Biological low-dose radiation effects

Mutatton Research, 258 (1991) 191-205

191

L 1991 Elsevier Science Pubhshers B V 0165-1110/91/$03 50 ADONIS 016511109100067

MUTREV 07304

Biological low-dose radiation effects Per Oftedal Dwtston of General Genetws, Untverstty of Oslo, Oslo (Norway) (Received 8 December 1990) (Accepted 19 December 1990)

Keywords lomzang radtat~on, B~ologtcal response

Summa~ It ts theorized that biological responses to ionizing radiation in the low dose range are determined according to a doubly dichotomous pattern. Energy depositions fall into 2 categories: events at thermal energy levels where they may be experienced by cells as r a t e s even at background exposure conditions, and events at energy levels of the order of 10-100 eV where damage to D N A may be caused. Variations in background exposure intensity may or may not lead preemptively to changes m the cell's capacity for response to radiation damage. High-level energy depositions lead post hoc to an initial stabilizing reaction largely leading to the fixation of the imtial DNA damage, and to a subsequent restorative or palliative repair process. This model entails reinterpretation of some experimental results. The model has implications for the relationship between scientific analysis of low-dose effects and the regulatory needs for simplicity and homogeneity in risk evaluation. This represents a new challenge for the acceptability of radiation protection norms.

This paper presents a model for the interpretation of low-dose radiation effects in eukaryotes. The model is based more on molecular and cellular physiology and repair functions than on physical and mlcrodosimetric considerations. The model has implications for the pnnclples of radiation management. Analyses of biologtcal radiation effects have been dominated by interpretations in relatively stmple terms, i.e., single- or multiple-hat formulations, largely accepted or discarded on the basis of

Correspondence Dr P Oftedal, D~ws~on of General Genetics, Umverslty of Oslo, P O Box 1031, Bhndern, N-0315 Oslo (Norway).

their usefulness in practical applications. The bases for the discussion have been mainly data from tugh-dose experiments and population exposures giving easily recognized and quantified sequelae, in relation to physical variables such as dose, dose rate, and type of radiation, wtuch are relatively easily measured and controlled (Lea, 1956; Zimmer, 1960; Kellerer, 1987; Elkand, 1987). Documentattons, reviews and derived policies have been presented m publications from the United Nations Scientific Committee on Atomic Radiation (UNSCEAR), Advisory Committee on the Biological Effects of Ionizing Radiation (US Acad. Sci.) (BEIR), the International Commission on Radiological Protection (ICRP), and others. Far less has been said or deduced regarding the

192 reactions per se of a cell or orgamsm to energy deposmon from ionizing radiations within its metabolic volume. The present discussion will be concerned mainly with the role of the biologtcal system itself m determining the outcome of results due to ionizing radiation. The study of effects of small doses is difficult for several reasons. Above all, the effects are correspondingly small and the demands on experimental capacity and specificity correspondingly great. But also, there are consistent and reproducible complex dose-effect curves m the low dose range that are not easy to interpret. In these various reactions may lie the key to an understanding of the mechanisms that determine the outcome of low-dose exposures. A model for low-dose irradiation effects Is faced with several sets of information which it must encompass. At the present stage and in the present context, they are, briefly, as follows. (a) Irregular low-dose and low-dose-rate effects. A selection of these data sets is accepted as valid and informative observations and is analyzed further in the following. Their characteristic similarities and differences are the bases for identffmation of the processes underlying the radiation effects. (b) The rich flora of 'adaptive responses' and inducible repair systems (Samson and Cairns, 1977; Setlow, 1985; Hickson and Harris, 1988; Ohvierl et al., 1984). Many of them are well defined and well understood, others are still poorly understood, such as agents with uncertain functions (HSP) (Pelham, 1986; Alberts and Sternglanz, 1977), or ill defined, such as mechanisms without a specific agent, e.g., mitotic inhibition (Canti and Spear, 1929). (c) Since life began on earth, organisms have been exposed to certain levels of natural background radiation, not constant and not homogeneous or uniform, but varying within rather narrow limits. Adequate protective m e c h a m s m s against damage to important structures, i.e., D N A , exist and have evolved with ancient genetic foundations conserved across widely different species (Bootsma et al., 1987). As noted by Pohl-Riiling et al. (1983), 'it is logical to expect cells to have the capacity to repair small amounts of radiation damage'. (d) One must assume that quantitative aspects

of repair will be consonant with the characteristics of natural background radiation and that the various links in the repair reaction network will be mutually balanced in capacity. Many features of repair systems will appear distorted and incomprehensible when examined m experiments with radiation sources m a n y orders of magnitude more intense than those found in nature. In the following, an outline of a model for low-dose radiation responses and effects is described first. The model is then used for the re-interpretation of some published and commonly accepted data. Finally some implications for risk assessment and radiation protection are discussed. The main elements of the model have been presented m Oftedal (1990a) and a complete but brief outline is given in Oftedal (1990b). Low-dose radiation response mechanisms General

