2
Molecular and Cellular Responses to Ionizing Radiation

Since the early years of radiobiology the cellular effects of ionizing radiation have been studied in the context of induced chromosomal aberrations, and early models of radiation action were largely based upon such studies (Savage 1996). In the 1970s, somatic cell genetic techniques were developed to allow the quantification and characterization of specific gene mutations arising in irradiated cultures of somatic cells. In more recent years, findings of persistent postirradiation genomic instability, bystander effects, and other types of cellular response have posed additional questions regarding the mechanisms underlying the cytogenetic and mutagenic effects of radiation and their potential to contribute to radiation tumorigenesis.

This chapter considers the general aspects of dose-response relationships for radiobiological effects and subsequently reviews the largely cellular data on a range of radiobiological end points. The main focus of the review is the issue of cellular effects at low doses of low-LET (linear energy transfer) radiation. Many of the conclusions reached from this review, when aggregated with those of Chapters 1 and 3, contribute to the judgments made in this report about human cancer risk at low doses and low dose rates.

GENERAL ASPECTS OF DOSE-RESPONSE RELATIONSHIPS

Any effect of radiation exposure must be quantified in relation to the effect observed in a control population. In this way the dose to an irradiated population is considered in the context of, among other factors, the natural background radiation received. For low-LET radiation an absorbed dose of 1Gy1 (1000 mGy) corresponds to an equivalent dose of 1 Sv (1000 mSv). Because this report focuses on low-LET radiation, reference is mostly to grays and not to sieverts. Low-LET background radiation worldwide is responsible for an average annual effective dose per person of about 0.9 mGy per year (UNSCEAR 2000b). This includes an estimated 0.48 mGy from external terrestrial radiation to the body, 0.28 mGy from cosmic radiation (excluding the neutron component), and 0.17 mGy from radioisotopes in the body. For the purposes of this report, it does not include background radiation of about 1.2 mSv delivered to the lungs from radon and radon progeny or other high-LET radiation. Radon is the subject of the BEIR VI report (NRC 1999).

The maximal permissible levels that are recommended in the United States by the National Council on Radiation Protection and Measurements (NCRP) for people exposed to radiation other than background radiation and from medical applications are 1 mSv per year for the general population and 50 mSv per year for radiation workers employed by nuclear-related industries (Federal Register 1987). Considering the levels of background radiation, the maximal permissible levels of exposure of radiation workers now in effect, and the fact that much of the epidemiology of low-dose exposures includes people who in the past have received up to 500 mSv, the BEIR VII committee has focused on evaluating radiation effects in the low-dose range <100 mGy, with emphasis on the lowest doses where relevant data are available. Effects that may occur as the radiation is delivered chronically over several months to a lifetime are thought to be most relevant.

An effect (E) (for example chromosomal aberrations, mutations, or animal carcinogenesis) induced by an acute dose of low-LET radiation delivered over a few minutes has been described by the relationship E = αD + βD2, where D = dose; this is a linear-quadratic dose-response relationship curving upward (Lea 1946; Cox and others 1977). Theoretically, the α term represents the single-hit intratrack component, and β represents the two-hit intertrack component. An alternative interpretation is that the D2 term may arise from multiple tracks that would increase the overall burden of damage in a cell and thereby partially saturate a repair

1  

Because the older dose term “rad” is used in some figures, the committee notes here that 1 Gy = 100 rads.



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2 Molecular and Cellular Responses to Ionizing Radiation Since the early years of radiobiology the cellular effects LET background radiation worldwide is responsible for an of ionizing radiation have been studied in the context of in- average annual effective dose per person of about 0.9 mGy duced chromosomal aberrations, and early models of radia- per year (UNSCEAR 2000b). This includes an estimated tion action were largely based upon such studies (Savage 0.48 mGy from external terrestrial radiation to the body, 1996). In the 1970s, somatic cell genetic techniques were 0.28 mGy from cosmic radiation (excluding the neutron developed to allow the quantification and characterization component), and 0.17 mGy from radioisotopes in the body. of specific gene mutations arising in irradiated cultures of For the purposes of this report, it does not include back- somatic cells. In more recent years, findings of persistent ground radiation of about 1.2 mSv delivered to the lungs postirradiation genomic instability, bystander effects, and from radon and radon progeny or other high-LET radiation. other types of cellular response have posed additional ques- Radon is the subject of the BEIR VI report (NRC 1999). tions regarding the mechanisms underlying the cytogenetic The maximal permissible levels that are recommended in and mutagenic effects of radiation and their potential to con- the United States by the National Council on Radiation Pro- tribute to radiation tumorigenesis. tection and Measurements (NCRP) for people exposed to This chapter considers the general aspects of dose-re- radiation other than background radiation and from medical sponse relationships for radiobiological effects and subse- applications are 1 mSv per year for the general population quently reviews the largely cellular data on a range of radio- and 50 mSv per year for radiation workers employed by biological end points. The main focus of the review is the nuclear-related industries (Federal Register 1987). Consid- issue of cellular effects at low doses of low-LET (linear en- ering the levels of background radiation, the maximal per- ergy transfer) radiation. Many of the conclusions reached missible levels of exposure of radiation workers now in from this review, when aggregated with those of Chapters 1 effect, and the fact that much of the epidemiology of low- and 3, contribute to the judgments made in this report about dose exposures includes people who in the past have received human cancer risk at low doses and low dose rates. up to 500 mSv, the BEIR VII committee has focused on evaluating radiation effects in the low-dose range <100 mGy, with emphasis on the lowest doses where relevant data are GENERAL ASPECTS OF DOSE-RESPONSE available. Effects that may occur as the radiation is delivered RELATIONSHIPS chronically over several months to a lifetime are thought to Any effect of radiation exposure must be quantified in be most relevant. relation to the effect observed in a control population. In this An effect (E) (for example chromosomal aberrations, way the dose to an irradiated population is considered in the mutations, or animal carcinogenesis) induced by an acute context of, among other factors, the natural background ra- dose of low-LET radiation delivered over a few minutes has diation received. For low-LET radiation an absorbed dose of been described by the relationship E = αD + βD2, where 1 Gy1 (1000 mGy) corresponds to an equivalent dose of 1 Sv D = dose; this is a linear-quadratic dose-response relation- (1000 mSv). Because this report focuses on low-LET radia- ship curving upward (Lea 1946; Cox and others 1977). Theo- tion, reference is mostly to grays and not to sieverts. Low- retically, the α term represents the single-hit intratrack com- ponent, and β represents the two-hit intertrack component. An alternative interpretation is that the D2 term may arise 1Because the older dose term “rad” is used in some figures, the commit- from multiple tracks that would increase the overall burden tee notes here that 1 Gy = 100 rads. of damage in a cell and thereby partially saturate a repair 43

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44 BEIR VII system and reduce the probability of repair of particular dam- shown (Cornforth and others 2002) to be equal to the dose- age from a track (UNSCEAR 1993). However, there is no rate effectiveness factor (DREF). Therefore, the term experimental evidence to support this model. As the dose is DDREF is used to estimate effects for either low doses or reduced, the β term becomes less important, and the dose- low dose rates. This value for DDREF can be estimated from response relationship approaches linearity with a slope of α. a fit of the acute data using the relationship described above For doses delivered in multiple fractions or at low dose rates, (i.e., E = αD + βD2). Thus, the DDREF = [(αD + βD2)/D] / in which case the effects during the exposure period are in- (αD/D) = (αD + βD2)/αD, which equals 1 + Dβ/α or 1 + D/ dependent and without additive or synergistic interactions, (α/β). D is the dose at which the response for acute irradia- the dose-response relationship should also be linear with a tion is divided by the response for low-dose-rate irradiation slope of α. Theoretically, the value of α should be the same to obtain the DDREF, and the relationship shows that for high and low dose rates and for single or multiple doses, DDREF will increase with the dose at which the curves A and there should be a limiting value, α1, so that reducing the and D are compared. Note, the contribution from the β term dose rate further would not reduce the α term (see Figure 2-1 (βD2) equals the contribution from the α term (αD) (i.e., for an illustration of these concepts). βD2 = αD, when D = α/β). For this dose equal to α/β, the For extrapolating data from acute high-dose-rate experi- incidence for curve D is equal to the difference between the ments to results expected for low doses and low-dose-rate incidence for curve A and the incidence for curve D; thus, experiments, the dose and dose-rate effectiveness factor, curve A intersects the linear curve B at the dose equal to α/ DDREF, is estimated (see Figure 2-1). The DDREF is esti- β. For example, if α/β equals 1 Gy, the DDREF for a dose of mated by comparing the linear extrapolation (curve B) of the 1 Gy would theoretically equal (1 + 1/1) or 2; for a dose of induced incidence for a set of acute dose points (curve A) 0.5 Gy, the DDREF would equal 1.5, and for a dose of 2 Gy, with the linear curve (D) for low dose rate. The DDREF is it would equal 3. If α/β equals 2 Gy, curves A and B would equal to the slope αL for curve B divided by the slope α1 for intersect at 2 Gy where the DDREF equals 2; at doses less curve D. If only acute high-dose data are available, the slope than or greater than 2 Gy, the DDREF would be less than or (α1) for the linear extrapolation of the data for acute doses greater than 2, respectively. This concept is illustrated with that approach zero (tangent to curve A) is used. This is the experimental data in Figure 2-8; for the induction of HPRT dose effectiveness factor (DEF), which is assumed and (hypoxanthine-guanine phosphoribosyl transferase) muta- FIGURE 2-1 Schematic curves of incidence versus absorbed dose. The curved solid line for high absorbed doses and high dose rates (curve A) is the “true” curve. The linear, no-threshold dashed line (curve B) was fitted to the four indicated “experimental” points and the origin. Slope α1 indicates the essentially linear portion of curve A at low doses. The dashed curve C, marked “low dose rate,” slope αEx, represents experimental high-dose data obtained at low dose rates. SOURCE: Reproduced with permission of the National Council on Radiation Protection and Measurements, NCRP Report No. 64 (NCRP 1980).

