The response of the key immune cells involved in inflammation is strongly dependent on the basal inflammatory status of these cells and the systemic inflammatory (micro)-environment. Further, the monocytic cells are central in all phases of the inflammatory process from initiation to termination and are characterized by an initial high plasticity that is weakened by prolonged tissue residency. Their phenotype is strongly influenced by the microenvironment, and radiation responses are therefore manifold and dose dependent [168]. Regarding inflammatory cytokine expression by macrophages, particularly TNF-alpha and IL1-beta, secretion is reduced following a single radiation exposure of 0.3–0.7 Gy without affecting the immune cell’s viability. Further, decreased expression of the inducible nitric oxide synthase (iNOS) protein and, as a consequence, nitric oxide (NO) production in inflammatory macrophages after radiation exposure are observed in inflamed joints. Radiation exposure causes stress in cells via the production of reactive oxygen species (ROS), and a dose of 0.5 Gy, being routinely applied for low-dose radiotherapy of benign chronic inflammatory and destructive diseases, resulted in the strongest reduction of ROS by activated endothelial cells. Besides affecting immune cells and endothelial cells, low/intermediate-dose radiation exposure has osteoimmunological modes of action by reducing the activation of bone-resorbing osteoclasts and by fostering bone construction by osteoblasts [169]. Epidemiological, clinical, and experimental data regarding the effects of low-dose radiation on the homeostasis and functional integrity of immune cells was just recently comprehensively summarized [170]. Finally, particularly in the interactions of radiation with immune cells and cells of the inflammatory process, nonlinear dose relationships are prominent and may reflect a nonlinearity and complexity of immune responses. Figure 3.47 summarizes the key immune cells that are involved in inflammation and are modulated together with the endothelium by radiation in a dose range of 0.1–1.0 Gy. Finally, in polymorphonuclear leukocytes (PMN), irradiation with doses between 0.5 and 1.0 Gy resulted in a discontinuous reduction of chemokine CCL20 secretion that parallels a hampered PMN adhesion to endothelial cells [171].

A diagram depicts the adverse effects of low-dose irradiation of 0.1 to 1.0 gray units on macrophages, lymphocytes, polymorphonuclear cells, and endothelial cells.

Fig. 3.47
Radiation affects key cells involved in initiation and maintenance of inflammation


A diagram depicts the adverse effects of low-dose irradiation of 0.1 to 1.0 gray units on macrophages, lymphocytes, polymorphonuclear cells, and endothelial cells.


Ionizing radiation causes phenotypic changes in endothelial cells, resulting in endothelial activation and ultimately endothelial dysfunction [172]. In vitro, this activation triggers an increase in the expression of the adhesion molecules vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), platelet endothelial cell adhesion molecule (PECAM-1), and E- and P-selectins involved in the recruitment of circulating lymphocytes. In vivo, increased expression of endothelial ICAM-1 and VCAM-1 was demonstrated in a model of radiation-induced intestinal inflammation. ICAM-1 knockout mice showed less severe pulmonary and intestinal inflammation than wild-type mice, suggesting that cellular infiltration may be deleterious in this situation. In humans, the endothelium may be activated by RT via the transcription factor nuclear factor kappa B (NF-kB) pathway, which is likely critical in the development of RT-induced cardiovascular diseases [173]. Overall, these studies demonstrated that radiation-induced increase in adhesion molecule expression by endothelial cells plays a crucial role in circulating cell recruitment and radiation-induced inflammation of the tissue and/or tumor, with a potential deleterious effect on normal tissues. Therefore, the vascular endothelium can be considered as a main control point of radiation-induced inflammatory and immune processes in normal tissues and tumors and may thus cover an ideal target to improve the therapeutic efficacy of radiotherapy of malign diseases. Furthermore, low-dose irradiation was demonstrated to result in a nonlinear expression and activity of major compounds of the antioxidative system in endothelial cells. This might contribute to anti-inflammatory effects in these stimulated cells and be beneficial in low-dose radiotherapy for benign diseases [174]. The effects of higher doses on the immune system in healthy tissue and tumors differ from those of low and intermediate doses and are covered in Chap. 4 (Box 3.30).


Inflammation is intrinsincally linked to the immune response.

Monocytes/macrophages are key immune cells in the initiation and resolution of inflammation.

Radiation in a dose range of 0.1–1.0 Gy ameliorates inflammation by mainly affecting macrophages, PMN, lymphocytes, and endothelial cells.

Ionizing radiation causes several phenotypic changes in endothelial cells

Vascular endothelium can be considered as a main control point of radiation-induced inflammatory and immune processes.


Monocytes/macrophages are key immune cells in the initiation and resolution of inflammation.


Radiation in a dose range of 0.1–1.0 Gy ameliorates inflammation by mainly affecting macrophages, PMN, lymphocytes, and endothelial cells.


Vascular endothelium can be considered as a main control point of radiation-induced inflammatory and immune processes.


Clustered regularly interspaced palindromic repeats (CRISPR)-CAS (CRISPR-associated protein) system is a defense mechanism that has been identified in prokaryotes that effectively acts to fight viruses. The five homologous sequences of 29 nucleotides separated by spacers of 32 nucleotides were observed initially in 1987 by a Japanese research group. The group identified a gene responsible for the conversion of alkaline phosphatase isozyme in Escherichia coli [175]. In 2002, another grouping of genes adjacent to the CRISPR locus was revealed which was termed CRISPR-associated system, or Cas. The system has been found in diverse species of bacteria and archaea, however with slightly different composition and mechanism of action. Since this time, new forms of CRISPR systems have been discovered that can be classified into six types and grouped into two classes [176]. Types I–III are well studied, while other types IV–VI, which have more recently been discovered, need further research to fully understand their mechanism of action. These systems have now been realized to be important breakthroughs for modern genetic engineering and are revolutionizing science.


CRISPR are fragments of RNA that are cloned from the DNA of viruses that have infected a bacterium. Together with other sequences, it forms an adaptive immune system that stores memory of viral DNA within the bacterial host chromosomes. It is comprised of three main components: an RNA sequence made from the relevant CRISPR gene (crRNA) that contains within it a 20-base pair-long sequence complementary to the target DNA sequence; a DNA endonuclease that can edit genes and is referred to as Cas9; and a tracrRNA that acts to help bind the crRNA and Cas9 together. All three components are well studied [177]. In concert, the CRISPR-Cas9 system works to fight virus invasion in prokaryotes. When a bacterium comes across a virus that it was previously exposed to, it produces an RNA copy of the CRISPR that contains that virus’ genetic information. The crRNA then binds with the tracrRNA to form a single-guide RNA (sgRNA) that leads the enzyme Cas9 to the correct DNA sequence. The sgRNA binds to the target site in the genome that matches the viral sequence on the crRNA and directs the Cas9 protein to create a double-stranded break. Next to the viral sequence is a protospacer adjacent motif (PAM), which also helps to align the enzyme. Once broken, the strand will experience a change in the viral DNA sequence through the activation of a DNA repair method, either nonhomologous end joining or homology-directed repair [177, 178]. The process shown in Fig. 3.48 is very efficient and effective and shown to be a valuable tool for researchers to study gene function and uncover biological mechanisms.

A flow diagram depicts that t r a c r R N A and c r R N A form s g R N A. The s g R N A and Cas 9 form the C R I S P R Cas 9 forward slash s g R N A complex, which binds to D N A and causes D S B, or double-stranded break.

Fig. 3.48
Mechanism of CRISPR-Cas9 to produce a DNA double-strand break. The CRISPR-Cas9/single-guide RNA (sgRNA) complex consists of the Cas9 protein, which is coupled to the sgRNA, consisting of the transactivating crRNA (tracrRNA), responsible for binding of the RNA complex to Cas9 and the CRISPR RNA (crRNA) which encodes the target sequence. The CRISPR-Cas9/sgRNA complex binds to the specifically targeted DNA sequence and induces a DSB. (Adapted with permission (CCBY) from Zhao et al. [178])


A flow diagram depicts that t r a c r R N A and c r R N A form s g R N A. The s g R N A and Cas 9 form the C R I S P R Cas 9 forward slash s g R N A complex, which binds to D N A and causes D S B, or double-stranded break.


The CRISPR-Cas9 system is unique due to its ability to induce double-strand breaks in almost any type of organism or cell type. The system is more accurate, providing an alternative to previous genome editing tools, such as zinc finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs) [178]. The technology is an efficient genome editing system that can detect, manipulate, and annotate from diverse species-specific DNA sequences. The system is mainly used for studying DNA because manipulating RNA is difficult due to the lack of a PAM sequence, requiring efficient RNA targeting tools. The most widespread application of the CRISPR-CAS system has been in the context of genome editing of DNA, achieved through three mechanisms: (1) nonhomologous end joining, (2) single-base editing enzymes, and (3) homology-directed repair for DNA repair. The system can be delivered virally (adenovirus or lentivirus) or through nonviral mechanisms (hydrodynamic injection, electroporation, nanoparticles, and transposon carriers) and combined [178].


The technology can be applied to develop a better understanding of a specific gene function or the manipulation of genetic material, as genetic sequences can be removed or edited. For example, a select tissue type can undergo multiplex mutagenesis for high-throughput analysis to identify cancer drivers or correction of a loss-of-function mutation; likewise, gene knockout could be used to enhance a specific cell type. Beyond gene editing, researchers have also used the Cas9 unit for targeting purposes instead of catalytically, known as the dead Cas9 (dCas9) [179]. For instance, epigenetic editing involves the alteration of the chromatin structure without modifying the individual’s genomic sequence. The dCas9 is fused to a functional DNA methylation or demethylation enzymes or DNA modifiers [179]. The same idea follows CRISPRi and CRISPRa, which repress and inhibit gene expression. The CRISPRi uses the dCas9 to bind to the DNA-blocking RNA polymerase and transcription factor binding, while CRISPRa combines the dCas9 unit and selects transcription factors targeting activating sequences.


Overall, these advancements provide new avenues to study genetic mechanisms and demonstrate the applicational value of CRISPR-Cas-based tools. It is being used with success in the field of agriculture, therapeutics, food industries, and more. The success of CRISPR has inspired efforts to discover new systems for targeting nucleic acids, including those from Cas9, Cas12, and Cas13 orthologues. The approach is gaining traction for use across multiple fields of research.


As the field of CRISPR-Cas rapidly evolves, challenges have emerged which have also been the focus of much research. This is particularly in the context of development of treatment modalities for cancer. Some hurdles that have been identified are in relation to methods for effectively delivering the technology into the host that ensures suppression of the innate immune responses. Injection methods are traditionally used to deliver CRISPR-Cas9 components to cells via delivery vectors; however, the efficiencies of these injection methods are dependent on the target cells and tissues. Traditional delivery methods targeting cancer cells are not yet efficient enough to be applied clinically. For CRISPR-Cas9 to be applied as a therapeutic tool in cancer treatment, delivery must be more efficient and accurate which may require novel delivery methods [180].


