The biologic effects of IR are unique in that the induced damage is clustered due to the local deposition of energy in radiation tracks. The distance between the depositions of energy is biologically very relevant and unique to the energy and the type of radiation. The term linear energy transfer (LET) denotes the energy transferred per unit length of a track of radiation. Electromagnetic radiation, such as x-rays or gamma rays, are sparsely ionizing and therefore classified as low LET radiation, whereas particulate radiation, such as neutrons, protons, and alpha particles, are examples of high LET radiation.¹

Radiation-induced damage to cellular target molecules, such as DNA, proteins, and lipids, can be either direct or indirect (Fig. 8.1). The direct action of radiation, which is the dominant mode of action of high LET radiation, is due to the deposition of energy directly to the target molecule, resulting in one or more ionization events. The indirect action of radiation is due to the radiolysis of water molecules, which, after initial absorption of radiation energy, become excited and generate different types of radiolysis products where the reactive hydroxyl radical (•OH), can damage both DNA and proteins. About two-thirds of the damage induced by low LET radiation is due to the indirect action of radiation. Since the hydroxyl radical is very reactive (half-life is 10^-9 seconds), it does not diffuse more than a few nanometers after it is formed before it reacts with other molecules, and, thus, only radicals formed in close proximity to the target molecule will contribute to the damage of that target.² However, by chemical recombination of the primary radiolysis products, hydrogen peroxide (H₂O₂) is formed, which in turn can produce hydroxyl radicals at a later time through the Fenton reaction, involving free metals. Because H₂O₂ is not very reactive, it can diffuse long distances away from the initial site of energy deposition.

Radical scavengers normally present in cells, such as glutathione, can protect target molecules by reacting with the hydroxyl radical (see Fig. 8.1). Even after the target molecule has been hit and ionized, glutathione can contribute to cell protection by donating a hydrogen atom to the radical, allowing the unpaired electron present in the radical to pair up with the electron from the hydrogen atom. This is considered the simplest of all types of repair and is called chemical repair.³ However, if oxygen molecules are present, they will compete with scavenger molecules for the ionized molecule, and if oxygen reacts with the ionized target molecule before the hydrogen donation occurs, the damage will be solidified as a peroxide, which is not amendable to chemical repair. Instead, this lesion will require enzymatic repair for the restoration of DNA. This augmenting biologic effect of oxygen is called the oxygen effect and is considered an important factor for the effectiveness of radiation therapy.¹

The direct and indirect effects of radiation induce more or less identical types of lesions in DNA. However, the density of lesions induced in a stretch of DNA is higher for high LET radiation, and this increased complexity is thought to complicate the repair of these lesions. Radiation-induced lesions consist of more than 100 chemically distinct base lesions, such as the mutagenic lesions thymine glycol and 8-hydroxyguanine.^2,4,5 Furthermore, damage to the sugar moiety in the backbone of DNA and some types of base damage can result in single-strand breaks (SSB). Because the energy deposition of radiation is clustered even for low LET radiation, it is possible that two individual strand breaks are formed in close proximity on opposite strands, resulting in the formation of a double-strand break (DSB). It has been estimated that 1 Gy of ionizing radiation gives rise to about 40 DSBs, 1,000 SSBs, 1,000 base lesions, and 150 DNA-protein cross-links per cell.² For a similarly lethal dose of UV light, about 400,000 lesions are required, demonstrating that the lesions induced by IR are much more toxic
than lesions induced by UV light. It is believed that DSBs are the critical lesions that lead to cell lethality following exposure to ionizing radiation.⁶

Although proteins and lipids are subject to damage following exposure to IR, the common belief is that DNA is the critical target for the biologic effects of radiation. Indeed, abrogation of DNA damage surveillance or repair processes in cells results in the enhanced induction of mutations and decreased cell survival following radiation.⁵ However, studies of radiation-sensitive and radiation-resistant bacteria imply that mechanisms that suppress protein damage may also play important roles in radiation resistance.⁷ Deinococcus radiodurans is a bacterium that can survive radiation exposures of up to 17,000 Gy, and its extreme radioresistance has been linked to high intracellular levels of manganese, which protect proteins from oxidation. The thought is that if a cell can limit protein oxidation, then its enzymes will remain active, and cellular functions such as DNA repair will be able to restore the integrity of DNA even after severe DNA damage.⁸ It would be interesting to explore whether the concentration of manganese can be manipulated to sensitize tumor cells to radiation therapy. Furthermore, because protein damage due to reactive oxygen species (ROS) accumulate during the aging process, could supplements of manganese turn back the clock on aging?