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MOLECULAR AND CELLULAR RESPONSES TO IONIZING RADIATION 45 tions in mouse splenic T lymphocytes, the DDREF was ~1.5 the following paragraphs, a brief outline is provided of the at 1 Gy and ~4 at 5 Gy. Also, in Figure 10-2, dose-response current state of knowledge of the mechanisms that are be- curves for the incidence of solid cancers in Japanese A-bomb lieved to play a role in the induction of chromosomal aberra- studies were constructed over the dose range of 0–1.5 Sv, tions (see Bedford and Dewey 2002 for a detailed discus- assuming α/β = 1.45 Sv and α/β = 3.33 Sv, and DDREF val- sion). Aberrations formed following irradiation of cells in ues were calculated by dividing the slope of curve B by the the G0/G1 phase of the cell cycle are dicentric exchanges, slope of curve D. These slope ratios give DDREF values of centric rings, and monocentric exchanges (translocations). 1.8 for α/β = 1.45 Sv and 1.3 for α/β = 3.33 Sv. The vast majority of studies show that the dose-response for Several factors may affect the theoretical dose-response low-LET radiation is curvilinear and fits well to the equation relationships described above, namely: variations in radi- αD + βD2. At high doses, saturation effects occur, and the osensitivity during the cell cycle; induction of an adaptive dose-response tends to turn down; for human lymphocytes, response to an initial exposure, which can reduce the effect saturation occurs at doses greater than 4–5 Gy. The linear of later exposures; a bystander effect that causes an irradi- coefficient α, representing the initial slope of the dose-re- ated cell to have an effect on a nearby unirradiated cell; the sponse, increases with the LET of the radiation, reaches a induction of persistent genomic instability; and hyper-radia- maximum at ~70 keV µm–1, and then falls. The quadratic tion sensitivity in the low-dose region. Except for the cell coefficient β is approximately constant up to around 20 keV cycle, these factors have been identified and studied since µm–1 but reduces at higher LET (>100 keV µm–1). A reduc- the BEIR V report (NRC 1990). These factors, together with tion in low-LET dose rate reduces aberration yields in a dose- data on the induction of gene/chromosomal mutations in so- dependent manner; the value of α is unaffected, but the value matic cells are discussed in subsequent sections of this of β decreases (Edwards and others 1989). chapter. A current explanation of the above dose-response charac- teristics is that DNA DSBs are the principal causal events for aberration induction and that these are induced with lin- INDUCTION OF CHROMOSOME ABERRATIONS ear kinetics at around 30 DNA DSBs Gy–1. Correct repair Early studies on the mechanisms of chromosome aberra- and misrepair processes operate in competition for these tion induction summarized by Savage (1996) lead to the fol- DNA DSBs, with the majority of breaks restituting correctly lowing conclusions: Primary radiation-induced break-type and a small fraction taking part in misrepair-mediated chro- lesions can (1) reconstitute without morphological change mosomal exchanges (Hlatky and others 1991). The fraction to chromosomes; (2) rejoin illegitimately with another break of misrepair events is suggested to be dose dependent, with close in time and space to produce an intra- or interchromo- the close proximity of DNA DSBs promoting exchanges and somal aberration visible at the subsequent mitosis; or (3) re- thereby imposing curvature on the low-LET dose response. main “open,” leading to a simple break at mitosis. These The two-track component of DNA lesion production and early conclusions, based primarily on work with plant cells, interaction increases as a quadratic function of dose and pro- are supported by subsequent studies with mammalian cells. duces biophysical curvature on the dose-response. However, The quantitative cytogenetic systems developed over the the concept of proximity-promoted interaction of lesions years, particularly in G0 human lymphocytes, have been uti- gives more weight to lesions arising along the path of single lized in studies on the effects of dose, dose rate, and radia- tracks. Such proximity effects have been reviewed (Sachs tion quality. From a mechanistic viewpoint there is compel- and others 1997). Modeling procedures of this type, while ling evidence that the induction and interaction of DNA providing a coherent explanation of low-LET dose-response, double-strand breaks (DNA DSBs or, more correctly, are insufficient to account fully for high-LET effects double-stranded lesions) is the principal mechanism for the (Moiseenko and others 1997). An additional factor consid- production of chromosome aberrations. The fundamental ered in some modeling of dose- and LET-dependent re- arguments supporting this widely accepted conclusion have sponses is the possibility that some exchanges might involve been discussed in depth (Bender and others 1974; Scott 1980; interaction of a DNA DSB with an undamaged DNA site Cornforth and Bedford 1993; Natarajan and Obe 1996). Of (i.e., recombinational-like DNA misrepair). It seems likely particular note are the data showing excess aberrations fol- that a variety of repair and misrepair options are available to lowing the introduction of DNA DSB-inducing restriction the cell and that their relative importance is LET dependent; endonucleases into cells (Bryant 1984; Obe and others 1985; this feature may relate to the complexity of a significant frac- Morgan and Winegar 1990). The increased chromosomal tion of initial DNA DSBs (see Chapter 1). radiosensitivity in cells genetically deficient in processes Dose and LET dependence also apply to the morphologi- associated with DNA DSB repair, reviewed by ICRP (1998), cal complexity of the induced chromosomal aberrations also supports this conclusion. themselves. The development of fluorescence in situ hybrid- The biophysical modeling of the dose-response and LET ization (FISH) methods of chromosome painting has allowed dependence for chromosome aberration induction has been a aberration complexity to be studied in detail. In brief, aber- major focus in radiobiological research for many years. In ration complexity reflects the number of DNA DSBs in-

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46 BEIR VII volved in a given chromosomal exchange event; not surpris- (1973) predicted that the dicentric aberration frequency will ingly, aberration complexity becomes most apparent at high fall by a factor of around 2 per cell division on the basis that doses of low-LET radiation and at all doses of high-LET at each mitotic anaphase, a given dicentric has an equal radiation (Finnon and others 1995, 1999; Griffin and others chance of falling free or producing a lethal anaphase bridge. 1995; Anderson and others 2000). The precise mechanism This prediction has been tested as part of a recent study (Pala of formation of these complexes remains uncertain, but mul- and others 2001) that showed dicentric yields falling by up tiple pairwise exchanges involving the same chromosomes to a factor of 4 between the first and second postirradiation play some part (Edwards and Savage 1999). However, cy- cell division. It seems therefore that the vast majority of ini- clic exchanges involving three and four breaks are not un- tial unrepaired and misrepaired lesions are expressed as chro- common, implying that the interaction of multiple DNA mosomal damage at the first division. Cells carrying unbal- DSBs can occur. Recent studies using multicolor mFISH anced chromosomal exchanges (dicentrics) or substantial analyses further emphasize the complexity of many radia- chromosomal losses are not expected to contribute to the tion-induced chromosomal exchanges produced after high viable postirradiation population. By contrast, cells carrying acute doses of radiation (Loucas and Cornforth 2001). These small deletions or balanced exchanges such as reciprocal mFISH analyses also show that even after exposure at very translocations are likely to remain viable, and some may have low dose rates, the formation of complex chromosomal ex- the potential to contribute to tumor development. changes is not completely eliminated (Loucas and others Later in the chapter this conventional view is contrasted 2004). with data implying that in some circumstances, a certain frac- Combining FISH painting and premature chromosome tion of irradiated cells can express chromosomal damage condensation techniques (Darroudi and others 1998) has also over many cell cycles (i.e., persistent genomic instability). facilitated studies on the rate of formation of aberrations. In The proposition that this induced instability phenotype can these studies (Darroudi and others 1998; Greinert and others contribute to tumorigenesis is explored in Chapter 3. 2000) a substantial portion of exchanges have been shown to form rapidly, although some require several hours. There is INDUCTION OF GENE MUTATIONS IN some evidence that those aberrations forming rapidly tend to SOMATIC CELLS be incomplete exchanges, which suggests a time dependence for pairwise exchange (Alper and others 1988) of DNA Ionizing radiation is known to induce a broad range of DSBs. The general picture that emerges from these biophysi- potentially mutagenic lesions in DNA ranging from dam- cal studies is that the misrepair events of radiation-induced aged DNA bases to frank DNA breaks and chemically com- DNA DSBs that lead to chromosome aberrations are prob- plex lesion clusters (see Chapter 1). Not unexpectedly, mo- ably associated with the dominant postirradiation function lecular analyses of radiation-induced somatic mutations at a of the nonhomologous end joining (NHEJ) repair processes number of loci provide evidence of induction of point muta- described in Chapter 1. tions in single genes and of small and large deletions that Overall, biophysical approaches to the modeling of dose- may encompass a number of physically linked genes response for chromosomal aberrations, although not without (Sankaranarayanan 1991; Thacker 1992). An important fac- some uncertainties on mechanisms, imply that the single- tor in the induction and recovery of deletion-type, multilocus track component of radiation action will dominate responses mutations is the degree to which multiple gene loss may be at low doses and low dose rates (i.e., the dose-response for tolerated by the cell. There is good evidence that such toler- all forms of aberrations will be linear at low doses and low ance is highly dependent on the genetic context of the muta- dose rates). Considerable effort has been expended to test tion (i.e., its position in respect to essential genes and, for this proposition, and in a very large multicenter study using autosomal loci, the genetic status of the second gene copy on assays of dicentric aberrations in human lymphocytes, the the homologous chromosome). These issues are discussed in linearity of the response was evident down to at least 20 mGy depth elsewhere (Thacker 1992); here it is sufficient to note of low-LET radiation (Lloyd and others 1992), which is il- that genetic context can result in up to a twentyfold change lustrated in Figure 2-5. Below that dose, the statistical power in induced mutation frequencies in autosomal genes (Brad- of the data was not sufficient to exclude the theoretical pos- ley and others 1988; Amundson and Liber 1991). There is sibility of a dose threshold for radiation effects. strong molecular evidence that in most circumstances, a Another important feature of the chromosomal response DNA deletion mechanism dominates mutagenic response to radiation is the postirradiation period during which initial after ionizing radiation (Sankaranarayanan 1991; Thacker DNA damage is fixed and then expressed in the form of 1992), and it is for this reason that the genetic context of the aberrations such as dicentric chromosomes. On the basis of mutation is of great importance. In illustration of this, radia- direct observation and theory, the conventional cytogenetic tion mutagenesis in cells hemizygous (one gene copy de- view is that all such chromosomal damage sustained within leted) for autosomal APRT (adenine phosphoribosyltrans- a given cell cycle will be fixed and then expressed at the first ferase) is constrained by the proximity of an essential postirradiation mitosis. Accordingly, Carrano and Heddle sequence; induced mutation frequencies are relatively low,

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MOLECULAR AND CELLULAR RESPONSES TO IONIZING RADIATION 47 and only ~20% of induced mutations are of the deletion or tion of the dose-response down to ~200 mGy (Thacker 1992) rearrangement type (Miles and others 1990)—many de- and, from limited data, at lower doses. The exceptions to this letions will have led to cell death. By contrast, radiation are the data from a particularly sensitive in vivo system that mutagenesis at the X-linked HPRT gene is much less con- scores reversion mutations (as hair color changes) at the strained by neighboring sequence; induced mutation fre- pink-eyed unstable (Bonassi and others 1995) locus in the quencies are substantially higher, and ~70% of induced mu- mouse. Using this system, a linear nonthreshold low-LET tations show HPRT deletion or rearrangement (Thacker dose response has been obtained at doses down to 10 mGy 1986)—many more will have been tolerated (Bedford and (Schiestl and others 1994), but as discussed later in this Dewey 2002). Stated simply, gene loss mutations are char- chapter, that system is probably reflecting a mutagenic com- acteristic of radiation, but their recovery in viable cells can ponent from the induction of genomic instability. be a major limiting factor. Also, gene amplification can re- Studies of radiation-induced gene mutation in radiosensi- sult from the process of DSB repair (Difilippantonio and oth- tive mutant cell lines indicate that increased mutability can ers 2002). As shown later, these features are important for be associated not only with defective repair of DNA DSB consideration of carcinogenic mechanisms and are also dis- but also with processes that affect the regulation of DNA cussed in respect of germline mutagenesis. repair (Thacker and others 1994). Finally, in studies on the Deletion and rearrangement of APRT, HPRT, and other effects of low-dose-rate, low-LET radiation and other cellu- target genes do occur spontaneously but are generally less lar repair-related factors (Thacker 1992), there is consistent frequent than point mutation; in the case of most chemical evidence for potentially increased efficiency of repair of pre- mutagens, there is a strong bias toward the induction of point mutagenic lesions at low dose rates, but none of these stud- mutations (Thacker 1986; Miles and others 1990; Sankaran- ies specifically suggest the presence of a low-dose thresh- arayanan 1991). old. The following sections consider specific aspects of Studies of the effect of radiation quality on the induction cellular response relating to cell cycle effects, adaptive re- of gene mutations show a relationship similar between rela- sponses to radiation, the transfer of damage signals between tive biological effectiveness (RBE) and LET to that noted cells (bystander effects), induced and persistent genomic in- for chromosome aberration induction. Mutagenic effective- stability, low-dose hyper-radiation sensitivity, and other as- ness peaks at a LET of 100–200 keV µm–1, with maximum pects of dose-response. RBE values usually in the range of 7–10 based largely on initial slopes of the dose-response (Cox and Masson 1979; RADIATION-INDUCED GENOMIC INSTABILITY Thacker and others 1979; Thacker 1992). Molecular analy- ses broadly suggest that a DNA deletion mechanism pre- Radiation-induced genomic instability has been defined dominates for all radiation qualities (Thacker 1986; Gibbs as the manifestation of genetic damage in a certain fraction and others 1987; Aghamohammadi and others 1992; Jostes of irradiated cells over many cell cycles after they were irra- and others 1994), but there are some conflicting data on this diated (Little 2003). This persistent instability is expressed issue. as chromosomal rearrangements, chromosomal bridge for- DNA sequence data for radiation-induced intragenic de- mation, chromatid breaks and gaps, and micronuclei (Gro- letions in APRT and larger deletions encompassing HPRT sovsky and others 1996; Murnane 1996; Poupon and others indicate the frequent involvement of short direct or inverted 1996; Limoli and others 1997a; Suzuki and others 1998) in DNA repeats at deletion breakpoints (Miles and others 1990; the progeny of cells that survive irradiation. Reduction in Morris and Thacker 1993). The presence of these short re- cell cloning efficiency several generations after irradiation peats is highly suggestive of an important role for illegiti- is called delayed lethality; it is supposedly a manifestation mate recombination processes in mutagenesis and, as for of genomic instability associated with an increase in lethal chromosome aberration induction, the involvement of DNA mutations (Seymour and Mothersill 1997). Also, gene muta- DSBs and error-prone NHEJ repair. Evidence for a close tions, such as HPRT mutations, that arise de novo several relationship between gene mutations and chromosome aber- generations after irradiation are thought to be another mani- rations is that several induced gene mutations are associated festation of genomic instability. The spectrum of these de with macroscopic region-specific chromosomal deletions or novo mutations resembles that of spontaneous mutations rearrangements (Cox and Masson 1978; Thacker and Cox (i.e., primarily point mutations instead of deletions that are 1983; Morris and Thacker 1993). induced directly by irradiation; Little and others 1997). If, as molecular data suggest, error-prone NHEJ repair of There is controversy, however, as to whether all of these DNA DSBs is the principal source of radiation-induced gene different end points represent the same fundamental chro- mutations, then a linear dose-response would be anticipated mosomal alterations that result in genomic instability (Chang at low doses. For technical reasons, dose-response relation- and Little 1992; Morgan and others 1996; Limoli and others ships for gene mutations are far less precise than those for 1997a; Little 1998; Mothersill and others 2000a). However, chromosome aberrations. In general, however, a linear or the similarity in the frequencies of genomic instability linear-quadratic relationship provides a satisfactory descrip- induced in X-irradiated cells, (3 to 19) × 10–5 per cell/mGy,