Apart from limitations with delivery methods, the delivery vehicle itself also prevents a challenge, as delivery vectors hold a limited amount of genomic material. The most used delivery vehicle is adeno-associated virus (AAV) as it is relatively safe and effective; however, this method has a limited packaging capacity due to its size, which restricts the amount of genetic information that can be transferred to the target cell or tissue. AAVs can contain roughly 5 kB of information, while information for the Cas9 protein and the sgRNA which must be included on the plasmid is roughly 4.2 kB in size. To offset this, current research is being done to find smaller Cas9 orthologues, which in the future may allow for more helpful elements to be added such as reporter genes or fluorescent tags to support more successful gene editing [179].


Immune responses to the Cas9 protein have also been well documented in animal models, which presents an added challenge to the clinical application of the CRISPR-Cas system. A high prevalence of the human population has been exposed to the bacteria from which the Cas9 protein originates, meaning that there is likely a large population with preexisting immunity. While the implications of this are not yet entirely clear, testing of Cas9 orthologues may be required before CRISPR-Cas technology can be applied as a therapeutic to prevent T-cell responses. Alternatively, immunosuppressant drugs could potentially be used during treatment [179]. Off-target effects of the CRISPR-Cas system, such as mutations at undesired sites, also present a challenge. Extensive research has been done to minimize these effects; however, further investigation on increasing precision is required to improve safety [181]. As these hurdles become addressed, CRISPR-Cas9 will play a crucial role in medical treatments, including the treatment of cancers, and will effectively support gene therapy modalities (Box 3.31).


CRISPR-Cas system is a defense mechanism that has been identified in prokaryotes that effectively acts to fight viruses.

CRISPR are fragments of RNA that are cloned from the DNA of viruses that have infected a bacteria.

Cas9 is a DNA endonuclease that can edit genes.

Together, CRISPR-Cas9 is an efficient genome editing system that can detect, manipulate, and annotate from diverse species-specific DNA sequences.

It can be applied to develop a better understanding of a specific gene function or the manipulation of genetic material, as genetic sequences can be removed or edited.


CRISPR-Cas system is a defense mechanism that has been identified in prokaryotes that effectively acts to fight viruses.


CRISPR are fragments of RNA that are cloned from the DNA of viruses that have infected a bacteria.


Together, CRISPR-Cas9 is an efficient genome editing system that can detect, manipulate, and annotate from diverse species-specific DNA sequences.


It can be applied to develop a better understanding of a specific gene function or the manipulation of genetic material, as genetic sequences can be removed or edited.


DNA methylation, histone modifications, and incorporation of histone variants are chemical alterations of the cellular DNA. Such changes are not necessarily permanent and can be influenced by endogenous and exogenous stressors. One of these stressors is radiation. Radiation induces various alterations in these epigenetic modifications, mainly affecting gene expression and DNA repair.


MicroRNAs are small, highly conserved noncoding RNA molecules that regulate gene expression. They are single-stranded RNA transcripts with a length of 21–25 nucleotides that are derived from hairpin loop precursors. miRNAs affect the cellular radiation response via regulation of vital genes involved in DNA damage repair, cell cycle checkpoints, autophagy, and apoptosis.


Long noncoding RNAs (lncRNAs) are defined as RNA transcripts with a length of more than 200 nucleotides missing a distinct protein-coding region. lncRNAs regulate gene expression on multiple levels, including transcription, RNA stability, and translation. Radiation exposure deregulates lncRNA expression, which affects radiosensitivity by interfering with canonical radiation response pathways, such as cell cycle control, DNA repair, and cell death induction.


Circular RNAs (circRNAs) are a recently described class of RNA molecules that are derived from precursor mRNA (pre-mRNA) in a process called backsplicing. Despite increasing attention, the number of studies investigating the direct effect of ionizing radiation on circRNA expression is still very limited. However, it is now evident that circRNAs are affected by irradiation and that they are important players in the cellular radiation response and sensitivity.


Extracellular vesicles (EVs) are generated by all cells within our body and are important communicators in normal and cancer cells. EVs have different sizes ranging from 40 nm up to several μm and are produced via different biogenesis routes. EVs’ cargo contains various RNA species, DNA fragments, proteins, and lipids partly reflecting their cell of origin. EVs may influence neighboring cells via their cargo but also act in distant tissue as illustrated in cancer, where they play a role in both carcinogenesis and metastasis. Exosomes are a particular type of EVs formed by viable cells via the endosomal system, and there are specific cellular mechanisms that determine their cargo. Upon radiation, EVs are generated by both normal and cancer cells and transmit effects in irradiated and nonirradiated cells (e.g., bystander or non-targeted effects). EVs may constitute a source of biomarkers for diseases and stress conditions, including radiation.


Modifications of DNA bases and histone proteins, including the incorporation of histone variants, have important functions in the epigenetic control of gene expression. Both types of alterations add further information to the DNA molecule in addition to the genetic code, which contribute to phenotypic changes without altering the DNA sequence. Importantly, such changes are not necessarily permanent and can be influenced by endogenous and exogenous stressors. Enzymes that add, recognize, and dislodge DNA and histone modifications are called writers, erasers, and readers. The generation of modifications is facilitated by writers. Erasers modify and/or remove labels. Readers recognize and associate to modifications [182].


The methylation of DNA is a heritable epigenetic label in dividing cells. Methylation of DNA segments typically induces its silencing, while demethylation is characteristic for actively transcribed regions. Possible mechanisms for these effects are the binding of methyl-DNA-binding proteins, which affect gene activity or alterations of the chromatin structure. In mammals, DNA methylation patterns are retained or established by DNA methyltransferases (DNMTs) that catalyze the addition of methyl groups to nucleotides. S-Adenyl methionine (SAM) acts as a methyl group donor. DNMT1 function is the maintenance of methylation, and DNMT3a/b is responsible for de novo methylation. On the other hand, DNA demethylases can catalyze active demethylation. Most of DNA methylation takes place at cytosines, which are succeeded by a guanine nucleotide (CpG sites). Regions (>200 nucleotides and CG content >50%) with a high frequency of CpG sites are called CpG islands. Promotor sequences are often located within such CpG islands. Methylation of CpG islands silences gene expression, for example by impeding the binding of transcription factors or by recruitment of repressive methyl-binding proteins. The most common modification of DNA bases is the methylation of cytosine on carbon position 5, leading to 5-methylcytosine (5-mC). This modification accounts for approximately 1% of all bases and is therefore sometimes designated as the fifth base of the DNA. Further less abundant modifications are for example 5-hydroxymethylcytosine, 5-carboxycytosine, and 6-methyladenine.


Histone modifications are covalent modifications joined to histone proteins. These modifications impair DNA-histone interactions, thereby changing chromatin architecture and gene expression. Some reduce DNA-histone interactions leading to nucleosome unwinding, chromatin opening, and increased accessibility for the transcription machinery leading to the activation of gene expression (euchromatin). Others increase DNA-histone interactions, leading to tightly packed chromatin followed by reduced access of the transcription machinery and thus gene silencing (heterochromatin). Currently, acetylation, methylation, phosphorylation, and ubiquitination are the most well understood, while others, like GlcNAcylation, citrullination, crotonylation, and isomerization, are more recent discoveries. All of these modifications are highly dynamic and added to or removed from histone amino acid residues by specific sets of enzymes. A well-described posttranslational histone modification is the trimethylation of histone H3 on the lysine located at position 4 of the protruding N-terminal tail (H3K4me3), which is correlated with promoters of actively transcribed genes. In contrast, the trimethylation of lysine on positions 9 (H3K9me3) and 27 (H3K27me3) is a heterochromatin mark, associated with repressed genes.


In addition to posttranslational modifications, the histone structure of the chromatin can also be influenced by the incorporation of histone variants. Histone variants are low abundant and differ only in one or a few amino acids with their canonical counterparts. They are produced throughout the cell cycle and can be deposited into chromatin independent of replication by rapid exchange processes. Histone variants seem to be especially important for protecting genome integrity by the regulation of damaged chromatin accessibility and restoration.


Both DNA methylation and histone modifications are essential components in the cellular stress response. Therefore, it is not surprising that various alterations are reported after radiation exposure. On a molecular level, radiation-induced alterations in histone and DNA modifications either are required for the efficient detection and repair of DNA damage to avoid chromosomal instability or lead to changes in transcriptional activity and thereby alter a variety of cellular processes, including cell cycle regulation, DNA repair, and cell death induction. At organism level, epigenomic alterations were reported in various radiation-induced cancer and non-cancer diseases. As epigenomic alterations can be transferred to the offspring also, the contribution to radiation-induced transgenerational effects was recently suggested [183, 184].


Altered DNA methylation patterns were found in in vitro and in vivo studies in response to irradiation. The majority of studies showed global hypomethylation, which was often linked with a reduced expression of enzymes involved in DNA methylation. As global hypomethylation is connected to malignant transformations, radiation-impaired DNA methylation may contribute to cancer development. However, hypomethylation is not always evenly dispersed across the genome and also radiation-induced hypermethylation was reported for specific loci. Interestingly, new studies imply that low- and high-LET radiations affect methylation differentially.


A variety of radiation-induced histone modifications affecting transcriptional regulation and particularly DNA repair are described. In regard to DNA repair, an intensively studied histone modification is the phosphorylation of the histone H2A variant H2AX at serin-139 (phosphorylated H2AX is designated as γ-H2AX) at sites of DNA double-strand breaks (DSBs). Formation of γ-H2AX is an initial response after exposure and facilitates a cascade of further histone modifications, including ubiquitination of H2A/H2B as well as changes in the acetylation of H3 and H4. Together, these posttranslational histone modifications contribute to chromatin relaxation that enables the accession of DNA repair factors and influences the repair pathway choice. Moreover, the γ-H2AX modification is widely used as a biomarker for DSBs, and a lot of methods were developed to use γ-H2AX counting for DSB quantification.


With the knowledge about DNA methylation and histone modification in radiation response, the targeted modulation of these features is investigated as a novel strategy to radiosensitize tumor cells during radiotherapy. For example, radiosensitizing activity was shown for DNA demethylation agents, like cytidine analogs. In addition, small-molecule inhibitors of histone deacetylases changing histone acetylation showed the potential to alter radiosensitivity.


