1
Background Information

This report focuses on the health effects of low-dose, low-LET (low linear energy transfer) radiation. In this chapter the committee provides background information relating to the physical and chemical aspects of radiation and the interaction of radiation with the target molecule DNA. The committee discusses contributions of normal oxidative DNA damage relative to radiation-induced DNA damage and describes the DNA repair mechanisms that mammalian cells have developed to cope with such damage. Finally, this chapter introduces a special subject, the physical characteristics that determine the relative biological effectiveness (RBE) of neutrons, estimates of which are required in the derivation of low-LET radiation risk estimates from atomic bomb survivors.

PHYSICAL ASPECTS OF RADIATION

The central question that must be resolved when considering the physical and biological effects of low-dose ionizing radiation is whether the effects of ionizing radiation and the effects of the free radicals and oxidative reaction products generated in normal cellular metabolism are the same or different. Is ionizing radiation a unique insult to cells, or are its effects lost in the ocean of naturally occurring metabolic reaction products? Can cells detect and respond to low doses of ionizing radiation because of detectable qualitative and quantitative differences from endogenous reaction products?

Different Types of Ionizing Radiation

Ionizing radiation, by definition, contains enough energy to displace electrons and break chemical bonds. Charged particles, such as high-energy electrons, protons, α-particles, or fast heavy ions, are termed directly ionizing because, while they traverse the cell, they ionize numerous molecules by direct collisions with their electrons. Electromagnetic radiations, such as X- and γ-rays, consist of photons that can travel relatively large distances in tissue without interaction. Once an interaction with one of the electrons in the material occurs, part or all of the photon energy is transferred to the electron. The energetic electrons released in this way produce the bulk of ionizations. X- and γ-rays are accordingly termed “indirectly ionizing” radiation. This term is also applied to fast neutrons, because they too traverse large distances in tissue without interaction but can, in occasional collisions, transfer much of their energy to atomic nuclei that in turn produce the main part of the ionizations.

In addition to the distinction between indirectly ionizing and directly ionizing (i.e., uncharged and charged radiation) a distinction is made between sparsely ionizing, or low-LET, and densely ionizing, or high-LET, radiation. The (unrestricted) LET of an ionizing charged particle is defined as the average energy lost by the particle due to electronic interactions per unit length of its trajectory; it is expressed in kiloelectronvolts per micrometer (keV/μm).1 High-energy electromagnetic radiations, such as X-rays or γ-rays, are sparsely ionizing since, in tissue, they release fast electrons that have low LET. Neutrons are densely ionizing because in tissue they release fast protons and heavier atomic nuclei that have high LET.

Figure 1-1 gives the LET of electrons as a function of their kinetic energy and compares it to the considerably higher LET of protons. It is seen that electrons are generally sparsely ionizing while protons are, at moderate energies, densely ionizing. However it is also noted that very energetic protons, as they occur in altitudes relevant to aviation and in space, are sufficiently fast to be sparsely ionizing.

1  

Restricted linear energy transfer, LΔ, results when, within the charged particle tracks, secondary electrons (δ-rays) with energies in excess of Δ are followed separately. It is important to distinguish between track average LET and dose average LET. Dose average LET represents more realistically the high local energy densities that can occur in a track even for low-LET radiation, and it therefore can assume larger values. For example, the track average of L100eV for cobalt-60 γ-rays is 0.23 keV/μm, and the dose average is 5.5 keV/μm (ICRU 1970).



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1 Background Information This report focuses on the health effects of low-dose, low- Once an interaction with one of the electrons in the material LET (low linear energy transfer) radiation. In this chapter occurs, part or all of the photon energy is transferred to the the committee provides background information relating to electron. The energetic electrons released in this way pro- the physical and chemical aspects of radiation and the inter- duce the bulk of ionizations. X- and γ-rays are accordingly action of radiation with the target molecule DNA. The com- termed “indirectly ionizing” radiation. This term is also ap- mittee discusses contributions of normal oxidative DNA plied to fast neutrons, because they too traverse large dis- damage relative to radiation-induced DNA damage and de- tances in tissue without interaction but can, in occasional scribes the DNA repair mechanisms that mammalian cells collisions, transfer much of their energy to atomic nuclei have developed to cope with such damage. Finally, this chap- that in turn produce the main part of the ionizations. ter introduces a special subject, the physical characteristics In addition to the distinction between indirectly ionizing that determine the relative biological effectiveness (RBE) of and directly ionizing (i.e., uncharged and charged radiation) neutrons, estimates of which are required in the derivation of a distinction is made between sparsely ionizing, or low-LET, low-LET radiation risk estimates from atomic bomb and densely ionizing, or high-LET, radiation. The (unre- survivors. stricted) LET of an ionizing charged particle is defined as the average energy lost by the particle due to electronic in- teractions per unit length of its trajectory; it is expressed in PHYSICAL ASPECTS OF RADIATION kiloelectronvolts per micrometer (keV/µm).1 High-energy The central question that must be resolved when consid- electromagnetic radiations, such as X-rays or γ-rays, are ering the physical and biological effects of low-dose ioniz- sparsely ionizing since, in tissue, they release fast electrons ing radiation is whether the effects of ionizing radiation and that have low LET. Neutrons are densely ionizing because in the effects of the free radicals and oxidative reaction prod- tissue they release fast protons and heavier atomic nuclei ucts generated in normal cellular metabolism are the same or that have high LET. different. Is ionizing radiation a unique insult to cells, or are Figure 1-1 gives the LET of electrons as a function of its effects lost in the ocean of naturally occurring metabolic their kinetic energy and compares it to the considerably reaction products? Can cells detect and respond to low doses higher LET of protons. It is seen that electrons are generally of ionizing radiation because of detectable qualitative and sparsely ionizing while protons are, at moderate energies, quantitative differences from endogenous reaction products? densely ionizing. However it is also noted that very ener- getic protons, as they occur in altitudes relevant to aviation and in space, are sufficiently fast to be sparsely ionizing. Different Types of Ionizing Radiation Ionizing radiation, by definition, contains enough energy to displace electrons and break chemical bonds. Charged 1Restricted linear energy transfer, L , results when, within the charged ∆ particles, such as high-energy electrons, protons, α-particles, particle tracks, secondary electrons (δ-rays) with energies in excess of ∆ are or fast heavy ions, are termed directly ionizing because, followed separately. It is important to distinguish between track average LET and dose average LET. Dose average LET represents more realisti- while they traverse the cell, they ionize numerous molecules cally the high local energy densities that can occur in a track even for low- by direct collisions with their electrons. Electromagnetic ra- LET radiation, and it therefore can assume larger values. For example, the diations, such as X- and γ-rays, consist of photons that can track average of L100eV for cobalt-60 γ-rays is 0.23 keV/µm, and the dose travel relatively large distances in tissue without interaction. average is 5.5 keV/µm (ICRU 1970). 19

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20 BEIR VII 100 LET / keV / µm 10 protons 1 electrons 0.1 0.01 0.1 1 10 100 1,000 10 4 energy / MeV FIGURE 1-1 Linear energy transfer of protons and electrons in water. SOURCE: Data from ICRU (1970). The effects of high-LET particles (i.e., protons and atom releasing photons of discrete energy. Conventional X- heavier ions) are outside the scope of this report. However, rays, used for diagnostic radiology, are commonly produced neutrons and their high relative biological effectiveness must with accelerating voltages of about 200 kV. For mammogra- be considered in the context of low-LET risk estimates de- phy, where high contrast is sought and only a moderate thick- rived from the observations on delayed health effects among ness of tissue must be traversed by the X-rays, the low accel- A-bomb survivors. The reason is that a small fraction of the eration voltage of 29 kV is usually employed. absorbed dose to A-bomb survivors was due not to the pre- There are three different types of energy-transfer pro- dominant high-energy γ-rays, but to fast neutrons. Because cesses whereby photons of sufficient energy eject electrons of the greater effectiveness of these fast neutrons, this small from an atom, which can then interact with other atoms and dose component must be taken into consideration. molecules to produce a cascade of alterations that ultimately lead to observable biological effects. These are the photo- electric process, Compton scattering, and pair production. Photon Spectral Distributions At low energies (<0.1 MeV), the photoelectric process The absorption and scattering of photons depends on their dominates in tissue. A photon interacts with and ejects an energy. The γ-rays from radioactive decay consist of electron from one of the inner shells of an atom. The photon monoenergetic photons that do not exceed several million is extinguished, and most of its energy is imparted to the electronvolts (MeV) in energy; γ-rays that result from the ejected electron as kinetic energy. fission of uranium or plutonium have a spectrum of energies At medium photon energies (about 0.5–3.5 MeV), with a maximum of 2 MeV. Higher-energy γ-rays, up to Compton scattering is the most probable event. Compton 7 MeV, can be generated by inelastic scattering, as occurred scattering occurs when an incoming photon’s energy greatly in the neutron-nitrogen interaction from the atomic bomb exceeds the electron-binding energy of the affected atom. In explosions in Hiroshima and Nagasaki. this case the energy of the incoming photon is converted into Artificially produced X-rays have a wide spectrum of the kinetic energy of an ejected electron and a secondary energies resulting from the deceleration of electrons as they “scattered” photon. The scattered photon has less energy than traverse high-atomic-number materials. A continuous distri- the primary photon and can undergo further Compton scat- bution of photon energies is generated, with a mean energy tering until its energy is sufficiently degraded for the photo- of about one-third the maximal energy of the accelerated electric process to occur. electrons. Added filtration selectively removes the “soft” At energies greater than 1.02 MeV, pair production can (i.e., less energetic) photon component and, thus, hardens occur. A photon interacts with an atomic nucleus, and the the X-rays. Discrete energy “spikes” also occur in the X-ray photon energy is converted into a positron and an electron. spectrum; these spikes originate in the ejection of electrons The photon energy above 1.02 MeV is converted into the from atoms of the affected element, which is followed by the kinetic energy of the newly created particles. The electron transition of electrons from outer shells to inner shells of the and the positron interact with and can ionize other molecules.