What are the characteristics of background radiation, as seen from a cell's or organism's viewpoint? The phenomenon of ionizing radiation is an integral part of any environment, as a stress factor of within-limits variable magnitude over time and space. The flux of energy is low, but concentrated in packets - photons or particles with initial energies far above those needed to break chemical bonds and cause lomzations. Energy absorption occurs through a degradation and d~stributlon process and has its m a x i m u m biologacal efficiency when the energy of individual events is of the order of 10-100 eV (Goodhead, 1987; Paretzke, 1987). The energy is dissipated further and finally - but within microseconds - becomes thermalized and distributed h o m o g e n e o u s l y throughout the system (Magee and Chatterjee, 1987). The biologically ~mportant damage is llmated to DNA. Accordingly, one would expect protective mechanisms to develop in response to stimuli which may be classified into 2 categories: general changes in the environment, indicative of an increase in background radiation rate and thus future adverse conditions, and catastrophic events, i.e., manifest D N A damage caused by energy packets in the 10-100-eV range (Goodhead, 1987; Paretzke, 1987).

193 W~th regard to the ommpresent character of background radlaUon, a ' b a c k g r o u n d sensing' capability is needed governing a preemptive adjustment of the expression to an approprmte level of the numerous genes involved in repmr (Setlow, 1985; Hlckson and Harris, 1988). The amount of energy m normal background radmtion, about 1 m G y / y e a r , is so low that the number of 100-eV events is only 1-10 per cell nucleus per year (Feinendegen, 1988). It seems probable that cellular memory of smgle events does not extend to years and months in mitotically actwe t~ssues, and so a cell will not experience background radmtlon in terms of a 'rate of ionization'. However, the thermallzed events will be 102-103 times more frequent and more evenly dissipated, and may thus be a base for the sensmg of the level of background radlauon. Stimuli of this category may be of a less specific type and may derive also from other noxes, chemical or phys~ologlcal, opening the possibility of seeing the preemptive change in capacity for response to ionizing radiation as a facet of a more general physiological pattern (Ames, 1989). It ~s interesting that among the genes governing the production of some stress proteins there Is a diwsion m activity levels, one set being consututlvely activated, while the other is dependent upon induction, as discussed by Morimoto et al. (1991) There will be variation in the natural radiation background related to local geology, elevation above sea level, sunspot actiwty, etc. (Grhneberg et al., 1966; Pohl-Rtihng et al., 1978; Ujeno, 1983). Repmr capacity adjustment cannot be instantaneous and proportionate, and the sensing apparatus would be expected to tolerate a certain varlauon before adjustment is reduced. Consequently there will be a threshold for the reaction to changes m radmtlon intensity (Ehrenberg and Eriksson, 1966; Shadley and Wiencke, 1989).

Molecular mechantsms Reaction of the ~onizing energy deposmons is of a different character. The 10-100-eV event cannot be foreseen, and the cell has to react post hoc. If there is D N A damage, ~t must be repaired. The preemptwe sensing has set the stage and prepared the countermeasures that are needed. In the normal activity of the cell, there are

m a n y processes revolving D N A that are going on continuously. These processes - rephcat~on, transcription, refolding, etc. - have to be stopped so that the damaged s~tes will not become permanent. This must take place rapidly. In other words, it must make use of matermls already available in the cells, and cannot be dependent upon b~osynthes~s. Secondarily, the damaged s~tes must be repaired, e~ther by the low level of constituent repair enzyme systems, or w~th the help of newly induced and synthesized enzymes. The reactions in D N A immediately following the primary energy deposition - wxthin femto- to rmcro-seconds - will be determined by phys~cochemical factors such as cage effects, valency binding strengths, presence of radical scavengers, etc. (Magee and Chatterjee, 1987; Goodhead, 1990). After this period, the present mterpretatton proposes that the cell will respond along 2 hnes: partly by rapid intervention w~th nuclear stabilizing elements (s~rmlar or related to heat-shock proteins, chaperonins, and other stress proteins (Aiberts and Sternglanz, 1977: Pelham, 1986: Ellis and Hennlngsen, 1989)), preventing further processmg of damaged D N A into permanent structures, and partly by repair mechamsms being activated, derepressed or induced (Samson and Cmrns, 1977; Pohl-Rfihng et al., 1978; Olivien et al., 1984; Hlckson and H a m s , 1988). There are perhaps a hundred such genes that may be stimulated. The processes of induction, transcription and translation take some 4 - 5 h. By this time, the stabihzing elements are again freed, to fulfill their normal function, or available for new stabdizat~on events A typical example of response on a macroscoplc scale is the rmtot~c inhibition which follows acute irradiations with relatively low doses (Canti and Spear, 1929). This presumably reflects the period needed to mobilize or synthesize the enzymatic apparatus needed for repmr functions.