The first studies in this field also demonstrated an exchange of histone variants in response to radiation exposure. For example, it was shown that the histone variant H2A-Z.2 is incorporated into chromatin immediately after DSB induction, where it contributes to recombinational repair by assisting RAD51 foci formation. In line, H2A-Z.2 U2OS tumor cells were shown to be more radiosensitive than controls. H2A.J, another histone variant, accumulates during radiation-triggered senescence processes in the vicinity of 53BP1 foci and affects the expression of inflammatory genes (Box 3.32).


DNA methylation, histone modifications, and incorporation of histone variants are chemical alterations of the cellular DNA.

Radiation induces various alterations in these epigenetic modifications, mainly affecting gene expression and DNA repair.

Phosphorylation of histone H2AX (γ-H2AX) is the most prominent radiation-induced epigenetic alteration with significant impact on DNA repair.


DNA methylation, histone modifications, and incorporation of histone variants are chemical alterations of the cellular DNA.


Radiation induces various alterations in these epigenetic modifications, mainly affecting gene expression and DNA repair.


Phosphorylation of histone H2AX (γ-H2AX) is the most prominent radiation-induced epigenetic alteration with significant impact on DNA repair.


MicroRNAs are small, highly conserved noncoding RNA molecules that regulate gene expression. They are single-stranded RNA transcripts with a length of 21–25 nucleotides that are derived from hairpin loop precursors. The basic mode of action of miRNAs is competitive partial binding with the 3′ UTR of the target mRNA, which inhibits translation and/or leads to mRNA destruction. MiRNAs have also been shown to interact with the 5′ UTR, coding regions, and gene promoters via binding complementary sequences [185]. Because each miRNA can act on multiple different target genes, and one target gene can be regulated by many different miRNAs, the miRNA-mediated regulation of cellular phenotype is highly complex. miRNA-mediated regulation is thought to affect roughly 60% of all protein-coding genes, according to estimations. To regulate miRNA abundance at the levels of transcription, maturation, and stability, cells have evolved various sophisticated methods to govern such extensive miRNA-mediated functions. miRNA actions have been linked to the regulation of a variety of cellular processes, including cellular homeostasis and stress responses. Furthermore, they have been linked to a variety of diseases. miRNAs, in addition to their intracellular roles, are also found in the extracellular environment. miRNAs can be identified in physiological fluids such as plasma, saliva, and urine. This extracellular miRNA population is varied and heterogeneous. Although the activities of extracellular miRNAs are not completely understood, it has been demonstrated that extracellular microvesicle-embedded miRNAs can be transferred and incorporated into destination cells [186].


Ionizing radiation (IR) disturbs cellular equilibrium in a variety of ways. Cellular stress pathways shield cells from the harmful consequences of genotoxic assault. Cells respond to ionizing radiation-induced stress by activating several pathways ranging from DNA damage processing, signal transmission, altered gene expression, cell cycle arrest, genomic instability, and cell death. The available evidence implies that radiation exposure causes cellular responses that are influenced in part by gene expression networks. miRNAs govern several intracellular processes involved in the response to cellular stress and have been demonstrated to regulate gene expression [187].


Radiation exposure, whether accidental or intentional, is a serious public health issue that demands immediate attention for correct diagnosis and clinical planning. Exposure to large doses of ionizing radiation in a short time causes acute radiation syndrome (ARS), often known as radiation sickness or radiation poisoning. ARS involves a total dose of over 0.7 Gy (70 rad) from an external source, administered in a few minutes. Radiation sources might be accidental or deliberate. Several animal species were used to study the effects of radiation on miRNA expression. For ARS, miRNA analysis has been done in murine and nonhuman primate (NHP) models. Several studies employing different mouse strains (CD2F1, C57BL/6J, C57BL/6, and CBA/J) have identified miRNAs as biomarkers for radiation injury and countermeasure efficacy.


While several miRNAs have been proven to be modulated by radiation, not all studies have showed the same miRNAs. However, most studies have shown downregulation of miR-150 and overexpression of miR-30 and miR-126. Exposure to 60Co γ-radiation, high LET, and high-energy particles reduced miR-150 expression (56Fe, iron-56) [188]. In addition to total-body irradiation, miR-150 downregulation was observed in the lung and blood of female WAG/RijCmcr rats irradiated (15 Gy at 1.43 Gy/min), indicating the potential of employing miRNAs for partial-body exposure and the impact on miRNA expression in organs and biofluids [189]. A profile of seven significantly changed miRNAs (miR-150-5p, miR-215-5p, miR-30a-5p, miR-126-5p, miR-133a-3p, miR-133b-3p, and miR-375-3p) was discovered in rhesus macaques 24 h after exposure to ionizing radiation. Differences in the expression of three miRNAs (miR-133b, miR-215, and miR-375) were used to accurately discriminate between irradiated and nonirradiated NHPs. Two miRNAs (miR-30a and miR-126) were able to predict radiation-induced mortality in NHPs in this study. Another study utilizing rhesus macaques found miR-126-3p upregulated and miR-150-5p downregulated. Unlike rhesus macaques, miR-342-3p was shown to be most affected (tenfold persistent downregulation) at 24 and 48 h postirradiation in baboons [190].


miRNAs strongly affect the cellular radiation response via regulation of vital genes involved in DNA damage repair [187], cell cycle checkpoints [191], and apoptosis [187]. Several important miRNAs, as well as their mRNA targets and signaling pathways implicated in radioresistance and radiosensitivity, are depicted in Fig. 3.49a, b, respectively.

Two diagrams depict the cellular pathways related to molecules and the clinical manifestations caused by the adverse effects of radio resistance and radiosensitivity.

Fig. 3.49
(a) miRNAs and cellular radioresistance: a summary representation of miRNAs in different cancers (outer circle) that regulate various mRNA targets (middle circle). These mRNA targets in turn influence various crucial biological pathways (inner circle) responsible for cellular radioresistance. Data for the figure acquired and modified from Ebahimzadeh et al. [192] (data taken with permission); [193] (CCBY). Gene names: P21 cyclin-dependent kinase inhibitor 1, AIFM3 apoptosis-inducing factor mitochondria-associated 3, APAF1 apoptotic peptidase-activating factor 1, BRCA1 breast cancer gene 1, p53 TP53 gene and tumor protein p53 gene, RB retinoblastoma protein, TCEAL7 transcription elongation factor A-like 7, PTEN phosphatase and tensin homolog, APAF1 apoptotic peptidase-activating factor 1, MTOR mechanistic target of rapamycin kinase. miR microRNA, NSCLC non-small cell lung cancer, GBM glioblastoma, CRC colorectal cancer, HCC hepatocellular carcinoma, NPC nasopharyngeal carcinoma, OSCC oral squamous cell carcinoma. (b) miRNAs and cellular radiosensitivity. A summary representation of miRNAs in different cancers (outer circle) that regulate various mRNA targets (middle circle). These mRNA targets in turn influence various crucial biological pathways (inner circle) responsible for cellular radiosensitivity. Data for the figure acquired and modified from Ebahimzadeh et al. [192] (data taken with permission); [193] (CCBY). Gene names: STAT3 signal transducer and activator of transcription 3, CDK4 cyclin-dependent kinase 4, MCL1 MCL1 apoptosis regulator, BCL2 family member, SIRT1 sirtuin 1, E2F1 E2F transcription factor 1, P21 cyclin-dependent kinase inhibitor 1, EGFR epidermal growth factor receptor, BCL2 BCL2 apoptosis regulator, LDHA lactate dehydrogenase A, ATM ataxia-telangiectasia mutated, AKT AKT serine/threonine kinase 1, H2AX H2A histone family, member X, Beclin-1 coiled-coil, moesin-like BCL2-interacting protein, ATG12 autophagy-related protein 12, TP53INP1 tumor protein p53 inducible nuclear protein 1, DRAM1 DNA damage-regulated autophagy modulator 1, UBQLN1 ubiquilin 1, DUSP10 dual-specificity phosphatase 10, STMN1, stathmin 1, c-MYC Myc-related translation/localization regulatory factor, WNT2B wingless-type MMTV integration site family, member 2B, WNT wingless-type MMTV integration site family, member, PKM2 pyruvate kinase isozymes M1/M2, LDHA lactate dehydrogenase A, MTOR mechanistic target of rapamycin kinase. miR microRNA, NSCLC non-small cell lung cancer, NK/T-cell lymphoma natural killer/T-cell lymphoma, SCC squamous cell carcinoma, ESCC esophageal cancer, GBM glioblastoma; CRC colorectal cancer, HCC hepatocellular carcinoma, NPC nasopharyngeal carcinoma, OSCC oral squamous cell carcinoma, DSB double-strand breaks


Two diagrams depict the cellular pathways related to molecules and the clinical manifestations caused by the adverse effects of radio resistance and radiosensitivity.


The expression of RAD51 and the subsequent formation of RAD51 foci in response to IR are a critical stage in HR. Following IR, RAD51 was revealed to be a direct target of miR-34a, miR-107, miR-155, and miR-222. Overexpression of miR-34a in lung cancer cells prevented the formation of radiation-induced RAD51 foci. Greater miR-155 levels were associated with lower RAD51 expression and better overall survival in a large dataset of triple-negative breast cancer patients. IR-induced damaged DNA is sensed by ataxia-telangiectasia mutated (ATM), which can initiate the signaling pathway, leading to checkpoint activation and DNA repair. ATM was shown to be downregulated by miR-18a in breast cancer, miR-26a in glioma, and miR-421 in squamous cell carcinoma (SCC), making the cells more sensitive to radiation.


Additionally, in response to IR, two miRNAs, miR-24 and miR-138, have been discovered to directly control H2AX. miR-182 suppressed BRCA1, another important protein in HR, in breast cancer cells. miR-875 also hampered the HR pathway by directly targeting the epidermal growth factor receptor (EGFR) and disrupting the EGFR-ZEB1-CHK1 axis.


PI3K/AKT is one of the key downstream targets of EGFR. The PI3-K/AKT pathway is crucial for establishing radiation resistance and intrinsic radiosensitivity of the cell. It is a critical regulator of normal and malignant development and cell fate decisions via activities such as proliferation, invasion, apoptosis, and activation of hypoxia-related proteins in cell signaling cascades. Several miRNAs are known to target and regulate key components of this pathway and help elicit the cellular response to radiation. AKT, an immediate downstream effector of the PI3K cascade, has been found to be directly targeted by miR-150 in natural killer (NK) and T-cell lymphoma cells. In a xenograft mouse model, miR-150 overexpression increased IR-induced apoptosis by decreasing PI3K/AKT signaling and sensitized NK/T-cell lymphoma cells to radiation. Furthermore, through blocking the AKT/GSK3/Snail signaling pathway, miR-203a-mediated ATM downregulation promoted apoptosis and cell cycle arrest in G1 phase in ovarian cancer cells. The tumor suppressor protein phosphatase and TENsin homolog (PTEN) is the central negative regulator of the PI3K/AKT pathway by dephosphorylation of PIP3 at the plasma membrane.