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BACKGROUND INFORMATION 21 100 10 photons Range or mean free path in water / cm 1 neutrons 0.1 electrons 0.01 protons 0.001 0.0001 0.01 0.1 1 10 100 energy / MeV FIGURE 1-2 Mean free path of photons and neutrons in water and range of electrons and protons. SOURCE: Data from ICRU (1970). The positron ultimately interacts with another electron, and sparsely ionizing) radiation. There are, nevertheless, differ- this results in an “annihilation” event in which the mass is ences in effectiveness and possibly also differences in the extinguished and two 0.51 MeV photons are emitted in op- risk for late effects due to these radiations. posite directions. The annihilation photons can themselves produce further ionizations. Track Structure Figure 1-2 shows the mean free path for monoenergetic photons (i.e., the average distance in water until the photon The passage of fast electrons through tissue creates a track undergoes an interaction). To compare the penetration depth of excited and ionized molecules that are relatively far apart. of photon radiation with that of electron radiation, the mean X- and γ-rays produce electrons with relatively low linear range of electrons of specified energy is given in the same energy transfer, (i.e., energy loss per unit track length) and diagram. It is seen that the electrons released by photons are are considered low-LET radiation. For example, the track always considerably less penetrating than the photons them- average of unrestricted LET of the electrons liberated by selves. cobalt-60 (60Co) gamma rays is about 0.25 keV/µm, which Figure 1-3 compares in terms of the distributions of pho- can be contrasted with an average LET of about 180 keV/ ton energy fluence the γ-rays from the A-bomb explosions µm for a 2 MeV α-particle, a high-LET radiation. LET is an with the distributions of photon energy for orthovoltage X- important measure in the evaluation of relative biological rays and low-energy mammography X-rays. These different effectiveness (ICRU 1970; Engels and Wambersie 1998) of electromagnetic radiations are all classified as low-LET (i.e., a given kind of radiation.

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22 BEIR VII 2.5 30 kV 200 kV x-rays 2 x-rays energy fluence rel to log-scale 1.5 γ -rays Hiroshima 1 0.5 0 0.01 0.1 1 10 photon energy, E /MeV FIGURE 1-3 Distributions of photon energy fluence for mammography X-rays, orthovoltage X-rays, and γ-rays from the atomic bomb explosion in Hiroshima. The distributions of the energy fluence relative to the logarithmic scale of energy are plotted, because they represent roughly the fractional contribution of incident photons of specified energy to the dose absorbed by a person. SOURCE: Data from Seelentag and others (1979) and Roesch (1987). Different Effectiveness of γ-Rays and X-Rays X-rays) produce less energetic Compton electrons with higher LET. This explains the substantial difference between LET and Related Parameters of Radiation Quality the mean LET of high-energy γ-rays and conventional X- rays. For lower-energy X-rays the photon energy is further While γ-rays and X-rays of various energies are all reduced, and the photo effect (i.e., the total transfer of pho- sparsely ionizing, in the body they generate electrons with ton energy to electrons) begins to dominate. Accordingly, somewhat different spectra of LET values (ICRU 1970). To the average energy of the electrons begins to increase again, quantify the differences, reference is usually made to the which explains the relatively small difference in average dose average LET or to the mean values of the related LET between 200 kV X-rays and soft X-rays. At very low microdosimetric parameter dose-averaged linear energy, y. photon energies (i.e., less than about 20 keV) the LET val- Figure 1-4 gives the dose average LET values for the elec- ues increase strongly, but these ultrasoft X-rays are of little trons released by monoenergetic photons (solid curves) and concern in radiation protection because of their very limited compares these values to the averages for 29 kV mammog- penetration depth. raphy X-rays and 200 kV X-rays (solid circles and squares, The dose average, LD,∆, of the restricted LET is a param- respectively; ICRP 2003). In addition to the dose average, eter that correlates with the low dose effectiveness of photon LD, of the unrestricted LET, the diagram contains the dose or electron radiation. With a cutoff value ∆ = 1 keV, the nu- averages, LD,∆, of the restricted LET, L∆. The restricted LET merical values of LD,∆ are consistent with a low-dose RBE of treats the ∆-rays beyond the specified cutoff energy ∆ as about 2 for conventional X-rays versus γ-rays. A similar de- separate tracks. This accounts in an approximate way for the pendence on photon energy is seen in the related micro- increased local energies due to ∆-rays and therefore provides dosimetric parameter dose lineal energy, y, which has been larger values that are more meaningful than those of unre- used as reference parameter by the liaison committee of the stricted LET. International Commission on Radiological Protection High-energy photons (e.g., 60Co γ-rays) release Compton (ICRP) and the International Commission on Radiation Units electrons of comparatively high energy and correspondingly and Measurements (ICRU) in The Quality Factor in Radia- low LET. Photons of less energy (e.g., conventional 200 kV tion Protection (ICRU 1986). Figure 1-5 gives values of its

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BACKGROUND INFORMATION 23 10 / (keV/ µm) 8 L 100eV,D 6 D, ∆ / (keV/ µ m), L 4 L 1keV,D 2 L D L D 0 0.01 0.1 1 photon energy, E / MeV ph FIGURE 1-4 The dose mean restricted and unrestricted linear energy transfer for electrons liberated by monoenergetic photons of energy Eph. The dots and squares give the values for the 29 kVp and the 200 kVp X-rays. They are plotted at the weighted photon energies of the X- ray spectra. SOURCE: Data from Kellerer (2002). 10 8 0.24 mm y / (keV/ m m) 6 0.96 mm 4 D d = 3.9 mm 2 0 0.01 0.1 1 photon energy / MeV FIGURE 1-5 Measured dose average lineal energy, yD, for monoenergetic photons and for different simulated site diameters, d. SOURCE: Data from Kliauga and Dvorak (1978).

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24 BEIR VII dose average, yD, as measured by Kliauga and Dvorak (1978) from synchrotron radiation. The lower panel gives analo- for various photon radiations and for different simulated site gous data obtained by Schmid and others (2002). diameters, d. The diagram demonstrates that there is a substantial de- The γ-rays from the atomic bomb explosions had average crease of the yield of dicentrics from conventional X-rays to energies between 2 and 5 MeV at the relevant distances γ-rays. The photon energies below 20 keV are of special in- (Straume 1996). Figures 1-4 and 1-5 do not extend to these terest with regard to biophysical consideration, but are less energies; however, it is apparent from Figures 1-4 and 1-5 relevant to exposure situations in radiation protection. They that the mean values of the restricted LET or the lineal en- are included here to show the full trend of the energy depen- ergy do not decrease substantially beyond a photon energy dence. of 1 MeV. There is, thus, little indication that the hard γ-rays It is seen that the low-dose RBE for dicentrics for moder- from the atomic bombs should have an RBE substantially ately filtered 200 kV X-rays is about 2–3 relative to γ-rays, less than unity compared to conventional 60Co γ-rays. while the RBE of mammographic X-rays (29 kV) relative to the moderately filtered 200 kV X-rays is somewhat in excess of 1.5. Information from In Vitro Studies The data for dicentrics in Figures 1-6 are reasonably con- It has long been recognized in experimental radiobiology sistent with the LET values in Figure 1-4 for a cutoff value that low-LET radiations do not all have the same effective- in excess of 1 keV. The difference by a factor of 2–3 in the ness at low doses. With regard to mutations in Tradescantia, low-dose effectiveness of conventional X-rays and γ-rays has aberrations in human lymphocytes, and killing of mouse been known and, even if it should apply equally to radiation- oocytes (Bond and others 1978), conventional 200 kV X- induced late effects, would not necessarily require a depar- rays have been found to be about twice as effective at low ture from the current convention for radiation protection, doses as high-energy γ-rays. Fast electrons may be even less which assigns the radiation weighting factor unity to all pho- effective than γ-rays. These differences are most clearly ton radiations. However, the difference has to be noted when- documented in cell studies and, especially, in studies on ever risk estimates are derived from exposures to γ-rays and chromosome aberrations (Sinclair 1985; ICRU 1986). The then applied to X-rays. most reliable and detailed data on photon RBE exist for chro- Apart from these considerations it is uncertain whether mosome aberrations in human lymphocytes. Edwards and the marked dependence of the low-dose RBE on photon en- others (1982) have obtained the data for dicentrics in human ergy for chromosome aberrations also is representative for lymphocytes listed in Table 1-1 for 15 MeV electrons, 60Co late radiation effects in man. The dependence of RBE on γ-rays, and 250 kV X-rays. New data have since confirmed photon energy for dicentric chromosomes reflects the fact these substantial differences of effectiveness for different that the dose dependencies have large curvature for 60Co γ- types of penetrating low-LET radiations. rays (α/β = 0.2 Gy in the data reported by Schmid and oth- Sasaki and colleagues (1989; Sasaki 1991) have deter- ers 2002), but little curvature for 29 kV X-rays (α/β = mined the yields of dicentrics in human lymphocytes over a 1.9 Gy). If there were no curvature below 1 Gy in the dose broad range of photon energies. The upper panel of Figure 1- relations for chromosome aberrations, the low-dose RBE of 6 gives the linear coefficients (and standard errors) from lin- 29 kV X-rays would be only 1.65 compared to 60Co γ-rays. ear-quadratic fits to the dose dependencies. The closed Since the dose dependence for solid tumors among A-bomb circles relate to γ-rays and to broad X-ray spectra; the survivors indicates little curvature, the dependence of risk squares, to characteristic X-rays and monoenergetic photons on photon energy may be similarly weak for tumor induction in man. It is of interest to compare the biophysical informa- tion and the experimental results to the radioepidemiologic evidence for health effects. TABLE 1-1 Low-Dose Coefficients (and standard errors) Information from Radioepidemiology for Induction of Chromosome Aberrations in Human Lymphocytes by Low-LET Penetrating Radiation Numerous epidemiologic studies on medical cohorts have provided risk estimates that exhibit considerable variation. Radiation Type Dicentrics per Cell per Gray Many of these studies on patients relate to X-ray exposures, but there is no consistent epidemiologic evidence for higher 15 MeV electrons 0.0055 (± 0.011) risk factors from X-rays than from γ-rays. In fact, while the 60Co γ-rays 0.0157 (± 0.003) risk estimates from medical studies are not inconsistent with 250 kV X-rays 0.0476 (± 0.005) those for atomic bomb survivors, they tend to be, as a whole, NOTE: The low-dose coefficients represent the linear component of a lin- somewhat lower (UNSCEAR 2000b). The radiation-related ear-quadratic fit to the data. SOURCE: Data from Edwards and others increase in breast cancer incidence can serve as an example (1982). because it has been most thoroughly studied.