Re-interpretation of some experimental results Experimental and epldemtologlcal data from verj'low-dose sttuattons We are looking for experimental evidence (1) for a threshold in the very low dose range where the cell/organism does not sense the need for

194 repair m o b d l z a t i o n , (2) for a stabihzang f u n c t i o n i m p l e m e n t e d rapidly following a significant radiation stress, (3) for i n d u c t i o n of a n increase in repair capacity, f u n c t i o n a l l y or as synthesLs of new enzymes, a n d finally (4) for a q u a n t i t a t i v e interrelationship b e t w e e n these processes.

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Below and above the mductlon threshold I n a recent p a p e r (Oftedal, 1990a), examples of data relevant to various aspects of the interpretation presented above are discussed m some detail. A m o n g these are 3 examples of very-low-dose exposures having u n e x p e c t e d l y high effects, pres u m a b l y because the dose rates are too low to reduce the ' r e q u i s i t e ' repair levels (Stokke et al., 1968. Oftedal a n d Lund, 1983; Oftedal, 1989a,b). The cases are p r e s e n t e d m Figs. 1, 2 a n d 3. I n the first case, that of cellular depletion of the rat b o n e m a r r o w (Fig. 1), the doses have been calculated o n the basis of measured r e t e n t i o n of injected radioactive s t r o n t i u m (Stokke et al., 1968). The lrradiatton appears to have a n efficiency of a p p r o x i m a t e l y constxlog dose for the dose interval of a b o u t 0 . 0 1 - 1 2 m G y . The m e a n dose rates are 1 0 - 4 - 1 m G y per week, or 0 . 0 0 5 - 5 0 times the n o r m a l b a c k g r o u n d . Cellularity begins to be reduced at a b o u t a 5% increase of the b a c k g r o u n d dose rate. It is totally u n e x p e c t e d that there should be a 25% reduction m cellularity caused by a dose of 10 m G y over 12 weeks, even if physiological

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Fig 2 Number of cases of thyroid cancer regtstered m 19531985 by the Cancer Registry of Norway m females in the agc group 30-34 years according to year of birth ( © and ordinate on left) Birth cohorts 1953 and later were not complete at the time of analysis Annual mean content of 131] measured fortnightly in milk from 10 dames ( - - - z x - - and ordinate on right) Contamination occurred predormnantl3 dunng 6-8 weeks every fall Updated and redrawn from Ofteda and Lund (1983)

m e c h a m s m s other t h a n cell death are involved F l i e d n e r et al. (1961) n o t e d rapid smusoidal dilatation m the rat f e m u r after acute a n d high doses 500 rad a n d more. It seems d o u b t f u l whether thL, m e c h a n i s m should be of relevance after low dose., a n d several weeks' o b s e r v a t i o n time. Peterson et al. (1989) f o u n d complete recovery of nucleated cell n u m b e r per femur m rmce a b o u t 12 weeks and later after acute exposures with 2 G y n e u t r o r i r r a d i a t i o n or 5 G y ~/ irradiation. However, botl7 c o l o n y - f o r m i n g units ( C F U ) a n d p r o h f e r a u v e ability were reduced b y 10-30% at 12 weeks, anc showed further r e d u c t i o n over the r e m a i n i n g ob. servation period up to a year. Immediately, the most relevant c o m p a r i s o n with Stokke et al. (1968. appears to be the cell n u m b e r . Conceivably, the C F U or the proliferating abthty m a y be relevant as an alternative basis for c o m p a r i s o n , in wtuclcase the effect m the Stokke et al. (1968) experv m e n t s c o r r e s p o n d s to a n effect of 1 G y or more The e x p e r i m e n t a l differences make exact c o m p a n . sons difficult. I n the second case, that of thyroid cancer ir 3 0 - 3 4 - y e a r - o l d w o m e n m N o r w a y (Fig. 2), the doses are less certain (Oftedal a n d Lund, 1983) O n the basis of m e a n iodine-131 c o n t e n t in milk due to atmospheric nuclear b o m b tests, a m e a t

