Effect of Radiation on Biological Structures. Radiation Mutagenesis

Chapter 35

Effect of Radiation on Biological Structures. Radiation Mutagenesis Chapter Outline 35.1 Damages in Genetic Apparatus: Mutations 35.2 Apoptosis and Necrosis 35.3 Mutation Production 35.3.1 Spontaneous Mutations 35.3.2 Radiation Effect 35.3.3 Chemical Effect 35.3.4 Viruses 35.4 Adaptive Mutations 35.5 Epigenetics in Radiation Biology 35.6 Radiobiological Paradox 35.7 Direct Action 35.8 Indirect Action 35.8.1 Effects of Free Radicals 35.8.2 Oxygen Effect

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35.8.3 Other Molecules (Except Water) as Mediators 35.8.4 Radiotoxins 35.9 Radioadaptive Response 35.10 Hyper-Radiosensitivity and Increased Radioresistance 35.11 Bystander Effects 35.12 Survival Rate 35.13 The Dependence of Radiation Exposure on Linear Energy Transfer 35.14 Radiosensitivity of Tissues, Organs, and Organisms 35.15 Long-Term Consequences 35.16 Radiation Sickness References

457 458 458 459 460 461 464 466 466 467 470

35.1 DAMAGES IN GENETIC APPARATUS: MUTATIONS The information about the cell activity is stored in the genome, i.e., in the set of genes, which are located in a linear molecule DNA, and also in the epigenome, which controls the switching on and off of genes. Damages in the genome and epigenome are called mutations. Because of their importance in the life of an organism, one needs to consider that it is the DNA molecule that is the main subcellular target for radiation damage. The damage might occur directly within the DNA molecule (such as strand breaks or modification of nucleotides) and/ or the radiation can affect an epigenetic mechanism, controlling the gene expression. Actually, according to the definition first given by the Dutch botanist and geneticist Hugo de Vries, who rediscovered Mendel’s laws in 1901, mutations (from Latin mutatiod“change”) are any changes in the genetic apparatus, throughout the genome, in individual chromosomes, their parts, or in a particular gene. Genomic and chromosomal mutations (the latter are called chromosomal aberrations [CAs]) lead to major alterations in the structure of the genetic apparatus. Gene mutations responsible for violations of the nucleotide sequence in the DNA molecule are most likely to occur. These mutations, being the main result of the effects of low doses of radiation, are discussed in this section. For a physicist, it might be easy to identify classification of mutations with the classification of structural defects of the crystal lattice. Defects can be point, associated with a particular lattice site, or extended, affecting several sites. Generally, point defects of crystal structure are vacancies (i.e., absence of an atom in a lattice site), insertions (i.e., interstitial atoms), and substitutions (an atom occurs in the site or an interstitial atom is replaced by an impurity atom). Thus, if a crystal is made of one kind of atoms, e.g., the typical semiconductor (such as silicon), the range of simple defects is limited to those listed above. If the crystal is more complexly arranged, like a typical ionic crystal of NaCl, it expands the set of defects. Anion and cation vacancies are two different defects that have significantly different properties. Extended defects are

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dislocation, block boundaries, cracks, blisters, and other similar defects that disrupt the structure of the crystal much more significantly than point defects. Similarly, in DNA one can distinguish a group of point mutations. Point mutations are minor; they often affect just one base. This does not mean that they are of little significance. As we can see below, point mutation can significantly affect a cell’s function. Point mutations can be of the following types: l l

l

Deletions (vacancies): an absence of one or more bases in one site. Substitution: a nucleotide pair substitution. In this case, substitutions may be simple (they are called transitions), those, when purine is substituted by purine (A 4 G) and pyrimidine by pyrimidine (T 4 C), or complex (they are called transversions), when pyrimidine substitutes purine and purine substitutes pyrimidine. Insertions (or additions): inserting one or more nucleotides at new locations in the DNA molecule. Unlike the similar crystal lattice defects, interstitial atoms, wherein an atom is not in a regular position but between them, DNA insertion implies appearance of a new place in the DNA chain.

Point mutations can be performed in alphabetic letters. Considering the example, we have to remember the following: first, the three-letter codons in the DNA molecule occur in a row, with no gaps, as shown in the first row. Second, the genetic code consists of four letters, A, G, T, and C, and we are forced to use more for illustration. Therefore, THEFATCATATETHEFATRAT .: a normal gene THE FAT CAT ATE THE FAT RAT: a normal gene with distinct codons THE FAT SAT ATE THE FAT RAT: substitution (S instead of C in the third word) THE FAT CAT THE FAT RAT: deletion of a number of bases, divisible by three (ATE - fourth word) THE ATC ATA TET HEF ATR AT .: deletion of a single base (F - in the second word) THE FAT DOG CAT ATE THE FAT RAT: insertion of a number of bases, divisible by three (DOG) THE FAT CAL BTA TET HEF ATR AT .: insertion of two bases (LB - in the third and fourth words). In these examples, deletion and insertion of mutation types are shown in two variants, when deletion or insertion of the bases is divisible or not divisible by three. Because the genetic code is a triplet, examples with insertion and deletion of bases by a number not divisible by three demonstrate the type of mutation called “frame shift.” A shift in codons’ partitioning completely changes the content of the phrase and, hence, changes at least the part of the protein that comes after the shift. The alphabetic example presented above illustrates very clearly the changes in codons (three-letter words) caused by different types of mutations. However, it is useful to show point mutations and the changes caused by them using the example of a flat DNA model. At least certain “mutations” make a phrase within the literal example shown above completely meaningless, unreadable. In DNA, the situation is somewhat different. Among the 64 codon variants (Section 34.2.7), only three codons are meaningless, i.e., they have no matching amino acid (UAA, UAG, UGA). They stop protein synthesis. Therefore, the code resulting from a mutation corresponds to some protein, but it is very unlikely that such a protein would be neutral or even beneficial. Regarding mutations that alter only one codon, there can be different scenarios. Because the code is degenerate, i.e., several codons correspond to one amino acid, it may accidentally happen that a codon replacement does not alter the type of amino acid and, therefore, does not change the protein. Sometimes, the replacement of one amino acid has little effect on the properties of the protein. It is possible that a mutation occurs in an area that does not encode a protein, i.e., in an intron. It also does not affect the structure of the protein, although it may affect its expression. But there are times when this replacement plays a huge role. Often cited is an example of mutation associated with the replacement of the sixth amino acid in the hemoglobin’s beta chain. There must usually be a Glu (glutamic acid), but as a result of mutation, a Val (valine) takes its place. This mutation is responsible for a severe disease called sickle cell anemia, wherein the oxygen-carrying cells in the blood (red blood cells) change their shape and they pretty much lose their ability to carry oxygen. Another example is substitution of one amino acid (replacing lysine by glutamate) of 487 amino acids arranged sequentially in a ferment aldehyde dehydrogenase, which is responsible for removing from an organism the acetaldehyde that accumulates during oxidation of ethanol. This leads to an increased sensitivity to alcohol, common in many Asian nations. A deletion of three nucleotides in the definite gene (CFTR) results in the loss of the amino acid phenylalanine and causes an incorrectly folded protein and a disease called cystic fibrosis. This disease is associated with thick, sticky mucus

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in the lungs and trouble breathing, salty sweat, infertility in certain individuals, and a shortened life expectancy. This deletion does not shift a reading frame because three bases next to each other are deleted, and all the other amino acids in the chain remain the same. TayeSachs disease is another recessive disorder caused by point mutations in the HEXA gene on chromosome 15. TayeSachs causes nerve cells to deteriorate over time, which in turn results in the decline of physical and mental functioning. Some well-known inherited genetic disorders include phenylketonuria and color blindness, among many others. All of these disorders are caused by the mutation of a single gene. The list of diseases, caused by point mutations and by more complex chromosome abnormalities as well, one can find, e.g., on the site [1]. Practically, in Wikipedia, there is an article devoted to each chromosome, e.g., “Chromosome 1” [2], and others up to X and Y, and each article has a section “Diseases and disorders” or “Role in diseases.” Thus, point mutations may produce three effects: 1. Preservation of a codon meaning (silent mutation). Example: DNA codon TTC turns into codon TTT, after transcription mRNA codon AAG turns into AAA. Both, AAG and AAA correspond to the same amino acid Lys (lysine). 2. Changing a codon meaning (missense mutations). Example: DNA codon TTC turns into TCC, and then mRNA codon turns into codon AGG, that codes the amino acid Arg (Arginine). 3. Forming a meaningless codon (nonsense mutations). Example: DNA codon TTC turns into ATC, and then mRNA codon turns into codon UAG, that means termination (stop codon). Point mutations are also called simple mutations, otherwise single-site or single-base mutations. Not only bases can be damaged by various factors but also sugarephosphate backbones of the DNA molecule can be damaged. Rupture of one strand is usually relatively easily fixed by the repair mechanisms of a cell. Rupture of two strands is much harder to repair, and it leads to very serious consequences. Modern methods of research, in particular method of gene sequencing, let to determine the ratio of different types of mutations, at least in cells of cancer tumors. Studies to date indicate that the number of point mutations is roughly 10 times greater than the number of chromosomal changes. About 95% of point mutations are single-base substitutions. And of the single-base substitutions, w90% result in missense changes, w8% result in nonsense changes, and w2% result in alterations of splice sites or untranslated regions immediately adjacent to the start and stop codons [3]. In various tissues, the relation of mutations can slightly differ, but the order of values remains the same. It is useful to note that in addition to direct mutations discussed above, there may also occur reverse mutations, i.e., a return to the original order. For example, the famous Ames test, now the main test of chemical carcinogenicity, is based on reverse mutations. Mutations in somatic cells have been already discussed above. But mutations can also occur in gametal cells. In this case, if the mutations are not removed by a repair mechanism or do not lead to immediate cell destruction, they are inherited by descendants. In this case, the possible options are the following: 1. These mutations have no impact on the phenotype. We have already pointed out why some mutations may not affect the life of the cell. 2. Minor harmless changes occur in the phenotype. One of genetics reference gives an example of such a change: a kitten’s ear is dangling a little bit. 3. Mutations lead to serious changes in the offspring’s phenotype. For example, a single mutation can make an insect be insensitive to the action of a well-known pesticide, called DDT. Mutations play crucial role in changing species, but usually they turn out to be lethal and lead to the death of the organism. It is obvious that in this case, such a mutation does not get inherited and will cause no more damage. Some sections of DNA encode genes that control other genes, indicating when and where they should start operating. Mutations in this part of the genome may have a much more serious impact on the way the organism is formed. Difference in mutations in control and normal genes can be compared with the difference between instructions given to a trumpeter or to the conductor of the orchestra in which this trumpeter plays. Obviously, the instructions to the conductor will have a much more serious impact on the performance than those to just one member of the orchestra. Thus, mutations in control genes may entail a cascade of changes in behavior of the genes controlled by them. The mutations in special control genes that strongly affect the cell life in proto-oncogenes and in tumor suppressor genes are discussed in Chapter 36. Mutations can be broadly categorized as beneficial, neutral, deleterious, or lethal with respect to their effects on biological fitness.