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48 BEIR VII and the frequencies of chromosomal aberrations induced di- Because chromosomal instability has been associated rectly by irradiation may suggest that the induction of chro- with breakage-fusion-bridge (B/F/B) cycles (Fouladi and mosomal aberrations is a primary event that plays a major others, 2000; Gisselsson and others 2000; Lo and others, role in radiation-induced genomic instability (data presented 2002a, 2002b; Little 2003), the roles of telomeres may be in section “Observed Dose-Response Relationships at Low particularly relevant. See Mathieu and colleagues (2004) and Doses”). Murnane and Sabatier (2004) for reviews. Chromosome There is controversy concerning the fundamental radia- instability can also be initiated by DSBs that result in the tion target and lesions that result in genomic instability. Evi- loss of a telomere that protects the chromosome end and dence that the nucleus is the target (Limoli and others 1997b; prevents chromosome fusion. A single DSB introduced at a Kaplan and Morgan 1998) is that 125IdU (iododeoxyuridine) telomere with the I-SceI endonuclease in mouse embryonic disintegrations in the DNA resulted in chromosomal insta- stem (ES) cells (Lo and others, 2002a) and spontaneous bility, whereas 125I disintegrations in the cytoplasm and telomere loss in a human tumor cell line (Fouladi and others, cellular membrane did not. Furthermore, incorporation of 2000; Lo and others, 2002b) were found to result in sister- BrdU (bromodeoxyuridine) into DNA increased the amount chromatid fusion and chromosome instability. Chromosome of radiation-induced chromosomal instability (Limoli and instability can be associated with prolonged B/F/B cycles; others 1999), which argues for DNA as the target. However, these cycles arise as a consequence of breakage of fused since restriction enzymes that produced DSBs in DNA sister chromatids when their centromeres are pulled in oppo- (Kinashi and others 1995), mutations (Phillips and Morgan site directions during anaphase, with subsequent re-fusion in 1994), and chromosomal aberrations (Bryant 1984) did not the next cell cycle. These B/F/B cycles result in extensive induce chromosomal instability (Limoli and others 1997b), DNA amplification and cease only when the chromosome the hypothesis was presented that DSBs themselves are acquires a new telomere, often by nonreciprocal transloca- insufficient and that complex clustered damage in the DNA, tions from another chromosome. However, because the such as that from 125I disintegrations, is required. There is nonreciprocal translocations provide telomeres that stabilize also some evidence that genomic instability results from the marker chromosome, those chromosomes that donate the complex chromosomal abnormalities created de novo by nonreciprocal translocations can become unstable due to the rearrangements that generate unstable combinations of DNA loss of their telomeres. Then, a subsequent nonreciprocal sequences (Murnane 1990), such as inverted repeats or asso- translocation can serve to transfer instability to another chro- ciations of euchromatin with heterochromatin (Grosovsky mosome (Murnane and Sabatier 2004; Sabatier and others and others 1996). Nevertheless, since the amount of insta- 2005). Thus, the loss of a single telomere can result in trans- bility induced by 125I disintegrations in the DNA was rela- fer of instability from one chromosome to another, leading tively low (maximum of 4–9% unstable clones; Kaplan and to extensive genomic instability. Morgan 1998; Griffin and others 2000), the possibility was The importance of telomere loss as a mechanism for chro- suggested that targets in addition to DNA might be involved mosome instability through B/F/B cycles in cancer has been (Limoli and others 2001). At the least, damage and/or error- emphasized by the demonstration that telomerase-deficient prone repair in DNA is probably involved in radiation- mice that are also deficient in p53 have a high cancer inci- induced genomic instability because mutant cells deficient dence (Artandi and others 2000; Chang and others 2001; in the repair enzymes needed for NHEJ are most sensitive to Rudolph and others 2001). The analysis of the tumor cells the induction of radiation-induced instability (Little 2003) from these mice demonstrated the presence of chromosome and especially genomic instability induced by DNA DSBs rearrangements typical of B/F/B cycles, including gene am- (Difilippantonio and others 2002). plification and nonreciprocal translocations commonly seen There are also data indicating that reactive oxygen spe- in human cancer. It is possible that the genomic instability cies (Limoli and others 2001; Little 2003), potentially per- observed for chromosomal aberrations, HPRT mutations, sistent over several generations, may play an important role and longer telomere terminal restriction fragment lengths in in ongoing genomic instability. In addition, alterations in sig- X-irradiated CHO cells (Romney and others 2001) is also a nal transduction pathways may be involved (Morgan and manifestation of nonreciprocal translocations that lead to te- others 1996), and alterations in nucleotide pools have been lomere loss. shown to lead to genomic instability (Poupon and others A question that has to be addressed is the relevance of 1996). Another possibility is that damage to centrosomes radiation-induced genomic instability for radiation-induced might be an important target because centrosome defects are cancer, and a corollary of this question is the relationship thought to result in genomic instability through missegre- among expression of p53, radiation-induced apoptosis, and gation of chromosomes (Pihan and others 1998; Duensing radiation-induced genomic instability. The “guardian-of-the- and others 2001) that would result in aneuploidy (Duensing genome” hypothesis postulates that either cell cycle arrest and Munger 2001). However, as reported recently (Hut and allows additional time for repair of DNA damage or, alterna- others 2003), centrosomal damage can result from incom- tively, apoptosis eliminates damaged cells, thereby prevent- pletely replicated or damaged DNA. ing progeny from manifesting genomic instability and ulti-

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MOLECULAR AND CELLULAR RESPONSES TO IONIZING RADIATION 49 mately carcinogenesis (Lane 1992; Kemp and others 1994; ship for genomic instability and especially why, in some cel- White and others 1994; Levine 1997; Lengauer and others lular systems, the induction frequency saturates with only 1998). Evidence has been presented that radiation-induced about 10–30% of the surviving cells manifesting genomic apoptosis can occur via p53-dependent and p53-independent instability (Little 1998; Limoli and others 1999) (data pre- mechanisms (Strasser and others 1994) initiated by damage sented in Table 2-1). It may be that only a certain fraction of in the nucleus (Guo and others 1997) or cytoplasm-mem- the cells, or those in a certain part of the cell cycle, are sus- brane (Haimovitz-Friedman 1998). This damage results in ceptible to radiation-induced genomic instability. Until the cells undergoing apoptosis either during interphase without molecular mechanisms responsible for genomic instability attempting division (Endlich and others 2000), several hours and its relationship to carcinogenesis are understood, the after they have divided a few times (Forrester and others extrapolation of dose-response data for genomic instability 1999), or during an aberrant mitosis (Endlich and others to radiation-induced cancers in the low-dose range 2000). The signal transduction pathways (White and Prives <100 mGy is not warranted. 1999) resulting in radiation-induced apoptosis involve the nucleus and cytoplasm with alterations in mitochondrial CELL CYCLE EFFECTS electron transport (Voehringer and others 2000) and release of cytochrome c from the mitochondria, which initiates In a number of mammalian cell lines, cells irradiated in caspase cleavage (Finucane and others 1999) and terminates mitosis or late G2 are most susceptible, cells in G1 are inter- in activation of a nuclease responsible for internucleosomal mediate in susceptibility, and cells in middle to late S phase digestion of DNA (Wyllie 1998). and early G2 are most resistant to the induction of cell lethal- In accord with the guardian-of-the-genome hypothesis, ity, chromosomal aberrations, and mutations (Sinclair and mouse tumors undergoing apoptosis in a p53-independent Morton 1963; Terasima and Tolmach 1963; Dewey and manner contained abnormally amplified centrosomes, aneu- others 1970; Burki 1980; Jostes and others 1980; Watanabe ploidy, and gene amplification (Fukasawa and others 1997). and Horikawa 1980; Chuang and Liber 1996; Leonhardt and Also, a decrease in radiation-induced apoptosis associated others 1997). Also, cells irradiated at the G1/S transition are with nonfunctional p53 or expression of Bcl2 correlated with often observed to be more radiosensitive than cells in G1 or an increase in mutagenesis (Xia and others 1995; S. However, exceptions have been observed, such as little Cherbonnel-Lasserre and others 1996; Yu and others 1997). variation in radiosensitivity during the cell cycle (Henderson However, the latter correlation might be due not to p53- and others 1982) and greater sensitivity of cells in late S than mediated’s enhancement of radiation-induced apoptosis (Xia of cells in G1 (Thompson and Humphrey 1968; Guo and and others 1995) but instead to p53-mediated’s suppression others 1997; Furre and others 1999). Since radioresistance of homologous recombination (Sturzbecher and others during late S phase has been attributed to error-free repair of 1996), which in turn might suppress genomic instability and DNA DSBs by homologous recombination when sister chro- a hypermutable phenotype. However, there is evidence that matids have been replicated (Rothkamm and Lobrich 2003; radiation-induced genomic instability is independent of p53 Rothkamm and others 2003), the lack of radioresistance dur- expression (Kadhim and others 1996). Furthermore, when ing late S phase in some cell lines may be attributed to their the guardian-of-the-genome hypothesis was tested in lym- inability to carry out repair by homologous recombination. phocyte cultures that were irradiated under different dose- Those effects have been observed in connection with rela- rate and mitogen-treatment conditions, postradiation incu- tively high acute doses of 1.5–10 Gy (1500-10,000 mGy), bation allowing apoptotic processes to remove damaged cells but how such variations in radiosensitivity during the cell did not prevent the development of chromosomal instability cycle may affect responses to low doses up to 100 mGy is during long-term cell proliferation over 51–57 days not known. Also, there are no reports of studies to determine (Holmberg and others 1998). Thus, the relationship between whether there may be variations in radiosensitivity during radiation-induced genomic instability, radiation-induced the cell cycle for induction of genomic instability. However, apoptosis, and radiation-induced cancer is uncertain (dis- studies with cell lines have indicated that cells are most sus- cussed at length in Chapter 3). Furthermore, radiation-in- ceptible to malignant transformation in vitro when they are duced genomic instability could not be induced in normal irradiated with high-LET radiation or low-energy X-rays in diploid human fibroblasts (Dugan and Bedford 2003) and late G2/M (Cao and others 1992, 1993; Miller and others may be related to confounding in vitro stress factors 1992). (Bouffler and others 2001) or to the cells being partially The inverse dose-rate effect (Crompton and others 1990; transformed. Finally, as discussed in Chapter 3, it may be Amundson and Chen 1996), in which cells at first become that genomic instability plays a more important role in tumor more radioresistant and then more radiosensitive again as progression than in tumor initiation. the dose rate of low-LET radiation is decreased below about Data are critically needed for the definition of molecular 1–10 mGy/min, has been attributed to the arrest of cells in a targets and processes responsible for genomic instability in radiosensitive G2 phase of the cycle (Mitchell and others order to define and understand the dose-response relation- 1979; Furre and others 1999). However, evidence has been

