Radiation biology applied to clinical radiotherapy is concerned with the relationship between a given absorbed dose of radiation and the consequent biologic response; of particular interest are factors that modify this relationship. With increasing radiation dose, radiation effects may increase in severity (i.e., grade), in frequency (i.e., incidence), or both. In most cases, it is the relationship between dose and incidence that is important. Such dose-response curves have a sigmoid (S) shape, with the incidence tending to zero as dose tends to zero and the incidence tending to 100% at very large doses. This applies to both tumor control and normal tissue complications.

A simple example is shown in Figure 19.1. Tumor control probability (TCP) is plotted as a function of total dose, and the incidence of normal tissue complications is also plotted as a function of dose. What is illustrated is a favorable situation where the tumor is more radiosensitive than the normal tissue. In the case of tumor control, the shape can be explained solely from the random nature of cell killing (or clonogen survival) after irradiation and the need to kill every single cell to achieve a cure.

For most normal tissue end points, the biologic interpretation of the S shape of the relationship is not obvious. Some researchers have evoked a hypothetical tissue rescue unit (TRU), arguing that tissue breakdown occurs when the number of TRUs falls below a critical level; however, this explanation is questionable.

Mammalian cells exposed to ionizing radiation can die through various mechanisms: mitotic-linked cell death, necrotic cell death, apoptotic cell death, autophagic cell death, and bystander induced cell death. The underlying mechanisms and importance of each of these forms of cell death have been described in Chapters 3, 4, and 18 in detail and will only be briefly reviewed here. Regarding normal tissues that are exposed to ionizing radiation, mitotic-linked cell death and apoptotic cell death have been studied the most and are responsible for most cell killing by ionizing radiation. The other forms of cell death may also contribute to cell killing, but their mechanism and importance are still under investigation. One additional point to consider is that ionizing radiation also induces a form of senescence or permanent growth arrest in which cells are still metabolically active, but reproductively inhibited. This is best exemplified by fibroblasts that when irradiated in cell culture, stay attached to plates for weeks but never divide. However, they are able to secrete growth factors and mitogens that promote the growth of tumor cells. Senescence has been largely studied in cell culture in the laboratory, and only recently has evidence been accumulating that it occurs in tissues. Senescence is also discussed in more detail in Chapter 18.

Most cell lines cultured in vitro die a mitotic death after irradiation; that is, they die attempting to divide. This does not necessarily occur at the first postirradiation mitosis; the cell may struggle through one, two, or more mitoses before the damaged chromosomes cause it to die, attempting the complex task of cell division. Time-lapse films of irradiated cells cultured in vitro clearly show this process of mitotic death, which is the dominant cause of death if reproductive integrity is assessed in vitro as described in Chapter 3.

It is not, however, the only form of cell death. Programmed cell death, or apoptosis, occurs in normal tissues and neoplasms, in mammals and amphibians, in the embryo, and the adult. It is implicated, for example, in tissue involution such as the regression of the tadpole tail during metamorphosis. It is the programmed cell death that is common during embryonic development. It also can occur after irradiation. Apoptosis, like mitosis, comes from the Greek word meaning “falling off,” as of petals from flowers or leaves from trees.

Apoptosis is characterized by a stereotyped sequence of morphologic events, which take place in two discrete phases. In the first phase, cells condense and bud to produce many membrane-enclosed bodies. In the second phase, these bodies are phagocytized and digested by nearby tissue cells. The characteristic “laddering” of DNA that occurs during apoptotic death is illustrated in Chapter 3. Apoptosis characteristically affects scattered individual cells. If apoptosis affects cells in tissues, the resulting apoptotic bodies are squeezed along the intercellular spaces and are either shed from the epithelial surface or rapidly phagocytized by nearby cells. The cells surrounding those being deleted merely close ranks, and there is no tissue disorganization that occurs after necrosis.