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BACKGROUND INFORMATION 25 0.5 human lymphocytes dicentrics per cell / Gy 0.1 137 Cs 60 220kV Co 0.01 1 10 100 1000 mean photon energy / keV 0.1 10kV 29kV dicentrics per cell / Gy 60kV 137 Cs 60 Co 220kV 0.01 1 10 100 1000 mean photon energy / keV FIGURE 1-6 Data points are linear coefficients (and standard errors) of the dose dependence for dicentric chromosomes in human peripheral blood lymphocytes. Squares are for monoenergetic photons; circles are X-ray spectra or γ-rays. The two data points in the lower panel labeled 220 kV both had 220 kV generating voltage, but the filtration was different. SOURCE: Upper panel: Data from Sasaki and others (1989; Sasaki 1991). Lower panel: Data from Schmid and others (2002). Figure 1-7 gives risk estimates from major studies on ra- The uncertainties are large, and the risk estimates vary diation-induced breast cancer. The estimated risk coeffi- widely because the patient treatment regimes differed not cients (and 90% confidence intervals) are expressed in terms only in the type of radiation but also in the various exposure of the excess relative risk (ERR) per gray and the excess modalities, such as acute, fractionated, or protracted expo- absolute risk (EAR) per gray per 10,000 person-years (PY). sure; whole- or partial-body exposure; exposure rate; and

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26 BEIR VII FIGURE 1-7 Excess relative risk (and 90% confidence interval) from various epidemiologic studies of breast cancer. The upper panel shows the excess relative risk per gray, the lower panel, the absolute risk per 10,000 person-years per gray. (For the description of individual studies, see UNSCEAR 2000b and Preston and others 2002a.) The confidence limit for the study of cervical carcinoma patients is recalculated. Cohorts: LSS: Life Span Study of atomic bomb survivors; MasTb: Massachusetts tuberculosis patients; PPMast: New York postpartum mastits patients; SwBBD: Swedish benign breast disease patients; CervCa: cervical cancer patients (case-control study); RochThym: Rochester infants with thymic enlargement; SwHem: Swedish infants with skin hemangioma. magnitude of the exposure. Furthermore, there are ethnic ies and biophysical considerations suggest a low-dose RBE differences, including those related to life-style, that are as- for conventional X-rays versus hard γ-rays of about 2–3, this sociated with greatly different background rates of breast difference cannot be confirmed at present through epidemio- cancer. Populations with low spontaneous rates tend to ex- logic investigations. hibit comparatively high ERR, while their EAR tends to be low. This complicates the comparison of risk estimates, since Effects of Radiation on DNA, Genes, and Chromosomes it remains uncertain whether relative or absolute excess inci- dence is the more relevant measure of risk. The probability that a low-LET primary electron will in- The various exposed cohorts also differ considerably in teract with a DNA molecule along its track is low, but a the duration of follow-up and, especially, the age at expo- direct interaction of this sort is possible (Nikjoo and others sure. The last two studies (RochThym, SwHem) relate to 2002). Along the primary electron track, secondary electrons exposures in childhood, while the remainder refer to expo- with lower energies are also formed, producing clusters of sures at intermediate or higher ages. The last factor is espe- ionizations (see Figure 1-8, panel A). If such an ionization cially critical, because both ERR and lifetime integrated cluster occurs near a DNA molecule, multiple damages can EAR decrease substantially with increasing age at exposure. occur in a very localized segment of the DNA (Figure 1-8, The dominant influence of the various modifying factors panel B). These clusters have been referred to as as clus- makes it impossible on the basis of epidemiologic data to tered-damage or locally multiply damaged sites (LMDS) confirm the difference in effectiveness between γ-rays and (Ward and others 1985; Goodhead 1994). X-rays or the difference between X-rays of different ener- Figure 1-8 illustrates two typical structures of electron gies. Studies related to other types of cancer are even further tracks produced by low-LET photons (e.g., γ-rays). The removed from providing an answer. Thus, although cell stud- wavy lines outside the sphere represent primary and second-

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BACKGROUND INFORMATION 27 material, which directs the structure and function of the or- ganism. This genetic material is made up of DNA organized Low-LET tracks in cell nucleus into genes and chromosomes (for a brief description, see for example, Appendix A). Radiation can damage DNA as described in from gamma rays this chapter, and the damage can result in cell lethality, im- paired cell function, or may produce damage involved in the carcinogenic process. Radiation has also been shown to pro- duce heritable gene mutations in animals. For a basic de- scription of gene mutations, see Appendix A. A dose of 1 Gy corresponds to about 1000 tracks Relative Biological Effectiveness of Neutrons This report assesses the biological effects of low-LET radiation, that is, photons and electrons. It does not deal with densely ionizing radiation, such as heavy ions (including α- particles) and fast neutrons. Although neutrons need not be considered here on their own account, they must be ac- counted for in the analysis of the most important source of information on radiation risks, observations on the atomic bomb survivors of Hiroshima and Nagasaki. Such analysis requires consideration of the relative biological effective- ness of neutrons. The following remarks deal with the RBE of neutrons in general terms. According to the 1986 dosimetry system, DS86, only a Tracks in chromatin fiber small fraction of the absorbed dose to atomic bomb survi- vors was due to neutrons—about 2% in Hiroshima in the Cluster most relevant dose range and 0.7% in Nagasaki (Roesch Low-LET tracks 1987). The current reevaluation of the Hiroshima and Nagasaki dosimetry, DS02, is in general agreement with these observations. However, although the absorbed dose fraction of neutrons was small in both cities, it is known FIGURE 1-8 Panel A: Illustration of primary and secondary elec- from a multitude of radiobiological investigations that the tron tracks producing clusters of ionization events. The calculated RBE of small neutron doses can be large enough for even the number of tracks is based on a nucleus with a diameter of 8 µm. small absorbed dose fraction to add appreciably to the late The track size is enlarged relative to the nucleus to illustrate the theoretical track structure. Panel B: Illustration of clustered health effects among atomic bomb survivors. damage. The arrow identifies an ionization cluster near a DNA mol- Fast neutrons interact with exposed tissue predominantly ecule to represent the possibility of locally multiply damaged sites. by releasing recoil nuclei. At neutron energies up to a few Only a segment of the electron track is illustrated in Panel B. million electronvolts, the energy transfer is predominantly to protons. On the average, a neutron transfers half its en- ergy to a recoil proton in a collision. Neutrons of 1 MeV therefore produce recoil protons with an average initial en- ergy of 500 keV. At a neutron energy of 0.4 MeV, the typi- ary photons; the straight lines represent the paths of ejected cal recoil proton energy is 200 keV, enough to allow the electrons. For clarity of presentation, the size of the tracks is proton to go through its maximal LET of about 100 keV/µm, increased relative to the cell and is not drawn to scale. As the which is reached at its Bragg peak energy of 0.1 MeV. The energetic electron interacts with atoms of the material, sec- ionization density in such proton tracks is far greater than ondary electrons are produced and kinetic energy is lost. that in an electron track, as depicted in Figure 1-1. It is evi- Such collisions can result in deflection of the primary elec- dent that the resulting high local energy concentration will tron from its original path (Figure 1-8, panel A). Important produce far more clusters of closely spaced ionizations than components of the track structure are the clusters of second- do low-LET photons and thus more LMDS (clustered dam- ary ionizations that occur in a very small volume (see Fig- age) that may remain unrepaired or misrepaired. In addition, ure 1-8, panel B). These clusters, acting directly or indirectly recoil protons have track lengths of a few micrometers, so on the DNA molecule, may produce clustered damage, critical damage can, with fairly high probability, be caused LMDS, that may in turn be refractory to repair. The likely in neighboring chromosomal structures. The interaction of site of health effects of low-dose radiation is the genetic closely spaced chromosomal damage has long been noted to