195

total dose of about 5 m G y may be estimated for the relevant age group, but this dose was received during several episodes, each of a few weeks, over the years 1955-1962. There will be regional differences in fallout intensity, and individual differences due to variable milk consumption. But ~t ~s h~ghly unexpected that a dose of some fraction of 5 mGy should lead to a doubhng of the rate of thyroid cancer in one particular cohort. Males show a similar anomalous distribution at a shghtly earher age, and with 3-fold lower numbers than m females as is normally found for thyroid cancer. A skewed dose dlstribut~on rmght mean a sigmficantly higher dose in part of the population, but would also reduce the number of persons at risk and so would not make the phenomenon more understandable. The effect spdls over into the cohorts 2 - 3 years younger and 1 - 2 years older. It may be a biologically important feature that the cohorts were of pre-puberty or puberty age at the t~me of exposure. The third case - that of reduced scholastic

capacity in Norwegian children born during 1965, exposed to radioactive fallout m the fetal stages (Fig. 3) - has no real dose estimate to which one might relate, beyond the variation m the regional mean content of 137Cs m milk, but this is broadly a not unreasonable proxy for fetal exposure (Oftedal, 1989a,b). In any case it seems improbable that the exposures will be much tugher than 2 - 3 times the normal annual background, but concentrated to the 2 - 3 months where the effect appears to be caused. Again, the dose of some few m G y seems out of order for tlus level of effect, which over 3 scholastic subjects and 2 age levels on the average appears to correspond to about 1 year's development m 15-year-olds. These 3 cases are then interpreted as exposures at dose levels or rates below the presumed threshold for repair mducuon, and therefore of exceptionally high damage capacity. A further example is found in the study by Ehrenberg and Ertksson (1966) of mutation m d u c u o n in barley by means of 9°Sr-9°Y ~rradmtion. O F T E D A L 1989 Mdk

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There are two aspects of these results winch deserve discussion. (In view of the scarcity of data xn the lowest dose range, it seems justified to explore even the most tenuous explanations.) The results are shown m F~g. 4, in terms of mutations observed per pollen grain. (This differs somewhat from the authors' presentation, whach is based on the number of mutational events observed. However, since in their calculations only the numerator and not the denormnator have been corrected for mulupliclty of mutants (clusters), the numbers come out slightly too low. The error estimates are, on the other hand, correct.) According to the present model, the break in the dose-effect curve at 5 r a d / d a y may be taken to mdlcate the presence of a threshold for mducuon of additional repmr acuvity. The efficiency of radmtion is reduced by a factor of 10 when the dose rate is increased above 0.05 r a d / d a y . Since we do not know the persistence of the radmt~onreduced signals, or the time pattern of sensitivity variations, it is ~mpossible to say if there is a relevant threshold dose, or if the important aspects ~s the momentary dose rate. It is interesting to note that the present 'threshold' dose rate ~s more than twice the maximum rate used in the Stokke et al. (1968) experiments referred to earlier (Fig. 1) (1000 mrem m 6 weeks = 24 m r e m / d a y ) . It should also be noted that the Ehrenberg and Enksson doslmetry is based on measured isotope content and calculated fraction of energy ab-

sorbed, which comes out at 40% of the total kerma. In a similar experimental situation in Drosophila (Oftedal, 1959), the fraction of absorbed radiation from 91y injected into fhes was shown to be about 6%, based on the mutagemc efficiency. There will be differences between 9°Sr (0.54 MeV fl), 90y (2.25 MeV fl) and 91y (1.53 MeV 13) radiations, and between the geometries of the developing barley grain and the adult Drosophila male, so comparisons are not straightforward. The other interesting point as the variation in muluphclty, or clustering of mutants. The lowest dose-rate range, 0-0.05 r a d / d a y , is characterized by low multiplicity (1.0-1.17), while the higher doses have a range of 1.25-1.60. It is important that the con'rol value lies within this low range, winch thus appears as the normal low dose level, with very few clusters of mutations. This is somewhat surprising, since one would expect that the background mutations would have a 'fluctuation test' pattern, if the early stages of somato-germhne growth collected and carried forward a random sample of induced mutants. Since this is not the case, nearly all mutants being singletons, it is tempting to conclude that this is connected w~th the non-reduced state of the repair apparatus. The reason for the lack of clusters of mutants must be that the early damage is left unrepalred or less efficiently repaired, leading to the death and elimination of the damaged cell. Presumably, it may be more economic for the organism to 'accept' the occasional loss of a cell, rather than to actwate the repair apparatus and in addition run the risk of an increased load of mutants to be selected against at a later stage. In the above-mentioned paper (Oftedal, 1990), I also discuss a well documented case of hormet~c effect after one small acute dose found in a mululaboratory multi-author collaborate study of the chromosome-breaking effects of acute X-ray doses on human lymphocytes in wtro (Pohl-Riihng et al., 1983), as an example of repair function being triggered to a higher degree than warranted by the damage inflicted. That stabihzang elements m this case appear to be released and react with D N A s~tes of pertinent character - breaks or disarrangements of the strands - over and beyond those caused by radmt~on indicates that the event of the normnally 10-100-eV energy absorption itself and