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50 BEIR VII presented that the inverse dose-rate effect can be observed to dose at low dose rates was much lower than that at high when cells do not arrest in G2 and, instead, correlates with dose rates (Ullrich 1984). How these data on high-LET fis- low-dose hyper-radiation sensitivity (HRS; Mitchell and sion neutrons can be extrapolated to low-LET radiation is others 2002). This conclusion may be consistent with recent unknown, especially because the RBE for these carcinogenic results from the same research group (Marples and others effects has been estimated to be as high as 10 or more. This 2003), which reported that HRS for acute radiation doses means that the equivalent doses and equivalent dose rates was attributed to cells in radiosensitive G2 failing to arrest mentioned above, when expressed in millisieverts, would be before mitosis. For high-LET radiation, the inverse dose- at least 10 times greater than the values expressed in rate effect has been attributed to the traversal of cells through milligrays. a radiosensitive G2 phase (Brenner and others 1996; Elkind Furthermore, when the same tumors were induced in mice 1996; Tauchi and others 1999). Such an inverse dose-rate by low-LET radiation at doses of 0.1–6.0 Gy, no inverse effect has been reported for cell lethality and mutations dose-rate effect was observed between 0.04 and 0.6 mGy/ induced by low-LET radiation and for transformation min; these low dose rates always had a dose-response rela- induced by high-LET radiation. tionship significantly below that observed for acute high- Vilenchik and Knudson (2000) hypothesized that the in- dose-rate irradiation (Ullrich and others 1976, 1987; Ullrich crease in mutability observed below a dose rate of 1 mGy/ and Storer 1979a, 1979b, 1979c; Ullrich 1983). Similar ob- min for mouse spermatogonia and 10 mGy/min for cells servations were reported for neoplastic transformation of in vitro is not caused by variations in radiosensitivity during C3H 10T1/2 cells by low-LET radiation, for which the dose- the cell cycle but rather by a diminished activation of error- response relationship for a low dose rate of 1 mGy/min was free repair at very low dose rates inasmuch as the rate of much below that observed for an acute high dose rate of induced DNA damage (signal) is lower than the background 1.0 Gy/min (Han and others 1980). The lack of a low-LET rate of spontaneous DNA damage (noise). This interpreta- inverse dose-rate effect for tumor induction and neoplastic tion of the data remains controversial, particularly since there transformation in vitro contrasts with the inverse dose-rate is evidence that argues against the inducibility of DNA re- effect seen for cell killing and induction of mutations that is pair genes. However, Collis and colleagues (2004) reported sometimes attributed to perturbations in cell cycle progres- recently that DNA damage introduced at a very low dose sion. However, results obtained with mammalian cell lines, rate of 0.33 or 1.5 mGy/min produced less activation of the in particular those for neoplastic transformation, should be radiation damage sensor ATM (ataxia-telangiectasia-mu- interpreted with great caution if they are to be used in esti- tated), as detected by H2AX foci, than activation at a high mating radiation risk to humans. dose rate of 750 mGy/min. Furthermore, this reduction of ATM activation was observed after irradiation in Go/G1, S, ADAPTIVE RESPONSE and G2/M, and correlated with enhanced cell killing. For a discussion of the expression of particular genes involved in Organisms, such as bacteria, that live in a highly change- DNA repair and controlling checkpoints in the cell cycle, able environment have multiple mechanisms for adapting to see “DSB Signal Transduction and Inducible Repair” in environmental stress. The bacterium Escherichia coli has Chapter 1, along with Figure 1-10. two distinct, inducible, redox-regulated transcriptional Although some small transient effects on cell cycle pro- switches involving the soxRS and oxyR transcription fac- gression have been reported for doses of 20–100 mGy (Puck tors, which respond to exposure to superoxide and hydrogen and others 1997; Amundson and others 1999b), no inverse peroxide, respectively (Demple 1991; Choi and others dose-rate effect would be expected at these dose levels 2001). After exposure to ionizing radiation, these factors re- (Brenner and others 1996), and if it did exist, it would be program the cellular transcription pattern with increased ex- difficult to demonstrate. However, at approximately pression of proteins that inactivate reactive oxygen species 100 mGy, an inverse dose-rate effect of fission-spectrum and some DNA repair enzymes that process oxidative DNA neutrons has been observed between 4 and 100 mGy/min for damage. As a consequence, E. coli cells exhibit a distinct neoplastic transformation of C3H 10T1/2 cells (Hill and oth- adaptive response to oxidative stress: exposure to a low dose ers 1982, 1984) and between 10 versus 250 mGy/min and of active oxygen makes the cells more resistant to later ex- 0.0083 versus 0.083 mGy/min for induction of lung adeno- posures for some finite period. In that situation, there is a carcinomas and mammary adenocarcinomas in mice (Ullrich clear threshold value for deleterious effects of ionizing ra- 1984). Apparently, these inverse dose-rate effects could not diation. However, the soxRS and oxyR gene regulons have be explained by perturbations in the cell cycle, and for mam- not been conserved during evolution, and human cells, mary tumors, the effect was associated with an increased which exist in a much more stable cellular environment than probability of progression of carcinogen-altered cells rather bacteria, do not appear to have counterparts. Thus, humans than an increased number of initiated cells (Ullrich 1986). do not have an adaptive response to oxidative damage simi- Furthermore, an inverse dose-rate effect was not observed lar to the well-characterized systems in bacteria. for the induction of ovarian tumors, for which the response

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MOLECULAR AND CELLULAR RESPONSES TO IONIZING RADIATION 51 A broad perturbation of DNA transcription is observed late to the genetic variation reported for radiation-induced in human cells after exposure to ionizing radiation; it in- transcriptional changes (Correa and Cheung 2004). volves the activation of transcription factors, such as NF- Adaptive responses to radiation observed in other cellu- kappaB and c-jun/c-fos. After exposure of human lympho- lar systems for induction of cell lethality, chromosomal ab- blastoid cells to 5 Gy of radiation, 2–3% of the genes exhibit errations, mutations (Zhou and others 1993; Rigaud and oth- more than a 50% change in induction or repression (Tusher ers 1995), and defects in embryonic development provide and others 2001). These genes include several involved in little information that can be used to suggest that the dose- cell cycle control. No genes involved in repair of DNA response curve in the dose range 0–100 mGy will be less DSBs generated by ionizing radiation were induced (Tusher steep than that described by the limiting value of α men- and others 2001; Wood and others 2001). It should also be tioned above. When mouse embryos were exposed to a noted that the base-excision repair enzymes involved in the priming dose of about 10 mGy and evaluated for chromo- removal of oxidative damage are not induced by low doses somal aberrations or defects in development induced by a of ionizing radiation in human cells (Inoue and others 2004). challenge dose several hours later, the results were highly These studies have provided no support for a general adap- variable for the induction of an adaptive response (Muller tive repair response in human cells to counteract DNA DSB and others 1992; Wojcik and others 1992; Wolff 1996; formation that can result in cell death or mutagenesis. Wang and others 1998). Studies of radiation-induced mu- A different type of apparent adaptive response has been tagenesis also had variable results. Adaptation not only de- well documented for the induction of chromatid-type breaks creases the frequency of mutants induced by a challenge and mutations in human lymphocytes stimulated to divide. dose but also appears to alter the types of mutants. Adapta- In most studies, a priming or adaptive dose of about 10 mGy tion of human lymphoblastoid cells to a challenge dose of significantly reduces the frequency of chromosomal aberra- 4 Gy 6 h after 20 mGy decreased the proportion of HPRT tions (Shadley and others 1987; Wolff 1992a, 1996) and mutants of the deletion type relative to small point muta- mutations (Kelsey and others 1991) induced a few hours tions (Rigaud and others 1995). In contrast, adaptation of later by 1–3 Gy. However, when the priming dose was human-hamster hybrid AL cells to a challenge dose of 3 Gy 10 mGy, the adaptive response for chromosomal aberrations after a priming dose of 40 mGy increased the proportion of was reduced significantly as the priming dose rate was re- complex unstable mutations (Ueno and others 1996). An duced from 50 mGy/min to 6.4 mGy/min (Shadley and extensive study (Sasaki 1995) of chromosomal aberrations, Wiencke 1989). Adaptive responses of this type were re- HPRT mutations, and cell killing demonstrated adaptation viewed by UNSCEAR (1994). in quiescent cultured m5S mouse embryonic skin cells Although alterations in cell cycle progression have been preexposed in G1 to 10–50 mGy; cells exposed 4 h later to implicated in the mammalian cell adaptive phenomenon doses greater than 2 Gy were significantly more resistant (Aghamohammadi and Savage 1991), carefully controlled than nonadapted cells for all three end points (see Figure 2-2 studies indicate that the priming dose induces radioresis- for cell-killing results). The adaptation phenomenon ap- tance for induction of chromosomal aberrations in human peared to involve a protein kinase C signaling pathway. In lymphocytes (Wolff 1996); priming doses less than 5 mGy, addition, the lack of an adaptive response in a tumorigenic or greater than about 200 mGy, yield very little if any adap- variant, clone 6110, and restoration of the adaptive response tation (Wolff 1992b). The induction and magnitude of the obtained by introducing human chromosome 11 (five other adaptive response in human lymphocytes are highly vari- chromosomes had no effect) further suggested that interfer- able among people (Bose and Olivieri 1989; Sankar- ence of signaling pathways may alter adaptive responses in anarayanan and others 1989; Shadley and Wiencke 1989; malignant cells. The observation (Broome and others 2002) Hain and others 1992; Vijayalaxmi and others 1995; Upton that a priming dose as low as 1 mGy induced an adaptive 2000), and the adaptive response could not be induced when response in a nontransformed human fibroblast cell line for lymphocytes were given the priming dose during G0 (Shad- micronuclei induced by a challenge dose of 2 Gy has to be ley and others 1987). Although inhibitor and electrophoretic confirmed for other systems and end points, such as muta- studies (Youngblom and others 1989; Wolff 1992b) suggest tion induction. Also, the large variation in adaptive response that alterations in transcribing messenger RNA and synthe- for radiation-induced micronuclei in human lymphoblastoid sis of proteins are involved in the adaptive response in lym- cell lines must be considered (Sorensen and others 2002). phocytes, no specific signal transduction or repair pathways Most important, the adaptive response has to be demon- have been identified. Finally, humans exposed occupation- strated for both priming and challenging doses in the low- ally (Barquinero and others 1995) or to iodine-131 (131I) for dose range <100 mGy, and an understanding of the molecu- treatment of thyroid disease (Monsieurs and others 2000) or lar and cellular mechanisms of the adaptive response is as children after Chernobyl (Tedeschi and others 1995) var- essential if it is to have relevance for risk assessment. ied in their ability to demonstrate an apparent adaptive re- Studies of adaptation for malignant transformation sponse for chromosomal aberrations (Padovani and others in vitro provide conflicting information and might not be 1995; Tedeschi and others 1996). This variability may re- relevant to malignant transformation in vivo. Although the

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52 BEIR VII The reduction was observed only when the cells were trypsinized and replated 24 h after irradiation for the trans- formation assay; trypsinization and replating immediately after irradiation did not alter the frequency. Similar results have been reported by Redpath and coworkers (Redpath and Antoniono 1998; Redpath and others 2001): the malignant transformation frequency was reduced by about half when human hybrid cells approaching confluence were trypsinized and replated 24 h after a priming dose of 10 mGy; again, no statistically significant reduction in transformation frequency was observed when the cells were trypsinized and replated immediately after irradiation. The validity of extrapolating any of the results from in vitro neoplastic transformation systems to malignant transformation in vivo may be questioned for the following reasons. First, the effects associated with variations in time of trypsinization and replating after irradiation must be un- derstood (Schollnberger and others 2002). Second, the mea- sured neoplastic transformation frequency depends on both the density of viable cells plated (Bettega and others 1989) and the number of generations before the cells become confluent (Kennedy and others 1980). Third, when priming doses of 1–100 mGy resulted in a decrease in the neoplastic transformation frequency, the spontaneous transformation frequency was unusually high in one case (Azzam and oth- FIGURE 2-2 Effects of preirradiation on clonogenic survival of mouse m5S cells. Closed symbols represent results in cells in G1 ers 1994), and a Hela X skin fibroblast human hybrid cell preirradiated with 20 mGy of X-rays 5 h before graded doses of system was used in the other (Redpath and Antoniono 1998). acute radiation. Open symbols represent results in cells in G1 given Fourth, studies of malignant transformation in immortalized graded doses of acute radiation only. Statistical errors are standard (already-transformed) cell lines may have little relevance to errors of the mean based on variation in the number of recovered malignant transformation of normal nonimmortalized cells, colonies in irradiated dishes (this does not include propagation of especially in vivo, where complex interactive processes can error in plating efficiency of nonirradiated controls). SOURCE: occur (Harvey and Levine 1991; Kamijo and others 1997). Sasaki (1995). For several mammalian cell lines in culture, adaptive re- sponses for cell lethality after doses of 200–600 mGy (Marples and Joiner 1995; Joiner and others 1996; Marples and Skov 1996; Wouters and others 1996; Skov 1999) and morphologic transformation frequency of m5S adapted for enhanced removal of thymine glycols after a dose of 2 Gy mouse embryonic skin cells that had received 20 mGy was (Le and others 1998) have been observed 4–6 h after a prim- about half the spontaneous frequency of 3 × 10–5 observed in ing dose of 200 mGy. In Chinese hamster V79 cells, the rate nonprimed cells, the adapted cells exposed 5 h later to a chal- of repair of DNA DSBs induced by 1.5 or 5.0 Gy was lenge dose of 1 Gy were more susceptible to morphologic increased 4 h after a priming dose of 50 mGy (Ikushima and transformation than the nonadapted cells (Sasaki 1995). others 1996). These transformation results, however, contrast with results The adaptive responses of mammalian cells described in mouse C3H 10T1/2 cells that were exposed in plateau above, at least for cell survival and repair of DNA strand phase to a challenge dose of 4 Gy 5 h after a priming dose of breaks (Robson and others 2000), may be associated in part 100 or 670 mGy (i.e., adapted cells were more resistant to with the downregulation of a gene DIR1 90 min after doses malignant transformation than nonadapted cells; Azzam and of 50–1000 mGy. This gene codes for proteins (Robson and others 1994). Furthermore, the priming dose of 100 or others 1997, 1999, 2000) similar to a family of heat shock- 670 mGy caused an increase by a factor of 2–5 in the trans- related proteins (HSPs) known as immunophilins with tet- formation frequency relative to the frequency of about 3 × rapeptide repeats (TPRs). TPR-containing proteins, such as 10–4 observed for nonirradiated cells. When the same group cell cyclin proteins cdc23, cdc27, and cdc16, have been re- of investigators exposed the same C3H 10T1/2 cells in pla- ported to form complexes in vivo, and the TPR domain is teau phase to priming doses of 1, 10, or 100 mGy, the neo- thought to be involved in binding HSP90 and HSP70. Less plastic transformation frequency was lower by a factor of 3– binding of HSP70 and the induction of other members of the 4 than the spontaneous frequency (Azzam and others 1996). HSP70 family by low doses of radiation (Sadekova and