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28 BEIR VII be a critical factor in the production of chromosomal aberra- tal studies, the numerical values of RBE vary, and the varia- tions (Lea 1946). tion appears to be largely a matter of the different magnitude Recoil protons with energy of a few hundred kiloelectron- of the linear dose component for photon radiation. volts appear, in line with the above biophysical consider- Cell survival curves usually exhibit pronounced initial ations, to be the particles that produce maximal cellular dam- slopes, and the observed maximal neutron RBE rarely ex- age per unit energy imparted. This is confirmed by various ceeds a factor of about 10. For dicentric chromosomal aber- experimental studies that consistently demonstrate the maxi- rations in human lymphocytes, values of about 70 are ob- mal effectiveness of neutrons at a neutron energy of about tained for the maximal RBE of 0.5 MeV neutrons against 0.4 MeV (Kellerer and Rossi 1972b). γ-rays (Dobson and others 1991; Schmid and others 2000). The dose-effect relationship, E(D), for photons can in This large maximal value might be seen as an indication of many radiobiological investigations be described as a linear an exceptionally high effectiveness of neutrons at low doses. quadratic function of absorbed dose: In fact the dose-effect relationship for neutrons is simply linear, and the high maximal RBE of neutrons is merely a E(D) = aDγ + bDγ2. (1-1) reflection of the very shallow and imprecisely known (stan- dard error, 30–40%) initial slope in the dose-effect relation- In experiments with fast neutrons, the effect is typically ship for γ-rays. The RBE of neutrons versus a γ-ray dose of proportional to the absorbed dose, Dn, of neutrons over a 1 Gy is only about 12 (Bauchinger and others 1983; Schmid variable dose range depending on the tissue and effect: and others 2000). In the context of risk estimation, the major interest is in E(D) = anDn. (1-2) neutron RBEs that have been evaluated in animal experi- ments with regard to tumor induction. A multitude of results The linear dose coefficient, an, for neutrons is always sub- have been reported in the literature for many tumor systems stantially larger than the linear dose coefficient, a, for pho- (NCRP 1990). Experiments with rodents show considerable ton radiation. The RBE of neutrons is defined as the ratio of variation, especially in female mice and rats, and this varia- a γ-ray dose to the neutron dose that produces the same tion reflects the decisive influence of hormonal status. In effect: experiments with female Sprague-Dawley rats, Shellabarger and others (1980) found that 4 mGy of fast neutrons pro- RBE = Dγ / Dn, with: E(Dγ) = E(Dn). (1-3) duced as many mammary neoplasms as 0.4 Gy of X-rays, which implied an RBE of 100. Broerse and Gerber (1982) In terms of Equations (1-1) and (1-2), RBE can be ex- used female Sprague-Dawley rats, which have a much lower pressed as a function of the neutron dose or the photon dose. spontaneous incidence, and found substantially lower values The latter expression is somewhat simpler: of neutron RBE. However, considerable differences in neu- tron RBE at higher doses were observed for different tumor RBE(Dγ) = an /(a + bDγ). (1-4) types. As an extreme example, one may refer to lung ad- enomas in female RFM mice, in which there is a clear reduc- This implies that RBE assumes its maximal value, tion in age-adjusted incidence after γ-ray exposures up to RBEmax = an/a, at low doses, whereas it decreases with in- about 2 Gy, but neutron doses of 0.2 Gy cause a substantial creasing dose and then tends to be inversely proportional to increase (Ullrich and others 1976). The simple assumptions the photon dose. made in the calculation of RBE do not seem to be applicable in such a case. In view of this complexity, it appears best to refer to ex- Experimental Observations periments with male mice or rats that determine the overall Indeed, numerous experimental investigations of chromo- incidence of solid tumors. In an extensive series of studies of somal aberrations, cellular transformations, and cell killing the French Commissariat a l’Energie Atomique using male have confirmed that maximal RBE values of neutrons occur Sprague-Dawley rats, a fission neutron dose of 20 mGy was at low doses and that, at somewhat higher doses, RBE varies consistently found to be equivalent to an acute γ-ray dose of inversely with increasing reference dose (i.e., the photon 1 Gy with regard to both nonlethal tumors (Lafuma and dose). The same has been observed for more complex ef- others 1989) and lethal tumors (Wolf and others 2000). This fects such as opacification of the lens and, more important in comparison corresponds to a neutron RBE of 50 against a the context of risk assessment, induction of tumors in ani- reference γ-ray dose of 1 Gy. When the experiments were mals. A synopsis of such findings was provided in the con- evaluated in terms of life shortening as a proxy for tumor text of the microdosimetric interpretation of the neutron RBE mortality, the inferred RBE was closer to 30 (Wolf and others (Kellerer and Rossi 1972b). 2000). Smaller values of the RBE—around 20 compared to Although the general features of the dependence of neu- a γ-ray dose of 1 Gy and about 15 compared to X-rays—are tron RBE on dose are brought out consistently in experimen- suggested by major studies with mice that were evaluated in

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BACKGROUND INFORMATION 29 terms of life shortening, again as a reflection of increased logical effects. Signal transduction from cell membrane mortality from tumors (Storer and others 1988; Carnes and phospholipids damaged by free radicals and oxidizing reac- others 1989; Covelli and others 1989). tions is an important natural process. This is one set of bio- In all experimental studies with rodents, it was difficult or chemical pathways by which the effects of ionizing radia- impossible to determine excess tumor rates at γ-ray doses tion may overlap with the effects of endogenous processes, substantially less than 1 Gy. For the purpose of risk estima- such as macrophage oxidative bursts. These processes may tion, it is therefore assumed in this report that the relevant underlie those seen in irradiated cells that have been charac- animal experiments with rodents indicate a neutron RBE for terized as “bystander effects” and “adaptation” (see Chap- solid tumors of 20–50 compared to a reference γ-ray dose of ter 2). 1 Gy. Experimental evidence suggests lower neutron RBEs Nikjoo and colleagues (1997, 2002) have modeled the for leukemia; in experiments with RFM mice (Ullrich and probability of electron and OH• radical interaction with Preston 1987), an RBE of about 3 was seen versus a γ-ray DNA. In a 1997 publication, they modeled the spectrum of dose of 0.5 Gy; at lower γ-ray doses, statistical uncertainty DNA damage (direct energy deposition and reactions with did not permit the specification of a neutron RBE. diffusing OH• radicals) induced by low-energy secondary electrons (0.1–4.5 keV). They note that to extrapolate avail- able epidemiologic and experimental data from high-dose CHEMICAL ASPECTS OF RADIATION and high-dose-rate studies to the relevant low levels of single isolated tracks, it is essential to develop a more molecular Electron Ionization of Water Molecules and Indirect and mechanistic approach based on the amounts, types, and Effects on DNA repairability of the early molecular damage that results from As previously described, free electrons can be produced the initial physical and chemical processes. Their calcula- by X- and γ-ray interactions with atoms in tissue. These elec- tions for secondary electrons show that most (about 66–74%) trons can then interact with the DNA molecule and create low-energy electron interactions in DNA “do not lead to damage in the form of strand breaks or damaged bases; these damage in the form of strand breaks and when they do occur, are known as direct effects. Indirect effects can occur after a they are most frequently single strand breaks” (SSBs). Al- photon interacts with a water molecule. Water molecules though the data are complex, SSB percentages in their study make up 70% of human tissue. Ejection of an electron from range from about 22 to 27% in the electron energy range of a water molecule by an incoming photon produces an ion- 0.1–4.5 keV and double-strand break (DSB) percentages ized water molecule, H2O+. Trapping of the electron by po- range from about 1.4–2.4% in the same energy range. How- larizing water molecules produces a so-called hydrated elec- ever, more than 30% of DSBs are of a more complex form; tron, e-aq. When the ionized water molecule collides with these complex breaks are somewhat analogous to LMDS, another water molecule, it reacts to produce a highly reac- but Nikjoo and colleagues do not include base damage in tive hydroxyl radical, OH•, according to the reaction their model. Their calculations also indicate that the DNA damage tends to be along short lengths of DNA: 1–34 base H2O+ + H2O → OH• + H3O+. pairs (bp) for 0.3 and 1.5 keV electrons. The authors con- clude that the large deletions seen in radiation-induced mu- Other reactions produce a hydrogen radical (H•), hydro- tations may have other mechanisms, such as nonhomologous gen peroxide, and water. Thus, these reactions produce three recombination (Nikjoo and others 1997). important reactive species—e-aq, H•, and OH•, which have In the case of energetic electron interactions with DNA initial relative yields of about 45%, 10%, and 45%, respec- (0.1 eV to 100 keV electrons), Nikjoo and others (2002) es- tively, in the case of γ-radiation. The reactive species can timate that more than 80% of the interactions do not cause damage DNA, and such damage is termed an indirect effect. damage in the form of DNA SSBs. Of the interactions that The relatively long-lived (about 10–5 s) OH• radical is do cause strand breaks, the authors calculate that a small believed to be the most effective of the reactive species; as percentage (about 0.5–1.4%) produce DSBs. They note, an oxidizing agent, it can extract a hydrogen atom from the however, that there is still a considerable contribution deoxyribose component of DNA, creating a DNA radical. (>20%) to the DSB yield from complex DSBs in which a Early experiments demonstrated that about 70% of the DNA simple DSB is accompanied by at least one additional strand damage can be prevented by the addition of OH• scavengers break within 10 bp. As in the low-energy study just de- (Roots and Okada 1972). Because OH• is so highly reactive, scribed, this model does not include any contribution to the it has been estimated that only the radicals formed within yield of strand breaks from damaged bases. about 3 nm of DNA can react with it (Ward 1994). Although Another recent study suggests that single low-energy DNA is deemed the most important target for biological electrons can produce DNA SSBs and DSBs at energies be- damage that leads to health effects, other sites—such as the low ionization thresholds (Boudaiffa and others 2000). The nuclear membrane, the DNA-membrane complex, and the authors speculate that these breaks are initiated as direct outer cell membrane—may also be important for some bio- damage by resonant electron attachment to DNA compo-