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The stabthzmg functton The hypothes~s of prompt release of stabilizing elements, subsequently followed by repmr processes, provides a bas~s for d~scusslon of relevant experiments in greater depth than does the ordinary dose-effect curve analysis. One of the best examples of such data appears to be the Borek and Hall (1974), Miller and Hall (1978), Miller et al. (1979), and Hall and Miller (1981) investigation and discussion of transformation m eternalized mouse fibroblast cells reduced by 300 kV X-rays, as single doses or as 2 or more doses within a 5-h period (F~g. 5A, B). The experiments were performed by irradmting C 3 H / 1 0 T 1 / 2 cells m vitro 18 h after seeding, m concentrations leadmg to survival of about 400 cells per petri dish. After 6 weeks clones of transformed cells were scored (irregular growth pattern, no contact inhibition). Survival was estimated from cultures where indwidual survwing clones were counted prior to confluence. The resulting dose-effect curve for transformatmn frequency has an abrupt increase at the lowest doses, followed by a plateau, leading into a subsequent 'conventional' increase with higher doses. Similarly 'plateaued' dose-effect curves are known from a number of other materials, e.g., Miiller et al. (1962) observing Ephestm wing-scale changes, Pohl-Riihng (1978) looking at chromosome breaks in human circulating lymphocytes m vwo, and Luchntk (1983) scoring chromosome breaks m human lymphocytes exposed m vitro Pohl-Riiling et al. (1978) refer to some 22 investigations of chromosome aberrations in circulating human lymphocytes, the totality giving an impression of a plateau stretctung from about 2 to about 300 mGy. The data are dffficuh to analyze, being a collectton of acute and chronic a and 7 irradmtions and with few dose pomts in each series. The authors mterpret the data as indicatwe of inductble repair actiwty in the plateau region. It should be noted that there are different processes that lead to curves that resemble each other superficially. The peaked curve - the G r a y - U p t o n curve for lymphoma mduction in mice - ~s a result of competing induction and cell-kilhng effects, and

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turns down because the number of survwmg cells at risk per mouse becomes so low that it ts less probable that an induced l y m p h o m a will be picked up (Gray, 1965). The humped curve is related to

198

mutation reduction m heterogeneous cell populations (e.g., those that are mitotically actwe) where sensttxvity to mutation m d u c u o n and to cell killing are correlated (Oftedal, 1964a, 1968a). The reahty of this mechamsm may be demonstrated by increased mutation yields following dose protraction (Oftedal, 1964b), allowing greater survival of senstove stages, or by looking for sensitivity varlat~ons m synchronously dividing cell populations (Oftedal, 1968b). In the Miller et al. experiments (1979) (Fig. 5A, B), the inittal steep mcrease m transformation frequency ts seen at doses so low that cell killing is unimportant, survwal is given as better than 90%. In the plateau region cell death becomes discernxble. It is interesting to note that the killing effect of the second half of the split dose, though somewhat variable, is about as great as that of the first half. Thus there ~s no indication of reduction of a repair of cell-killing damage by the first half dose, as rmght be expected on the bas~s of the results of Wlencke et al. (1986) and Wolff et al. (1989). (The present interpretation implicitly rests on the assumption that the sum total of repair is dtrected towards all types of D N A damage, and that transformation ~s the result of an redirect process, i.e., nusrepam and therefore may be used as an indicator of repair activity. This argument zs supported in tugh-dose experiments by the findings of Favor et al. (1987) of higher mutation rates in a repaircompetent mouse strata than m a strain less competent. On the other hand, Ehling and Neuh~iuserKlaus (1988) fred m a thard mouse strain that mhtb~t~on of repair by cyclophosphamide leads to higher yields of mutation. In prokaryotes, the relationship between repair and mutation induction has been studted at the molecular level (Seeberg and Fuchs, 1990), and clearly, the process is complex ) According to the proposed model, the initial abrupt increase m transformation frequency observed by Mdler et al. (1979) would represent damage which is caused by energy deposition events of the order of 10-100 eV in cell nuclei before the cell has mobdlzed the relevant stabdizmg elements to a sufficient degree. After a single dose or dose fraction amounting to about 0.3 Gy, the full complement of stablhzing elements is able to cope with most of the further damage mfhcted