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35.2 APOPTOSIS AND NECROSIS Apoptosis is the genetically determined process of programmed cell death. Using apoptosis, the body gets rid of unnecessary, “used,” or defective cells. Apoptosis is activated by external signals and triggers enzymes, which dismantle the cell into parts and turn them out into the intercellular space and, further, out of the organism by excretory system. Signals that activate apoptosis are confronted by other signals that block apoptosis. Failure to produce the stimulatory signal is essential for unlimited growth of cancerous tumors. By violating production of inhibitory signals, viruses prevent the premature death of the host cell by apoptosis. In the process of apoptosis, the cell membrane collapses, cytoplasmic and nuclear skeletons break apart, cytosol displaces, chromosomes degrade, and the nucleus gets fragmented. As a result of apoptosis, a cell gets broken down into fragments in 30e120 min. Fragments are absorbed by neighboring cells and disappear, which normally takes 24 h. Biologists call apoptosis an internal police officer or biological flusher. Another possible way of cell death is known that is not programmed in an organism, and that is necrosis. Necrosis can be caused by external influence on a cell, by exposure to pathogenic bacteria or viruses, by stopping its blood supply, or by some other reasons. Necrosis in contrast to apoptosis is accompanied by inflammatory reactions.

35.3 MUTATION PRODUCTION The process of mutation is called mutagenesis. There are several sources of mutations. They are spontaneous mutations and mutations that can be caused by external factors, i.e., radiations (ultraviolet [UV] and ionizing), chemicals, and viruses.

35.3.1 Spontaneous Mutations Spontaneous genetic mutations are determined by errors arising from thermal motion of atoms and molecules during DNA replication and repair, as well as from some endogenous oxidative processes. These mutations account for background mutations, exceeding those that may be caused by other external factors. Two well-known and most common types of spontaneous damages of DNA are depurination and deamination. Depurination consists of the interruption of the glycosidic bond between the base and deoxyribose and the subsequent loss of a guanine or an adenine residue from the DNA. The deamination of cytosine yields uracil. Unrepaired uracil residues pairs with adenine in replication, resulting in the conversion of a GeC pair into an AeT pair. The both reactions are shown in Fig. 35.1.

FIGURE 35.1 Spontaneous DNA damage: (A) depurination (loss of purine bases) resulting from cleavage of the bond between the purine bases and deoxyribose, leaving an apurinic site in DNA; (B) deamination (converts cytosine into uracil; adenine to hypoxanthine).

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The frequency of spontaneous mutations is quite high. In the case of depurination, every mammalian cell spontaneously loses about 10,000 purines from its DNA in a 20-hour cell-generation period. As a result of deamination, every cell loses up to 500 pyrimidine bases per cell in a day. In both cases, these mutations would result in significant genetic damage. Fortunately, efficient repair systems almost completely remove damaged sites, or they could be eliminated by apoptosis. The remaining unrepaired or misrepaired DNA are just a natural mutation background. One more source of the spontaneous point mutations is the tautomeric shift, i.e., the spontaneous changes of canonical tautomeric forms of nucleobases into their rare forms. In normal nucleic acids, nucleobases have their canonical forms: amino forms for adenine and cytosine and lactam (keto) forms for guanine and thymine. Nevertheless, their rare tautomers, imino, and lactim (enol) forms are also possible (Section 33.7.2.3). In normal DNA, cytosine (C) is paired with guanine (G), and thymine (T) is paired with adenine (A). However, the pairing can be impossible when the tautomeric preferences change. For example, the rare isomer of cytosine can be paired with adenine, and during DNA replication it can be replaced by thymine leading to the GC / AT transition. The reason for the tautomerism is not only heat motion but also proton tunneling [4,5]. For a long time, the frequency of spontaneous mutations has been estimated indirectly or from theoretical considerations. In particular, the frequency of mutations was evaluated by the result of the activity of the genetic apparatus revealed by the number of errors in the amino acid composition in protein synthesis. The analysis of fibrinopeptides (fibrinopeptides do not have any special function, and, therefore, they are tolerant to almost all amino acid substitutions and were not eliminated from populations by selection) shows that a protein of medium size, consisting of 400 amino acids, changes randomly as a result of one amino acid replacement approximately every 200,000 years. The development of the method of sequencing allows to determine the types and frequency of mutations per pair base or per genome that occur in replication or accumulated over a certain number of generations for different objects from bacteria to humans. The error rate of DNA replication in humans is close to 10
8 for replication complexes that are capable of proofreading. Many errors are fixed by repair enzymes, and this process is about 99% efficient. Thus, the overall error rate is close to 10
10 mutations per base pair [6]. The values of mutation frequency in various organisms are presented in Table 35.1. One can find the latest information about spontaneous mutations in the papers [3,6,8,9]. It is useful to consider that the background mutations that can be revealed in experimental tests of genome sequencing are a collection of real spontaneous mutations, mutations caused by natural background ionizing radiation and natural background chemicals in the environment. For a long time, it was almost impossible to separate mutations caused by various factors. But the set of mutation types for different methods of influence on the genome varies. The mutation spectrum induced by radiation is different from those of spontaneous mutations or mutations induced by chemical mutagens, the majority of the latter being consequences of point mutations. Ionizing radiations can induce a wider spectrum of mutations, from point mutations in single genes to the absence of several genes and chromosome abnormalities. Apparently, soon with the development of the genome sequencing, we could get the chance to determine the source of mutations. On the role of spontaneous mutations in induction of cancer, see Chapter 36.

TABLE 35.1 Frequency of Mutations in the Various Organisms. Numbers are Taken From the Site [7] Organism

Per base Pair/replication


10

E. coli Arabidopsis Drosophila Mouse Human

Per base Pair/generation 10

e10


10

7$10


10

10
8


10


8

2$10 3$10 2$10


10

10


9

Per Genome/replication 5$10
4e5$10
3


9

0.06

10

0.5
8

(1e4)$10

0.2e0.1

Genome sizes: E. colid5$10 base pairs; human haploid genomed3.2$10 base pairs, correspondingly the diploid genome consists of 6.4  109 base pairs. 6

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35.3.2 Radiation Effect The main object of research in this book is ionizing radiation, i.e., nuclear particles and X-ray quanta. Photons from the short wavelength part of the UV spectrum can also ionize at least some molecules. The large part of the spectrum can excite organic molecules. As excitation of nucleobases by the UV light produces a peculiar and important effect, it needs some attention to be paid. The destructive effect of UV radiation on the structure of DNA is well known. A quantum of UV radiation transfers energy to a nitrogenous base and it does transfers it to an excited state. Because of the structure of the electron shells of adenine, guanine, and cytosine molecules, the excitation energy gets converted into heat quickly in a nonradiative fashion. In the case of thymine (and only when it adjoins in the chain with other thymine), the excitation energy causes a chemical reaction between adjacent thymine molecules. As a result, a new chemical compound, thymine photodimer, appears somewhere in the DNA chain. Enzymes that control replication or transcription stop in this place, having met with a strange molecule and, depending on where in the genome it happened, either the further operation of the cell becomes impossible, and thus becomes likely to die, or the mutation persists, and after a few such mutations the cell degenerates into a cancerous one. To eliminate such mutations, evolution developed a special repair system. Interestingly, this system exists in all cells, including those that are never exposed to sunlight, for example, in intestinal epithelial cells. Ionization and excitation of DNA molecules after a set of relaxation processes can lead to molecule dissociation and result in the loss of some nucleobases or the breaking of one or both DNA strands. These induced by radiation effects are the primary events leading to mutagenic effects. Thus, mutations are produced as the result of damages caused by the radiation or other environmental agent action. Here, it is useful to clarify that biologists call mutations just the only damages that cannot be eliminated by DNA repair mechanisms. The estimations show that the number of spontaneous DNA damages that occurred in metabolic processes is many orders greater than the number of damages induced by natural radiation background. This is the important argument for the conclusion that radiation of natural background adds almost nothing to the spontaneous damages.

35.3.3 Chemical Effect It was determined that although chemical mutagens differ significantly in their structure, they have one thing in common, and that is electrophilicity. A genotoxic chemical entering the body with food, drinking, breathing, or through the skin passes through numerous barriers and undergoes metabolic transformations before hitting the target cell nucleus. The transfer of an electron from the nucleophilic portion of the DNA molecule to the electrophilic mutagen molecule can lead to a binding of electrophilic metabolites with DNA and to the formation of an adduct or to destruction of the DNA section via several processes after dissociative electron attachment reactions. The mutagenicity of chemicals can be determined with some bacterial mutagenicity tests. The most famous and widely used is the so-called the Ames test. B.N. Ames used a certain strain of Salmonella bacteria (Salmonella typhimurium), which as a result of mutation lost the ability to synthesize histidine for itself (biologists call this histidine auxotrophy). Therefore, in the control group, S. typhimurium do not grow on medium without histidine. The test substance is usually treated by a specially prepared fraction of rat liver for metabolic activation. To identify “direct” mutagens, the research can also be conducted without metabolic activation. Bacteria are treated with a test chemical and are incubated for a certain period. If the test chemical and/or its metabolites possess mutagenic activity, they will induce reverse mutations from auxotrophy to prototrophy for histidine, otherwise known as backward mutations. As a result, the bacteria recover the ability to produce this amino acid and live on nonhistidine medium.

35.3.4 Viruses First of all, let us recall what viruses are. Viruses (from Latin virus [poison]) are the smallest subcellular particles that can reproduce only inside a living cell. Outside the cell, viruses do not show signs of life and behave as particles of organic polymers. In terms of their size, viruses occupy the place between the smallest bacterial cells and the largest organic moleculesdfrom 0.02 to 0.3 mm. They are not visible in optical microscopes and pass through ultrathin filters. To define them, the term “filterable viruses” was used for a long time. Viruses contain only one type of nucleic aciddeither RNA or DNA (all cellular organisms contain both DNA and RNA at the same time). Viruses can be regarded as genetic elements, covered in a protein shell and capable of movement from one cell to another.

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For this section, it is significant that viruses can be incorporated into the genome of the host cell and, thereby, reprogram its systems. Therefore, with some exceptions, it can be assumed that viruses cause mutations.