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32 BEIR VII refractory to repair, the risk to humans posed by ionizing 1998; Waters and others 1999) protects the cytotoxic abasic radiation may be viewed as greater than that posed by en- residue and may delay the rearrangement of the base-free dogenous oxidative stress. deoxyribose into a reactive free-aldehyde conformation that could cause cross-linking and other unwanted side effects. The main human apurinic-apyrimidinic (AP) endonu- MOLECULAR MECHANISMS OF DNA REPAIR clease, APE1, occupies a pivotal position in BER of anoma- Ionizing radiation can cause a wide array of damage to lous residues, recognizing and cleaving at the 5′ side of individual DNA bases and SSBs and DSBs resulting from abasic sites generated by spontaneous hydrolysis, reactive deoxyribose destruction (for basic biological and genetic oxygen species, and DNA glycosylases. Abasic sites gener- concepts, see Appendix A). Damaged bases are repaired by ated by nonenzymatic depurination probably outnumber mechanisms that involve excision and replacement of indi- those generated by all of the DNA glycosylases; conse- vidual damaged bases (base-excision repair) or of larger oli- quently, APE1 and subsequent key proteins in the BER path- gonucleotide fragments (nucleotide-excision repair). SSBs way (XRCC1 and polymerase β) are essential, whereas mice are repaired in a process similar to base-excision repair with with knockouts of various DNA glycosylases so far investi- some of the same enzymatic components. DSBs potentially gated have been viable (Wilson and Thompson 1997). In a involve a number of repair processes, especially because or- substrate recognition process similar to DNA glycosylases, ganisms require the ability to distinguish between breaks APE1 flips out the base-free deoxyribose residue from the caused by damage and those associated with normal pro- double helix before chain cleavage (Gorman and others cesses, such as recombination, telomere maintenance, DNA 1997; Parikh and others 1998). When bound to DNA, the replication, and processing of genes encoding antibodies. APE1 protein interacts with the next enzyme in the BER Some DSBs are simply rejoined end to end in a process pathway, POL β, and recruits the polymerase to the site of called nonhomologous end joining (NHEJ). Others are re- repair (Bennett and others 1997). POL β has two distinct paired by a process of homologous recombination (HR) in domains that are well suited for DNA gap filling during BER. which the broken strand is repaired by crossing over with an The larger domain is the polymerase domain itself; a small adjacent identical DNA sequence; this generally occurs only basic NH2-terminal domain contains an AP lyase activity during or after chromosome duplication and before chromo- that excises the abasic sugar-phosphate residue at the strand some segregation. Damage, especially DSBs, also elicits a break (Matsumoto and Kim 1995; Sobol and others 1996). signal transduction process that uses a cascade of kinase and POL β also interacts with the noncatalytic XRCC1 subunit other protein modifications and changes in gene transcrip- of the XRCC1-DNA ligase III heterodimer. Consequently, tion, all of which contribute to a cellwide response to DNA XRCC1 acts as a scaffold protein by bringing the polymerase damage. and ligase together at the site of repair and interacts with poly(ADP-ribose) polymerase and polynucleotide kinase (Whitehouse and others 2001); further stabilization of the Base-Excision Repair complex may be achieved by direct binding of the NH2- Release of altered bases by base-excision repair (BER) is terminal region of XRCC1 to the DNA SSB (Kubota and initiated by DNA glycosylases that hydrolytically cleave the others 1996; Marintchev and others 1999). XRCC1 contrib- base-deoxyribose glycosyl bond of a damaged nucleotide utes to the normal X-ray resistance of mammalian cells, and residue (Figure 1-9). A present estimate would be that hu- mutant cells with a defective XRCC1 protein are hyper- man cell nuclei have ten to twelve different DNA glyco- sensitive to ionizing radiation. sylases, which have varied but overlapping specificities for When the terminal sugar-phosphate residue has a more different base damage. BER has two main pathways that re- complex structure that is relatively resistant to cleavage by sult in replacement of the damaged base with either a short the AP lyase function of POL β, DNA strand displacement or a long patch. may occur instead—involving either POL β or a larger poly- A common strategy for DNA glycosylases, deduced merase such as POL δ—for filling in gaps a few nucleotides largely from structural studies, appears to be facilitated dif- long (Fortini and others 1998; Dianov and others 1999). The fusion along the minor groove of DNA until a specific type FEN1 structure-specific nuclease removes the displaced flap, of damaged nucleotide is recognized. The enzyme then kinks and the PCNA protein stimulates these reactions (Wu and the DNA by compression of the flanking backbone in the others 1996; Klungland and Lindahl 1997), acting as a scaf- same strand as the lesion, flips out the abnormal nucleoside fold protein in this alternative pathway in a way similar to residue to accommodate the altered base in a specific recog- that of XRCC1 in the main pathway. Another replication nition pocket, and mediates cleavage (Parikh and others factor, DNA ligase I (LIG1), then completes this longer- 1998). The DNA glycosylase then may remain clamped to patch form of repair. An important property of FEN1 here, the damaged site until displaced by the next enzyme in the in addition to processing the 5′ ends of Okazaki fragments BER pathway, APE1 (also called HAP1), which has greater during lagging-strand DNA replication, is to minimize the affinity for the abasic site. This strategy (Parikh and others possibility of hairpin-loop formation and slippage during

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BACKGROUND INFORMATION 33 FIGURE 1-9 Base-excision repair. This pathway repairs single-base damage (from X-rays, reactive oxygen species, methylation, or deami- nation), apurinic sites, and SSBs (from X-rays). A damaged base is removed by glycosylases, leaving an apurinic site that is a substrate for apurinic endonuclease (APE1), which converts it into a SSB. X-ray breaks are modified by XRCC1, polynucleotide kinase (PNK), and poly(ADP-ribose) polymerase (PARP) to produce a cleaved substrate with 3′ and 5′ termini similar to those produced by APE1. The break is then patched by short- or long-patch BER. The short-patch pathway predominates in mammalian cells, and involves polymerase β, which can remove a 5′-deoxyribose moiety by its lyase activity and then insert a single base patch that is sealed by DNA ligase III. The long-patch pathway involves polymerase δ or ε, which is anchored to DNA by a PCNA collar and carries out strand displacement synthesis. The displaced flap is cleaved by the structure-specific endonuclease FEN1, and the patch is sealed by ligase I. XRCC1 is a nonenzymatic scaffold protein that interacts with many of the participants of BER and anchors them to the substrate and hands on repair intermediates through successive stages of BER. SOURCE: Reproduced with permission from J.H. Hoeijmakers (2001).