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MOLECULAR AND CELLULAR RESPONSES TO IONIZING RADIATION 53 others 1997) might result in adaptation through the same data are needed on delivery of the priming and challenge mechanisms. doses over several weeks or months at very low dose rates or The recent microarray expression studies (Yin and others with fractionated exposures. 2003) that demonstrated downregulation of the large HSPs Finally, we should be concerned about the cumulative 30 min after irradiating the mouse brain with 100 mGy may effect of multiple low doses of less than 10 mGy. Such data support these conjectures. Also, the radiation-induced have not yet been obtained, in particular those explaining the downregulation of CDC16, which belongs to the anaphase- molecular and cellular mechanisms of the adaptive response. promoting complex, was enhanced by an adaptive dose of Therefore, it is concluded that any useful extrapolations for 20 mGy (Zhou and Rigaud 2001). In fact, regulation of re- dose-response relationships in humans cannot be made from pair and cell cycle progression may be achieved by differen- the adaptive responses observed in human lymphocytes or tial complex formation (Eckardt-Schupp and Klaus 1999). the other cellular systems mentioned above. In fact, a study For instance, PCNA (proliferating cell nuclear antigen) ex- (Barquinero and others 1995) reporting that an average oc- pression, which is modulated by p53 in response to radia- cupational exposure of about 2.5 mGy per year over 7– tion, may play an important role in regulating and coordinat- 21 years resulted in a variable adaptive response for chro- ing cell cycle progression, DNA replication, translesion mosomal aberrations induced in human lymphocytes by a synthesis, and DNA excision repair, depending on its part- large challenge dose of 2 Gy also reported that the incidence ner proteins. Within minutes after ionizing radiation, the of spontaneous aberrations was increased significantly by immediate-response genes transcription factors such as c- the occupational exposure. Barquinero and colleagues (1995) jun, c-fos, and NF-kB are turned on, possibly thwarting the also cite six reports indicating that basal rates of chromo- general downregulation of transcription after irradiation and somal abnormalities are in general higher in exposed human allowing privileged transcription of special genes. The sen- populations; recent papers (Tanaka and others 2000; Tawn sors for these fast responses are in membranes, and they ini- and others 2000a, 2004; Burak and others 2001; Liu and tiate signal transduction by several cascades of protein ki- others 2002; Maffei and others 2004) present similar infor- nases (Eckardt-Schupp and Klaus 1999) that may involve mation. Therefore, based on current information, the as- reactive oxygen intermediates (Mohan and Meltz 1994; sumption is unwarranted that any stimulatory effects of low Hoshi and others 1997). Therefore, adaptation in mamma- doses of ionizing radiation substantially reduce long-term lian cells probably involves induction of signal transduction deleterious radiation effects in humans. pathways (Stecca and Gerber 1998) rather than induction of DNA repair enzymes. BYSTANDER EFFECTS There is much variability and heterogeneity in the ability to induce adaptive responses that usually require a priming A factor that could have a significant effect on the dose- dose of 10–200 mGy and a large challenge dose of 1–2 Gy. response relationship is the bystander effect that irradiated Challenge doses of this magnitude probably have little rel- cells have on nonirradiated cells. Recent comprehensive re- evance to risk assessment for low radiation doses of 1– views of bystander effects observed in vitro (Morgan 2003a) 100 mGy. Furthermore, the molecular pathways associated and in vivo (Morgan 2003b) emphasized their possible with the phenomenon have not been delineated. Available mechanisms, implications, and variability. In addition, re- data indicate that the adaptive response results from DNA views have been published recently on the relationship be- damage that can be induced by 3HTdR (triliated thymidine) tween the bystander effect, genomic instability, and car- incorporated into DNA, by H2O2, and by restriction enzymes cinogenesis (Little 2003; Lorimore and others 2003). (Wolff 1992b; Sasaki 1995; Belyaev and Harms-Ringdahl Observations that irradiated cells or tissues could have del- 1996). The ability to induce an adaptive response appears to eterious effects on nonirradiated cells or tissues were re- depend on the genotype (Wojcik and others 1992), which ported many years ago (Bacq and Alexander 1961) and were may relate to genetic variation reported for radiation-induced termed abscopal effects. As an example of such an effect, transcriptional changes (Correa and Cheung 2004). In fact, plasma from patients who underwent localized radiation the effect of the genotype on the adaptive response has been therapy induced chromosomal aberrations in lymphocytes demonstrated most conclusively in Drosophila melanogaster from nonirradiated patients (Hollowell and Littlefield 1968; (Schappi-Bushi 1994). Littlefield and others 1969). A bystander effect has been A priming dose has been reported to reduce chromosomal demonstrated conclusively for cells in culture exposed to damage in some chromosomes and increase it in others high-LET radiation, usually α-particles. Little and col- (Broome and others 1999). Data are needed, particularly at leagues estimated that a single α-particle traversing a cell the molecular level, on adaptation induced when both can induce HPRT mutations (Nagasawa and Little 1999), priming and challenging doses are in the low-dose range sister-chromatid exchanges (Nagasawa and Little 1992), <100 mGy; relevant end points should include not only chro- upregulation of p21 and p53, and downregulation of cyclin mosomal aberrations and mutations but also genomic insta- B1, cdc2, and rad51 (Azzam and others 1998) in unirradiated bility and, if possible, tumor induction. In vitro and in vivo cells. At least for the bystander effect on signal transduction

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54 BEIR VII pathways and induction of mutations, the irradiated and particles, there may be reduced phosphorylation of connexin nonirradiated cells had to be in contact with each other 43 by CDC2 and thus increased membrane permeability through gap junctions. Hall and colleagues demonstrated the (Azzam and others 1998). This hypothesis is supported by same bystander phenomenon for cell killing, induction of the observation that membrane signaling is involved in the mutations (Zhou and others 2000), micronuclei formation bystander effect for sister-chromatid exchanges and HPRT (Hall 2000), and malignant transformation (Sawant and oth- mutations induced indirectly by α-particles (Nagasawa and ers 2001a); the magnitude of the bystander effect increased others 2002). with the number of α-particles traversing the nuclei (Sawant Regardless of the molecular mechanisms involved, the and others 2001a). bystander effects observed with high-LET particles may For malignant transformation, the frequency when only have important implications for low doses of high-LET ra- 10% of the cells were traversed by an α-particle was as great diation. According to Sawant and others (2001a), “These as when every cell was exposed to an α-particle; further- results, if applicable in vivo, would have significant conse- more, nonirradiated cells did not have to be in contact with quences in terms of radiation risk extrapolation to low doses, irradiated cells. However, the same group subsequently re- implying that the relevant target for radiation oncogenesis is ported that gap junctions appeared to be required for another larger than an individual cell, and that the risk of carcinogen- bystander effect resulting in cell lethality in nonhit cells esis would increase more slowly, if at all, at higher doses— (Sawant and others 2002). The group also showed that irra- an effect seen in vivo, as well as epidemiologically. Thus, a diating the cytoplasm with α-particles (Wu and others 1999) simple linear extrapolation of radiation risk from high doses induced mutations (small deletion and base-pair alteration (where they can be measured) to lower doses (where they mutations) that resembled those occurring spontaneously, must be inferred) would be of questionable validity.” In other not the larger deletions observed when the nucleus was irra- words, it is speculated that there could be a convex, down- diated to induce mutations in both irradiated and non- ward-curving dose-response relationship at low doses, and irradiated cells (Zhou and others 2000). Lorimore and col- that extrapolation of data from high doses could lead to an leagues (1998) have observed a similar bystander effect: underestimate of the effect at low doses of high-LET delayed chromosomal aberrations associated with genomic radiation. instability when cells were exposed to α-particles. Prise and A most critical question, however, is whether these types colleagues (1998) have observed a bystander effect for ge- of bystander effects exist for low-LET radiation doses nomic instability associated with the formation of micronu- <100 mGy, which are the focus of this report. For α-par- clei 20–30 generations after individual cells were irradiated ticles and other high-LET radiation used in bystander stud- with a charged-particle microbeam. Their subsequent stud- ies, the dose to the nucleus was calculated to be 130– ies with primary human fibroblasts (Belyakov and others 500 mGy per α-particle traversal, depending on the size and 2001) showed that even though a single cell had been tar- shape of the cell and its nucleus (Azzam and others 1998); geted, an additional 80–110 cells had micronuclei; the yield that is, a flattened cell nucleus would have a much lower of cells that had excess micronuclei was independent of the dose from high-LET radiation than a spherical rounded cell number of charged particles delivered to the targeted cell. nucleus because of the geometry of the nucleus in relation to The molecular mechanisms proposed for the bystander the radiation source (Clutton and others 1996a, 1996b). For effects described above are speculative (see Chapter 1 “DSB low-LET radiation (assuming an RBE of 3), the dose corre- Signal Transduction and Inducible Repair” for a discussion sponding to that from the high-LET radiation would be 0.39– of possible repair and signal transduction pathways that may 1.5 Gy. Because the bystander effect resulting from an α- be involved). Activation of the p53-mediated DNA damage particle traversal through an irradiated cell was lower by a response pathway in bystander cells has led to speculation factor of 3–5 than the direct effect on the irradiated cell and (Grosovsky 1999) that reduced replication fidelity or in- because the magnitude of the bystander effect appeared to creased recombinational activity might lead to the genetic increase as the number of traversals through the cell in- effects that occur in these cells. However, α-particle-induced creased (Sawant and others 2001a), one might expect that chromosomal instability was reported to be independent of the same type of bystander effect would not be observed in the p53 status of the cells (Kadhim and others 1996). The the low-dose range <100 mGy for low-LET radiation. In fact, bystander phenomenon may involve the diffusion of data indicate that the bystander effect for induced expression cytokines or long-lived reactive oxygen species (ROS; of p53 was much greater and persisted much longer after α- Narayanan and others 1997, 1999; Lorimore and others irradiation than after X-irradiation (Hickman and others 1998; Wu and others 1999; Azzam and others 2002; Morgan 1994). 2003a, 2003b) including any products formed by reaction In human keratinocytes, a bystander effect for cell lethal- with hydrated electrons or OH• radicals (Ward 2002). Also, ity that required cell-to-cell contact with gap junctions has the diffusion of paracrine proapoptotic or antiapoptotic fac- been reported for γ-ray doses of 500 mGy and above tors induced by upregulation of p21 (Chang and others 2000) (Mothersill and Seymour 1997). In the same dose range, a may be involved. Because CDC2 is downregulated by α- bystander effect that did not require cell-to-cell contact was