35.4 ADAPTIVE MUTATIONS For a long time, in biology, the principle dominated that all genetic mutations are blind and occur randomly. This principle was experimentally confirmed in the well-known fluctuation by the S. Luria and M. Delbrück test. This test significantly contributed to the general achievement of the authors was marked (in association with A. Hersehy) by the Noble prize in Physiology and Medicine in 1969. In 1988, a serious challenge arose versus generally accepted ideas of the random nature of the mutations. J. Cairns and his colleagues studied the bacteria E. coli, though with a specially derived strain FC40, in which as a result of mutation the gene encoding the enzyme needed to consume milk sugardlactosedwas deactivated. Placed on a nutrient medium containing only lactose, the cells did not have to develop because they could not consume lactose. However, experience has shown that at a rate greater than the established rate of spontaneous mutations, reverse mutations occurred in the culture of bacteria and bacteria appeared to be able to feed and grow on lactose-containing media [10,11]. The occurring mutations were beneficial and specific to starvation conditions in which the bacteria were placed. In response to the hostile environment, the cells are mutated in a way that allows them to survive. Such mutations have been called adaptive or directed mutations. The errors were tried to be found in this experiment, but so far without success. Similar results were obtained with other bacteria and yeast [12]. Results obtained in this and in a widening amount of following works challenged the Central Dogma of molecular biology. According to this Dogma, genetic information transfers only in one direction, from DNA to protein. Between genotype and phenotype, there is a barrier, so-called “Weisman barrier.” The existence of adaptive mutations means that the body can adjust its genetic apparatus under the influence of environment. This situation corresponds to the previously discarded the Lamarck theory of the inheritability of acquired characteristics (Jean-Baptiste Lamarck, 1809) [13]. Moreover, adaptive mutations gave a trump card to supporters of the teleological “Intelligent Design.” Several mechanisms that could explain adaptive mutations have been suggested, but no one could be accepted as final. It is interesting to point out that the approach to the problem is based on quantum mechanics [14e16]. The investigations of adaptive mutations continue. Scientists have observed that an apparent “back channel” for genetic information called retromutagenesis can produce conditions for adaptive mutation to happen in bacteria. The adaptive mutations may explain how bacteria develop resistance to some types of antibiotics under selective pressure, as well as how mutations in cancer cells enable their growth or resistance to chemotherapy drugs [17]. The last results of the investigation of adaptive mutations in yeast provide evidence that 35% of the mutations identified in experimentally evolved populations are advantageous, and that the distribution of beneficial fitness effects depends on the genetic background and the selective conditions. The authors have pointed out that the analysis of the recent publications shows that the list of mutations, associated with adaptation to different conditions, has dramatically increased [18]. Basic information about adaptive mutations has been obtained on bacteria and yeasts. Nevertheless, it is reasonable to believe that adaptive mutations can occur in the human genetic apparatus. Some data on this opportunity are discussed in Chapter 53.

35.5 EPIGENETICS IN RADIATION BIOLOGY To date, convincing evidence has been obtained that the disruption of the cell’s operation under the action of radiation is not limited to those effects that were taken into account earlier in the framework of classical genetics, and that the direct breakdown of the DNA structure is not always the cause of the malfunctioning or cell death. The investigations, mainly for last 10e20 years, have shown that phenomena, which are difficult to be explained within the framework of classical genetics, receive or promise to receive quite reasonable explanations from epigenetics. The connection of the epigenetics phenomena with the bystander effects (Section 35.11), remote consequences (Section 35.15), and radiation-induced genomic instability (RIGI) (Section 35.11) has been found. It is significant that the effects of irradiation can be manifested with a noticeable shift in time after irradiation, and not in the cells that are irradiated. In particular, the reaction to irradiation can be exhibited by the descendants of irradiated cells [19e26]. Radiation affects all components of epigenetics, but the methylation process has been most thoroughly studied (Sections 33.6.3 and 34.3.2). It is known that the methylation of embryonic cells is either very weak or nonexistent. This makes it possible to activate any necessary genes and makes cells pluripotent. With the development of the body, cells have already acquired their specific role and many genes must be turned off. The turn-off is achieved, in particular, by

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methylation of the corresponding DNA regions. Irradiation reduces the level of methylation. It has been shown that under the effect of radiation with a low linear energy transfer (LET) (X-ray and gamma) hypomethylation occurs. The detailed mechanism of the effect of irradiation on the state of methylation is not known, but it is assumed that irradiation affects the production of methyltransferase, an enzyme that performs the methylation procedure. Usually, a change in the methylation regime affects the expression of genes, but when exposed to radiation, these changes also dramatically affect the process of repair of DNA, which is damaged by radiation. Irradiation leads to an increase in the breakage of DNA chains and to a change in the content of key proteins responsible for the installation of methyl labels on DNA. Such effects were observed even in the parts of the body protected by lead and located at a distance of about 1 cm from the irradiated region. It was shown that when the rat’s cranial area is irradiated with the X-rays, hypomethylation takes place in the nonirradiated organ, which is far enough from the irradiated one, e.g., in the spleen. The effect was recorded 24 h after the irradiation but could be observed after 7 months. This is a very important result for the elucidation of the mechanism of carcinogenesis. The cancer develops very slowly and requires a long time for the damages to exist before they become a clinically definable tumor. Epigenetic changes caused by irradiation can persist for 10 years. They can also be observed in the offspring organisms. The danger of disrupting DNA methylation is confirmed, in particular, by the fact that hypomethylation is one of the well-known characteristics of cancer cells. However, in cancer cells, the hypomethylation of certain loci is often accompanied by hypermethylation of other DNA regions responsible for the activation of tumor suppressor genes (antioncogenes). The decrease in methylation of DNA caused by radiation correlates with histone methylation processes. It was shown, for example, that hypomethylation in human breast tumors is accompanied by a decrease in the trimethylation of the amino acid lysine in a particular histone. The development of epigenetics has led to the emergence of the “epigenome” idea, so that the phenotype of any organism is the total realization of the genome and epigenome. Breakings of the epigenome are associated with the notion of epimutation. The difference between epimutations and true mutations is in the fact that the new phenotypic variation transferred from somatic cells to the genitals can gradually “fade out” in a series of generations if the conditions that induce this variation are not subsequently maintained.

35.6 RADIOBIOLOGICAL PARADOX Before discussing the biological effects of ionizing radiation on living cells, the fundamental paradox of radiobiology should be discussed. Absorption of a negligible amount of radiation energy causes a grave shock to an organism or even its death. For example, irradiation by a dose of 10 Gy, i.e., 10 J/kg, received in a relatively short period of time (hours, days), kills any mammal. At the same time, it is easy to calculate that about 10 J of light energy fall to a human body in 1 min at noon on a sunny day. (However, sunlight is absorbed only by the surface of skin, but ionizing radiation is absorbed in the volume.) In literature, there are also other comparisons. For example, in biological tissue with a volume of 1 mm3 containing 1010 atoms, a dose of 10 Gy produces only about 1000 ionizations. In other words, the lethal dose of 10 Gy affects a negligible number of molecules in a given volume. This dose absorbed by a person weighing 70 kg is equivalent to the thermal energy of only 170 calories. The maximum heating of the human body will not exceed 0.001 C (the thermal effect is approximately equivalent to drinking a glass of hot tea). Attempts to solve the radiobiological paradox led to the target theory. Most of the 20th century physical concepts dominated in radiobiology, according to which a cell is a complex of targets. The German scientist, biophysicist, and philosopher Friedrich Dessauer was one of the first to provide a solution. He applied the principles of quantum mechanics and nuclear physics to the problems of radiobiology. He introduced the concept of discontinuity and quantization of energy absorption and the probabilistic nature of absorption events. Dessauer called these events “spot heating” in certain discrete microscopic volumes. Although ionizing radiations have a low average bulk density, individual charged particles have a very considerable energy compared with the energy of chemical bonds released in a relatively small volume. Probabilistic nature of the manifestation of the radiobiological effect in the cells was due to the statistical distribution of energy absorption events. Now, we understand that these local discrete events are molecule ionization acts. The event described, of local release of high energy, was called the hit principle. Further development of the principle of direct effect is associated with the works of N.V. Timofeev-Resovskij, K. Zimmer, M. Delbrück, J. Lee, and several other researchers (see Ref. [27]). The main question of direct radiation effect was the following: how many targets one needs to inactivate in a cell to kill it? The concept of one- and two-impact chromosome damage was proposed by Carl Sachs in 1938. Sachs, his contemporaries, and predecessors worked mainly with plant cells. Later, methods of work with mammalian cells in vitro were

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developed. This made it possible to move from working with plant cells or bacteria to obtaining clear quantitative results with animal cells. The main method of research was to analyze survival curves (Section 35.12). Note that reliable results are obtained from the survival curves only at relatively high doses. Effects of radiation on living organisms can be considered on three levels. First, the effect of radiation on molecules is discussed in Chapters 4, 8, and 16. Secondly, the effect of radiation on cells will be dealt with now. Finally, the effects of radiation on the entire organism are discussed in Section 35.16. Biological changes under the influence of ionizing radiation may occur immediately after irradiation, during the first few seconds. Such effects are called primary. But some processes can occur with a large time delay, years or even decades later. Such effects are called long-term effects. Let us start with the primary processes. These include direct and indirect effects of radiation.

35.7 DIRECT ACTION Direct action is direct transfer of charged particle energy to the functional molecules of a cell. As a result of the processes described for the first stages of radiation effect on a substance (Section 16.2), a molecule can be damaged and cease to perform its function. Molecule damages are mainly the breaking of bonds and dissociation. If a damaged molecule is a proteineenzyme or a cell organelle’s molecule, it is certainly sad, but possibly not fatal. In synthesis processes occurring in a cell, new molecules can be produced to replace the damaged ones. Therefore, if there are not too many such damaged molecules in the cell (i.e., the radiation dose was not very high), the cell may well recover and continue its normal activity. It is a different story if a DNA molecule were directly damaged. As a result, DNA mutation will arise that alters the program of synthesis of other important cell components. Depending on the gene in which the mutation has occurred, it can so substantially change the program that the cell will cease to function correctly. Usually, as a result, the mechanism of programmed defect cell removal, called apoptosis (Section 35.2), is turned on. Special enzymes dismantle the complex molecules into simpler compounds that are withdrawn through the membrane into the blood and then into the excretory organs. In principle, damage can be corrected by the repair mechanism. Only a small number of specific mutations does not prevent the cell from functioning normally and does not get fixed by the repair. Such mutations are stored and transmitted to the next generation of cells during mitosis. Still, most of the damage is corrected by the reparations mechanism. It is very important to note that a cell is a dynamic system in which the processes of damage and recovery are competing. Hence, the main intracellular target of ionizing radiation is DNA. A single event of DNA ionization can seriously disrupt the cell’s functioning or even become fatal for it because it is the DNA that contains information on the working program of the cell. This program shows itself in transcriptione translation and replication (Sections 34.2.3 and 34.2.4). If a cell does not divide (and there are tissues in a living organism in which cells divide very rarely), then isolated defects caused to the DNA will not reveal. So one can assume that dividing cells are mostly affected by radiation. Indeed, this fact was found at the dawn of radiobiology and was named the BergoniéeTribondeau rule (Section 36.4.1). Currently, we know that fast upgrading normal tissuesdgametal cells, cells of blood and the hematopoietic system, epithelial cells of the gastrointestinal tract and skindare most sensitive to radiation. Radiation damage of these tissues reveals quickly. Neurons and muscle cells show minimal sensitivity. In slowly renewing tissues, radiation damage develops much later, and sometimes, only after additional pathogenic effects. For example, radiation damage of long tubular bones may only reveal in slow fracture healing. In particular, J. Bergonié and L. Tribondeau concluded that because tumor cells divide often, they should be more sensitive to radiation and, therefore, they can be killed by irradiation. This laid the foundation for radiotherapy. However, the latter statement is true only in part. In fact, cancer cells located in the middle of a tumor may be poorly supplied with oxygen because of the problems with circulatory system development inside the tumor. Such cells remaining in a hypoxic state are less sensitive. This is due to the special role of oxygen during irradiation, which will be discussed in Section 35.8.2. Proteins can become another important target within a cell. They make up between one-half and two-thirds of a cell’s dry weight. It is proteins that act as enzymesdas catalysts of biochemical reactions. Not only the so-called primary structure of the proteins, i.e., sequence and type of amino acids in the polypeptide chain, is important to perform their enzymatic function but also the complex conformation of the molecule, which is called the secondary and tertiary structure of the protein molecule. As a result of exposure to radiation, destructions of amino acids in the chain, chain rupture, and violation of the three-dimensional molecular structure are possible. This leads to loss of the enzymatic ability. However, it