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34 BEIR VII strand displacement and subsequent DNA synthesis, which spontaneous sister-chromatid exchanges and sensitivity to might otherwise result in local expansion of sequence re- ionizing radiation. Extracts of cells from such mice contain peats (Tishkoff and others 1997; Freudenreich and others low concentrations of other PARP enzymes, which may have 1998). The temporary inefficiency of this process during distinct unknown roles but could also have backup functions. early mammalian development could explain the origin of Crossing PARP1 knockout mice with severe combined im- several human syndromes that are associated with expansion munodeficient disease knockout mice that lack DNA- of triplet repeats in relevant genes. dependent protein kinase, which is required for VDJ recom- A series of pairwise interactions between the relevant pro- bination during lymphocyte development, alleviates the teins in BER seem to occur in most cases without any direct DNA-processing defect in the latter and allows some low- strong protein-protein interactions in the absence of DNA. fidelity recombination (Morrison and others 1997). PARP1 The XRCC1-LIG3 heterodimer is the only preformed com- plays no clear role in the BER process itself, as POL β and plex, and no large preassembled multiprotein BER complex LIG3 do, but it interacts with the scaffold protein XRCC1 is likely to exist. Nevertheless, the consecutive ordered in- and may in this way accelerate the recruitment of these re- teractions may protect reaction intermediates and ensure ef- pair enzymes for strand interruptions (Mackey and others ficient completion of the correction process after initial DNA 1999). damage recognition. Repair of Double-Strand Breaks Nucleotide-Excision Repair of Cyclodeoxynucleosides Exposure of DNA to ionizing radiation produces about The great majority of endogenous DNA lesions produced 5–7% as many DSBs as SSBs (e.g., see earlier discussion of by reactive oxygen species are corrected by the BER path- Nikjoo and others 1997, 2000). DSBs are sites at which a way, and the contributions of the different pathways of nucle- surprisingly large number of proteins can bind, carry out otide-excision repair (NER) and mismatch repair are very strand-break repair, and initiate a complex series of cellular minor. However, exposure of DNA or cells to ionizing ra- signals that regulate cell cycle progression and the induction diation under hypoxic conditions causes the formation of 5′, and activation of many downstream genes. Cells often en- 8-purine cyclodeoxynucleosides. This chemically stable and counter DNA DSBs under natural circumstances. These in- distorting form of DNA damage, in which the purine is at- clude termini (e.g., telomeres at chromosome ends); recom- tached by two covalent bonds to the sugar-phosphate back- bination intermediates; and immunoglobulin rearrangement bone, can be removed only by NER (Heyer and others 2000; during the processing of antibody genes (which leads to in- Kuraoka and others 2000). Similarly, a major lipid peroxi- creased versatility in the repertoire of immature immuno- dation product, malondialdehyde, reacts with G to produce cytes), during the processing of stalled or collapsed replica- an exocyclic pyrimidopurinone (M1G) that requires NER for tion forks arrested by damage on the template strand and repair. These are not the major mutagenic or cytotoxic le- during topoisomerase action on DNA. DSB repair enzymes sions that occur as a consequence of exposure to ionizing have been suggested as playing an essential role in telomere radiation, but they could be critical in individuals with im- maintenance in normal undamaged cells (Blackburn 2000). paired ability to perform NER. One critical difference between metabolically generated DSBs and those generated by ionizing radiation is that some fraction of the latter contain complex radiochemical damage Repair of Single-Strand Breaks that results in LMDS. LMDS (clustered damage) involve Reactive oxygen species cause DNA strand breaks by frank breaks, radiolytic fragments as termini, and base destroying deoxyribose residues. Such SSBs are processed damage that is processed into breaks by cellular glycosylases and repaired by the same enzymes responsible for the later (Blaisdell and Wallace 2001). DSBs thus are not inherently stages of BER, sometimes with the additional steps of novel, although substantial differences between natural and exonucleolytic removal of base pairs and phosphorylation of radiation-induced breaks are likely. Cells contain many 5′ termini by DNA kinase. In contrast to the continuous pro- genes that code for DNA-binding proteins and signal trans- tection of DNA reaction intermediates when an altered base duction pathways that respond specifically to DNA double- residue is replaced however, the initial strand break is fragile strand breakage. Consequently, cells can distinguish between and attracts unwelcome recombination events. An abundant a naturally occurring end of DNA at a telomere or recombi- nuclear protein, poly(ADP-ribose) polymerase-1 (PARP1), nation structure, for example, and a DSB at an unusual loca- appears to have as its main role the temporary protection of tion with atypical chemistry. This suggests that metabolic DNA single-strand interruptions (Le Rhun and others 1998; responses to DSBs and LMDS are highly evolved in most Lindahl and Wood 1999). PARP1 rapidly shuttles strand cell types and that cells are not completely unprepared and breaks in DNA on and off, with NAD-dependent synthesis unequipped for these kinds of lesions, but are in fact able to of poly(ADP-ribose) as its release mechanism. PARP1 exercise considerable discrimination in their detection and knockout mice are viable but show increased numbers of repair. Cells can also repair damage by novel chemicals, such

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BACKGROUND INFORMATION 35 as cisplatinum, which was newly synthesized in the twenti- eth century, an indication that novelty or uniqueness is no barrier to the repair of DNA damage. Repair of DSBs involves a number of biochemically dis- tinct processes. Direct rejoining of the broken ends occurs by several mechanisms, generally described as NHEJ. A fast NHEJ process involves end-binding proteins (Ku70, Ku80, and DNA-PK; Baumann and West 1998; Critchlow and Jackson 1998; Zhao and others 2000), and a slower process involves the hMre11/hRad50/Nbs1 DNA-binding and exo- nuclease complex that appears to act on refractory, complex breaks (Haber 1998; Petrini 1999). A more complicated re- joining process—homologous recombination—depends on matching damaged DNA with its identical sequence in a sis- ter chromatid after DNA replication or in the homologous chromosome in diploid cells. This process depends on the hRad51 protein, which facilitates homologous pairing, and accessory proteins, such as hRad52, hRad54, XRCC2, and XRCC3 (Thompson 1996). How cells coordinate these pro- cesses and determine which should be used under various circumstances is unknown. Coordination may be under the control of the Brca1 and Brca2 proteins. Brca1 binds to unusual DNA structures (Parvin 2001) and is found in a large complex that contains many repair and replication proteins (Wang and others 2000). The proteins directly involved in DNA strand-break re- pair do not appear to be inducible (Tusher and others 2001) or to be strongly influenced by p53 functions, except where recombination is involved. Radiation-induced genes repre- sent predominantly cellular signaling molecules, particularly those induced by transactivation by p53. Radiation does, however, activate a series of protein kinases, of which ATM (ataxia-telangiectasia-mutated) is the most prominent, that modify the activity of many other proteins in the repair path- ways (Bakkenist and Kastan 2003). Nonhomologous End Joining—Fast Reaction DSBs begin to rejoin rapidly after irradiation, with half- times of about 10 min or less (Ward and others 1991). This rapid rejoining involves accumulation of the end-binding proteins Ku70 and Ku80, DNA-PK kinase, the DNA ligase FIGURE 1-10 Nonhomologous end joining: this repair pathway IV-XRCC4 heterodimer, PARP, and others (Figure 1-10). re-ligates DNA DSBs by using the end-binding proteins Ku70 and The same factors are also an integral part of the normal pro- Ku80 to maintain alignment, and p450 kinase acts as a binding cess of immunologic rearrangement (Labhart 1999). Con- factor. The region across the break is then sealed by ligase IV and ceivably, if the LMDS contains damaged bases, the ends will its cofactor XRCC4. The sealed break often gains or loses a few also require repair steps involving glycosylases, apurinic nucleotides, especially if the break is an LMDS. In some cases, endonuclease, and DNA polymerase β. Attempted repair by nonhomologous end joining appears to be responsible for large these BER enzymes can enhance DSB formation and loss of DNA deletions and chromosome aberrations. In these cases, con- base pairs, which then must be repaired by NHEJ (Blaisdell siderably more than a few nucleotides can be lost. SOURCE: Re- produced with modifications and with permission from Hoeij- and Wallace 2001). Attempted BER of LMDS in human makers (2001). lymphoblastoid cells produces lethal and mutagenic DSBs (Yang and others, 2004). Small deletions associated with NHEJ have been mapped by sequencing techniques and range up to about 10 nucleotides (Daza and others 1996).

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36 BEIR VII The histone protein H2AX is phosphorylated rapidly over produces two intact double-strand DNA molecules with or large regions of DNA around sites of DSBs by ATM kinase without exchanges according to the orientation of the resolu- (Burma and others 2001). Loss of H2AX phosphorylation tion nuclease actions. The activity of hRad51 is enhanced by occurs rapidly with the repair of DSBs, but the biochemical other factors, such as hRad52, XRCC2, and XRCC3, and details of dephosphorylation remain to be ascertained. A re- suppressed by p53, which binds to both Holliday junctions cent study showed that in human cells, a background level of and hRad51 (Buchhop and others 1997). HR is much more H2AX phosphorylation occurred in about 5% of the cells. efficient and important for repair in yeast and somatic chick After low doses of X-rays that initially increased the level to cells than in normal (nonmalignant) mammalian (human) 10%, most cells eliminated this phosphorylation, except for somatic cells, where NHEJ is the dominant mechanism for a small fraction in which it persisted unless the cell entered DSB repair (Sonoda and others 1998). However, there are DNA synthesis (Rothkamm and Lobrich 2003). Whether this exceptions, and there may be times in the cell cycle, such as means that a small fraction of cells cannot repair some late S, when HR assumes greater importance because of the classes of LMDS or that dephosphorylation of H2AX can be proximity of sister chromatids (Thompson 1996). The low slower than repair itself in a subset of cells remains to be level of sister-chromatid exchange, a form of HR, induced determined. by X-rays and high-LET radiation indicates that, in absolute The DNA-PK kinase is a member of a class of phosphati- terms, HR remains a minor pathway for the repair of damage dyl-3-inosityl enzymes that includes ataxia-telangiectasia- caused by ionizing radiation in somatic cells. mutated (ATM) and ataxia-telangiectasia-related (ATR) ki- There is some question about the source of an identical nases, all of which are involved in signaling the presence of matching sequence for repair by HR in somatic human cells. DNA damage (Shiloh 2001, 2004; Figure 1-11). Although A homologous sequence may be the other allele on a chro- DNA-PK kinase can phosphorylate many proteins in vitro, mosome of a recently replicated sister-chromatid sequence it is unclear which proteins it usually phosphorylates in vivo. on a daughter chromatid or a similar sequence in a repetitive Early cytologic evidence of X-ray damage is phosphoryla- region along the same chromosome. In the latter case the tion of a histone protein to create γ-H2AX foci that are vis- sequences may not be identical over long regions, and the ible microscopically within minutes of irradiation. mechanism is known as “homeologous” recombination. Recombination between alleles on separate chromosomes occurs at much lower frequency than between identical Nonhomologous End Joining—Slow Reaction sequences on sister chromatids or arranged in tandem on the After the rapid phase of rejoining is complete, the repair same chromosome. In general, HR between sister chroma- of DSBs slows to a second phase with a half-time of several tids may occur at higher frequencies late in the cell cycle hours. Foci containing the hMre11/hRad50/Nbs complex (e.g., late S; Thompson and Schild 1999), and homeologous form or persist and reach a maximum at about 4–6 h. Be- recombination is likely to result in the loss of intervening cause this complex has endonuclease and DNA-binding ac- sequences with the production of deletion mutations. tivity, it may be involved in the slower repair of refractory The HR involving hRad51 can be visualized immuno- DSBs that cannot be repaired by the earlier, fast mechanism. histochemically: foci containing hRad51, Brca1, and other The complex is not active unless the Nbs1 protein is phos- proteins can be seen microscopically soon after irradiation phorylated on several sites by ATM kinase (Figure 1-11), (Scully and others 1997). Cells generally exhibit either which is itself activated by DNA breaks (Shiloh 2001; hRad51 foci or hMre11/hRad50/Nbs foci, but not both, and Bakkenist and Kastan 2003). The precise DNA structures the choice of which of the mutually exclusive pathways an involved in these refractory breaks are unknown. However, irradiated cell follows may be determined by Brca1 (Parvin one model suggests that nuclease action by the Mre11 com- 2001). plex resects single DNA strands and that short regions of sequence identity (microhomologies) can be used for align- DSB Signal Transduction and Inducible Repair ment and rejoining of DNA strands (Figure 1-12). Bacteria live in a highly variable environment and have evolved efficient inducible DNA repair processes to deal Homologous Recombination with sudden challenges of DNA damage from oxygen free Repair of a DSB by HR involves matching the two broken radicals, ionizing radiation, chemicals, and ultraviolet radia- ends of a DNA strand with identical sequences of intact DNA tion. These inducible repair pathways are now mechanisti- (Figure 1-12). The broken and intact molecules are aligned cally well understood. In Escherichia coli, the regulatory according to their sequences and encompassed by a toroid of genes soxR, ada, and lex control transcription of DNA repair hRad51 molecules that facilitate repair by having DNA functions, and increased amounts of relevant DNA repair single strands invade their homologues, producing an enzymes can be produced in response to environmental chal- X-shaped four-armed structure called a Holliday junction. lenges. In mammalian cells, the same types of DNA damage Resolution of this structure by specific junction nucleases are recognized by similar DNA repair enzymes. However, a