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MOLECULAR AND CELLULAR RESPONSES TO IONIZING RADIATION 55 observed when cell culture medium from irradiated cells was tions or cell killing (Wolff 1992b; Mothersill and Seymour added to nonirradiated cells (Mothersill and Seymour 1998a). Third, an adaptive response induced by irradiating a 1998a). The observed bystander effect is specific for cell directly may cancel out at least part of the bystander keratinocytes because it was not observed for fibroblasts. effect; this was observed for cell lethality when mouse C3H The effect is eliminated by heating the medium at 70°C for 10T1/2 cells were irradiated with 20 mGy of X-rays 6 h be- 30 min, and there is some evidence that an alteration in en- fore α-particle irradiation (Sawant and others 2001b). ergy metabolism and induction of apoptosis are involved Fourth, molecular mechanisms responsible for the bystander (Mothersill and others 2000b). Furthermore, the bystander effect of low-LET radiation, as well as high-LET radiation, effect from transfer of medium varies among cell lines that may include genetic variation in transcriptional response (Mothersill and others 2000b; Seymour and Mothersill to radiation exposure (Correa and Cheung 2004), have not 2000), and its contribution to cell lethality has been reported been delineated. Fifth, recent results (Prise and others 2003) either to plateau with about 40% of human keratinocytes suggest that a bystander effect for cell lethality from soft X- killed at 30–60 mGy (Seymour and Mothersill 2000) or to ray irradiation (LET of 25–30 keV/µ) might be observed increase at doses over 1 Gy delivered to CHO (Chinese ham- down to 50 mGy but not below. Sixth, until molecular ster ovary) cells (Mothersill and others 2000b). Finally, by- mechanisms of the bystander effect are elucidated, especially stander cell killing reported for a dose as low as 10 mGy as related to an intact organism, and until reproducible by- appears to be greater for delayed cell lethality quantified by stander effects are observed for low-LET radiation in the cloning efficiency at about 14 d after irradiation than for ini- dose range of 1–5 mGy, where an average of about one elec- tial cell lethality quantified by cloning efficiency determined tron track traverses the nucleus, a bystander effect of low- immediately after irradiation (Seymour and Mothersill dose, low-LET radiation that might result in a dose-response 2000). Delayed lethality is supposedly a manifestation of curving either upwards or downwards should not be genomic instability associated with an increase in lethal assumed. mutations in cells that survive irradiation (Seymour and Mothersill 1997). HYPER-RADIATION SENSITIVITY AT LOW DOSES In another study, a low-LET radiation bystander effect that required gap junctions was observed in a three-dimen- Another factor that can cause the dose-response to devi- sional Chinese hamster culture model (Bishayee and others ate from the alpha-beta model is HRS that has been reported 1999). The bystander effect that caused cell lethality in the for cell lethality induced by low-LET radiation at doses up nonirradiated cells became apparent only after the irradiated to 200 mGy (Joiner and others 1996; Skov 1999; Figure 2-3). cells had undergone 1000–2000 disintegrations of 3HTdR in In this dose range, survival can decrease to 85–90%, depend- the DNA, that is, at a very high dose of about 2.5–5.0 Gy ing on the cell line, which is significantly lower than sur- (Dewey and others 1965). vival predicted by the value of α determined from survival Several issues should be considered in relation to the by- values above 1–2 Gy. HRS might be associated with a by- stander effect. First, in contrast with the results summarized stander effect, but a recent study (Mothersill and others 2002) above that involved enhancement of damage, a bystander suggests that it is not. Although the magnitude of HRS varies, effect was reported to increase survival (Dent and others there is some evidence that it also occurs for fractionated 1999) when medium from γ-irradiated mammary carcinoma doses of about 400 or 500 mGy both in vitro (Smith and cells was transferred to nonirradiated cells 120 min after others 1999; Short and others 2001) and in vivo for kidney a dose of 2 Gy. Apparently, the soluble TGF-α (transform- and skin (Joiner and others 1996) and for glioma cell lines ing growth factor-α) that was released induced secondary irradiated with multiple fractions of 700–800 mGy activation of EGFR (epidermal growth factor receptor), (Beauchesne and others 2003). Furthermore, an observed MAPK (mitogen-activated protein kinase), and JNK (c-jun inverse dose-rate effect was attributed to HRS seen for low N-terminal kinase), which resulted in an increase in survival. acute doses (Mitchell and others 2002), and recent cell cycle Thus, as reviewed by Waldren (2004) both beneficial and studies (Mitchell and others 2002; Marples and others 2003; detrimental effects may result from the bystander effect. A Short and others 2003) suggest that HRS may be related to similar observation was reported for normal human diploid cells not arresting in radiosensitive G2. Since a high propor- lung fibroblasts exposed to low doses of α-particles; the tion of the target stem-like cells in humans would be observed enhancement of cell growth was hypothesized to noncycling G0 cells (see Chapter 3, “General Aspects of result from an ROS-caused increase in TGF-β (Iyer and Dose-Response”), the last two observations, if generally true, Lehnert 2000). Second, there is a suggestion that an adaptive would suggest that neither HRS nor the inverse dose-rate response induced by a priming dose of 1 mGy for reducing phenomenon should have any significant effect on the dose- radiation-induced micronuclei was due in part to a bystander response for cancer induction in humans. effect (Broome and others 2002). However, the bystander Molecular mechanisms involved in HRS have been de- effect of a priming dose has not been found to induce a ra- scribed in only a preliminary way. However, HRS for cell dioprotective or adaptive response for chromosomal aberra- lethality up to 200 mGy was not observed in radiosensitive

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56 BEIR VII Studies of other end points have provided some additional evidence of HRS. In a signal transduction study that used γ- ray doses of 20, 50, 100, 250, and 500 mGy, there was a suggestion of HRS up to 200 mGy for radiation-induced transcription of MDM2, ATF3, and BAX in a human my- eloid tumor line (Amundson and others 1999b; Figure 2-4). Similar observations over the same dose range were re- ported (Yang and others 2000) for X-ray induction of pro- tein-8 (XIP8) in human MCF-7:W58 breast cancer cells; this protein as it complexes with Ku70/Ku80 appears to be an important cell-death signal. HRS was also observed in mice as gene deletions that reverted unstable mutations in mel- anocytes exposed to 10 mGy of X-radiation (Schiestl and others 1994); that is, there was a threefold effect at 10 mGy and a twelvefold effect at 1 Gy. The frequency of gene dele- tions was about 100 times higher than the frequency of other FIGURE 2-3 Illustrative example of hyper-radiation sensitivity for low doses. Example is from HT29 cells given graded doses of X- rays. SOURCE: From Joiner and colleagues (1996). AT and XR-V15B cell lines (Skov 1999) or with high-LET radiation (Lambin and others 1993). For doses above 50– 200 mGy, for which HRS is no longer observed, the flatten- ing of the survival curve between 500 and 1000 mGy may be related to DNA PKcs activity (Marples and others 2002) or to the downregulation of the DIR1 gene (Robson and others 1997, 1999); this downregulation has been shown to corre- late with an increase in rate of repair of DNA single-strand breaks (Robson and others 2000; Collis and others 2004; Marples and others 2004). DNA damage introduced at a very low rate may not acti- vate the radiation damage sensor ATM (Collis and others 2004). Consequently, exposure to low levels of chronic ra- diation may cause more cell damage than estimated from extrapolation of higher doses. This hypersensitivity to kill- ing could serve to eliminate cells that have received DNA damage and potentially carcinogenic changes to their ge- nome. Alternatively, it cannot presently be excluded that FIGURE 2-4 Maximal induction of CDKN1A ( ), GADD45 ( ), some of these cells may survive and proliferate as clones of MDM2 ( ), ATF3 ( ), and BAX ( ) by low doses of γ-rays. Points mutated cells. It is important to note that the effect of cellu- are averages of four independent experiments; error bars are stan- lar hypersensitivity to killing by very low chronic doses of dard errors. Dashed line indicates basal level in untreated controls; ionizing radiation is a modest effect that has been detected solid lines were fitted by linear regression through the data. only in some, but not all, human cell lines investigated. SOURCE: From Amundson and colleagues (1999b).

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MOLECULAR AND CELLULAR RESPONSES TO IONIZING RADIATION 57 recessive mutations at other coat color loci; therefore, the in quantifying the frequency of chromosomal aberrations in authors speculated that the deletions resulted from non- human lymphocytes exposed to eight different acute doses targeted effects, such as increased recombination frequencies from 3 to 300 mGy; a linear dose-response relationship was (i.e., genomic instability) in the proliferating melanocytes. observed above 20 mGy, with a slope of 2.9 × 10–5 chromo- In summary, there are data suggesting HRS for cell le- somal aberrations per cell per milligray (Lloyd and others thality and signal transduction up to 200 and some data sug- 1992; Figure 2-5). gesting HRS for mutagenesis or genomic instability at up to Below 20 mGy, however, the data could not distinguish 50 mGy. However, it is not clear (Malaise and others 1994; between a linear and a threshold model. When immortalized Skov 1999) whether HRS for cell lethality would cause an human lymphocytes were irradiated in G2 with four different increase in deleterious effects in surviving cells or would doses from 50 to 500 mGy, a linear dose-response relation- actually decrease deleterious effects by increased killing of ship was observed, with a slope of 2.5 × 10–5 chromosomal damaged cells. Also, it is not known what effect HRS for aberrations per cell per milligray (Puck and others 1997). signal transduction pathways (such as that illustrated in Fig- These results are similar to those obtained with primary hu- ure 2-4) will have in mitigating or increasing deleterious ef- man skin fibroblasts (Cornforth and others 2002), irradiated fects. Most important, it is not known if HRS plays a role while the cells were arrested in G0. For total aberrations per when radiation doses <100 mGy are delivered over weeks to cell, an α-component of (5.8 ± 2.4) × 10–5/mGy for acute months, which could be relevant for low doses of low-LET radiation corresponded to a linear dose-response relation- radiation delivered to radiation workers. ship of (4.9 ± 2.0) × 10–5/mGy for low-dose-rate irradiation (0.5 or 1 mGy/min) between 300 and 6000 mGy. For dicen- trics, the frequency was (1.9 ± 1.2) × 10–5/mGy for the low OBSERVED DOSE-RESPONSE RELATIONSHIPS AT dose rate (LDRs). These LDR coefficients correspond to the LOW DOSES limited slope (curve D) in Figure 2-1. At the time of publication of the BEIR V report (NRC An extensive aberration study was conducted in which 1990) and during the next several years, dose-response rela- mice were irradiated daily for 21, 42, or 63 d at doses of 6.4, tionships for induction of chromosomal aberrations and gene 18.5, or 55 mGy; lymphocyte cultures set up two weeks af- mutations by acute doses of low-LET X-irradiation were ter irradiation was completed yielded a linear dose-response described quite satisfactorily down to ~200 mGy by the lin- (1.2 × 10–5 chromosomal translocations per cell per milligray), ear quadratic (alpha-beta) relationship discussed earlier. In with no evidence of either an adaptive or a supralinear re- general, low dose rates and fractionated doses reduced the sponse (Tucker and others 1998) (see Figure 2-6 in which induction frequencies by factors of about 2 or more, but the the frequencies determined for painted chromosomes were results were variable and included a few reports of inverse corrected for the whole genome). The DDREF for acute dose-rate effects (Thacker 1992). In this section, more re- exposures of 1–3 Gy was about 4–6 (see “General Aspects cent experiments conducted with mammalian cellular sys- of Dose-Response Relationships” for definition of DDREF). tems that have measured frequencies of various events re- Most important, the induced frequency of chromosomal sulting from relatively low doses and low dose rates of translocations was not significantly different from that re- X-rays or γ-rays are reviewed (Table 2-1). The objective is ported in workers at the Sellafield Nuclear Facility who were to summarize data acquired primarily since the 1990 BEIR V occupationally exposed to lifetime cumulative doses of more report that provide information on the shape of the dose- than 500 mSv, that is, (1.0 ± 0.25) × 10–5/mGy for smokers response curve down to 100 mGy. Whenever possible, these (Tawn and others 2000a). A subsequent analysis by Tawn data will be related to human exposures, although caution and colleagues (2004) reported a linear dose-response should be exercised whenever attempting to extrapolate from between 50 and 1000 mSv of (1.11 ± 0.19) × 10–5 transloca- in vitro systems to the human. tions per cell per millisievert. Normal human fibroblasts irradiated in plateau phase with In addition, the α-component is 1.9 × 10–5/mGy for the doses of 109–6000 mGy gave a linear dose-response rela- frequency of chromosomal translocations in lymphocytes of tionship for the induction of chromosomal aberrations de- cleanup workers of the Chernobyl nuclear accident who tected by premature chromosomal condensation immediately received an estimated average dose of 95 mGy over 6– after irradiation (Darroudi and others 1998); the slope was 6 13 years (Jones and others 2002). These values are similar to × 10 –3 fragments per cell per milligray (Cornforth and the frequency of dicentrics (1.4 × 10–5/mGy) observed in Bedford 1983). When the cells entered metaphase and were people who were exposed to 100 ± 124 mGy of cobalt-60 scored for chromosomal dicentrics and rings after repair or over about 10 years (Liu and others 2002) and for Mayak misrepair of DNA damage had occurred (released from nuclear workers exposed over 1–5 years (0.5–0.9 × 10–5/ confluence after potentially lethal damage repair had mGy for translocations; Burak and others 2001). Note that in occurred), a β-component was apparent, and the α-compo- the seven studies above, the dose-response relationships are nent decreased to 5.8 × 10–5 aberrations per cell per milligray consistent with a linear no-threshold model in which the ab- (Cornforth and Bedford 1987). Six laboratories collaborated erration frequencies per milligray are similar.