up to a dose level of about 1.0 Gy, causing the presence of the plateau in the dose-effect curve. After even higher doses (starting at about 1-1.5 Gy) the reservoir of stablhzing elements appears to become exhausted, and there follows an increase in transformations with dose with sensitivity similar to the initial steep slope (Fig. 5A, B). In the split-dose experiments the 2 half doses act independently, as noted by the authors, indicating that wittun 5 h the situauon reverts to ~mtial conditions. After the interval, the stabdizang effect of the first dose has disappeared, as shown by the double dose having very close to twice the effect of a single dose (e.g., 0.5 G y × 2 compared to 0.5 G y × 1). Following a second half dose of 0.15 G y or more, new stabilizing capacity is avadable, leading to a plateau at about twice the earher level (3 vs. 1.5 transformations per 1 0 4 survivors), and extending a hit beyond twice the single dose (1.0 G y × 2 vs. 1.0 G y x 1). The similarities and differences between the effects of single and split-dose exposures become clearer if one plots the effect of the second half dose (Dx2) by itself. This effect can be estimated by subtracting the effect of a single dose D x from the effect of the double dose (Dxl + Dx2)5 h. One may thus treat the exposures as independent and plot them on the same abscissa (F~g. 6). It is then seen that there is no general enhancing effect of

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Fzg 6 Low-dose secuon of Ftg 5B zx, single dose points, v, spht dose points, ©, interpolated values, ~ , spht-dose effect n u n u s observed or interpolated effect of single half dose. v, one-third of the effect of dose spht into 3, A, one-fourth of the effect of dose spht into 4 Experimental data from Mdler et al (1979) and Hall and Mdler (1981) Abscissa dose m Gy Ordinate transformations per 104 survwmg cells

199

Effect rati o]~/ Dx2 Dx 20 10C,--~ 0

I I L ] ] I ] I~

Hall and Mxller (1981) in order to become informative.

Induction of repair capactty

-

10 [

I

20Gy [

Fzg "7 Effect of dose fractions m spht-dose experiments,

relative to single dose Values from Figs 5B and 6 o, dose spht into 2, v, into 3, A, into 4 Experimental data froth Miller et al (1979) and Hall and Miller (1981). Abscissa dose in Gy Ordinate ratio between effects of the vanous dose fracuons and the corresponding single dose.

splitting the dose (as claimed by the authors). Except for the 0,15 G y dose point, the Dx2 effect is quite close to the D x effect (rauo 0.70-1.15) (Fig. 7). An enhancement by dose sphtting cannot be excluded at 0.15 G y (ratio 2.15). However, at this point there is an added uncertainty, since there is no real observation of the effect of the 0.15 G y single dose, this value being obtained by interpolation. For the rest of the dose range 0.052.0 Gy, the differences in effects of D x and Dx2 do not appear to be meaningful. In a subsequent paper (Hall and Miller, 1981) it ]s shown that division of the dose of 1 G y into 3 and 4 fractions within the period of 5 h leads to increasing yields of transformations (Fig. 4). However, the yields of all the fractions used, i.e., 1 Gy, 0.5 Gy, 0.33 G y and 0.25 Gy, all have approximately the same or possibly a slightly decreasing yield of about 1.8-1.4 × 10 -4 transformations per fraction (Fxg. 5). The increase in total yield with fractxonation is obviously to be expected with all doses wltinn the plateau region, provided the interval permits a return to the initial conditions. What is being shown is really that the radiation is almost totally inefficient for that part of dose winch exceeds 0.25 Gy. The intervals in the 4-fold fractionated exposure support the authors' earlier conclusion that the xnitlal condition is largely reestablished in less than 1 h 40 min. The effect of protraction of exposures needs more observation in addition to the single dose point presented by

After the d e m o n s t r a u o n of the so-called adaptive response (Samson and Cairns, 1977, Jeggo et al., 1977), inducible repair has been found in m a n y systems. Of particular relevance m the present context are the experiments on reduction of chromosomal damage m human lymphocytes by a very small conditiomng dose (5-10 m G y ) revealed by a challenging dose of 1.5 G y (Ohvlen et al., 1984; Wolff et al., 1989). The damage is slgmftcantly reduced, there is cross-reaction with other chromosome-breaking agents, and there is synthesis of new proteins m the interval of 4 - 5 h between conditioning and challenging doses. The adaptive response has been demonstrated following low-dose-level protracted exposures as well as acute ones, in a number of organisms and for a number of chemicals as well as radiation. Wojclk and Taschl (1990) have shown that the response persists for up to 12 days in vivo m mice. The observed cross-reaction between different agents seems to indicate that the reaction mechanism is of a general nature, determined by the pattern of damage rather than the type of noxus. However, in h u m a n cells at least, there appears to be considerable variability m the capacity to respond positively to a condluoning dose, and this capacity is not constant for a given individual (Olivierl and Rosi, 1990). In mice, no effect of a small condlUoning dose was seen on the LD~0,30 (Oftedal, unpublished).