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is known that damage to enzymes requires irradiation at doses much higher than for occurrence of changes in the cell that lead to its destruction. This means that a cell has targets more sensitive to radiation than enzymes. In conclusion, note that direct immediate action is responsible for only 10%e20% of radiation damage.

35.8 INDIRECT ACTION As a result of research of biological effects of cell and organism exposure to radiation, the understanding accumulated that only direct action of radiation cannot explain the observed effects. On this basis, there appeared a concept of a special mechanism for an indirect effect of radiation. One of the reasons for the introduction of the indirect effect mechanism was comparing the effects from radiation exposure to vital macromolecules in solutions and in the dry state. When irradiating enzyme ribonuclease with 60Co gamma radiation, the cell survival markerdthe dose D37dis 42 Mrad in the dry state and 0.42 Mrad in an aqueous solution, i.e., they differ by a factor of 100. This suggests that in the aqueous solution an additional mechanism steps in, that increases the efficiency of the irradiation. In this example, only 1% of the ribonuclease molecules get inactivated directly by absorption of radiation energy, whereas indirect impacts are responsible for 99% of the effect. Thus, in the dissolved state in water, macromolecules are by several orders of magnitude more sensitive to radiation than in the dry state. It is natural to assume that products of water radiolysis contribute significantly to damage. A similar effect occurs with respect to the irradiation of more simple organic compounds, for example, aqueous solutions of formic acid. An important role in accepting the concept of so-called indirect action was played by the “Dilution effect” (or as it was later called, the “Dale effect”). The essence of this effect is as follows: in irradiation of aqueous solutions of different molecules (e.g., molecules of simple organic compounds or enzymes), the absolute number of affected molecules does not depend on their initial concentration in a certain concentration range. For the first time, experiments were carried out in the 1930s by G. Fricke, using solutions of simple organic compounds and in the 1940s by W. Dale with enzymatic solutions. It is clear that in the case of direct action of radiation, the number of affected molecules should increase with increasing concentration of solute molecules because of the higher probability of a quantum of radiation to affect them. At the same time, the percentage of the affected molecules should remain unchanged. The experiment although showed that in a certain range of concentrations, the radiation effect did not depend on the concentration of solute molecules. Typical graphs of dissolution influence on the damage nature are shown in Fig. 35.2. Dependence on concentration of the damaging effect of radiation correlates well with the fact that while irradiation in vivo, a certain level of damage of DNA molecules is observed at doses, exceeding by two to three orders those needed to damage these molecules when irradiated in dilute solutions. Independence of the part of affected molecules from the solute molecule concentration in the solution is attributed to the fact that at a given concentration not all solute molecules “receive” active products of radiolysis of water. These products are formed in a certain amount at a given radiation dose, but some get intercepted by cellular metabolites and do not go to biologically active macromolecules. That is, the concentration of solute molecules is not the limiting parameter, but the amount of active water radiolysis products formed at a given dose.

FIGURE 35.2 Typical graphs of dissolution’s influence on damage nature. (A) Percentage of inactivated molecules, %; (B) the absolute number of inactivated molecules. For direct (1) and indirect (2) effects of radiation.

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35.8.1 Effects of Free Radicals Because water is the main component of cell content, first of all let us look at the products of water radiolysis. Section 16.4 shows that radiolysis of water produces free radicals in large concentrations, a hydrogen radical H , hydroxyl radical OH , the superoxide H2O2, and the hydronium ion H3Oþ. Peroxide radical HO2 is formed in low concentrations with gamma irradiation, which dramatically increases with increasing LET. These radicals are strong oxidants. In addition, an active role is played by nonradical products: hydrated electron e
aq, which is highly reactive as a reducing agent, and hydrogen peroxide H2O2, which, though not a radical, is a very unstable compound and is a source of radical products. Entering the compounds with organic substances, radiolysis products cause significant chemical changes in cells and tissues, denaturation of protein and other organic structures, and depolymerization of macromolecules, disrupt the permeability of cell membranes, and cause mutations in the DNA and RNA. It is mainly water radiolysis products that determine the indirect effect of radiation. Note that radiolysis products also form in cells during the normal metabolic processes. As most of these products include oxygen, they are called reactive oxygen species (ROS). To prevent excessive accumulation and to correct the damages occurring, there is a complex set of protective antioxidant systems in the body. Therefore, the damaging effect of radiolysis products is shown at doses at which antioxidant systems can no longer cope with their work. 







35.8.2 Oxygen Effect Presence of oxygen enhances the effectiveness of buildup of radiolysis products with oxidizing properties: hydroperoxide radical HO2 and hydrogen peroxide H2O2. In addition, as a result of reactions with radiolysis products, atomic oxygen appears. Because of this, oxygen enhances the damaging effect of radiation. Radiobiological studies on irradiated cells of various tissues under the presence of oxygen in them showed that the same effect is achieved with much lower dosages than in the case of irradiation in the absence of oxygen. The correlation of doses in these two cases is called the oxygen enhancement ratio (OER) of radiation damage. The concentration of oxygen in the cell corresponding to its normal content in the atmosphere (w21% ¼ 159 mmHg) results in an OER of approximately 3 and no longer increases with increase of oxygen concentration up to 100%. With oxygen partial pressure decreasing to 30 mm of mercury, radiosensitivity decreases very slowly and then more sharply. In the range of 3e4 mmHg, the OER is 2 and then it goes down to 1. It is convenient to show the oxygen effect using as an example the survival curves discussed in Section 35.12. Survival curves illustrating the role of oxygen are shown in Fig. 35.3, and the connection between OER and the partial pressure of oxygen is shown in Fig. 35.4. Oxygen effect is typical for irradiation of low-LET radiation (X-rays and gamma rays). With increasing LET, the oxygen effect decreases rapidly and completely disappears in alpha-radiation exposure with high LET. 

35.8.3 Other Molecules (Except Water) as Mediators Originally, in the 1940s, after the emergence of the idea of indirect effects of radiation, only water molecules were considered as molecule mediators. However, in the 1950e1960s, it was suggested, and confirmed by experimental data, that lipid molecules (primarily those of unsaturated fatty acids) can also act as molecule mediators. They also form active radical products under the effect of radiation, capable of damaging critical structures and biologically important macromolecules in a cell.

FIGURE 35.3 Typical cell survival curves under X-ray exposure: 1, in air; and 2, in nitrogen; doses causing equal survival rate in nitrogen and air are 15 and 5 Gy, respectively; oxygen enhancement ratio w3.

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FIGURE 35.4 Dependence of oxygen enhancement ratio on the partial pressure of oxygen at 37 C. OER, oxygen enhancement ratio.

35.8.4 Radiotoxins If the products of cell molecule oxidation by free radical are sufficiently long-lived, they can produce a damaging effect not only on the cell, where they were formed, but also, by getting into the blood, they can be transported far away from the place where they were formed and have a pathogenic effect there. Such radiolysis products are called radiotoxins. Lipid radiotoxins, violating the barrier properties of membranes, have the greatest importance in radiation exposure pathogenesis. Radiotoxins formed from certain amino acids may inhibit the activity of many enzymes. In conclusion, we note that although accurate data on the relative role of direct and indirect effects still cannot be obtained, many researchers believe that the total contribution of indirect effect reaches 90% and is the determining factor in cell inactivation by ionizing radiation.

35.9 RADIOADAPTIVE RESPONSE Radioadaptive response is a phenomenon in which small conditioning doses of ionizing radiation, called the “priming dose” or “adapting dose,” reduce detrimental effects of subsequent higher doses, called the “challenge dose,” and increases resistance of cells or even an organism to an exposure up to that relatively high challenging doses. The event was discovered in 1984 by G. Olivieri et al. Radioadaptive response is realized in a variety of systems ranging from yeasts to animal models. The end points are such effects as radiation-induced DNA damage, CAs, cell transformation, cell death, mutation in in-vitro experiments, and also prenatal death, malformation, hematopoietic death, and carcinogenesis in in-vivo experiments [28]. Adaptation is observed in response to both low and high-LET radiation. Reduction in the extent of measured end points was found to range between about 5% and 60% [29]. In addition, the duration of protection varies and can last a few hours and sometimes for longer periods of time after the priming. The manifestation of radioadaptive response depends on the end point and considerably varies among individuals. In the study analyzing radioadaptive response in human lymphocytes from numerous individuals, it was reported that radioadaptive response with the end points of chromatid or chromosome damage was observed in 50%e78% of cases, and its extent (magnitude of reduction of challenge dose effects after priming dose exposure) ranged from 11% to 32% [29]. The order of the values of priming and challenging doses one can find in the following example [30]. Investigations in the high background area in Ramsar, Iran, have showed that a frequency of CAs exposed to 1.5 Gy of gamma rays lymphocytes, collected from residents from a high background radiation area (w260 mGy/year), is significantly lower than a frequency of CAs collected from the normal background radiation area residents. This result confirms that long-term exposure of human individuals to lowedose rate radiation induces a steady radioresistance. The priming irradiation can be continuous long-term lowedose rate exposure to environmental or occupational radiation, or short-term low-dose accidental, or special irradiation. In the last case, the radioresistant effect of priming radiation is restricted in time. The in-vitro studies revealed that radioresistance is typically elevated during a limited time period of about 20 h following the time interval of about 4 h after the priming irradiation [31]. The radioadaptive response is strongly connected with the bystander effect (Section 35.11). In many cases, bystander effect described to date is detrimental to the bystander cell. But sometimes it can play a protective role [32].