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58 BEIR VII TABLE 2-1 Dose-Response Relationships at Relatively Low Doses Frequency of Events per System (including exposure conditions Dose Range, Curve Viable Cell and acute αa or LDRb) End Point mGy Shape per Milligray Comments and References Human fibroblasts in G0 Immediate 109–6000 Linear 6 × 10–3 LNTc extrapolates to 5 mGy PCC fragments (acute) (Cornforth and Bedford 1983) Human fibroblasts in G0 Chromosome 1000–12,000 Upward 5.8 × 10–5 (Cornforth and Bedford 1987) α-component-metaphase dicentrics and (acute) curvature rings Immortal human lymphocytes in G2 Chromatid 50–500 Linear 2.5 × 10–5 LNT > ~50 mGy (Puck and others 1997) gaps (acute) Human lymphocytes in G0 (six Chromosome 3–300 Linear 2.9 × 10–5 LNT > ~20 mGy (Lloyd and others 1992) laboratories) dicentrics (acute) Human primary fibroblasts in G0 Chromosome 1000–6000 Upward 5.8 × 10–5 α-Component for acute corresponds to (acute α-component) aberrations (acute) curvature linear dose-response for LDR (Cornforth and others 2002) Human primary fibroblasts in G0-0.5 or Chromosome 300–6000 Linear 4.9 × 10–5 LNT > 300 mGy 1 mGy/min aberrations (LDR) (Cornforth and others 2002) Mice—daily doses of 6.4, 18.5, or Chromosome 100–3500 Linear 1.2 × 10–5 LNT > ~100 mGy DDREFd of 4–6 for 55 mGy for 21, 42, or 63 d, respectively translocations (LDR) 1–2 Gy acute exposure (Tucker and others 1998) Nuclear workers at Sellafield— Chromosome 50–1000 Linear 1.1 × 10–5 LNT > 50 mGy lymphocyte cultures translocations (LDR) (Tawn and others 2000a, 2004) Cleanup workers at Chernobyl— Chromosome ~95 (LDR) ? 1.9 × 10–5 Increase of 30% (10–53% p < .002) lymphocyte cultures translocations relative to controls (Jones and others 2002) Chinese hamster cells with human Loss of 250–1500 Linear 7 × 10–6 LNT > ~ 250 mGy chromosome 11 antigen on (acute) (Puck and Waldren 1987) chromosome 11 TK6 human lymphoblasts—daily doses HPRT 50–2000 Linear 6 × 10–9 LNT > ~50 mGy of 10, 25, 50, or 100 mGy for 1 month mutations (LDR) (Grosovsky and Little 1985) Mice—T lymphocytes in spleen— HPRT 300–6000 Linear 3 × 10–9 LNT > ~300 mGy DDREF of ~1.5 for chronic at 0.69 mGy/min or 0.1 mGy/min mutations (LDR) acute <2 Gy (Lorenz and others 1994) Cleanup workers at Chernobyl— HPRT ~95 (LDR) ? 5 × 10–8 Increase of 41% (19–66% p < .001) lymphocyte cultures mutations relative to controls (Jones and others 2002) Chinese hamster cells with human Genomic 1000–10,000 Linear 3 × 10–5 Based on percent unstable clones with chromosome 11 instability (acute) BrdU saturates at 30% Translocations (Limoli and others 1999) on chromosome 11 Chinese hamster cells (CHO) Genomic 2000 ? 5 × 10–5 Based on percent unstable clones; from instability 4 to 12 Gy saturates at 20% (Little 1998) de novo HPRT mutations Melanocytes in irradiated mice Genomic 10–1000 Linear 8 × 10–5 LNT > 10 mGy, but supralinear from 0 to instability gene 10 mGy (Schiestl and others 1994) deletions continues

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MOLECULAR AND CELLULAR RESPONSES TO IONIZING RADIATION 59 TABLE 2-1 Continued Frequency of Events per System (including exposure conditions Dose Range, Curve Viable Cell and acute αa or LDRb) End Point mGy Shape per Milligray Comments and References Human blood lymphocytes stimulated Genomic 1000–3000 ? (3–10) × 10–5 Analyzed at 51–57 d after irradiation with PHA instability (acute) (Holmberg and others 1998) chromosomal aberrations Hamster embryo cells Malignant 30–1500 Linear 4 × 10–6 LNT > ~30 mGy (Borek and others 1983) transformation (acute) C3H 10 T1/2 mouse cells (six labs) Malignant 250–5000 Linear 8 × 10–8 LNT > ~250 mGy (Mill and others 1998) transformation (acute) Hela X skin fibroblast human hybrid cell Malignant 0–1000 Sigmoid 4 × 10–8 Threshold at ~300 mGy dependent on time system transformation (acute) of trypsinization after irradiation (Redpath and others 2001) NOTE: LDR = low dose rate; PCC = premature chromosome condensation; PHA = phytohemagglutinin. aAcute indicates that doses were delivered at high dose rate (e.g., 0.1 Gy/min.), and α-component signifies the value of α in the linear-quadratic relationship. bLDR indicates that the doses were delivered at low dose rates less than 0.01 Gy/min. cLNT signifies a linear, no-threshold dose-response relationship. dDDREF is defined and illustrated in Figures 2-1 and 2-8. Three mutation experiments have yielded a linear dose- antigen marker and essential genes on chromosome 11 (see response relationship. First, the loss of an antigen marker on “Induction of Gene Mutations in Somatic Cells”). human chromosome 11 integrated in Chinese hamster cells Second, human lymphoblast cells (TK6) exposed to one and exposed to four different doses from 250 to 1500 mGy acute dose or to daily doses of 10, 25, 50, or 100 mGy for up yielded a linear dose-response relationship with a slope of 7 to one month, with samples taken every 5 d, yielded a linear × 10 –6 mutants per viable cell per milligray (Puck and dose-response relationship for induction of HPRT or TK Waldren 1987). The relatively high frequency is due to the mutations (Figure 2-7). Over a total dose range of 50– large target size because of the large distance between the 2000 mGy, the slope for HPRT mutations was 6 × 10–9 mu- FIGURE 2-5 Dicentric yields as a function of dose; , Pohl-Ruling and others (1983); ✖, Lloyd and others (1992), experiment 1; experi- ment 2. SOURCE: From Lloyd and colleagues (1992).

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60 BEIR VII FIGURE 2-6 Induced translocations (observed frequency less control value) per dose fraction as a function of radiation dose per fraction. The line is the least-squares regression fit, with Y = 0.0121X1.367; R2 = 0.98. Five points on the upper part of the line represent the acute exposures (DDREF of 4–6), and the three sets of values on the lower portion of the line are from mice that received fractionated exposures. SOURCE: From Tucker and others (1998). tants per viable cell per milligray (Grosovsky and Little By dividing the HPRT mutation frequencies for acute ir- 1985). radiation by the frequencies for LDR irradiation (obtained in Third, mice were irradiated with total doses of 300– the mouse T-lymphocyte experiment of Lorenz and others 6000 mGy applied at an acute dose rate of 500 mGy/min or 1994 described above), the DDREF was 3–5 for acute doses at a low dose rate of 1000 mGy/d (0.69 mGy/min) or greater than 3 Gy and about 1.5 for acute doses less than 1000 mGy per week (0.1 mGy/min; Lorenz and others 2 Gy (Figure 2-8). The DDREF points (averages of 1.0) 1994). At 8–10 weeks after irradiation was completed, the plotted for each of the LDRs were obtained by dividing the frequency of HPRT mutants in splenic T lymphocytes for mutation frequencies for each total dose by the product of the LDRs was described by a linear dose-response relation- 3 × 10–6 mutants per viable cell per gray (value for LDRs) ship that had a slope of 3 × 10–9 mutants per viable cell per times the total dose. The range of DDREF values for acute milligray. This is about one-tenth the frequency of HPRT doses are similar to those obtained for the same dose ranges mutants observed in lymphocytes of cleanup workers of the in transformation in vitro (Han and others 1980) and animal Chernobyl nuclear accident who received an estimated aver- carcinogenesis and life-shortening experiments (Ullrich and age dose of 95 mGy over 6–13 years (Jones and others 2002). Storer 1979a; Ullrich and others 1987). (For definition and An interesting observation in the mouse experiments was illustration of DDREF, see “General Aspects of Dose- that an inverse dose-rate effect was not observed; the muta- Response Relationships.”) tion frequency for 0.1 mGy/min was the same as that for Overall, the dose-response for radiation-induced genomic 0.69 mGy/min. From a summary of data for radiation- instability is quantitatively similar to that for radiation-in- induced mutations as function of dose rate (Vilenchik and duced chromosomal aberrations, with the exception that the Knudson 2000), an inverse dose-rate effect would not be frequency for genomic instability saturates between 4 and expected if the induction of HPRT mutations in T lympho- 12 Gy (Little 1998; Limoli and others 1999), while the fre- cytes in the spleen corresponded to the induction of specific quency for chromosomal aberrations continues to increase locus mutations in spermatogonia. However, if they corre- with dose. After 10 Gy, 30% of the CHO clones were un- sponded to the induction of HPRT mutations in cells in vitro, stable for chromosomal aberrations, which was the satura- the mutation frequency for 0.1 mGy/min should have been tion level reached after 4 Gy when the cells had incorporated about half that for 0.69 mGy/min. BrdU (Limoli and others 1999). Furthermore, when induc-

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MOLECULAR AND CELLULAR RESPONSES TO IONIZING RADIATION 61 human blood lymphocytes stimulated with PHA (phytohe- magglutinin) and analyzed 51–57 d after irradiation, the fre- quency of de novo aberrations was (3 to 10) × 10–5 chromo- somal aberrations per cell per milligray (Holmberg and others 1998). Genomic instability was also observed in mice as gene deletions in melanocytes exposed to X-irradiation (Schiestl and others 1994a), with a threefold increase at 0.01 Gy and a twelvefold increase at 1.0 Gy. The frequency of gene de- letions was about 100 times higher than mutation frequen- cies; therefore, the authors speculated that the deletions re- sulted from nontargeted effects, such as an increased recombination frequency or genomic instability in the pro- liferating melanocytes. The dose-response was linear be- tween 0.01 and 1.0 Gy and had a slope of 8 × 10–5 events per cell per milligray. Note that the three values listed above for the frequencies of radiation-induced instability (3–10 × 10–5 events per cell per milligray) are of the same order of magnitude as the frequency of chromosomal aber- ration induced directly by irradiation (1–4 × 10–5 events per cell per milligray; Table 2-1). A malignant transformation experiment with primary hamster embryo cells exposed to five different doses from 0.03 to 1.5 Gy yielded a linear dose-response curve that had a slope of 4 × 10–6 transformants per viable cell per milligray (Borek and others 1983). An extensive collaborative study involving six laboratories that quantified malignant transfor- mation of immortalized mouse C3H 10T1/2 cells exposed to rads FIGURE 2-7 Frequency of 6TGR cells induced by 1–10 rads (0.01– 0.1 Gy) of X-rays in TK6 human lymphoblastoid cells. Data points (with standard deviations) are from regression analyses of muta- tions induced per day at various dose rates (1–10 rads/d; 0-30 d) as described in Grosovsky and Little (1985). DDREF tion of genomic instability was assayed as chromosomal ab- errations in mammary epithelial cells at 25 population doublings after the cells had been irradiated in vitro or in vivo (Ullrich and Davis 1999), a downward-curving dose-re- sponse curve was observed between 0 and 0.25 Gy, with the response saturating between 1 and 3 Gy at about 0.35 aber- ration per cell. The percentage of CHO clones (containing a human chromosome 4) that were stable for chromosomal translocations in chromosome 4, had a linear dose-response of 3 × 10–5 events per irradiated cell per milligray between 1 and 10 Gy (Limoli and others 1999). For HPRT mutations in Total Dose (Gy) CHO cells, the percentage of clones that were unstable for de novo HPRT mutations was 5 × 10–5 events per irradiated FIGURE 2-8 DDREF for low-LET 137Cs γ-rays: ( ) dose rates cell per milligray, based on 10% being unstable after 2 Gy 0.5 Gy/min; ( ) dose rates 1 Gy/d (0.69 mGy/min) to 1 Gy per (Little 1998). Between 4 and 12 Gy, the percentage of un- week (0.10 mGy/min). SOURCE: From Lorenz and colleagues stable clones remained the same at 10–20%. For irradiated (1994).