Several miRNAs generate pro-survival signals in response to IR by targeting PTEN. Activation of the PI3K/AKT pathway, suppression of apoptosis, and improved radioresistance were seen when miR-17, miR-20a, miR-106b, miR-205, miR-221, miR-222, and miR-498 were overexpressed. Regulation of PTEN expression is crucial for cell cycle maintenance. In colorectal cancer cells, miR-106b is known to target the CDK inhibitor p21 as well as PTEN. Overexpression of miR-106b promoted the G1-to-S transition in CRC cells, which was blocked by overexpression of either PTEN or p21. miR-17-mediated PTEN inhibition, like miR-106b, boosted G2-to-M progression and increased NPC cell proliferation through its effects on AKT signaling.


After IR exposure, apoptotic regulatory pathways are activated to remove cells with a high burden of DNA damage. Several miRNAs, including miR-133a, miR-125b, miR-124, miR-320a, and miR-634, are known to exert their effects on IR response via targeting components of crucial survival pathways, i.e., extracellular signal-regulated kinase (ERK), Janus kinase/signal transducer, and activator of transcription (JAK/STAT) as well as PI3K/AKT pathway. These pathways are initiated in response to IR-dependent activation of EGFR. In the JAK/STAT pathway, STAT3 is a direct target of miR-124, 320a, and 634, and the regulatory effect of these miRNAs on STAT3 upon IR is known to promote radiosensitivity. p53 is a critical tumor suppressor that is activated in response to IR to cause cell cycle arrest or apoptosis. Apoptosis mediated by p53 was abolished in IR-treated gastric cancer cells when miR-375 was overexpressed. In lung cancer cells, miR-300 directly regulates Apaf-1, the structural core of the apoptosome. Ectopic miR-300 expression caused radioresistance via reduced Apaf-1-induced apoptosis. P21 (Waf1/Cip1) is a p53 transcription target implicated in both major functions of the tumor suppressor, apoptosis, as well as cell cycle arrest. In oral squamous cell carcinoma (OSCC) cells, miR-17 has been shown to inhibit p21. In xenograft tumors, suppressing miR-17 boosted p21 expression, apoptotic rate, and radiosensitivity. miR-210 improved radioresistance in hypoxic hepatoma cells by targeting AIFM3. The retinoblastoma (Rb) tumor suppressor protein is an important component in the protection of cells from apoptosis. miR-622 was shown to prevent apoptosis by inhibiting the Rb gene in colorectal cancer cells. Another miRNA that reduced IR-induced apoptosis was miR-212, which directly targeted BRCA1 in glioma cells.


IR-induced autophagy is important in defining cell fate and determining whether cells survive or die, and it also impacts radiosensitivity. A multitude of proteins, including Beclin-1, LC3B-II, mTOR, and other autophagy-related proteins, are crucial for the regulation of this multistep process. Beclin-1 is an autophagy central regulator that regulates autophagosome nucleation and maturation. Beclin-1 is known to be directly regulated by miR-216a and miR-199a, which inhibit autophagy and promote radiosensitivity in response to radiation. mir-199 has also been shown to regulate the DNA damage-regulated autophagy modulator protein 1 (DRAM1) in response to IR. In breast cancer cells, miR-26b also targets DRAM1. miR-23b reduced IR-induced autophagy by targeting ATG12, a ubiquitin-like protein involved in the production of autophagy vesicles. miR-214 also targets ATG12, which enhances radiosensitivity while blocking IR-induced autophagy in CRC both in vitro and in vivo. Several additional miRNAs that target autophagy activators have been demonstrated to suppress IR-induced autophagy. These include miR-200c, which targets ubiquilin-1 (UBQLN1), an autophagosome formation promoter; miR-101, which targets autophagy activator stathmin 1 (STMN1) in NPC cells; miR-30a and miR-205 in prostate cancer cells; and miR-450, which targets DUSP10. miR-1246 was one of the miRNAs that enhanced autophagy in NSCLC cells. In vitro and in vivo, ectopic expression of miR-1246 reduced mTOR activity and radiosensitivity in lung cancer cells.


miRNAs have been proven to be valuable diagnostic and prognostic biomarkers in the clinic for over three decades. miRNAs are found in plasma, serum, blood, and urine and even retrieved from formalin-fixed tissues. These benefits make it a biomarker that is persistent after IR exposure and allow for less invasive testing. Two miRNAs (miR-30a and miR-126) were found as predictors of radiation-induced death in nonhuman primates. Another study suggested that serum miRNAs could be utilized as functional dosimeters to detect early hematopoietic radiation harm. After 2 Gy total-body irradiation, miR-130a-3p expression increased, but miR-150-5p, -142-5p, -706, and -342-3p expression dropped. Determining the sublethal dose of 6.5 Gy required five miRNAs (miR-136-5p, -173p, -126-3p, -322-3p, and -34b-3p), while miR-30a-3p/30c-5p discriminated the lethal (8 Gy) and sublethal (6.5 Gy) groups. miRNAs can be used as clinical biomarkers to predict prognostic irradiation effects, in addition to radiation harm biomarkers. Some miRNAs show sensitivity or resistance to IR in cancer patients who have already received radiotherapy (Fig. 3.47). These miRNAs may be utilized as radiosensitivity or radioresistance biomarkers. miRNAs may soon be acknowledged as biomarkers at the level of proteins, which will be utilized to promptly classify harm from radiation exposure, as well as treatment responses, adverse reactions, and personalized radiotherapies.


Research conducted thus far shows a relevant role for miRNAs in the future of radiation oncology, which may offer the basis for predicting patient response to radiotherapy and aid in developing miRNA-based individualized treatments to improve radiosensitivity. Early research indicated that the use of miRNAs as a biomarker for therapeutic monitoring and prognosis, and hence for more precise and individualized patient treatment, is feasible. Applications of miRNA for treatment as radiosensitizers are currently limited to cell culture or xenograft model systems and will need to be expanded into in vivo applications in the future. The role of extracellular miRNAs is still unknown. A thorough examination of radiation-induced mechanisms for secretion, transfer, and activity in recipient cells may aid in the understanding of major RT issues such as abscopal effects and radiation-induced secondary cancers (Box 3.33).


miRNAs are small, highly conserved noncoding RNA molecules that regulate gene expression.

miRNAs can be identified in physiological fluids such as plasma, saliva, and urine.

miRNAs have been identified as biomarkers for radiation injury and countermeasure efficacy.

miRNAs affect the cellular radiation response via regulation of vital genes involved in DNA damage repair, cell cycle checkpoints, autophagy, and apoptosis.


miRNAs are small, highly conserved noncoding RNA molecules that regulate gene expression.


miRNAs can be identified in physiological fluids such as plasma, saliva, and urine.


miRNAs have been identified as biomarkers for radiation injury and countermeasure efficacy.


miRNAs affect the cellular radiation response via regulation of vital genes involved in DNA damage repair, cell cycle checkpoints, autophagy, and apoptosis.


Long noncoding RNAs (lncRNAs) are defined as RNA transcripts with a length of more than 200 nucleotides missing a distinct protein-coding region. In the genome, they are located in intergenic, intronic, and exonic regions as well as sense, antisense, and bidirectional with transcripts overlapping sometimes genes [194]. In humans, 30,000–60,000 long noncoding transcripts are estimated compared to 20,000–25,000 protein-coding mRNA transcripts. Details about the biogenesis and functions of lncRNAs are very well summarized by Statello and colleagues [195]. In principle, the biogenesis of most lncRNAs corresponds to the production of mRNAs with transcription by RNA polymerase II and subsequent 5′-end capping and 3′ poly-A-tailing. In comparison to mRNAs, lncRNAs are less efficiently processed and often remain in the nucleus. As mechanisms for nuclear retention, tethering, or degradation on chromatin, weak splicing signals and cis- and trans-acting motifs are suggested. However, a substantial proportion of lncRNAs is distributed to the cytoplasm, where they can be sorted to specific organelles (e.g., mitochondria and exosomes) or they associate with diverse RNA-binding proteins. A considerable amount of lncRNAs assembles with ribosomes.


Initially, lncRNAs were considered as transcription by-products, but meanwhile important cellular functions are described for an accumulating number of lncRNAs. In general, lncRNAs are regulators of gene expression, which interact with DNA, RNA, and proteins on various levels. There are examples for both lncRNAs acting locally at the site of transcription (cis-acting) and lncRNAs leaving the site of transcription (trans-acting). To activate or suppress gene transcription, lncRNAs can regulate chromatin structure to change their accessibility or sequester chromatin-modifying proteins from or to the promoters of target genes. In addition to their roles in transcription regulation and nuclear organization, lncRNAs are involved in posttranscriptional regulation. This can occur by the association between lncRNAs and RNA processing proteins, resulting in altered mRNA splicing and turnover. Other lncRNAs can directly base pair with RNAs and subsequently recruit proteins involved in mRNA degradation or they support translation by promoting polysome association. Also, the binding between lncRNAs and microRNAs can regulate gene expression as miRNAs are sequestered from their target mRNAs by binding to an lncRNA [=sponge or competitive endogenous (ce) RNA] and thus abolish the inhibitory effect of miRNAs on mRNAs.


Through their manifold impacts on the regulation of gene expression, lncRNAs affect widespread aspects of physiology, including differentiation, growth, and responses to diverse stimuli and stresses.


lncRNAs are involved in many aspects of cellular response to radiation. For a detailed overview, see May et al. [196] and Podralska et al. [193]. Firstly, radiation affects the expression levels of a plethora of lncRNAs in cancer and non-cancer tissues and both up- and downregulation are reported. Radiation-triggered changes are also reported for a wide dose range including low doses (below 100 mGy) as well as therapeutically relevant doses and for single and chronic treatments. The functional relevance in radiation response was shown for a considerable number of lncRNAs, where some enhance radiosensitivity and others increase radioresistance. The affected pathways cover crucial pathways of cellular radiation response, such as cell cycle control, DNA damage repair, and apoptosis. As the mechanism during radiation response of action, frequently, the sponging of microRNAs by lncRNAs and thereby promoting of the expression of target genes are described.


The broad effects of irradiation on lncRNAs suggest valuable applications of this class of RNAs. Applications of biomarkers for radiation exposure may be important for biodosimetry or markers for normal tissue effects and radiotherapy response. Moreover, in vitro and in vivo studies demonstrated that modulation of the levels of lncRNAs can significantly enhance radiosensitivity of tumor cells. This suggests that lncRNAs may be used as targets to improve the outcome of radiotherapy in the future.