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Two basic types of mechanisms underlying radioadaptive responses can be distinguished. On the one hand, an intracellular response can lead to the adaptation of a cell after it has been exposed to radiation. In Ref. [33], it is referred as “memory,” as it is the consequence of a previous radiation hit that leads to protection against future challenges. On the other hand, a cell exposed to radiation can emit signals and induce a state of adaptation in other cells that have not yet been hit by radiation. Increased levels of ROS and nitric oxide have been suggested as mediators that contribute to the development of adaptive responses. The role of priming radiation is in activation of effector factors that play direct roles in enhancement of DNA repair, induction of molecular chaperon, synchronization of the cell cycle, or induction of antioxidants. The signal in a radioadaptive response is meant to be transferred to the neighboring cells through growth factors, ROS, and/or nitric oxide (NO) [28,33,34].

35.10 HYPER-RADIOSENSITIVITY AND INCREASED RADIORESISTANCE Low-dose hyper-radiosensitivity (HRS) and increased radioresistance (IRR) are two linked events. HRS is more decreased than expected survival fraction at low doses; in the case of IRR, cells show more intense radioresistant response as the dose increases. Both regions of doses are shown in Fig. 35.5. From Fig. 35.5, one can see that the survival fraction is compared with a linear-quadratic back extrapolation of the values obtained at higher doses (>1 Gy). At doses less than 0.3 Gy, mammalian cells exhibit HRS. Over the 0.3e0.6-Gy dose range, a more radioresistant response (IRR) per unit dose is evident, as illustrated by the shallower slope of the radiation doseeresponse curve. In many laboratories, the deviation in the low-dose region of the cell survival curves from the linear or linear-quadratic behavior, using different biological systems irradiated both with low-LET (X/gamma rays) and high-LET (alpha particles, carbon ions) radiations was found. The transition dose from HRS to IRR ranges between 0.2 and 0.6 Gy depending on the cell line, the biological end point, and the quality of radiation [36]. It was found that the dose at which HRS transitioned to IRR decreased with increasing LET. Both linked effects, HRS-IRR, were observed in several normal cell lines, malignant cell lines, and human tissue explants. There is also clinical evidence of HRS of skin after low-dose fractions during radiotherapy. Similar biphasic survival responses, albeit at much higher doses, have also been reported previously in protozoa and in insect cell lines [37]. It is important that several phenomena can be observed during or after low-dose irradiation including hypersensitivity and induced radioresistance, adaptive response, bystander effect, and genomic instability. Because of these phenomena, cancer risk curves in the low-dose region show nonlinear behavior. In many studies, the question about the coexistence of these phenomena is investigated. The results demonstrate that bystander effects can occur at doses below 1 Gy in a cell line with strong HRS/IRR transition, whereas strong bystander effects are displayed across all doses in a cell line that is not hyper-radiosensitive. The authors of Ref. [37] found that bystander effects might only occur in the HRS region of the survival curve and might be lost as the dose increased to levels where IRR mechanisms predominated.

FIGURE 35.5 Typical cell survival curve with evidence of hyper-radiosensitivity (HRS) and increased radioresistance (IRR). Broken line shows lowdose extrapolation from linear-quadratic model applied to high-dose survival data. Solid line shows induced repair fit. Figure from B. Marples, S.J. Collis, Low-dose hyper-radiosensitivity: past, present, and future, Int. J. Radiat. Oncol. Biol. Phys. 70 (5) (2008) 1310e1318.

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The authors of Ref. [32] have built a mathematical model to examine the effect of radioadaptive response, HRS, and IRR on cellular responses to low-dose radiation. They found that all three phenomena and linear-quadratic dependence can be explained by the simple model that they call “memory” model.

35.11 BYSTANDER EFFECTS During the 20th century, radiobiology was dominated by the concept that a cell gets damaged only if the charged particle lost its energy in it. Damage could occur as a result of direct action on DNA or indirectly through free radicals, but necessarily in this cell, it was “target” paradigm of radiobiology. Damage could be fixed by repair mechanism and then the cell would survive, or this mechanism would fail and then the cells would die. Some of the damage could be identified as mutations that manifest themselves when a cell tries to divide. It was generally accepted that if a cell can pass through five divisions, one can assume that it escaped exposure and its offspring will behave as if it had never been subjected to irradiation. However, evidence that manifestations of radiation can also be observed in tissues that were outside the radiation field gradually began to accumulate in the middle of the century. Back in 1953, the term “abscopal effect” was introduced by R. J. Mole. The term is composed of the Latin words ab, prefix denoting removal, and scopus, mark or target. Somewhat later came another term, “bystander effect.” Both of these effects mean action of ionizing radiation on shielded tissues outside the irradiation zone, in neighboring or even distant parts of the body. Sometimes, these effects are referred to as “remote.” At the moment, it is assumed that the bystander effect corresponds to passing the signal on irradiation from irradiated cells to the nearby nonirradiated ones that were inside the irradiated volume. In the case of abscopal effect, the connection occurs between irradiated and nonirradiated cells that are outside of the irradiated volume. However, some authors use the names of these two effects as synonyms. In English literature, these effects are often referred to as radiation-induced bystander effect (RIBE). RIBE could be passed from one whole multicellular organism to another. Unirradiated fish swam in the same pool with irradiated. All fish were examined for bystander responses using a variety of assays. The data confirm transmission of signals from irradiated to unirradiated fish and raise the significance of the RIBE to a new hierarchical level [38]. Researchers are also finding evidence of information exchange between irradiated cells, that was called the cohort effect [39,40]. Remote effects also show in radiation therapy. For example, in a patient with advanced melanoma, local irradiation of one tumor also destroyed another that was outside the radiation field [41]. An idea of the difference of the bystander, abscopal, and cohort effects can be noted from Fig. 35.6. The value of bystander effect can be illustrated by the data from the report of Nagasawa and Little [42]. In their study, less than 1% of the nuclei in Chinese hamster ovary cells were traversed by a particles, but about 30% of the cells showed an increased frequency of sister chromatid exchanges. The doses in the experiments were as low as 0.31 mGy [43]. Detailed description of the history of these effects’ discovery and acceptance by the scientific community can be found in Ref. [38]. The title of the article [38] appearing in the magazine Mutation Research, “Changing Paradigm in

FIGURE 35.6 Schematic representation of the bystander, abscopal, and cohort effects. White cells, nonirradiated; gray cells (red cells in the web version), irradiated. 1, Bystander effect: the interaction between irradiated and nonirradiated cells in irradiated volume. 2, Abscopal effect: the interaction between irradiated cells and cells in nonirradiated volume. 3, Cohort effect: the interaction between irradiated cells in irradiated volume.

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Radiobiology,” is significant. The authors [38] claim that modern radiobiology has undergone a paradigm shift from the hit principle and target theory to the bystander effect. Existence of remote effects means that the irradiated cell can transfer a signal to nonirradiated cellsdbystanders. Three types of possible mechanisms were proposed based on previous experimental results [44]: 1. Apparently, such a signal may be a soluble factor capable of moving into the intercellular environment and transmitting information to nonirradiated cells. Possibly, the role of such a factor is played by radiotoxins. 2. The other one is transmission of signaling molecules through a gap junction assembly spanning plasma membranes of adjacent two cells. 3. The definite role in RIBE can play oxidative metabolism-mediated transmission of signals. Remote or bystander effects also include the effect of genomic instability (RIGI). This effect means that no apparent damage can be detected in irradiated cells but they show only in subsequent generations. Genomic instability is observed in vivo and in vitro. A detailed description of the contemporary state of research of all bystander effects is contained in the report by the UN Scientific Committee on effects of atomic radiation [45]. Bystander effects are the vivid examples of the intercellular communications.

35.12 SURVIVAL RATE A live cell has two purposes. First, it should perform a certain function in the organ, to which it belongs, and second, it should reproduce the offspring through the division process. Radiation can disrupt both of these functions. Large doses, i.e., many acts of ionization and excitation in the same cell, kill the cell and it ceases to perform its function. Smaller doses deprive the cell of its ability to divide. It continues to live and, in principle, can live a considerable time, but loses the ability to divide and eventually dies before mitosis. Even lower doses lead to a delay in the division. Division processes are organized in time in accordance with a specific cell cycle (Section 34.2.2.3). Cell death prior to entering the mitosis phase is called interphase death. If the cell is able to divide, but its descendants are defective and die after one or two divisions, then this is called reproductive death. We have already noted that in animals, cells of some tissues (hematopoietic, genital, and intestinal mucosa) actively divide to produce more of their kind. Cells of other tissues (kidney, liver, heart, muscle, neurons, etc.) divide seldom or never. For all dividing, and most nondividing, cells, an interphase death occurs only at doses of several hundred grays (Gy). Exceptions are lymphocytes and gametal cells at certain stages of their development. They die in an interphase at doses of tens of grays. As a result of exposure in the cell, one can register a wide variety of reactions. One of the most important ones is delayed division. Numerous studies of various cell cultures showed that on average the division delay is about 1 h per 1 Gy. However, the delay time in each particular case depends on the stage of the life cycle in which the cell got irradiated. The longest delay occurs from irradiation in the S-phase of DNA synthesis or postsynthetic stage G2; the shortest is during irradiation in mitosis M, when the vast majority of cells, having started mitosis, finish without delay. Experiments have shown that the delay of division depends on the radiation dose and is manifested in all cells of the exposed populations, regardless of the subsequent fate of the cellsdwhether it survives or dies. However, the delay time in different cell types varies. The typical graph of dependence of cell mitosis delay on the irradiation dose for cells in the different phase of a cell cycle is shown in Fig. 35.7. In principle, delay of division can be regarded as a direct effect of exposure or as a protective response of cells to damage. So far, there are insufficient data to select between these options. Now, let us analyze cell death in more detail. For many years, the main tools of this analysis were the so-called survival curves. A typical example of dependence of the percentage of surviving cells on the dose is shown in Fig. 35.8. In this figure, the y-axis shows the proportion of surviving cells in logarithmic coordinates and the x-axis shows the dose of gamma irradiation in linear coordinates. In such coordinates, exponential dependence should be demonstrated by a straight line. The graph shows that at low doses the relationship is curvilinear, and then with increasing doses it becomes linear, i.e., it turns into an exponential dependence. It should be noted that such curves are generally obtained in vitro from mammalian cell cultures. The percentage of surviving cells is determined based on the number of normal healthy colonies of cells grown in irradiated plates compared with the total number of colonies in the control plates. Obviously, in this case, only those cells were described as surviving that passed through multiple divisions, i.e., preserved their reproductive potential.