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BACKGROUND INFORMATION 37 FIGURE 1-11 Network of protein kinases activated by DNA DSBs. ATM is the primary kinase that phosphorylates downstream kinases. The specific activity of ATM is increased after introduction of DSBs in DNA through ionizing radiation or other means; this then activates other proteins by phosphorylation (denoted by amino acid symbol and number) and in a cell cycle-specific manner. G1 phase: Activated ATM (ATM*) directly phosphorylates three proteins involved in controlling p53 functions or levels—p53 (serine 15), CHK2 (threonine 68), and MDM2 (serine 395). CHK2 kinase may also be activated by ATM and in turn phosphorylate p53 on serine 20. This phosphorylation event and the phosphorylation of MDM2 seem to inhibit binding of MDM2 to p53 and should result in an increase in p53 protein. The increased p53 protein transcriptionally induces p21, which inhibits CDK2-cyclin E and causes arrest in the G1 phase of the cycle. S phase: Activated ATM also phosphorylates NBS1 (serine 343), and this phosphorylation event is required for the ionizing radiation-induced S-phase arrest. NBS1 exists in a complex with MRE11, RAD50, and BRCA1. The potential role of these proteins in S-phase arrest remains to be clarified; CHK2 may also be involved in this pathway, after activation by ATM, through phosphorylation of BRCA1 or NBS1. G2 phase: Details of the downstream targets of ATM at the G2 checkpoint have not been determined. CHK2 and CHK1 may be targets for ATM and ATR in the G2-M checkpoint pathway, respectively. CDC25C and 14-3-3 have been implicated in regulation of CDC2 kinase and progression through G2. Dashed arrows and question marks represent possible signaling steps; solid arrows represent reported phosphorylation events. SOURCE: Reproduced with permission from Kastan and Lim (2000). major difference from microorganisms is that mammalian Many reports have appeared about adaptive responses enzymes are constitutively expressed. Thus, there are no involving increased resistance or hypersensitivity in mam- transcription control or mammalian counterparts of soxR, malian cells in response to single or multiple doses of ionizing ada, and lex. This situation presumably reflects the much radiation (adaptive effects). There are also reports that the greater constancy of cellular environment in complex multi- effects of radiation on single cells can influence the response cellular organisms. Therefore, the work on inducible DNA of adjacent nonirradiated cells (bystander effect). These reports repair in bacteria offers no direct guidelines for the relative are discussed specifically in Chapter 2, but this chapter de- resistance of human cells repeatedly exposed to DNA-dam- scribes the general stress response and signal transduction aging agents. pathways that are known to occur after exposure to radiation.

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38 BEIR VII 3í ---NNN-----GATCC NNN---- 3í ---NNN CTAGG ------NNN---- 5í FEN1 3í FIGURE 1-12 HR- and microhomology-mediated DSB repair. These two pathways for repair of DSBs are driven by stretches of homolo- gous DNA. HR requires an identical sequence spanning the part of the DNA molecule containing the break and extensive remodeling of the broken DNA termini. Mre11/Rad50/Nbs1 resects individual strands by its 5′- to 3′-exonuclease activity and binds homologous double- stranded DNA by the Rad50 moiety. Exposure of single-stranded regions with only small regions of homology flanking the original break can allow microhomology-mediated strand-break rejoining coupled by cleavage of overhanging strands by FEN1 and resynthesis of any resulting gaps. The repair will, at the least, result in loss of one of the regions of microhomology. Exposure of single-stranded regions homologous to adjacent double-stranded DNA can lead to strand invasion and HR. Single-stranded regions are coated with single-strand binding protein (RPA); homology search and strand invasion are mediated by Rad52, 54, Brca 1 and 2, and Rad51. The complex structure produced forms a Holliday junction that is cleaved by junction-specific nucleases (resolvases), and associated polymerase and ligases complete an error-free exchange of DNA strands. SOURCE: Modified reproduction and reproduced with permission of J. Hoeijmakers (2001).

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BACKGROUND INFORMATION 39 Damage to cells elicits increases and decreases in the ex- activate ATM and p53 and create a cell-wide response pression of many genes. Recent microarray analysis has through this cascade of protein modifications and alterations shown that these changes can involve hundreds of genes and in gene expression. that different stresses can invoke both a common set of genes These signal transduction pathways are also activated by and genes that are peculiar to particular kinds of stress extracellular signals working through specific receptors on (Amundson and others 1999a, 1999b). Despite the large the cell membrane that then activates kinases, such as number of affected genes, none appears to be directly in- MAPKs, which phosphorylate p53. Irradiated cells also gen- volved in repair of DSBs (Tusher and others 2001). Central erate extracellular signals that resemble cytokines released to most damage responses is stabilization of the tumor-sup- during normal in vivo cell-cell communication processes pressor gene p53, which occurs as a result of posttransla- (Herrlich and others 1992). These can, through receptors on tional phosphorylation or acetylation of the protein (Blattner adjacent cells or gap junctions, result in activation of the and others 1999; Figure 1-11). Multiple potential serine and signal transduction pathways in nearby cells. These multiple threonine residues in p53 are capable of being phosphorylated intracellular and extracellular pathways of protein modifica- by different kinases in response to cellular stress, and several tion and signal transduction may constitute the mechanisms thousand combinations of modifications are possible in an by which many of the transient alterations in cellular me- irradiated cell. Resolving the functional role of any particu- tabolism occur after exposure to ionizing radiation (Blattner lar site can be difficult (Blattner and others 1999). The and others 1994). kinases include ATM, ATR, Chk1, Chk2, DNA-dependent Some responses observed in particular regimes of expo- protein kinase, and casein kinase I and II (Blattner and others sure to ionizing radiation and given unique names (e.g., adap- 1999; Chehab and others 2000). (For the role that p53, pRb, tive response, bystander effect, genomic instability) may cdc25C, chk1, chk2, 14-3-3 proteins, bub1, and the various constitute particular manifestations of these general stress cyclins and cyclin-dependent kinases play in radiation- responses and signal transduction pathways. These appar- induced checkpoints in G1, G2, and mitosis, see Little 1994; ently distinct radiation responses have been described mainly Jacks and Weinberg 1998; Lengauer and others 1998; in cell biology experiments, and in no case do they have Schmidt-Kastner and others 1998; Chan and others 1999; solid biochemical support or mechanistic understanding. In Ford and Pardee 1999; White and Prives 1999). addition to controversy among laboratories, some of the re- ATM is a centrally important kinase for X-ray damage sponses described appear to be valid only within a limited that is activated by DNA DSBs (Bakkenist and Kastan 2003; dose range and under particular experimental conditions. It Figure 1-11). In X-irradiated cells, phosphorylation of serine is also unclear whether different types of cells, such as epi- 15 and 37 interferes with the association of p53 with another thelial cells, fibroblasts, and lymphoid cells, respond simi- protein mdm2 that also becomes phosphorylated and nor- larly or differently in this regard. Some of the inducible re- mally causes degradation of p53, extending its lifetime. The sponses appear to be complex in that they depend on increased stability of p53 in irradiated cells permits it to form participation of intercellular gap junctions in communicat- a tetramer and then act as a transactivating factor, increasing ing radiation responses to neighboring cells. Work on this the expression of many other genes. Clearly, this will result subject is in the preliminary, descriptive stage, and there is in large-scale alterations of the gene expression pattern of no understanding of what compounds or factors would be irradiated cells that can influence their behavior. One down- transferred between cells in the gap junction. Therefore, it is stream target for p53 is the cell cycle regulator protein p21; difficult to evaluate whether the phenomena are of any gen- increased transcription of p21 due to p53 results in delays in eral physiologic significance. the onset of DNA synthesis (the G1 checkpoint) and reduced DNA synthesis due to p21 binding the replication factor SUMMARY PCNA. The major response of cells to ionizing radiation is a reduction in initiation of the S phase and of replication In this chapter the committee has provided background origins during S. Another important radiation-responsive information relating to the physical and chemical aspects of gene is GADD45; both this and p21 showed a linear dose- radiation and the interaction of radiation with the target mol- response relation for induction from 20 to 500 mGy with no ecule DNA. The chapter describes the physics of electrons indication of a threshold (Amundson and others 1999b). and beta particles, which are important contributors to direct Most of the members of the signal transduction pathways DNA damage after ionizing radiation exposure, and intro- including ATM, p53, Chk1, Chk2, Brca1, and hMre11/ duces a special subject—the effect that neutron RBEs have hRad50/Nbs1 are protein products of tumor-suppressor on low-LET radiation risk estimates. Radical formation by genes. Loss of function of these members can result in ge- ionizing radiation and its contribution to DNA damage are nomic instability and in some instances may contribute to a also described. The committee has discussed the contribu- series of events resulting in malignancy. They influence cell tions of normal oxidative DNA damage relative to radiation- cycle checkpoints, DNA replication, DNA repair, and re- induced DNA damage and described the DNA repair mecha- combination. Thus, it is possible for a single DNA DSB to nisms that mammalian cells have developed to cope with