Prominent examples for lncRNAs with multiple roles in radiation response are HOTAIR, PVT1, and MALAT1. In breast cancer models, HOTAIR has been shown to increase radioresistance through interfering with DNA damage repair by targeting miR-218 and miR-449b-5p. In pancreatic ductal adenocarcinoma (PDAC), HOTAIR was induced by radiation, while a knockdown increased radiosensitivity. The knockdown increased the expression of Wnt inhibitory factor 1 (WIF-1), which was shown to enhance radiosensitivity. HOTAIR also promoted radiosensitivity of PDAC by increasing autophagosome formation through increasing LC3-II and ATG7A proteins. In cervical cancer, knockdown of HOTAIR increased radiosensitivity by the induction of a G1 cell cycle-phase arrest.


PVT1 contributes to NF90 transcription and HIF-1α stabilization in nasopharyngeal cancer, resulting in enhanced radioresistance. On the other hand, the knockdown of PVT1 resulted in reduced phosphorylation of ATM, p53, and CHk2 leading to increased radiosensitivity by decreased DNA damage signaling and increased apoptosis. In non-small cell lung cancer, PVT knockdown increases radiosensitivity by sponging miR-195.


lncRNA MALAT1 was downregulated after radiation in esophageal squamous cell carcinoma, and its overexpression enhanced radioresistance. It was shown that MALAT1 inhibited the downregulation of cyclin-dependent kinase subunit (Cks1), which resulted in a decrease in irradiation-induced apoptosis. MALAT1 also affected IR-induced apoptosis by interacting with miRNAs. In nasopharyngeal cancer cells, MALAT1 associated to miR-1, which led to increased levels of the anti-apoptotic protein SLUG. In cervical cancer cells, MALAT1 directly interacted with miR-145 to affect radiation-induced apoptosis (Box 3.34).


Long noncoding RNAs (lncRNAs) are transcripts >200 bp, which are not translated into proteins.

lncRNAs regulate gene expression on multiple levels, including transcription, RNA stability, and translation.

lncRNA expression is deregulated after radiation exposure, and they affect radiosensitivity by interfering with canonical radiation response pathways, such as cell cycle control, DNA repair, and cell death induction.


Long noncoding RNAs (lncRNAs) are transcripts >200 bp, which are not translated into proteins.


lncRNAs regulate gene expression on multiple levels, including transcription, RNA stability, and translation.


lncRNA expression is deregulated after radiation exposure, and they affect radiosensitivity by interfering with canonical radiation response pathways, such as cell cycle control, DNA repair, and cell death induction.


Circular RNAs (circRNAs) are a recently described class of RNA molecules that are derived from precursor mRNA (pre-mRNA) in a process called backsplicing. During this process, which is regulated by the spliceosome, a splice donor is joined to an upstream splice acceptor. This generates a covalently closed RNA molecule, which is typically resistant to degradation by exonucleases, and therefore circRNAs are in general biologically more stable compared to their linear counterparts. Although most circRNAs are expressed in relatively low levels, their increased stability can result in accumulation to levels far exceeding those of their cognate linear mRNAs [197]. This is for instance observed during aging, which led to the hypothesis that certain circRNAs may represent biomarkers for aging tissues (such as the brain) and aging-associated diseases. Recent studies even implicate circRNAs as causative factors in aging and cellular senescence [198]. Since irradiation and excessive DNA damage are often proposed as inducers of senescence and accelerated aging, radiation-responsive circRNAs may contribute to these longer term effects of radiation exposure.


A detailed description of the biogenesis and function of circRNAs is beyond the scope of this chapter; we therefore refer the readers to some excellent reviews about these subjects [198] and will only briefly discuss matters that may directly relate to DNA damage and radiation.


Unlike original views that circRNAs are no more than aberrant by-products of normal splicing, it has become increasingly clear that they are often generated and function independently from their linear cognates. One important mechanism of circRNA biogenesis acts via the RNA-binding protein quaking (QKI). QKI is an alternative splicing factor that belongs to the STAR family of KH domain containing RNA-binding proteins and binds to specific sequences (QKI-binding motifs) in pre-mRNA [199]. The proposed mechanism for the role of QKI in circRNA biogenesis is that it binds motifs in introns adjacent to the circle-forming exons and subsequently forms a dimer to bring these exons into close proximity for further processing by the splicing machinery [200]. Importantly, QKI is expressed at low levels in epithelial cells but is increased during epithelial-to-mesenchymal transition (EMT), when cells reprogram their gene expression profiles resulting in the loss of intracellular junctions, polarity, and cytoskeletal organization, ultimately leading to a more migratory and invasive mesenchymal phenotype. The increase of QKI during EMT triggers the expression of hundreds of circRNAs [200]. EMT is a process which can be induced by irradiation, and very often the mesenchymal cells display a more radiation-resistant phenotype, highlighting the relevance of EMT, and therefore QKI-regulated circRNAs, for radiation and cancer biology.


Different functions for circRNAs have been identified, including (1) binding and transportation of RNA-binding proteins; (2) generation of protein isoforms; and (3) regulators of transcription and alternative splicing (e.g., Xiao et al. [198]). However, the most established and investigated function of circRNAs is the regulation of microRNA expression and subcellular localization via a sponging mechanism (competing endogenous RNA, ceRNA). However, since most circRNAs are expressed at only low levels, and they usually contain only a limited number of microRNA-binding sites, it is now clear that the function of microRNA sponges or ceRNAs to regulate the expression of microRNA targets cannot be generalized for many circRNAs [198].


An important consideration here is that studies often perform gene ontology enrichment analyses based on the functions of the host genes of differentially expressed circRNAs. However, since there is currently little evidence that circRNAs in general function in the same pathways as their hosts, such analyses should be critically interpreted.


Despite increasing attention, the number of studies investigating the direct effect of ionizing radiation on circRNA expression is still very limited. On the other hand, there have been quite some studies in which the differential expression of circRNAs between radiation-sensitive and radiation-resistant cancer cell lines and patients was compared.


In HEK293-T cells, gamma irradiation (8 Gy, single dose) resulted in very big differences in the expression of circRNAs between control and irradiated cells. Here, the authors focused only on circRNAs detected under both experimental conditions and identified a total of 158 differentially expressed circRNAs. However, among 5592 detected circRNAs in total, 2205 were detected uniquely in control cells while 1026 circRNAs were uniquely found in irradiated cells. This indicates that the differences were actually larger than was reflected by the 158 that were considered to be differentially expressed.


A study by O’Leary and co-workers investigated differential circRNA expression at 4 h and 24 h after exposure of endothelial HUVECs to a medium (0.25 Gy) and high dose (2.5 Gy) of g-rays [202]. Radiation-responsive circRNAs were predominantly produced from genes involved in the p53 pathway, as is in general the case for the early transcriptional response to radiation. The authors furthermore focused on two circRNAs derived from the WWOX gene, showing that they are differently regulated by QKI in response to radiation depending on the cell type and that they are enriched in exosomes [202].


Another study focused on specific p53-dependent genes and their circRNA abundance in the embryonic mouse brain and primary neurons [203]. This study showed that the temporal induction of circRNA expression follows that of their linear mRNA hosts and that they remained more abundant for a longer time after irradiation compared to mRNA. This may have important implications for the use of circRNAs as long-term biomarkers of radiation exposure [203]. Indeed, gene expression changes at the level of mRNA are usually short-lived. Therefore, the increased stability of circRNAs may result in prolonged radiation-induced expression as was shown by Mfossa and co-workers [203].


One of the most extensively studied circRNA host genes related to radiation and cellular radiosensitivity is PVT1, a long noncoding RNA (lncRNA) gene from which different circRNAs can be generated. One of these, termed circPVT1 (CircBase ID: hsa_circ_0001821, consisting of the exon 2 of the PVT1 mRNA), is downregulated during both multiplicative and radiation-induced senescence in human diploid WI-38 fibroblasts. This leads to reduced sponging of the hsa-let-7 microRNA and a subsequent reduction of proliferative proteins encoded by let-7 targets (e.g., IGF2BP1, KRAS, and HMGA2) that prevent senescence. Thus, circPVT1 is a suppressor of (radiation-induced) senescence by acting as a decoy for let-7. Interestingly, linear PVT1 lncRNA was not decreased in senescent cells, indicating that the observed effects were exclusively regulated by circPVT1 [204].


Pvt1 was one of the p53 target genes investigated in the aforementioned study of Mfossa et al. [203]. Also, in human head and neck squamous cell carcinoma cells, circPVT1 expression was found to be dependent on p53 as it was enriched in tumors with p53 mutations and silencing of p53 resulted in a downregulation of circPVT1, but not linear PVT1 [205]. Several other studies have implicated circPVT1 as an oncogene in different cancers, and it enhances to chemotherapy resistance in gastric cancer cells and lung adenocarcinoma by acting as a ceRNA for miR-124-3p and miR-145-5p, respectively [201, 205]. In non-small cell lung cancer, circPVT1 expression is induced after irradiation, while it enhances radiosensitivity via inhibition of the PI3K/AKT/mTOR pathway through sponging of miR-1208 [206].


The PI3K/AKT/mTOR pathway plays a central role in cancer cell radioresistance in part via activation of EMT [207]. Inhibitors are currently being investigated as therapeutics to improve radiotherapy outcome. AKT is a serine-threonine kinase that exists in three isoforms, AK1, AKT2, and AKT3. The AKT3 gene hosts different circRNAs. Of these, circ-AKT3 (hsa_circ_0017250) is a protein-coding circRNA that competes with AKT phosphorylation, thereby reducing radiation resistance of different GBM cell lines. In contrast, another circ-AKT3 transcript (hsa_circ_0000199) increases chemoresistance of gastric cancer to cisplatin by upregulation of PIK3R1 (Huang et al. 2019). This suggests that different circRNAs originating from the same host gene can have opposite biological functions, as is sometimes also observed with linear splice variants. This furthermore highlights the importance of functional characterization of individual circRNAs. Several other circRNAs have been demonstrated to affect PI3K/AKT/mTOR signaling. Some of these have been described in the review papers by Cui et al. [201] and Jeyaraman et al. [208].


Altogether, it is now evident that circRNAs are affected by irradiation and that they are important players in the cellular radiation response and sensitivity. However, their exact functions in these processes, which furthermore may be cell type dependent, need to be investigated in a case-by-case manner. Novel methods for the genome-wide identification and functional characterization of circRNAs may prove to be useful tools for these future investigations [209].