Autophagic cell death has been classified with apoptotic cell death but, in fact, is distinct and is controlled by a unique set of genes. Apoptosis and autophagy may be linked because cells defective in some forms of apoptosis exhibit increased levels of autophagy. The term implies that cells cannibalize themselves in the hope of generating adenosine triphosphate (ATP) and macromolecule precursors to survive. Growth factor withdrawal, inhibition of protein synthesis, hypoxia, and ionizing radiation are all potent inducers of autophagy. Although autophagy can be found in cells dying by stress, it is unclear whether it represents a drastic means for the cell to survive by digesting part of itself, or whether it actually promotes cell death. Evidences for both possibilities exist in different cell types and will require further studies to clarify its role in irradiated cells.

Necrotic cell death occurs when cells are devoid of precursors to generate energy, such as glucose, are exposed to decreased pH, or exhibit changes in osmolarity. Ionizing radiation is also able to induce necrotic cell death. It differs from other forms of cell death because it does appear to have a definitive genetic program that controls it, although there have been reports that prove otherwise.

The long-standing concept that ionizing radiation kills cells through direct exposure requires amendment. The demonstration that cells not exposed to ionizing radiation can be killed by being close to irradiated cells has coined the term “bystander induced cell death.” Therefore, the possibility exists that ionizing radiation can influence cell killing outside the field of radiation. This has been experimentally shown for tumors and is known as the abscopal effect. Such effects for normal tissues probably also exist in lymphopenia found after radiotherapy to nonlymphoid organs, and most probably represent an immune response.

Most experimental techniques are available to obtain dose-response relationships for the cells of normal tissues. First, there are a limited number of clonogenic assays—techniques in which the end point observed depends directly on the reproductive integrity of individual cells. These systems are directly analogous to cell survival in vitro. The techniques developed by Withers and his colleagues are based on their observation of a clone of cells regenerating in situ in irradiated tissue. Skin colonies, regenerating crypts in the jejunum, testes stem cells, and kidney tubules are described briefly later in this chapter. It is also possible to obtain dose-response curves for the cells of the epithelial lining of the colon or stomach, but the method used is essentially the same as for the jejunum. Kember described a system for scoring regenerating clones in cartilage at about the same time as Withers’ skin colony system, but it has not been used widely and is not discussed here.

The assay system for the stem cells in the bone marrow or cells of the thyroid and mammary gland depends on the observation of the growth of clones of cells taken from a donor animal and transplanted into a different tissue in a recipient animal. In Till and McCulloch’s bone marrow assay, colonies of bone marrow cells are counted in the spleens of recipient animals. Dose-response curves for mammary and thyroid cells have been obtained by Gould and Clifton by observing colonies growing from cells transplanted into the fat pads of recipient animals.

Second, dose-response relationships that are repeatable and quantitative, but that depend on functional end points, can be obtained. These include skin reactions in rodents or pigs (e.g., erythema and desquamation), pneumonitis or fibrosis in mouse lungs reflected in an increased breathing rate, myelopathy of the hind limbs from damage to the spinal cord, and deformities
to the feet of mice. The end points observed tend to reflect the minimum number of functional cells remaining in a tissue or organ, rather than the fraction of cells retaining their reproductive integrity.

Finally, one can infer a dose-response curve, for a tissue, which cannot be observed directly by assuming the form of the dose-response curve (linear quadratic) and performing a series of multifraction experiments. This procedure, first suggested by Douglas and Fowler, has been used widely to infer values for α and β in the dose-response relationships for normal tissues in which the parameters cannot be measured directly.

This chapter includes assays for both early- and late-responding tissues. The skin, intestinal epithelium, and bone marrow cells, for example, are rapidly dividing self-renewal tissues and respond early to the effects of radiation. The spinal cord, lung, and kidney, by contrast, are late-responding tissues. This reflects the current philosophy that the radiation response of all tissues results from the depletion of the critical parenchymal cells and that the difference in time at which early- and late-responding tissues express radiation damage is a function simply of different cell turnover rates. Many older papers in the literature ascribe the response of late- responding tissues to vascular damage rather than to depletion of parenchymal cells, but this thesis is becoming increasingly difficult to accept.

The various types of normal tissue assay systems are described briefly. The reader who is content with the summary already given may wish to skip the remainder of this chapter.