Ever since organisms started to utilize atmospheric oxygen for metabolic respiration many millions of years ago, they have been forced to deal with the cellular damage induced by ROS. Base excision repair (BER) evolved to remove many of the different types of oxidative base lesions and DNA SSBs induced by ROS. However, ROS seldom induce DSBs unless the generation of hydroxyl radicals is clustered near the DNA molecule. A more important source of intracellular generation of DSBs may instead be the process of DNA replication, and it is possible that homologous recombination (HR) repair primarily evolved to overcome DSBs sporadically induced during the replication process. The other major pathway of DSB repair is the nonhomologous end-joining (NHEJ) pathway, which is utilized by immune cells in the process of antibody generation. Although the HR pathway has high fidelity due to the utilization of homologous sister chromatids to ensure that correct DNA ends are joined, the NHEJ pathway lacks this control mechanism and therefore occasionally rejoins ends incorrectly. Thus, the NHEJ pathway may contribute to the generation of mutations following radiation (Fig. 8.2). However, NHEJ is the only mechanism available for DSB repair in postmitotic cells and cells in the G₁ phase of the cell cycle because no sister chromatids are available in these cells to support HR repair.

Due to the enormous task of replicating the whole genome during the S phase and segregating the chromosomes during mitosis, proliferating cells are generally much more vulnerable to radiation than stationary cells. To prevent cells with damaged DNA from entering into these critical stages of the cell cycle, cells can activate cell cycle checkpoints (see Fig. 8.2). The major sensor of radiation-induced damage in cells is the ataxia-telangiectasia mutated (ATM) kinase, which, following activation, can phosphorylate more than 700 proteins in cells.⁹ Two ATM substrates, p53 and Chk2, are critical for the activation of cell cycle arrests at multiple sites in the cell cycle.^10,11 The kinase p53 regulates the gene expression of specific genes such as p21, which inhibits cyclin-dependent kinase (CDK)2- and CDK4-mediated phosphorylation of the retinoblastoma protein, resulting in a block in the progression from the G₁ phase to the S phase of the cell cycle.^12,13 The Chk2 kinase promotes checkpoint activation in G₁ by targeting the cell division cycle 25 homolog A (CDC25A) phosphatase¹⁴ and, in G₂/M, by targeting the CDC25C phosphatase.¹⁵ The activation of a cell cycle arrest following DNA damage provides the cell with additional time to repair the DNA before entering critical cell cycle stages,
which promotes genetic stability. Loss or defects in the ATM or p53 genes result in abrogation of radiation-induced cell cycle checkpoints, which manifests itself as the highly cancer-prone human syndromes ataxia telangiectasia¹⁶ or Li-Fraumeni,¹⁷ respectively.

Terminally differentiated and stationary cells, such as kidney, lung, brain, muscle, and liver cells, are generally more resistant to radiation-induced killing than are cells with a high turnover rate, such as different epithelial cells, spermatogonia, and hair follicles. However, the spleen and thymus, which consist of mostly nondividing cells, are among the most radiosensitive tissues, implying that the rate of cell proliferation is not the sole determiner of the radiation sensitivity of a tissue. An important factor regulating the induction of programmed cell death (apoptosis) in tissues is the tumor suppressor p53.¹⁸ The p53 protein is activated in cells following exposure to IR by the ATM kinase (see Fig. 8.2). When activated, it regulates the expression of multiple genes that have roles in DNA repair, cell cycle arrest, and apoptosis. p53 can also localize to mitochondria following irradiation, where it triggers apoptosis through the inactivation of antiapoptotic regulatory proteins.¹⁹ Not all tissues induce the p53 response to the same degree after similar doses of IR, nor do they activate downstream pathways, such as DNA repair, cell cycle arrest, and apoptosis, in a similar way. For example, thymocytes have an intrinsic setting that favors apoptosis over cell cycle arrest following IR, whereas fibroblasts rarely induce apoptosis, but instead activate a strong and lasting cell cycle arrest.¹⁸

IR can induce cell death in tissues by many different mechanisms. Apoptosis can occur rapidly in a p53-dependent manner or later in a p53-independent manner. This later wave of radiation-induced apoptosis is often initiated by mitotic catastrophe, which occurs as a result of complications during chromosome segregation. Cell death induced by IR may in some cases be associated with autophagy, also called autophagocytosis, in which cells degrade cellular components via the lysosomal machinery. Whether autophagy is a programmed cell death or occurs in parallel with cell death is not clear. Interestingly, for some cell types, autophagy has been shown to actually protect the cells from radiation-induced death. Finally, tissue can undergo necrotic cell death following exposure to IR. Necrosis is a clinical problem following radiation therapy that can occur in normal tissues many months after treatment and can contribute to the inflammatory response.