Extracellular vesicles (EVs) are particles generated by all cells in our body by different routes and differ in diameter from <50 nm up to several μm [210]. EVs can based on their physical and molecular characteristics be divided into exosomes, ectosomes, microvesicles, microparticles, oncosomes, and apoptotic bodies. Size and expression of certain proteins reflecting their biogenesis and cellular origin are used for their classification (Table 3.15) [211]. Physical properties, e.g., size, density, and solubility of EVs, are often used for the isolation by differential high-speed centrifugation, size-exclusion chromatography, and precipitation. However, due to overlapping characteristics, pure preparations of individual EV species are challenging.Table 3.15
Characteristics of different extracellular vesicles (EVs)

Type of vesicle [size (nm)]

Description of characteristics

Microvesicles
100–1000 nm

A subgroup of EVs generated at the cell membrane. Found in both body fluids and tissues

Apoptotic bodies
500–2000 nm

A subgroup of EVs composed of cellular organelles and cytoplasm. Formed during apoptotic cell death by budding after the plasma membrane has undergone blebbing

Ectosomes
100–1000 nm

Membrane microvesicles produced by neutrophils or monocytes formed by direct budding from cell membrane. Vesicles larger than 350–400 nm are not always considered as true ectosomes

Oncosomes
100–500 nm
Large oncosomes
1–10 μm

EVs of different sizes generated by tumor cells which function as transmitters of oncogenic signals (RNA, protein complexes) between cells

Exosomes
40–150 nm

Membrane-bound EVs formed by the endocytic pathway. These EVs are first formed at the plasma membrane and subsequently transformed into early endosomes. These subsequently mature into late endosomes where they bud off to the ER intracytoplasmic lumen. The formed multivesicular bodies thereafter are unified with the cell membrane, and exosomes are released to the extracellular surroundings of the cell. Exosomal markers include CD63, CD9, CD81, and TSG101 among others


EVs are enclosed by a lipid-bilayer membrane, and their cargo includes coding and noncoding RNAs, genomic and mitochondrial DNA fragments, proteins, metabolites, and lipids. Initially, EVs were discovered as “garbage bins” to remove unwanted materials. Now, it is clear that most of the cells in our bodies utilize EV secretion into its close or distant microenvironment as a way of communication [212]. Thus, EVs can transfer functional biological molecules to recipient cells either by direct fusion with the plasma membrane or by internalization but also via interaction with cell surface receptors triggering downstream signaling (Box 3.35).


Extracellular vesicles (EVs) can be of different sizes and are generated via different biogenesis routes from all cells within our body.

EVs cargo RNA, DNA fragments, lipids, and proteins partly reflecting their cell of origin.

EVs are important communicators in health and disease.

EVs regulate carcinogenesis and metastasis.

Exosomes are generated via the endosomal system and are released from viable cells.


Extracellular vesicles (EVs) can be of different sizes and are generated via different biogenesis routes from all cells within our body.


EVs cargo RNA, DNA fragments, lipids, and proteins partly reflecting their cell of origin.


Exosomes are generated via the endosomal system and are released from viable cells.


EVs are found to be an integrated part of cell-to-cell communication, thereby contributing to regulation of the immune as well as the nervous system but also to tissue regeneration after damage [213]. Also, in the carcinogenesis and cancer metastasis fields, EVs have been demonstrated to be important communicators. Thus, EVs regulate the tumor and the tumor microenvironment signaling including angiogenic promotion, conversion of fibroblast into cancer-associated fibroblasts, and interplay with the immune system, thereby providing a good milieu for disseminated tumor cells to grow as well as establish themselves as metastases.


Given that EVs can influence a multitude of cell and tissue processes, it is not surprising that EVs today are considered an important source of biomarkers of different diseases including cancer. Thus, analyses of EVs and their cargo have been able to gain the US Food and Drug Administration (FDA) and international approvals to some extent [214]. With such diverse and varied roles of EVs in assisting cancer progression, it is essential that one can understand how IR, given its essential place in cancer therapy, can alter EVs cargo and/or function.


Exosomes are generated in the endosomal system of almost all cells (Fig. 3.50). These vesicles of nano size have membranes with parts from their cell of origin but also cargo membrane and cytosolic lipids, proteins, as well as various RNA species and DNA fragments [215].

A schematic diagram of a cell depicts that receptors form an early endosome that leads to a late endosome, which has I L Vs, R N A, D N A, and proteins that cause exocytosis. The exosomes released enter the recipient cell.

Fig. 3.50
Principal steps in exosome biogenesis. The early endosomes, which are generated at the plasma membrane (1), later undergo maturation, called late endosomes or multivesicular bodies (MVBs) (2). The MVBs’ membrane invagination results in the formation of intraluminal vesicles (ILVs). During the invaginating process, particular proteins are incorporated into the invaginating membrane. Other cytosolic biomolecules, i.e., nucleic acids and proteins, are engulfed and enclosed within ILVs. The release of exosomes into the extracellular environment happens after fusion of the MVB with plasma membrane (3)


A schematic diagram of a cell depicts that receptors form an early endosome that leads to a late endosome, which has I L Vs, R N A, D N A, and proteins that cause exocytosis. The exosomes released enter the recipient cell.


When exosomes were identified in the 1980s, they were seen upon as “garbage bins,” but later it was reported that exosomes generated from B lymphocytes could trigger a T-cell response. From that time, exosomes have been shown to participate in a multitude of cell–cell communication routes by carrying cargoes which are taken up by recipient cells close by or in a multicellular organism, in another tissue as exemplified in the cancer metastasis process [216]. The scientific community have gathered data on the exosome cargo, e.g., protein, lipid, and mRNA or miRNAs, into large databases, e.g., ExoCarta (http://www.exocarta.org/) and Vesiclepedia (http://www.microvesicles.org/), which are growing as more exosomes from cells of different origin and in different contexts are being deciphered and reported (Box 3.36).


Exosomes are EVs of endosomal origin, which contain nucleic acids, membrane and cytosolic proteins, metabolites, and lipids.

Once released, exosomes may act on cells in close vicinity or in a distant tissue.

Exosomes are involved in a multitude of human diseases including cancer where they regulate carcinogenesis, tumor-immune cell interplay, angiogenesis, and metastasis.


Exosomes are EVs of endosomal origin, which contain nucleic acids, membrane and cytosolic proteins, metabolites, and lipids.


Once released, exosomes may act on cells in close vicinity or in a distant tissue.


Exosomes are involved in a multitude of human diseases including cancer where they regulate carcinogenesis, tumor-immune cell interplay, angiogenesis, and metastasis.


It is still to some extent difficult to sort out plasma membrane-derived EVs from exosomes as their sizes are similar and given that there is a cell heterogeneity in the expression of the protein markers that define exosomes [210]. However, there are certain proteins that characterize exosomes including Rab GTPases, flotillin, heat-shock proteins (HSP70 and -90), tetraspanins (CD63, CD9, CD81, and CD82), Alix, flotillin, and TSG101. These markers are also suggested by the International Society for Extracellular Vesicles (ISEV) to be used to define exosomes [210].


The lipid membrane of EVs has a different composition relative to plasma membranes. Thus, EV membrane has higher level of sphingomyelin, cholesterol, ceramide, and phosphatidylserine while less expression of phosphatidylcholine. These lipids have been shown to have a profound effect on carcinogenesis and cancer progression including enhancing invasiveness, angiogenesis, and chemoresistance via transport of oncogenic elements.


There is clear evidence that cancer cells may have another rate of exosome release than non-transformed cells [217] while it is still a controversy as to what extent that is reflected in human liquid biopsies, e.g., plasma, and if it can be linked to therapy response. Also, it has been recognized that cancer and normal cells differ with respect to exosome cargo, e.g., miRNA, mRNA protein, and lipids. Exosomes have been found in plasma, serum, lymph fluid, bronchial fluid, cerebral spinal fluid (CSF), urine, saliva, tears, bile and gastric acid, amniotic fluid, breast milk, semen, and synovial fluid [218]. This has spurred an interest in their role as a source of biomarkers.


As indicated above, in human tissues, exosome can act near its cell of release or be transported in the blood to a distant tissue, e.g., site of metastasis in the context of cancer. It has been demonstrated in a large number of publications that once the exosome cargo reaches its target cell, several mechanisms cooperate for uptake as well as for altering signaling in the recipient cells [219]. Similar to EVs, exosomes participate in different processes of the immune system as well as in neurological signaling processes. Exosomes also have a clear function in cancer signaling. Exosomes are described to regulate tumor internal signaling but also tumor–tumor microenvironment interplay. For example, exosomes may promote angiogenesis as well as metastatic spread, and they are important communicators between tumor and different infiltrating immune cells.


Exosomes may exert these events by modulating paracrine, autocrine, and endocrine pathways in different cell types via their cargo. The exosome surface proteins are reported to resemble those of plasma membrane and endosome of a given cell yet with minor contribution of proteins from nucleus or Golgi. It has also been reported that EV membrane composition differs from plasma membrane concerning their lipids. Thus, EV membrane has higher level of sphingomyelin, cholesterol, ceramide, and phosphatidylserine while less expression of phosphatidylcholine.


Exosomes carry a wide range of cargoes, and it is currently thought that such cargoes, e.g., RNA species, are selectively loaded into exosomes and that loading is not a random process. This is supported by observed differences in miRNA abundances in cells compared to exosomes, which have been linked to 3′ uridylation in miRNAs of exosomes, while 3′ adenylated miRNAs are enriched in cellular fractions [220]. Moreover, certain sequence motifs are recognized by the nuclear ribonucleoprotein A2B1 (hnRNPA2B1), which then dictates the miRNA loading process into exosomes, possibly via interaction with cytoskeletal components. The protein AGO2 has also been shown to selectively package exosomes with miRNAs specifically miR-451. In addition, overexpression of the protein neutral sphingomyelinase 2 has been associated with an increase in exosome-associated miRNA. It has also been demonstrated that 3′ mRNA fragments are enriched in exosomes. The conserved 25-nucleotide sequence (also known as a zip code-like 25 nucleotide) is usually incorporated into mRNA’s 3′-untranslated region and expressed in many types of cells, leading to mRNA enrichment in the MVs/exosomes. It has been suggested that miR-1289 plays a crucial role in MV enrichment of the mRNA via binding to zip code sequence directly.


The release of exosomes requires the movement of late endosomes/multivesicular bodies (MVBs) to the cell surface, where they fuse with the cell membrane (Fig. 3.50). The actin cytoskeleton and microtubule network have been shown to be important in facilitating MVB movement towards the cell surface, while Rab GTPases facilitate the release of exosomes into the extracellular space. Interestingly, certain Rabs have been demonstrated to preferentially export exosomes with certain phenotypes; for example, Rab27A/B have been shown to release exosomes positive for CD63-, TSG101-, and Alix expression [221].