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100

Mitosis delay, hours

80

G2

60

S

40

G1 20

0 0

2

4

6

8

10

12

14

Dose, Gy

FIGURE 35.7 Dependence of cell mitosis delay on the irradiation dose for cells in the different phase of a cell cycle.

FIGURE 35.8 pointed out.

Typical survival curve. The shoulder Dq and extrapolation of the inclined part of the curve up to the intersection with the x-axis are

Curves under consideration show not the degree of cell damage (the degree of damage is always the samedcell death) but the probability of death. This makes an essential difference between the situation with death of cells and the situation with division delay, where the effect (as opposed to effect probability) depends on the dose. On a typical survival curve such as in Fig. 35.8, one can specify three parameters: shoulder Dq, and survival D0. At low doses, the curve has a shoulder. Then the intersection of the line parallel to the x-axis and coming out of the top of the curve with the extrapolation of the linear part of the curve gives a value of shoulder Dq. It is believed that this is the measure of the ability of cells to repair. Extrapolation of the linear portion of the curve to the intersection with the ordinate axis somewhat characterizes the number of targets (hits) that is necessary to destroy the cells. The linear portion corresponds to the exponent and, in principle, can be described by the relation NðDÞ=N0 ¼ expð
D=D0 Þ.

(35.1)

Here N0 and N(D) are the number of cells before and after irradiation, D0 is the parameter of the curve that defines its slope and is called the survival rate, and 1/D0 is the radiosensitivity. Another parameter used to describe the survival curves is D37dthe dose at which the percentage of surviving cells decreases by e times, so that 37% of them remain. If the survival curve was pure exponential, then D0 would be equal to D37. But they differ because of the shoulder; the value of D37 is greater than that of D0.

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FIGURE 35.9 Dose survival curves for objects with different target hit rates. (A) Death rate in the usual scale (S-shaped or sigmoidal curves); (B) Surviving rate in semilogarithmic scale (hits number is indicated on the curves). Curves in parts A and B correspond to each other.

For the majority of dividing cells, D0 ¼ (1.2
2.0) Gy, Dq w 1.5
5 Gy. The radiosensitive fraction of hematopoietic cells in bone marrow has a much greater radiosensitivity D0 ¼ 0.1 Gy, for the lymphocytes D0 ¼ 0.5 Gy. In particular, the survival curves reflect the radiosensitivity of cells (Section 35.14). Significantly, radiation sensitivity depends on the cell cycle phase in which the cell was irradiated. In other words, radiosensitivity varies over time quite noticeably, by a few dozen times. Cells prove to be most radiosensitive during mitosis. Further change in radiosensitivity is somewhat different in different types of cells. However, as a rule, it is minimal (and survival rate is maximal) when irradiated in the late S stage, when the radiosensitivity is approximately 10 times lower than in mitosis. In principle, the survival curves can also be built in other coordinates, where the ordinate shows not the number of surviving objects but of those that died. Then the well-known S-shaped curve appears. Both versions of curves are shown in Fig. 35.9. Curves in Fig. 35.9A and B correspond to each other. From this figure, it can be clearly seen how the forms of S-shaped curves change with the growth of the shoulder. The curves with the shoulder can be described on the basis of the so-called multitarget model: NðDÞ=N0 ¼ 1
½1
expðD=D0 Þ . n

(35.2)

Here, n is the number of targets that the particles must hit to inactivate the cell. With n ¼ 1, the relation (35.2) becomes (35.1). During gamma irradiation, one event of interaction of a fast Compton or photoelectron with a DNA molecule can lead to single chain rupture. Single ruptures themselves usually do not cause cell death. Repair system quickly and effectively corrects this damage. However, two different fast electrons can accidentally produce a double chain rupture, that is, a breakage of both DNA strands. In this case, the cell is, with high probability, doomed. In fact, sometimes one fast electron can produce a double rupture. It is believed that during gamma irradiation, for about every 100 single ruptures, there is 1 double one. With an appreciable probability, double ruptures occur during passing of a strongly ionizing particle. That is why highLET radiation is much more dangerous. Based on these concepts, cell survival rate in many cases can be described using the so-called linear-quadratic model of Chadwick and Leenhouts. According to this model, cell survival rate is described by the formula 
 NðDÞ=N0 ¼ exp
aD þ bD2 . (35.3) Here a and b are parameters characterizing the probability of DNA ruptures in an irradiated cell. In this model, it is believed that single ruptures are proportional to the first power of the dose and double ruptures to the square. The corresponding curve is shown in Fig. 35.10. Curves with shoulder are obtained using low-LET radiation: gamma- and hard X-ray radiation. With growing LET, the shoulder decreases. In the case of alpha-particle radiation, the curve turns into a proper exponent. In this case, one hit of a cell nucleus by a particle for DNA structure damage is enough.

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FIGURE 35.10 Survival curve in the case of linear-quadratic (a/b) model. LET, linear energy transfer.

Part of surviving colonies

10 1 0.1 0.01 0.001 0

5

10

15

20

25

Dose, Gy FIGURE 35.11 Survival in dependence on a dose with increasing radiation dose rate downward from w0.5 cGy/min up to w1 Gy/min.

If exposure to gamma or X-ray radiation occurs with low dose or after radiation exposure and a few hours’ pause is made, to enable a repair of damaged DNA structure, the percentage of surviving cells significantly increases. This is due to the creation of conditions for activation of enzymatic repair systems and increased DNA structure recovery time prior to synthesis before the entry of cells into mitosis. Depending on conditions, the recovery rate is typically of time intervals from 1 to 10 h. The role of repair processes is illustrated by the graph in Fig. 35.11, which shows the influence of the dose rate on the shape of the survival curves. To compare different exposure conditions, one uses the parameter “radiation exposure efficiency,” which is the relationship between dose LD50 (dose for 50% mortality) at short-term exposure and high-dose rate to the same value under different conditions of exposure. With decreasing dose rate, below a certain level, the radiation exposure efficiency falls and for irradiation taking several days it can drop several fold. It is believed that in this case, rapid recovery processes at the cellular and tissue levels begin to show. Repair of damage and increased percentage of surviving cells also occurs at the so-called fractionated irradiation. A typical pattern of possible survival curves in the case of short-term irradiation sessions separated by pauses lasting from several hours to several days is shown in Fig. 35.12.

35.13 THE DEPENDENCE OF RADIATION EXPOSURE ON LINEAR ENERGY TRANSFER In the history of research of the effects of ionizing radiation on biological objects, the most common and studied form of radiation was X-ray radiation with a maximum energy of 250 kV. However, when study of other types of radiation effects on cells and tissues began, it became clear that the same effects on the same cells are obtained at different doses. Particles with higher LET create more damage.

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FIGURE 35.12 Survival curves for the fractionated radiation exposure. Curves 1, single exposure; 2, 3, and 4, exposure is divided into two, four, and eight equal fractions, respectively.

FIGURE 35.13 Connection of relative biological effectiveness (RBE) of radiation, radiation weighting factors wR (quality factors), and lens epithelium chromosomal aberrations (CAs) with the linear energy transfer (LET). Empty diamonds, QF according to ICRP 26; circles, RBE; triangles, CA. The values for CA are from Ref. [47].

For proper consideration of the impact of various types of radiation, the relative biological effectiveness (RBE) was introduced RBE ¼

Dðany effect for X ray radiation with energy of 250 keVÞ Dðany effect for another type of radiationÞ

Different types of radiation having impact on biological objects mainly differ in their LET value. Therefore, the value of RBE depends on the LET. The connection between LET and RBE values is shown in Fig. 35.13. It is seen that the relationship between these two values is not strictly linear, but a positive correlation of LET and RBE is definitely observed to a certain maximum value of LET, corresponding to ionization density on the tracks of alpha particles. RBE decreases at high LET because of the fact that a particle with such a large local release of energy can kill more cells than it can reach on its track. The same figure shows the regulated values of quality coefficients for different types of radiation. Also shown are the results of experimental studies on the yield of CAs in the lens epithelium. In preparation of this figure, data from Ref. [47] were partly used. In dosimetry, to calculate equivalent doses, the so-called quality factors (or radiation weighting factors) of any given type of radiation are used. Values of quality coefficients are closely related to the RBE, but strictly speaking, they are not equal. It is important to understand that the RBE values strongly depend on the experimental conditions (type of cells, type of radiation, dose rate, etc.). Quality coefficients are fixed values that are used to calculate dose rates under any conditions.

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Quality coefficients are the operating parameters used to solve practical problems. On the other hand, RBE is a scientific parameter, taking into account measurement conditions. Officially accepted values of quality coefficients are shown in Appendix.

35.14 RADIOSENSITIVITY OF TISSUES, ORGANS, AND ORGANISMS Radiosensitivity is a measure of an organism’s reaction to ionizing radiation exposure. The reciprocal of radiosensitivity is radioresistance. Radiosensitivity, as a concept, is very important both from the theoretical and practical points of view. Knowledge of the mechanism controlling radiosensitivity and its regulation will enable conscious control of tissue response to irradiation, weaken it to protect the body, and strengthen it during radiation therapy of malignant tumors or bacteria inactivation. Different cells, tissues, organs, and organisms react differently to radiation and produce different specific effects. In summary, the existing rule states that cell radiosensitivity is proportional to division frequency and inversely proportional to the cell specialization degree. That is why frequently dividing and unspecialized cells, as a rule, are the most radiosensitive. Most obvious examples of such cells are marrow and embryo cells. On the other hand, central nervous system (CNS) cells of an adult are highly specific and have very low or zero division frequency. Thus, CNS cells are relatively radioresistant. An example of extremely low radiation sensitivity is bacteria found in a nuclear reactor channel. Under these conditions, the bacteria not only died but lived and multiplied. That is why they were called Micrococcus radioduransd radioresistant micrococcus. Values 1/D0 or LD50 (a dose at which 50% of organisms die) may serve as a quantitative measure of radiosensitivity. Usually, in this case, the typical time period of observation is also specified. The typical value of such a period is 30 days after irradiation and the corresponding parameter is denoted LD50/30. For a human, this value is of the order of 3.5e4.4 Gy. However, the active use of the achievements of modern medicine, especially bone marrow transplantation, enables pushing this limit toward higher doses. For example, here are several more values of LD50/30. For a triton, it is 30 Gy; for a turtle, 15 Gy; for a rat, 7.1 Gy; and for a dog, 2.75 Gy. A special feature of radiation damage to a living body is that such a body cannot be regarded as a simple collection of cells and organs. Being part of a tissue, cells are very dependent both on each other and on the surrounding environment. A response to the damaging effect of radiation involves all the body’s control systemsdnervous, endocrine, etc. Different organs have different radiosensitivity, which depends not only on the cells comprising the organ but also on their location, vascularity, and even irradiation time. Introducing the concept of critical organs enables slight simplification of the situation. Critical organs are the vital organs or systems that are the first ones to fail within the studied radiation dose range, resulting in death of the organism within a certain period after irradiation. For more about critical organs in humans, see Section 35.16. To consider different sensitivities of different organs and tissues in the calculation of effective dose, so-called weight coefficients are introduced. Values of weighting coefficients for different organs and tissues are shown in Appendix A3.6.