Quantttatwe aspects If it is the case that the response to low-dose radiation stress is the result of an evolutionary process determined by the character and amount of natural background radiation impinging upon cells and organisms, one would expect the various responses to be quantitatively coherent within a c o m m o n set of limitations. N o link m the chain should be much weaker than the others, and no part of the process should need a greater capacity than other parts have. The present model has at least 3 aspects winch should be so linked: a threshold level for release of conditional stablhzing and repair mechanisms; a limited quantity of

200 stablhzlng proteins rapidly accessible; and a quantitative hrmt to the repair capacity induced by a conditioning dose. These 3 aspects should be interrelated at least in terms of the amount of damage involved. The damage caused by the immediately sub-threshold dose should not be greater than (but quite close to) the damage which can be countered when the threshold is exceeded. It should also be comparable to the damage stabilized when a higher dose is applied. This should again be of the same order as that coped with by induced repair. The argument rests on the assumption of the most economic biological balance between processes. Intuitively, it relates to the kind of network of gene-controlled enzymatic functions discussed by Kacser and Burns (1981). The epidemlologlcal and experimental data provide a llrmted amount of information on these relationships. There are bound to be differences with regard to organisms and pathways, related to bmloglcal factors (e.g., cells vs. organisms) and radiation characteristics (e.g., ionization density, dose-rate variation). Nevertheless, a comparison brings out interesting features in those instances where analysis IS possible. Regarding thresholds, there is no proper d o s e effect curve for high doses with which to compare the results of the rat bone marrow cellularity experiments (Stokke et al., 1968), or the thyroid cancer observations (Oftedal and Lund, 1983). However, the deficiency m scholastic achievement may, under given assumptions on relationships between IQ and scholastic achievement as tested, amount to about 7 IQ points (Oftedal, 1989a,b). According to the data stemming from rather higher and acute exposures m Hlroshima/Nagasaka, this should correspond to about 0.4 G y worth of damage (Schull and Otake, 1986). Thus, it is hardly possible to say more than that the sub-threshold dose non-repaired damage corresponds to a dose of 0.1-1.0 Gy. Nevertheless, this range covers the values for stabilization capacity derived from the Miller et al. (1979) plateau lengths, about 0.7 Gy, and also the induced repair capacity in the Wolff et al (1989) studies, about 0.5-0.7 Gy. The threshold level of damage in the Ehrenberg and Enksson (1966) experiments, about 25 × 10 .6 mutations, appears to correspond to an increase in dose rate of about 28 r a d / d a y in repair-reduced

cells. This might indicate that postmelotic mutations accumulate over a period of less than 3 - 4 days, if the damage-eqmvalent dose is again about 1 Gy. If the absorbed fraction of kerma is smaller, the period of damage accumulation would be prolonged correspondingly. This consonance m damage-equivalent doses is taken to be meaningful, especially in view of the widely different endpomts as well as mechanisms under consideration: extraordinarily great effects of extremely low doses, lack of effects of quite high doses, and finally protective effects of small doses reflected in the effect of high doses. For a physicist it may be tempting to regard these reactions as so dissinular as to make compansons meaningless. For a biologtst it may be equally tempting to see them as meaningful indicators of a fundamental coherence in the reactions of living organisms to a type and level of stress which have been present since life on earth began. Inconststencles

The stabilizing consntuent in the Miller et al. (1979) experiments is obviously not the same as the newly induced proteins in the experiments of Wolff et al. (1989). In the Miller et al. experiments, the modification in radiation response appears immediately, and disappears within 1 h 40 min (Hall and Miller, 1981). In experiments made for that purpose, about half of the stabilizing effect disappeared within 1 h (Fig. 2 in Miller et al., 1979). In Wolff's system the new proteins make their appearance within 4 h after a very small (5-10 m G y ) c o n d m o n m g dose (Wolff et al., 1989). In the Hall group's split-dose experiments, no evidence of repair induction is seen (Fig. 3). This might indicate that higher doses somehow overwhelm or damage the induction process. However, the 0.05 G y × 2 results may speak against this interpretation, since it appears that no repair induction takes place even at that dose level. Conceivably, the differences between the Miller et al. and Wolff et al. results are caused by the dissimilarity m the systems used: eternalized mouse cell clones vs rmtotlcally stimulated human lymphocytes m vitro. On the other hand, a plateau effect was found by Luchnik (1983) using the same material and endpolnt as the Wolff group uses.