Exosomes may, via their cargo, induce both pro-survival and pro-death signaling in recipient cells. Thus, exosomes may promote tumor growth as well as induce inflammation through activation of mesenchymal stem cells (MSCs) and the subsequent secretion of IL-6 as well as IL-8. There is also evidence that exosomes carry inhibitors of apoptosis proteins (IAPs), e.g., survivin, XIAP, cIAP1, and cIAP2, the delivery of which is postulated to offer protection from a continually changing microenvironment, thereby helping tumor progression.


In the context of tumor and immune cell interplay, there is growing substantiation that tumor cells of different origin can via their exosome cargo impair infiltrating T-cell function as illustrated by PD-L1-expressing exosomes [222]. Similarly, CD73, an ecto-5′-nucleotidase and a master regulator in the tumor-immune microenvironment, has been reported in exosomes and also to have functional activity in this context, e.g., impairing T-cell function [222]. The effect exosomes have on tissue poses a number of interesting questions when it comes to radiation biology as radiation has tumor growth-inhibiting effects yet may negatively influence certain normal tissue in the radiation therapy field. Exosomes are also thought to offer some beneficial properties against different tissue damages. Thus, exosomes from MSCs have been reported to offer protection against diabetic nephropathy in the renal system by blocking apoptosis as well as promote vascular regeneration. Moreover, acute kidney injury caused by the DNA-damaging agent cisplatin was found to be blocked by microvesicles as a result of inhibition of apoptosis. Exosomes have moreover been shown to enhance recovery from ischemic brain injury through promoting angiogenesis and providing an extracellular milieu for appropriate brain remodeling.


As EVs are important regulators of multiple cellular signaling events, it is not surprising that EVs are also important communicators in the context of IR [223]. EVs are affected by IR on multiple levels, including alterations in subtype/size, release, cargo, uptake, and function. These changes facilitate the dissemination of IR signals to neighboring cells and to distant sites, which contributes to systemic effects in irradiated and nonirradiated areas. Therefore, EVs are potential mediators of IR-targeted and non-targeted effects, e.g., bystander and abscopal effects.


Several studies suggest increased EV release after irradiation in in vitro and in vivo models. As an example, it has been shown that IR may increase EV release in different tumor models including head and neck cancer and glioblastoma. Moreover, also in normal tissue after partial-body irradiation of mice, it has been reported that the EV content is altered in different tissues including the liver, brain, and heart. As the potential mechanism for the IR-increased EV release, p53-mediated induction of genes involved in the EV biogenesis and altered MAPK signaling were suggested. It was also shown that the cellular uptake of EVs is affected by radiation exposure. In mesenchymal stem cells, irradiation induced changes in the formation of cell surface CD29/CD81 complexes, which increased the cellular uptake of EVs [224].


IR also induces changes in the composition of EVs released from cancer and non-cancer cells. Alterations seem to be highly related to cell type, radiation dose, and also time postradiation exposure where both microRNA and protein changes have been described. Additionally, changes in lipids and metabolites in EVs from irradiated donor cells are reported. EV cargo changes were also shown for EVs isolated from blood during or after tumor RT. For example, differential expression of serum EV miRNAs was monitored in prostate cancer or glioma patients after RT. In the serum of HNC patients, it was shown that tumor-derived exosome (TDE) amount relative to total exosomes increased in patients that were refractory to a combination of radio-, targeted, and immune therapy while the opposite was found in the patients that responded. Moreover, in the same study, results demonstrated an increase in regulatory T-cell (Treg)-derived exosomes as well as in CD3(−)PD-L1+ exosomes in serum after treatment if the patients were refractory. Such alterations in EV cargo open up for potential applications of EVs/exosomes as a source of biomarkers for radiation exposure as well as for prognostic or predictive biomarkers of RT response in the context of cancer.


A substantial amount of studies suggest that EVs play a role in the progression, RT resistance, and metastasis of cancer cells. In glioblastoma, Mrowczynski et al. demonstrated a pro-survival function of EVs derived after IR, which may be triggered by elevated cargo levels of oncogenic miRNAs and mRNAs, while tumor-suppressive RNAs were reduced [225]. In the same cancer type, a pro-migratory role of radiation-related EVs was reported. Likewise, EVs from irradiated HNC and neuroblastoma cells were shown to stimulate survival, migration, and invasiveness. However, there are also studies reporting on an induction of harmful effects of EVs from irradiated cancer cells into recipient cells, like chromosomal damage and increased ROS levels.


EVs are also involved in the communication of radiation signals among normal cells. Early work by Jella et al. showed the transmission of cytotoxic effects between irradiated and nonirradiated keratinocytes in an in vitro model system. Thus, EVs from irradiated mice were able to increase DNA damage and reduce viability in co-cultivated mouse embryonic fibroblasts. On the other hand, several reports found beneficial effects of EVs released from irradiated human PBMCs. For example, EVs from irradiated blood cells were shown to reduce radiation-induced apoptosis in endothelial cells [226]. Accordingly, pro-angiogenic and tissue-regenerative capacities were attributed to EVs from irradiated PBMC. In this regard, it was shown that EVs (especially from mesenchymal cells) could be used for the treatment of radiation injury [227].


In summary, current knowledge indicates a vital role of EVs in the IR response of cancer and non-cancer cells. IR not only affects the production and the composition of EVs, but also alters the phenotypes of recipient cells. Therefore, these mechanisms can contribute to the communication between irradiated cells as well as to the systemic distribution of local radiation effects throughout an organism. Moreover, EVs may offer a source of biomarkers for monitoring RT responses in cancer patients (Box 3.37).


EVs, including exosomes, act as intercellular signaling components in response to IR.

IR may influence the EV release/uptake as well as cargo in normal as well as tumor cells contributing to both direct and bystander effects of IR.

EVs/exosomes may contribute to the distribution of systemic IR effects and offer a source of IR response biomarkers.


EVs, including exosomes, act as intercellular signaling components in response to IR.


IR may influence the EV release/uptake as well as cargo in normal as well as tumor cells contributing to both direct and bystander effects of IR.


EVs/exosomes may contribute to the distribution of systemic IR effects and offer a source of IR response biomarkers.


The term proteome was created to describe the set of proteins expressed by the genome [228]. Proteomics analyzes the proteome at a specific time and in a specific state. Proteome profiling provides information not only about the protein expression, but also about the function, structure, and interactions of proteins.


In the well-established paradigm of proteomics, protein mixture will be separated before digestion either by gel electrophoresis (gel-based approaches) or using liquid chromatography (gel-free approaches) to resolve the complexity of the protein mixture [228]. In the next step, proteins were fragmented into smaller units called peptides during digestion. The generated peptides were further separated and sorted in the mass spectrometry system based on the mass and charge, where the abundance of each peptide is translated into numerical values called intensity. To identify a protein, a certain number of good-quality peptides must be detected. Quantitative proteomics compares the peptide intensities for each protein between treated (e.g., irradiated) and non-treated (e.g., nonirradiated) samples. The alterations in peptide intensities represent the changes in the expression level of corresponding protein.


Protein quantification can be performed in two ways: either label based or label free. In label-free methods, protein expression in several samples is compared by measuring the intensity of the corresponding peptides or counting the number of correlated spectra for each protein. Label-based quantification is performed by labeling peptides or proteins with fluorescent dyes, chemical isotopes, radioisotopes, or affinity tags before mass spectrometry. Label-based proteomic approaches are classified into chemical labeling (ICPL, iTRAQ, and iCAT) and metabolic labeling techniques (SILAC).


Advanced proteomics approaches also offer an accurate platform to identify and quantify the posttranslational modifications (PTMs) such as phosphorylation, acetylation, methylation, or ubiquitination [229]. These modifications are crucial for the stability, localization, and conformation and functions of proteins. The analysis of phosphoproteome, acetylome, or ubiquitinome has revealed the regulatory role of these PTMs in cellular function and homeostasis [229].


A comprehensive combination of proteomics and advanced bioinformatics makes the complex biological processes in cells understandable. The bioinformatics tools provide a broad spectrum of information on protein functions, protein-protein interactions, protein interactions with other biomolecules (genes and metabolites), contribution to the signaling pathways, and predictions of diseases [230].


Different proteomics approaches were applied to investigate the biological effects of radiation exposure on normal and tumor tissues, cancer radiotherapy outcome, individual sensitivity, risk assessment, biodosimetry, and biomarker discovery; an extensive review can be found in the work of Azimzadeh et al. [231].


One of the main goals of cancer proteome profiling in radiation research has been to identify biomarkers that predict the tumor’s response to radiation exposure. The proteomes of different cancer cell lines such as nasopharyngeal carcinoma, head and neck cancer, oral squamous cell carcinoma, laryngeal cancer, breast cancer, and lung cancer have been analyzed in radiobiological studies to identify signatures of cancer radioresistance and potential prognostic markers for radiotherapy. Although the results of these studies are not uniform, the proteins identified and quantified belong mainly to the family of antioxidant proteins, heat shocks, and structural proteins.


The most challenging aspect of radiotherapy for cancer is to select the radiation dose so that the tumor is killed but the surrounding normal tissue is harmed as little as possible. The effect of radiation on normal tissue has also been analyzed by proteomics approaches. A number of studies have been carried out on in vitro and in vivo models to simulate the effects of radiation on normal tissue such as the heart, brain, and liver. These studies underlined the adverse effects of irradiation on tissue structure and function. The mitochondrial proteins, the metabolic enzymes, and the oxidative stress response proteins are the main groups of proteins affected in the irradiated heart. The structural proteins, proteins involved in cognition and learning function, and inflammatory response were impaired in the irradiated brain.


Biofluids such as serum, plasma, and urine are optimal biomaterials for biomarker discovery, mainly because of the relatively noninvasive collection methods. However, proteomic profiling in biofluids is still an analytical challenge due to the complexity and variable spectrum of protein abundance. Several studies have compared the biofluid proteome before and after radiation exposure. These studies provide a panel of proteins that serve as biomarkers of radiation exposure, radiation damage, cancer radiosensitivity and radiotherapy outcome, and biodosimetry.


Since cellular responses to irradiation are tightly regulated by PTMs, the analysis of these changes is becoming increasingly important in radiation research. PTM profiling is still a young field in radiation research, and only a few studies have analyzed the change in protein phosphorylation, acetylation, and ubiquitination in the context of cancer and normal tissue response to irradiation.