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40 BEIR VII such damage. Modeling of electron interactions with DNA by NHEJ were not represented among the sensitive mutants suggests that when more than one strand break occurs due to because this is a minor pathway in yeast. An additional an electron interaction, approximately 30% of the breaks will observation is that the set of genes whose expression was be multiple events (three or more) that occur over a very induced by damage differed from the genes required for small distance. These multiple events, sometimes referred to resistance against each agent, implying that repair genes as LMDS, would be expected to occur at the same average were not among those induced by damage (Birrell and others rate per electron traversal of the DNA, whether the overall 2002). dose is high or low. It is reasonable to expect that multiple The committee carried out a detailed comparison of the lesions of this sort would be more difficult to repair or might genes reported by each group, using publicly available data be prone to misrepair. This may explain the apparent incon- sets. One group (Birrell and others 2001, 2002; Game and sistency between the lethality and mutagenicity of agents others 2003) reported the response of the complete set of that principally cause DNA single-strand breaks and ioniz- 4800 genes and ranked them in sequence, from most sensi- ing radiation, which also produces double-strand breaks and tive to least sensitive. About 10% of all genes (470) showed LMDS. Furthermore, modeling of multiple damages in a some degree of sensitivity to ionizing radiation. The other small length of DNA suggests that the normal cellular oxida- group (Thorpe and others 2004) reported only those genes tive damage of DNA may differ qualitatively from that due that showed sensitivity to at least one oxidant (approximately to ionizing radiation. Recent information is presented as an 675 genes) and ranked them in categories 1–7, with the most annex to this chapter, about a significant disparity in the sensitive in category 1. genes that repair oxidative damage in yeast DNA and genes Comparison between these data sets is complicated by that repair radiation damage. different methods of reporting and different technical ap- proaches to determining sensitivity. Comparisons were therefore made in general terms rather than gene by gene, ANNEX 1A: IONIZING RADIATION AND OXIDATIVE and only those genes were considered that were reported by DAMAGE—A VIEWPOINT FROM SACCHAROMYCES both groups. The committee first compared the genes re- CEREVISIAE quired for resistance against hydrogen peroxide as reported Approximately 4800 deletion mutations have been made by two independent research groups, to establish the consis- in all the nonessential genes in the yeast Saccharomyces tency of the data (Figure 1A-1). A set containing about 200 cerevisiae. These have been used by two groups of investi- genes was common to both groups as necessary for resis- gators to identify the genes responsible for resistance against tance to hydrogen peroxide. Of these, 150 were also sensi- ionizing radiation, ultraviolet light, cisplatin, and a number tive to ionizing radiation. Since different methods were used of different oxidizing agents (hydrogen peroxide, diamide, to detect sensitivity and rank the strains, some differences linoleic acid 13-hydroperoxide, menadione, and cumene hy- are not surprising. The common set of 150 genes required droperoxide; Birrell and others 2001, 2002; Game and oth- for resistance to both ionizing radiation and hydrogen perox- ers 2003; Thorpe and others 2004; Wu and others 2004). The ide included those involved in postreplication repair and re- set of genes required for resistance against a particular agent combination, but the genes that ranked among the most sen- is an indication of the nature of the cellular biochemical path- sitive toward ionizing radiation were ranked lower on the list ways required to restore viability and, indirectly, of the kind for hydrogen peroxide (Birrell and others 2002). of damage generated by the agent. If a common set of genes The committee then compared the genes required for re- is required for several different agents, these will point to a sistance to different oxidizing agents with those required for common or overlapping chemical nature of the damage. The resistance to X-rays (Figure 1A-2). The overlap was small in striking observation about the results in S. cerevisiae is that comparison to the number of genes required for resistance to the sets of genes required for resistance against each agent ionizing radiation; conversely, more than half of the genes differed significantly from each other. When pairwise com- required for resistance to each oxidant were also required for parisons were made between ionizing radiation and each resistance to ionizing radiation. However, the same genes oxidant, the overlap was low: less than half of the genes were not involved for each oxidant. required for resistance against ionizing radiation were also The implication of these results is that each agent that is required for resistance to oxidative damage (Figures 1A-1, toxic to S. cerevisiae produces a unique spectrum of cellular and 1A-2). damage, with some overlap. The relevance of these com- Large numbers of genes not obviously involved in DNA parisons to this report lies in the attempts that have been repair fall within the list of sensitive mutants to ionizing ra- made to explain low-dose ionizing radiation as no more than diation and oxidants. Several genes whose deletion produced a special case of oxidative damage (Pollycove and Feinen- sensitivity to radiation and oxidants were involved in DNA degen 2003). If this were true, low doses of ionizing radia- replication and recombination, suggesting that this process tion would be insignificant compared to the levels of natu- was vulnerable to all kinds of cellular damage in yeast. In rally occurring reactive oxygen species and could therefore contrast, the most important genes in human cells for repair be ignored as having no detrimental health effects. How-

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BACKGROUND INFORMATION 41 Birrell and others (2002) Game and others (2003) IR -sensitive 470 genes total 8 298 150 Thorpe and others Game and others (2003) (2004) 57 H 2O 2-sensitive H 2O 2-sensitive 525 genes total 260 genes total FIGURE 1A-1 Venn diagram representing the overlap among genes involved in resistance against ionizing radiation and hydrogen peroxide as indicated in the reports cited. Numbers in regions of overlap represent the number of genes responsible for resistance against two agents as reported by one or another group. Cumene Linoleic acid Hydroperoxide 13-hydro Peroxide 161 total 237 total Thorpe and others Thorpe and others (2004) (2004) 105 172 Birrell and others (2002) Game and others Menadione Diamide 77 108 total 338 total (2003) 260 Thorpe and others Thorpe and others IR - sensitive (2004) (2004) 470 genes total 8 298 Thorpe and others Birrell and others (2004) (2002) H 2O 2-sensitive H 2O 2-sensitive 260 total 525 total FIGURE 1A-2 Venn diagram representing the overlap among genes involved in resistance against ionizing radiation and various oxidizing agents as indicated in the reports cited. Numbers in regions of overlap represent the number of genes responsible for resistance against two agents as reported by one or another group.

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42 BEIR VII ever, each oxidizing agent involved a significantly different These damage responses in S. cerevisiae are, however, set of genes, which also differed from those required for pro- dominated by the efficient homologous recombination that tection against X-rays, indicating that oxidative damage can- plays a major role in response to DNA damage (Kelley and not be considered a single entity, but is dependent on the others 2003). Homologous recombination may therefore chemical source of the oxidation. Mutants sensitive to hy- mask some of the effects caused by loss of genes on path- drogen peroxide included an overrepresentation of mito- ways that may be minor in yeast but more important in mam- chondrial respiratory functions, but those sensitive to dia- malian cells (Swanson and others 1999; Gellon and others mide encompassed genes involved in vacuolar protein 2001; Morey and others 2003). For example, mice that are sorting. This makes it especially difficult to predict what defective in apurinic endonuclease are embryonic lethals, kinds of damage would result from endogenous reactive oxi- and blastocysts derived from these nulls are radiosensitive dative species. Endogenous damage could present its own (Xanthoudakis and others 1996; Ludwig and others 1998). unique spectrum of genes required for resistance, different RNAi ablation of a pyrimidine-specific DNA glycosylase in from each of the exogenous sources as well as from ionizing mice confers radiosensitivity (Rosenquist and others 2003). radiation. Although the results described in yeast do indicate differ- These results must be confirmed and extended to human ences between ionizing radiations and oxidizing agents, the cells, because the genes known to be involved in repair of extent of differences or of overlap may not be the same in DNA DSBs by NHEJ (Ku70, Ku80, and DNA-PK) were mammalian cells. rarely found among those involved in resistance to ionizing These results in S. cerevisiae, however, provide no sup- radiation or oxidative damage in yeast, where they play a port for the attempts to equate low-dose ionizing radiation very minor role. The majority of genes required for resis- with endogenous oxidative reactions. The committee would tance to oxidative damage were, however, considered by one expect even greater divergence between ionizing radiation set of authors (Thorpe and others 2004) as more representa- and oxidative damage in human cells because of the higher tive of damage to the protein components of the cell than to ratio of cytoplasmic and nuclear proteins to DNA than in S. DNA. These included genes required for transcription, cerevisiae and the greater role of NHEJ. protein trafficking, and vacuolar function.