracellular trap, *ROS* reactive oxygen species, *LAMP1* lysosomal associated membrane protein 1, *Rab7* lysosomal Rab protein 7. (Adapted from Tang et al. [145])

addition, lipid peroxidation can be hindered by the various antioxidant systems such as the cystine/glutamate antiporter system, which consists of the catalytic subunit solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and pro-survival proteins, like nuclear factor erythroid 2-like 2 (NFE2L2). System xc- facilitates the exchange of cystine and glutamate in and out of the cell. The cystine which is taken up is reduced to cysteine in cells, which is needed for the synthesis of glutathione GSH. GSH is used by

**Fig. 3.42** Methodology for 2D (Puck) and 3D clonogenic curves. The clonogenic assay measures the ability of single cells to form colonies. A cancer cell that is not able to form a colony can be regarded as inactivated. Cellular monolayers are dissociated into single cells and counted and diluted to the required concentration, depending on the dose. The cells are then seeded in cell fasks/dishes for colony forma-

tion or in a 3D matrix for spheroid formation. After irradiation, the cells are incubated for 1–3 weeks depending on the cell doubling time of that particular cell line, before they are fxed, stained, and counted. The surviving fraction is calculated as the number of colonies in irradiated samples relative to the plating effciency of unirradiated control dishes

GPX4 to stop the generation of phospholipid hydroperoxides (PLOOH), the key mediator of chain reactions in lipoxygenases. The induction of ferroptosis can be determined by measuring lipid peroxides coupled with fow cytometry (Table 3.11).

# **<IP_ADDRESS> Neutrophil Extracellular Trap-Associated Cell Death (NETosis) and Methuosis**

NETosis is stimulated by various pathogens or other stimuli, which release neutrophil extracellular traps of mainly DNAprotein structures [148] in a process dependent on NADPH oxidase 4 (NOX4), the principal source of ROS (Fig. 3.41). NETosis also comes along with important increase of ROS conducting to the stimulation of protein-arginine deiminase 4 (PAD4). Then, PAD4 citrullinates (converts arginine to citrulline via deamination) the histones, promoting the nuclear chromatin decondensation. Further, the NET is released into the cytosol leading to the disruption of the neutrophil membrane. Then, neutrophil breaks up and the NETs are released into the environment. NETs can be generated by other forms of immune cells, e.g., eosinophils, mast cells, basophils, macrophages, and also epithelial cells and cancer cells as a response to various injuries [145]. NETosis can be studied using various techniques: immunofuorescence, transmission electron microscopy, scanning electron microscopy, ELISA tests, fow cytometry, as well as western blot analyses of NETosis markers (Table 3.11).

Methuosis (from Greek methuo—"drink to intoxifcation") is another type of caspase-independent regulated necrotic cell death that is induced by exposure to heat, trauma, and infection and which lead to cell swelling, lysis of plasma membrane, as well as infammation. Methuosis is correlated to macropinocytosis (referred to as "cell drinking") and is associated with the extensive accumulation of fuidflled cytoplasmic vacuoles stemmed from macropinosomes, which for example is observed in cancer cells driven by the oncoprotein Ras [149] (Fig. 3.41). Methuosis can be detected by electron microscopy, time-lapse fuorescence microscopy, visualization of vacuoles using fuorescent dyes, and metabolic fux analyses (Table 3.11) (Box 3.25).

#### **Box 3.25 In a Nutshell: Necrosis**

# **3.10.4 Autophagy**