35.15 LONG-TERM CONSEQUENCES Long-term consequences of exposure are phenomena that occur years and decades after exposure. They can be of two types: somatic, developing in exposed individuals, and genetic, hereditary diseases developing in the offspring of the exposed parents. The most dangerous and most widespread long-term consequence is cancer. The mechanism of the carcinogenic effects of radiation is described in Chapter 36. Besides cancer, there are other possible manifestations of long-term effects of radiation on the human body: physiological disorders (thyroid malfunction, etc.), cardiovascular diseases, allergies, chronic respiratory diseases, immune deficiency and associated increase in sensitivity to infectious agents, and temporary or permanent sterility. We note, in particular, two types of long-term effects: damage of cell fiber constituting the lens, and as a consequence clouding of the lens (cataract), and reduced life expectancy unassociated with cancer. Experimental studies on mammals suggest that a proportional relationship exists between the degree of shortening life expectancy and radiation dose. Every 0.01 Gy of single irradiation reduces life expectancy by 1e15 days, and at chronic exposure, by 0.08 days. Yet it should be clear that the likelihood of long-term effects is experimentally proven only at relatively high doses. Reliable detection at low doses is difficult because of problems with gathering enough statistical material and adequate control groups of animals. In addition, and most importantly, it is difficult because of the huge background of similar diseases in humans caused by other environmental factors, including carcinogenic and mutagenic.

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35.16 RADIATION SICKNESS In the mid-20th century, after the Second World War, humanity learnt about a new scary-sounding and dangerous radiation sickness. Various troubles associated with radiation became known immediately after the discovery of X-rays and radioactivity. But two fatal radiation events, that occurred at the end and immediately after World War II in Los Alamos, and are described in Section 46.3.3 were much more dangerous. At the same time, victims of the nuclear bombings of the Japanese towns of Hiroshima and Nagasaki on August 6 and 9, 1945, became known. It is impossible to identify the proportion of people that died in the explosion just from radiation because the people who were close to the epicenter got exposed to a blast wave similar to that of a very powerful but nonnuclear bomb, and to strong light radiation. Many died under the rubble and from many other explosion factors. But after the explosion, doctors could already distinguish between a death due to burns or wounds and due to radiation sickness. By December, 1945, 177 people died from atomic diseases [48]. It is unlikely that these data are accurate, although figures are given with an accuracy of one person. Those days medical care was not sufficient, so even atomic diseases may have been diagnosed as ordinary diseases. Radiation sickness struck hundreds of residents of the Marshall Islands and the crew of a Japanese fishing boat Fukury u-Maru as a result of thermonuclear bomb tests in 1954. Humanity was faced with radiation sickness, and because of sharp confrontation between the great powers during the “cold war” and the danger of global thermonuclear slaughter, people feared its further massive appearance. Hence, an intensive study of this new phenomenon began. Note that there is a certain convention: total body irradiation at a dose of less than 1 Gy, and locally at larger doses, is called “radiation injury” or sometimes “acute radiation reaction.” At doses higher than 1 Gy, radiation sickness develops. Up to this point, we have learnt about the effect of ionizing radiation on molecules, cells, and tissues. The problem of radiation sickness leads us to the analysis of the effects of radiation on the entire organism. It should be borne in mind that an organism is not simply a sum of cells but a complex dynamic system of interacting cells. In Section 35.11, it is shown that cells are able to pass each other information about irradiation (bystander effect, abscopal effect, genomic instability effect). In particular, cells transmit information to each other about the need to start active division (e.g., for healing wounds) or to stop active division (when the integrity of the body is restored). Activity of cells is run by whole-body hormonal and nervous systems. One of the manifestations of cell operation control is apoptosisda genetically programmed cell self-destruction in response, in particular, to external signals. To characterize interactions in a cellular society, the term “social behavior of cells” is even used. Hence, as a result of irradiation by a dose not lower than a particular level, radiation sickness starts. There are two variations of radiation sickness: acute radiation syndrome, after a short intense irradiation, and chronic radiation sickness, after long-term exposure at a relatively low dose rate. Radiation sickness is a multisymptom disease, and its specific development and dominating symptoms strongly depend on how the exposure occurreddwhat dose rate and how it was distributed in time, i.e., simultaneous, fractional, or continuousdover a period of time. If continuously, the dose rate is important. For radiation sickness development, it is important whether irradiation was external or internal, and whether the whole body or only part of it got irradiated. At doses above a certain threshold, radiation sickness is manifested by symptoms, the severity of which increases with increasing dose. These manifestations are called deterministic. The lower threshold of deterministic effects depends on many conditions as listed above. Each effect and each organ has its own threshold dose. With overall short-term irradiation in a human, the threshold dose for deterministic effects is from 0.2 to 1 Gy. As is known, dividing cells often are the most radiosensitive (the BergoniéeTribondeau rule). Therefore, organs with a large number of such cells get affected by irradiation primarily and at lower doses. These are hematopoietic organs, epithelium of intestines and stomach, ovaries, and testes. Organs primarily affected by radiation are called critical. Depending on which organs are mostly affected and which of them are the most critical, there are several forms of radiation sickness: bone, brain, intestinal, and cerebral. Various forms of radiation sickness can be easily illustrated by a curve showing the dependence of life span on the dose. Such a curve for a single X-ray irradiation of mice was developed by B. Rajewski in 1953 (Fig. 35.14) [46]. On the curve, one can clearly see stepwise changes in life expectancy from changing the dose, associated with the failure of critical systems. Similar curves were later obtained for other mammals (rats, hamsters, guinea pigs, monkeys, and humans) as well as for amphibians, insects, worms, and even plants. However, the specific dose rates at which the corresponding critical organ died differed for different animal species, pointing to the different radiosensitivity of the responsible systems. Thus, in this case, we are dealing with a general biological law. The portion of the curve corresponding to low doses (from a few to w10 Gy) is responsible for the death of animals due to failure of bone marrow (hematopoietic) system. In the dose range of w10e100 Gy, there is a plateau where the

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100

Life span, days

10

1

0.1

0.01 1

10

100

1000

Dose, Gy FIGURE 35.14 Dependence of the average life span on the dose for mice. Based on B. Rajewski, O. Heuse, K. Aurand, Weitere Untersuchungen zum Problem der Ganzkorper Bestrahlung der Weissen Maus. Sofortiger Tod durch Strahlung, Zeitschrift Naturf 86 (1953) 157e159.

average life expectancy is about 4 days. This section corresponds to the defeat of the epithelium of the small intestine. The survival time for gastrointestinal death is dose-independent. With further increase of the dose, the life expectancy drastically shortens, down to several hours, which is associated with damage to the CNS. The fact that some parts of the curve correspond to the specific system destruction is confirmed by conclusive and easily reproducible experiments. The presence of the plateau in the curve (Fig. 35.14) indicates that damage to a system incompatible with life occurs at a certain level of exposure, i.e., it has a threshold nature. Experts divide all the possible manifestations of radiation sickness in humans into periods, phases, variants, and stages in great detail. More detailed description of the radiation sickness can be found, for example, in the manual [49]. We will look into the most common variant of radiation sicknessdacute radiation sickness caused by relatively even irradiation relative to the body’s volume. Phases of acute radiation sickness are very clearly shown in Fig. 35.16, taken from Ref. [50]. As shown in Fig. 35.15, immediately after irradiation, there is a short delay; its duration depends on the dose and can take from several minutes to several hours. After the delay, the primary reaction phase occurs. Then, the painful symptoms subside and even disappear and the phase of apparent clinical well-being (hidden or latent phase) begins. The duration of this phase is also dependent on the dose; it takes from several hours to 3e4 days. Then again, the clinical manifestations return and the manifest illness stage starts. The disease ends with recovery or death. It is useful to know the indicators of primary reactions: l l l

nausea and vomiting; lack of appetite; dryness and bitterness in the mouth;

FIGURE 35.15 Stages of acute radiation sickness. The figure is from T. de Revel, Menace Terroriste Approche Medicale, Nucleaire Radiologique Chimique, 2005 with kind permission from Editions John Libbey Eurotext, Paris.

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l l l l l l l

469

a feeling of heaviness in the head, headache, weakness, and drowsiness; shocklike state; drop in blood pressure; brief loss of consciousness; diarrhea; fever; and neutrocytosis and lymphopenia in the peripheral blood during the first day after irradiation.

Type of damage is also affected by the method of exposure: the effect of an external ionizing radiation source (external radiation) or radionuclides that got inside the body (internal radiation). With a uniform distribution in the body of incorporated (internal) caesium-137, the disease picture is similar in clinical features, with radiation sickness arising due to external irradiation. Because of the difference in chemical properties of radionuclides, they can become very heterogeneously distributed in the body and cause selective organ failure: strontium-90 accumulates mainly in bones, plutonium-239 in lungs, iodine-131 in the thyroid gland, etc. It is obvious that with increasing doses, disease manifestations grow: increased temperature, the frequency of the urge to vomit, or the concentration of leukocytes or DNA fractions in the blood along with other indicators, accumulation in the cells of the oxidation products of biological membranes (peroxides and hydroperoxides of unsaturated lipids), etc. At low doses, this growth is almost linear; then it slows and soon stops altogether, the curve reaching saturation. This is the so-called S-shaped or sigmoidal curve corresponding to a quite natural, universal form of dependence. Indeed, at very small doses, the higher the dose, the greater its impact. But there are limits. Body temperature cannot exceed w42 C. Concentration of white blood cells is limited. Patient death is the ultimate event. Such an S-shaped curve describes the mortality of a living body depending on the irradiation dose as shown in Fig. 35.16. It can be seen that the curve showing the dose/effect relationship does not start at zero but is shifted to the right, toward higher doses. That is, there is a thresholddthe minimum lethal dose corresponding to death of the organism. Values of LD50 are different for different species of mammals, but the shape of the dose curve and causes of death are the same. At doses around LD50, the hematopoietic system is critical for the body; at high doses, the critical system is the mucosa of the small intestine. In the first case, part of the animals die within 10e14 days; in the second, 4e7 days after irradiation. At D > 1 Gy up to the absolutely lethal dose, radiation sickness of various severities is observed in survivors. At lower doses, no apparent clinical manifestations are observed, but there may be long-term effects (Section 35.15), mainly cancer (Chapter 36), cataract, and disorders of the nervous system and others. Furthermore, irradiation can affect both somatic and gametal cells; that is, it can cause damage to the genetic apparatus. In both cases above, at a certain dose, deterministic effects can be observed, and in the genetic apparatus, only stochastic. Stochastic effects are analyzed in detail in Chapter 54. Finally, one more type of radiation effects on the bodydthe so-called teratogenic (from the Greek teratos [monster]) effects of irradiationdshould be pointed out. Teratogenic effects of radiation are manifested in congenital malformations in children irradiated at the in utero development stage. These are not genetic but somatic effects arising from exposure of the fetus.