Archival formalin-fixed, paraffin-embedded (FFPE) tissues are the invaluable alternative of fresh frozen biomaterial in radiation research. Proteomic analysis of these samples is challenging, mainly due to the harsh conditions of tissue fixation and, in particular, biomolecule extraction method. The proteomics studies conducted on FFPE tissues from radiobiology archives have mainly investigated the predictive marker for radiotherapy resistance of cancer or adverse effect on normal tissue. They demonstrated the compatibility and applicability of FFPE tissues for proteomics studies [231].


The study of cellular lipid pathways and networks in biological systems is known as lipidomics [232]. Lipids are a necessary component of biological membranes and play essential roles in biological systems, such as the plasma membrane bilayer structure that separates the cell cytoplasm from the extracellular microenvironment, the provision of a hydrophobic medium for the functional performance and interactions of membrane proteins, and the generation of second messengers through enzyme reactions [233]. Lipidomics refers to the analysis of all lipids present in a sample using liquid chromatography (LC) and mass spectrometry (MS) techniques.


Glycerolipids, saccharolipids, sphingolipids, glycerophospholipids, sterols, polyketides, fatty acyls, and prenols are the eight types of lipids that can be classified based on their chemical structures and hydrophobic and hydrophilic aspects [233]. The most prevalent phospholipids (PLs) are glycerophospholipids, found in biological membranes and essential for numerous cellular activities. PCs and other related phospholipid derivatives like lysophosphatidylcholines (LPCs) are signaling molecules that play a role in regulating cellular death and proliferation. Triacylglycerides (TGs), sphingomyelins (SMs), phosphatidylinositols (PIs), diacylglycerides (DGs), and cholesteryl esters are also among lipids with key roles in cell physiology [234].


Reactive oxygen/nitrogen species (ROS/RNS) react extensively with lipid molecules following irradiation, causing lipid breakdown and eliciting both direct and indirect inflammatory responses. Lipid peroxidation and pro-inflammatory lipid intermediates can have immediate impacts on physiology and can lead to long-term consequences like CVD, lung damages, and even carcinogenesis. Apoptosis can also be triggered by the direct action of radiation or by lipid intermediates, such as the activation of sphingomyelinase, which produces ceramide from the hydrolysis of sphingomyelin. Ceramide is a direct apoptotic cell death [234]. Post-ionizing irradiation (IR) changes affecting lipids have been proven in preclinical investigations and may have biological consequences such as the acute radiation sickness (ARS) or lead to delayed effects of acute radiation exposure (DEARE).


When comparing sham or pre- and post-IR specimens, lipids examined in blood, such as PCs, LPCs, TGs, SMs, and CEs, exhibit modifications.


The link between lipid levels in serum/plasma and radiation has been studied in animal models in several publications. Phosphatidylethanolamine (PE) and phosphatidylserine (PS) levels in rat plasma following gamma irradiation exposure increased dramatically, thus indicating that IR may disrupt phospholipid metabolism [233]. Fatty acids, such as linoleic acid and palmitic acid, were found to be present at reduced levels in the blood following 137Cs exposure in mice, while phosphatidylcholines were among the most disturbed molecules in 137Cs-exposed mouse serum. A total of 67 biomarkers were discovered in some tissues and biofluids of mice exposed to radiation (6 Gy) (serum and urine). Among these, 3-methylglutarylcarnitine was found to be a unique metabolite seen in the liver, serum, and urine that might be employed as a marker of early radiation response.


Changes in lipid metabolism, including key lipid species such as free fatty acids, glycerolipids, glycerophospholipids, and esterified sterols, have also been observed in nonhuman primates exposed to IR. The results show that diacylglycerides decreased 1 day after IR, but triacylglycerides and lysophosphatidylcholines increased from 2 to 7 days after IR. At 7 days, after 10 Gy irradiation, the amount of polyunsaturated fatty acids, such as arachidonic acid and docosahexaenoic acid, increased significantly in the nonhuman primate model. Between 2 and 3 days after irradiation, an increase in LysoPCs and a decrease in SMs could be regarded as viable indicators (6.5 Gy). Compared to nonirradiated controls, recent research in nonhuman primates has discovered plasma-derived exosomal indicators of IR exposure related to the enrichment of N-acyl-amino acids, fatty acid esters of hydroxyl fatty acids, glycolipids, and triglycerides.


Radiation therapy caused blood lipidome disturbances, which were corrected within 1–2 months after IR treatment, according to lipid species quantification in individuals receiving radiation therapy. As a result, radiation-induced lipidome modifications could indicate changes in early time points and, perhaps, alternative damage pathways. Patients undergoing a complete body irradiation at the MSK Cancer Center (NYC), before hematopoietic stem cell transplantation, showed seven urine indicators with changes between pre- and postexposure at 4–6 h (1.25 Gy) and 24 h (three fractions of 1.25 Gy each) postirradiation, which involved trimethyl-l-lysine, acetylcarnitine, decanoylcarnitine, and octanoylcarnitine (carnitine conjugates involved in fatty acid transport), as well as hypoxanthine, xanthine, and uric acid (purine catabolism pathway end products).


During the period 2015–2019, the National Cancer Institute’s Radiation Research Program, in partnership with the Small Business Innovation Research Development Center, financed four small firms. This led to the development of a metabolomic/lipidomic assay for predicting late effects that have a negative impact on the quality of life in prostate cancer patients treated with radiation. Shuttle Pharmaceuticals (Rockville, MD) intended to move forward by developing and validating a metabolomic/lipidomic biomarker panel that could predict radiation toxicity. In a phase I experiment, metabolites in plasma from 100 patients were examined in order to develop a kit that could support metabolomic analysis and act as a biomarker panel to predict sensitivity to radiation late effects. A phase II SBIR project was set up for the multi-site analytic validation of the metabolite panel kit created in the phase I SBIR project [235].


Lipidomics has then emerged as a reliable technique for lipid identification and quantification and the search for biomarkers that can be used in radiation-related incidents such as nuclear and radiological hazardous occurrences. In this sense, easily available biofluids are critical, especially in the case of irradiated victims, as well as the use of biodosimetry techniques that are both quick and accurate. Huang et al. [233] discovered seven radiation-responsive lipids in the serum of mice and showed their utility in dose calculation. Lipid changes after whole-body exposures have been thoroughly documented in a variety of animals, including atomic bomb survivors [234]. Indeed, estimating the radiation dose has always been a priority in the medical treatment of these events.


Because of the combined effects of neutrons and photons, shielding from structures, and closeness to the epicenter, among other factors, radiation exposures from an IND can be complicated. Using lipidomics, Laiakis et al. [234] evaluated serum samples from mice exposed to varying percentages of neutrons and X-rays to a total dosage of 3 Gy. Several lipids including triacylglycerides, phosphatidylserines, lysophosphatidylethanolamines, lysophosphatidylcholines, sphingolipids, and cholesteryl esters exhibited a delayed increase in mixed exposures, while diacylglycerides declined and phosphatidylcholines (PCs) remained virtually unaltered.


The mammalian lipidome’s structural variety is so great that it necessitates a systematic nomenclature for precise categorization (identifying lipid subclasses, or the total number of carbons in the fatty acid chain and the number of double bonds). Because numerous lipid classes showed differences when comparing sham or pre- to post-IR samples, the variable sums and ratios within each metabolite class can increase and must be carefully considered.


Radiation exposure can cause a complex molecular and cellular response having an impact in metabolic processes and consequently change metabolite levels [236]. This approach aims to detect small molecules (<1000 Da) in biological samples, which occur downstream of genomic, transcriptomic, and proteomic processes, constituting a more complete picture of the system’s response to insult even prior to the onset of clinical symptoms [237]. The first use of metabolomics concerning radiation exposure studies was reported in the 1960s according to the experiences developed in human and animal samples; however, the understanding of cellular and molecular effects of ionizing radiation and the identification of radiation exposure biomarkers remain a challenge [236]. To obtain metabolic information, different methodologies can be used as nuclear magnetic resonance (NMR) or mass spectrometry (MS), including gas chromatography (GC) and liquid chromatography (LC). After sample collection, processing, and data acquisition, data analysis is the second step using principal component analysis (PCA) to have an initial perception about patterns and outliers, and if there are any easily discernible biomarkers. After, complex statistical data analysis should be employed in order to at the end perform biomarkers data interpretation and validation [236]. However, it is important to consider that there exist multiple analytical and clinical challenges that constitute an impediment for the successful translation of these biomarkers for clinical use [237].


Despite the few existing studies, some metabolite changes in small-molecule metabolites remain underexplored and underexploited concerning ionizing radiation effects [236]. The role of polyamine metabolism has been studied related to ionizing radiation effects. Present in all cells, polyamines, as putrescine, spermidine, and spermine, are aliphatic polycations with multiple functions related to cell metabolism, cell proliferation, and cell differentiation. These molecules have pleiotropic effects that allow their linkage to DNA, RNA, and proteins, identifying a regulatory role in cell metabolism [236]. Besides this, increased levels of these molecules are reported as healthy cell protectors against oxidative stress. Different studies using animal and patient samples have been performed reporting altered metabolites in response to ionizing radiation, namely creatine, creatinine, carnitine, hypoxanthine, citric acid, taurine, xanthine, threonine, uric acid, and citrulline. Besides these metabolites with high alterations, 2′-deoxyuridine, arginine, glycine, glutamine, hippuric acid, inositol, palmitic acid, uridine, lactic acid, leucine, linoleic acid, methionine, tyrosine, and sebacic acid are described as metabolites with moderate alterations in consequence of ionizing radiation. Therefore, and considering experimental data, among them exist strong candidates for diagnostic biomarkers being considered time- and dose-dependent measures [232]. Data obtained using T cells demonstrated that different metabolic pathways related to amino acid, nucleotide, fatty acid, and glutathione metabolism can be affected by in vivo radiation. Related with cancer and ionizing radiation response, metabolic profiling may help identify metabolites responsible for response to therapy, being the alteration of metabolite production, a feature that can influence tumor microenvironment and consequently cancer progression [237]. The complete characterization of the metabolome can be an opportunity to influence prognostic, predictive, and even pharmacodynamic biomarkers contributing to an individualized and targeted treatment [232]. Notwithstanding the capacity of metabolomics to detect alterations, it is necessary to be aware of the tumor-related responses, namely to the therapy, as inflammation and altered energy metabolism. Besides that, and considering cancer as a syndrome, there are also other cancer-associated conditions such as weight loss that can influence metabolism [238]. Having in consideration that ionizing radiation triggers a complex response influencing molecular and cellular processes and alteration of the metabolic processes and metabolite levels, more research work is necessary to identify biomarkers related to specific type and dose of radiation, genotypic differences, pathological conditions, and specific organs or tissues (Box 3.38).