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62 BEIR VII seven different doses from 0.25 to 5.0 Gy reported a linear Furthermore, the calculated value of 5 mGy for an aver- dose-response with a slope of 8 × 10–8 transformants per age of one electron track per nucleus depends on the size and viable cell per milligray (Mill and others 1998). A study con- shape of the nucleus, as well as on the energy of the radiation ducted with a human Hela hybrid cell system (Redpath and (Rossi and Zaider 1996; Edwards and Cox 2000). For ex- others 2001) reported a frequency of 4 × 10–8 transformants ample, the calculated doses for an average of one electron per viable cell per milligray beyond a threshold of ~0.3 Gy; track per nucleus are as follows: about 5 mGy for 60 keV however, the results were greatly dependent on the time the and a 6-µm diameter sphere, about 4 mGy for 60 keV and a cells were trypsinized and plated after irradiation for the 7-µm sphere, about 3 mGy for 300 keV and a 6-µm sphere, transformation analysis. Note that these results for transfor- and about 2 mGy for 300 keV and a 7-µm sphere. For the mation are quite variable and that the frequencies are ten- to very low doses for which important signal transduction a thousandfold lower than the frequencies for radiation-in- events may result from ionizations in either the nucleus or duced genomic instability. However, as discussed earlier the cytoplasm, the volume of the whole cell might be most under adaptive response, studies of malignant transforma- appropriate for these types of calculations. Possibly, the tion in immortalized (already-transformed) cell lines may shape of the dose-response relationship up to 5 mGy might have little relevance to malignant transformation of normal be determined with in vitro and in vivo experiments in which nonimmortalized cells, especially in vivo where complex in- multiple doses of about 1–5 mGy are delivered over a long teractive processes can occur. period. However, the question must be addressed rigorously In summary, results of experiments that quantified chro- by defining the molecular processes responsible for the end mosomal aberrations, malignant transformations, or muta- points in question at these very low doses. tions induced by relatively low total doses or low doses per fraction suggest that the dose-response relationship over a SUMMARY range of 20–200 mGy is generally linear and not affected significantly by either an adaptive or a bystander effect This chapter discusses the biological effects of the ranges (Table 2-1). No data are available in this dose range for ra- of radiation dose that are most relevant for the committee’s diation-induced genomic instability. The question of the deliberations on the shapes of dose-response relationships. shape of the dose-response relationship up to about 20 mGy Considering the levels of background radiation, the maximal remains, although several of the dose-response relationships permissible levels of exposure of radiation workers now in described above appear to be consistent with extrapolation effect, and the fact that much of the epidemiology of low- linearly down to about 5 mGy. As has been pointed out dose exposures includes people who in the past have received (Cornforth and Bedford 1983), a macroscopic X-ray dose of up to 500 mGy, the committee has focused on evaluating about 5 mGy would, on the average, result in one to two radiation effects in the low dose range of <100 mGy, with electron tracks crossing the nucleus of each cell. Since the emphasis on the lowest doses when relevant data are avail- tracks are produced randomly, the proportion of nuclei tra- able. Effects that may occur as the radiation is delivered versed by zero, one, or two electron tracks would be about chronically over several months to a lifetime are thought to 0.37, 0.37, and 0.18, respectively. For lower doses, a larger be most relevant. and larger proportion of cell nuclei would receive no dose Considerable emphasis has been placed on the dose-re- (track) at all. The nuclei that would receive a track would all sponse and mechanisms for inducing chromosomal aberra- receive (on the average) the same dose because the propor- tions and gene mutations because, as discussed in Chapter 3, tion receiving two or more tracks would diminish rapidly. there is evidence that the induction of cancer is associated Therefore, unless interactions among neighboring or sur- with these cellular responses. The general pictures that rounding cells influence the response, if 5 mGy produces an emerge from biophysical studies is that the misrepair of effect and if the effect is linear above 5 mGy, the dose- radiation-induced DNA DSBs that lead to chromosome response curve must also be linear from 0 to 5 mGy. In aberrations are probably associated with the dominant post- addition to the existence of biological information at these irradiation function of nonhomologous end joining repair very low dose levels, the committee concluded that the bio- processes described elsewhere is this report. Overall, physical characteristics of the interaction of low-LET radia- biophysical approaches to the modeling of dose-response for tion with DNA, coupled with the characteristics of DNA re- chromosome aberrations, although not without some pair, argue for a continuation of the linear response at lower uncertainties on mechanisms, imply that the single-track doses. However, if a single electron track traversing a cell’s α-component of radiation action will dominate at low doses nucleus could induce an adaptive or bystander effect, the and LDRs (i.e., the dose-response for all forms of aberra- dose-response relationship below 5 mGy might deviate from tions will be linear at low doses and LDRs). Also, as linearity depending on whether cellular effects are decreased observed, the response at LDRs and low doses, or after or increased. In the committee’s judgment, there is no evi- fractionated doses, should be lower by a DDREF; then the dence for either an adaptive response or a bystander effect response to a single acute high-dose-rate exposure for which for doses below 5 mGy. the two-hit β-component becomes important. In certain

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MOLECULAR AND CELLULAR RESPONSES TO IONIZING RADIATION 63 cases, an inverse dose-rate effect for cell lethality and muta- dose range <100 mGy; relevant end points should include tions has been reported for which the effect at very low dose not only chromosomal aberrations and mutations but also rates is as high or higher than for single, acute, high-dose- genomic instability and, if possible, tumor induction. Stud- rate exposures. The ability to demonstrate this phenomenon, ies of the adaptive response for malignant transformation in however, is variable, and no mechanisms have been clearly immortalized (already-transformed) cell lines may have little identified to explain such effects. relevance to malignant transformation of normal non- Several factors may affect the theoretical dose-response immortalized cells, especially in vivo, where complex inter- relationships described above: variations in radiosensitivity active processes can occur. In vitro and in vivo data are during the cell cycle; induction of an adaptive response to an needed on the delivery of priming and challenge doses over initial exposure, which can reduce the effect of later expo- several weeks or months at very low dose rates or with frac- sures; a bystander effect that causes an irradiated cell to have tionated exposures. Specifically, an adaptive response result- an effect on a nearby unirradiated cell; the induction of per- ing from the cumulative effect of multiple low doses of less sistent genomic instability; and HRS in the low-dose region. than 10 mGy should be determined. Such data have not yet Except for the cell cycle, these factors have been identified been obtained, particularly those explaining the molecular and studied since the BEIR V report (NRC 1990). These fac- and cellular mechanisms of the adaptive response. Thus, it is tors together with quantitative data on the induction of gene concluded that any useful extrapolations for dose-response or chromosomal mutations in somatic cells are discussed. relationships in humans cannot be made from the adaptive Radiation genomic instability has been demonstrated by responses observed in human lymphocytes or other mamma- the manifestation of chromosomal damage in a certain frac- lian cellular systems. Therefore, at present, the assumption tion of irradiated cells over many cell cycles after they were that any stimulatory effects of low doses of ionizing radia- irradiated. Data are critically needed for the definition of tion substantially reduce long-term deleterious radiation ef- molecular targets and processes responsible for genomic in- fects in humans is unwarranted. stability in order to define and understand the dose-response A bystander effect in which an irradiated cell induces a relationship, and especially why the induction frequency biological response in a neighboring unirradiated cell has saturates with only about 10–30% of the surviving cells been observed with high-LET radiation for inducing cell le- manifesting genomic instability. A possibility that has not thality, chromosome aberrations, sister-chromatid ex- been investigated is that only a certain fraction of the cells, changes, mutations, genomic instability, signal transduction such as those in a certain part of the cell cycle, are suscep- pathways, and in vitro transformation. There is some evi- tible to radiation-induced genomic instability. Because dence that long-lived reactive oxygen species or the diffu- chromosomal instability has been associated with breakage- sion of cytokines plays a role in the bystander effect. For fusion-bridge cycles, the role of telomeres may be particu- low-LET radiation, the bystander effect has been limited to larly relevant. Chromosome instability can also be initiated cell lethality and lethal mutations associated with reduced by DSBs that result in the loss of a telomere that protects the cloning efficiency. Recent results suggest that a bystander chromosome end and prevents chromosome fusion. Further- effect for cell lethality from soft X-ray irradiation might be more, from limited data, the similarity in the frequencies of observed down to 50 mGy but not below. Until molecular genomic instability induced in X-irradiated cells and the fre- mechanisms of the bystander effect are elucidated, especially quencies of chromosomal aberrations induced directly by as related to an intact organism, and until reproducible by- irradiation may suggest that the induction of chromosomal stander effects are observed for low-LET radiation in the aberrations is a primary event that plays a major role in dose range of 1–5 mGy, where an average of about one elec- radiation-induced genomic instability. There is also some tron track traverses the nucleus, a bystander effect of low- evidence that reactive oxygen species may play a role. How- dose, low-LET radiation that might result in modification of ever, until the molecular mechanisms responsible for the dose-response should not be assumed. genomic instability and its relationship to carcinogenesis are HRS is a phenomenon for which doses less than about understood, extrapolation of the limited dose-response data 200 mGy produce a dose-response for cell lethality that is for genomic instability to radiation-induced cancers in the steeper than that predicted from the classic D + D2 model. low-dose range <100 mGy is not warranted. There are data suggesting HRS for cell lethality and signal An apparent adaptive response has been well documented transduction at up to 200 mGy and some data suggesting for cell lethality, chromosomal aberrations, mutations, and HRS for mutagenesis or genomic instability at up to 50 mGy. in vitro transformation. The phenomena are illustrated by a Furthermore, from limited data from only one laboratory, an reduction in response to a challenge dose of about 1 Gy de- observed inverse dose-rate effect for cell lethality was attrib- livered a few hours after a low priming dose of about 10– uted to HRS seen for low acute doses, and cell cycle analysis 20 mGy. There is much variability in the ability to demon- suggested that HRS may be related to cells not arresting in strate the adaptive response, however. Data are needed, radiosensitive G2. Since a high proportion of the target stem- particularly at the molecular level, on adaptation induced like cells in humans would be noncycling, the last two obser- when both priming and challenging doses are in the low- vations, if generally true, would suggest that neither HRS

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64 BEIR VII nor the inverse dose-rate phenomenon should have any sig- data are available in this dose range for radiation-induced nificant effect on the dose-response for cancer induction in genomic instability. Furthermore, as stated previously, stud- humans. Furthermore, molecular mechanisms associated ies of malignant transformation in immortalized (already- with the two phenomena have not been delineated, and it is transformed) cell lines may have little relevance to malig- not known whether HRS for cell lethality would cause an nant transformation of normal nonimmortalized cells, increase in deleterious effects in surviving cells or would especially in vivo where complex interactive processes can actually decrease deleterious effects by increased killing of occur. However, the results from these in vitro transforma- damaged cells. Also, it is not known what effect HRS for tion studies may have relevance for effects involved in pro- signal transduction pathways will have in mitigating or in- moting the immortalization process, possibly through the creasing deleterious effects. Most important, it is not known induction of genomic instability. Thus, the question of the if HRS plays a role when radiation doses <100 mGy are de- shape of the dose-response relationship up to about 20 mGy livered over weeks to months, which could be relevant for remains, although several of the dose-response relationships low doses of low-LET radiation. Finally, until the molecular described above appear to be consistent with extrapolation mechanisms responsible for HRS are understood, its role in linearly down to about 5 mGy. The shape of the dose-re- low-dose radiation carcinogenesis is uncertain. sponse relationship up to 5 mGy might be determined with Results of experiments that quantified chromosomal ab- in vitro and in vivo experiments in which multiple doses of errations, malignant transformation in vitro, or mutations about 1–5 mGy are delivered over a long period. However, induced by relatively low total doses or low doses per frac- this question should be addressed rigorously by defining the tion indicate that the dose-response relationship over a range molecular processes responsible for the end points in ques- of 20–100 mGy is most likely to be linear and not affected tion at these very low doses. significantly by either an adaptive or a bystander effect. No