FIGURE 35.16 Dose curve of mammals’ death from gamma irradiation of the whole body. Crosses show the values of LD50 for different animals and a human. 1 - Pig, 2 - Dog, 3 - Man, 4 - Monkey, 5 - Mouse, 6 - Rat, 7 - Turtle.

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REFERENCES [1] List of Genetic Disorders. Wikipedia. https://en.wikipedia.org/wiki/List_of_genetic_disorders. [2] Chromosome 1 (Human). Wikipedia. https://en.wikipedia.org/wiki/Chromosome_1_(human). [3] B. Vogelstein, N. Papadopoulos, V.E. Velculescu, S. Zhou, L.A. Diaz Jr., K.W. Kinzler, Cancer genome landscapes, Science 339 (6127) (2013) 1546e1558. [4] P.-O. Löwdin, Proton tunneling in DNA and its biological implications, Rev. Mod. Phys. 35 (1963) 724e731. [5] A. Pérez, M.E. Tuckerman, H.P. Hjalmarson, O.A. von Lilienfeld, Enol tautomers of Watson
Crick base pair models are metastable because of nuclear quantum effects, J. Am. Chem. Soc. 132 (33) (2010) 11510e11515. [6] L.A. Moran, Human Mutation Rates e What’s the Right Number, Sandwalk, 2015. http://sandwalk.blogspot.de/2015/04/human-mutation-rateswhats-right-number.html. [7] What is the Mutation Rate during Genome Replication? Cell Biology by the Numbers. http://book.bionumbers.org/what-is-the-mutation-rate-duringgenome-replication/. [8] W. Huang, R.F. Lyman, R.A. Lyman, M.A. Carbone, S.T. Harbison, M.M. Magwire, et al., Spontaneous mutations and the origin and maintenance of quantitative genetic variation, Genom. Evol. Biol. eLife 5 (2016), e14625. [9] M. Lynch, Rate, molecular spectrum, and consequences of human mutation, Proc. Natl. Acad. Sci. U.S.A. 107 (3) (2010) 961e968. http://www. pnas.org/content/107/3/961.full.pdf. [10] J. Cairns, J. Overbaugh, S. Miller, The origin of mutants, Nature 335 (1988) 142e145. [11] J. Cairns, P.L. Foster, Adaptive reversion of a frameshift mutation in Escherichia coli, Genetics 128 (1991) 695e701. [12] A.J. Spiers, Review article. A mechanistic explanation linking adaptive mutation, niche change, and fitness advantage for the Wrinkly Spreader, Int. J. Evol. Biol. (2014) 10. Article ID 675432. [13] E.V. Koonin, Y.I. Wolf, Is evolution Darwinian or/and Lamarckian? Biol. Direct 4 (2009) 42. [14] J. McFadden, J. Al-Khalili, A quantum mechanical model of adaptive mutation, Biosystems 50 (3) (1999) 203e211. [15] V. Ogryzko, On Two Quantum Approaches to Adaptive Mutations in Bacteria, 2008. http://arxiv.org/ftp/arxiv/papers/0805/0805.4316.pdf. [16] M. Asano, A. Khrennikov, M. Ohya, Y. Tanaka, I. Yamato, Quantum Adaptivity in Biology: From Genetics to Cognition, Springer, 2015, p. 173. [17] J. Morreall, A. Kim, Y. Liu, N. Degtyareva, B. Weiss, P.W. Doetsch, Evidence for retromutagenesis as a mechanism for adaptive mutation in Escherichia coli, PLoS Genet. 11 (8) (2015) e1005477. [18] C. Payen, A.B. Sunshine, G.T. Ong, J.L. Pogachar, W. Zhao, M.J. Dunham, High-throughput identification of adaptive mutations in experimentally evolved yeast populations, PLoS Genet. 12 (10) (2016) e1006339. [19] D. Pan, Y. Du, B. Hu, The role of epigenetic modulation in the cellular response to ionizing radiation, Int. J. Radiol. 2 (1) (2015). [20] D. Holoch, D. Moazed, RNA-mediated epigenetic regulation of gene expression, Nat. Rev. Genet. 16 (2) (2015) 71e84. https://www.ncbi.nlm.nih. gov/pmc/articles/PMC4376354/. [21] S.P. Zielske, Epigenetic DNA methylation in radiation biology: on the field or on the sidelines? J. Cell. Biochem. 116 (2015) 212e217. [22] J.-G. Kim, M.-T. Park, K. Heo, K.-M. Yang, J.M. Yi, Epigenetics meets radiation biology as a new approach in cancer treatment. Review, Int. J. Mol. Sci. 14 (2013) 15059e15073. [23] M. Merrifield, O. Kovalchuk, Epigenetics in radiation biology: a new research frontier. Review article, Front. Genet. 4 (2013) 16. Article 40. [24] Y. Ilnytskyy, O. Kovalchuk, Non-targeted radiation effects e an epigenetic connection, Mutat. Res. 714 (1e2) (2011) 113e125. [25] O. Kovalchuk, J.E. Baulch, Epigenetic changes and nontargeted radiation effects e is there a link? Environ. Mol. Mutagen. 49 (1) (2008) 16e25. [26] Epigenetic events in tumorigenesis. in: UNSCEAR 2000 Report. Annex G Biological Effects at Low Doses, p. 143. [27] D.E. Lea, Action of Radiation on Living Cells, Cambridge Univ. Press, 1947. [28] M. Nenoi, B. Wang, G. Vares, In vivo radioadaptive response, Hum. Exp. Toxicol. 34 (3) (2015) 272e283. [29] S. Tapio, V. Jacob, Radioadaptive response revisited, Radiat. Environ. Biophys. 46 (2007) 1e12. [30] M. Ghiassi-nejad, S.M. Mortazavi, J.R. Cameron, A. Niroomand-rad, P.A. Karam, Very high background radiation areas of Ramsar, Iran: preliminary biological studies, Health Phys. 82 (2002) 87e93. [31] M.S. Sasaki, Y. Ejima, A. Tachibanà, T. Yamada, K. Ishizaki, T. Shimizu, et al., DNA damage response pathway in radioadaptive response, Mutat. Res. 504 (1e2) (2002) 101e118. [32] D. Wodarz, R. Sorace, N.L. Komarova, Dynamics of cellular responses to radiation, PLoS Comput. Biol. 10 (4) (2014) e1003513. [33] W. Morgan, Will radiation-induced bystander effects or adaptive responses impact on the shape of the dose response relationships at low doses of ionizing radiation? Dose Response 4 (4) (2006) 257e262. [34] N. Assadi, E. Zabihi, M. Khosravifarsani, S. Khafri, H. AkhavanNiaki, M. Amiri, et al., Radioadaptive response in human lymphocyte cells, Int. J. Mol. Cell Med. 3 (1) (2014) 57e60. [35] B. Marples, S.J. Collis, Low-dose hyper-radiosensitivity: past, present, and future, Int. J. Radiat. Oncol. Biol. Phys. 70 (5) (2008) 1310e1318. [36] R. Cherubini, V. De Nadal, S. Gerardi, Hyper-radiosensitivity and induced radioresistance and bystander effects in rodent and human cells as a function of radiation quality, Radiat. Prot. Dosim. 166 (1e4) (2015) 137e141. [37] C. Fernandez-Palomo, C. Seymour, C. Mothersill, Inter-relationship between low-dose hyper-radiosensitivity and radiation-induced bystander effects in the human T98G Glioma and the epithelial HaCaT cell line, Rad. Res. 185 (2016). [38] C. MotherSill, C. Seymour, Changing paradigms in radiobiology, Mutat. Res. 750 (2012) 85e95. [39] B.J. Blyth, P.J. Sykes, Radiation-induced bystander effects: what are they, and how relevant are they to human radiation exposures? Radiat. Res. 176 (2) (2011) 139e157.

Effect of Radiation on Biological Structures. Radiation Mutagenesis Chapter | 35

471

[40] US Department of Energy, Office of Science. Low Dose Radiation Research Program. Research Highlights. Radiation-Induced Bystander Effects and Relevance to Human Radiation Exposures. http://lowdose.energy.gov/radiation_bystandereffects.aspx. [41] G. Rattue, Abscopal Effect - When Radiation Also Destroys Non Targeted Tumors, 2012. http://www.medicalnewstoday.com/articles/242784.php. [42] H. Nagasawa, J.B. Little, Induction of sister chromatid exchanges by extremely low doses of a-particles, Cancer Res. 52 (1992) 6394e6396. [43] M. Tomita, M. Maeda, Mechanisms and biological importance of photon-induced bystander responses: do they have an impact on low-dose radiation responses, J. Radiat. Res. 56 (2) (2015) 205e219. [44] W. Han, K.N. Yu, Ionizing radiation, DNA double strand break and mutation (Chapter 7), in: K.V. Urbano (Ed.), Advances in Genetics Research, vol. 4, 2010, p. 13. [45] UNSCEAR Report 2006, Annex C. Non-targeted and Delayed Effects of Exposure to Ionizing Radiation. [46] B. Rajewski, O. Heuse, K. Aurand, Weitere Untersuchungen zum Problem der Ganzkorper Bestrahlung der Weissen Maus. Sofortiger Tod durch Strahlung, Zeitschrift Naturf 86 (1953) 157e159. [47] A.V. Shafirkin, V.V. Bengin, The Peculiarities of the Action of Cosmic Radiation on Biological Objects and Radiation Risk of Long-Term Space Missions. Institute of Nuclear Science of MSU. http://lib.qserty.ru/static/tutorials/01_textbook/index-908.htm. (in Russian). [48] Atomic Bomb Victims’ Appeal. http://www.ne.jp/asahi/hidankyo/nihon/rn_page/english/1988.htm. [49] R. Feldman, Radiation injury, in: J.J. Schaider, S.R. Hayden, R.E. Wolfe, R.M. Barkin, P. Rosen (Eds.), Rosen and Barkin’s 5-Minute Emergency Medicine Consult, third ed., Lippincott Williams & Wilkins, Philadelphia, PA, 2007. [50] T. de Revel, Menace Terroriste Approche Medicale, Nucleaire Radiologique Chimique, 2005.