201 Needless to say, there are numerous published results that do not conform to or support the interpretations d~scussed above. Suffice ~t to ment~on the results of Han and Elkind (1979), using the same cell type as Miller et al. (1979) ( C 3 H / 1 0 T 1 / 2 ) (but possibly another clone), the same endpomt (elimination of contact inhibition, multilevel growth), and a rather similar radmtion source (50 keV vs. 300 keV X-rays). And yet, the dose-effect curves are quite different. In the H a n et al. (1979) data there is no evidence of a plateau m the dose region down to 1 Gy, while there is a plateau in the range of 6 - 1 2 Gy. Correspondmgly, there is no evidence of a plateau m the Evans et al. (1979) analysis of chromosome breaks m nuclear dockyard workers' lymphocytes in vivo, a material sirmlar to that studied by Pohl-Riihng et al. (1978), who did find a plateau. No explanation for these discrepancies is at hand at present. But they may serve to show that the low-dose response patterns can be unravelled only under fortunate circumstances, and that a clear view may be particularly difficult under the intensive assaults of a normal radiation experiment. Testing the model There are a number of investigations where hitherto unexplained results may be interpreted in support of the proposed response model, but there are equally m a n y low-dose investigations where the features stressed m the above analyses are not obvious. Thus it is important that experiments be designed and made for the purpose of testing specific consequences of the proposed model. Here are some approaches.

Threshold The assumption of inducible repair implies the presence of a threshold below which repair is not reduced. The most straightforward test seems to be m line with those discussed above (Stokke et al., 1968; Oftedal and Lund, 1983; Oftedal, 1989a, b), where unexpectedly strong effects are seen after extremely low doses and dose rates, in experiment or in epidermology. Unfortunately, experiments with variable dose rates are difficult to

interpret quantitatively unless stage durations and sensitivities are known. Ehrenberg and Eriksson (1966) have demonstrated an unexpectedly high mutagenic effioency of 9°Sr-9°Y fl radiation at very low dose rates. Possibly, one other example may be the islands of higher leukerma frequency found in the neighborhood of some nuclear establishments (Forman et al., 1987; Cook-Mozaffari et al., 1989a,b; Gardner et al., 1990). That these effects cannot be caused by radiation because the doses are too low is not a scientifically valid stance if the complexity of the low dose-response relationship is still an open question. The most efficient expenmental design might be based on an exploration of the effects and efficiencies of variations of the condinomng dose via the influence it exerts on the effect of a much larger challenging dose. By testing the dose-effect relationship of a successively reduced conditiomng dose upon a constant challenging dose, the presence of a threshold in the reaction to the conditioning dose should be demonstrable. Results pointing in this direction have already been obtained by Shadley and Wiencke (1989), who found that reduction in the dose rate at which the conditioning dose was given led to a reduction and finally the absence of the adaptive response. The Ehrenberg and Enksson results (1966) may be interpreted in the same direction.

Hormesls Unless the repair induction effect of a conditioning dose is classified as an example of hormesis, there seems no way of demonstrating this type of effect except by careful and systematic work with low doses. It should be stressed that the hormesls effect should be seen m the context of a dose-effect curve, and that a continuum of doses need to be investigated. The task ~s difficult, and many ambiguous results are to be found in the literature (Sagan, 1987). Repair mductton The adaptwe response has become a fashionable corner of research of late (see Sankaranarayanan et al., 1989; Wojcik and Taschl, 1990). It is of course of interest to demonstrate the phenomenon in a broad array of biological systems, but it is of even greater importance to elucidate the

202 mechanisms involved. This would include the identification and dynamics of appearance of new proteins and their functions, and the cross-reactivity between chemical and radiation stresses. In addition, physiological reactions to animal handhng and experimental procedures may be ~mportant (Olivien and Rosi, 1990).

Plateau effects It Is obviously of importance to identify more instances of dose-effect curves exhlb~ting a plateau effect, and to analyze them by spht-dose procedures. This should include tests of cross-reactions between chermcals and irradiation, in order to analyze the relative ~mportance of different types of D N A damage for plateau formation. The concept of stabilizing proteins, as a mechanistic explanation for the plateau phenomenon, may conceivably be investigated by looking for unusual assocmtions of D N A and protein rapidly established after stress. The dynamics of the stabihzing process may be studied in exposures with multiple fractions, beyond the four used by Miller and Hall (1981). It might be relevant to consider the experimental weaknesses m the c o m m o n practice of perforrmng split-dose experiments by gwmg two half-doses separated by a gtven interval. Instead, the rmmmal dose for plateau formation should be estabhshed, and this should then be used to obtain a dose-effect curve for the second dose function. A flnetuning of the interval between doses would lead to more reformation on the dynamics of the stabilizing function.

Conclusions and implications Radmtton biology has developed mainly as a b~ophyslcal disciphne. Analysis of cause and effect has been dominated by physical and physicochemical phenomena connected with ~rradmt~on and energy absorption, and with the mathemaucal analysis of dose-effect curves (Lea, 1956; Zimmer, 1960; Haynes and Eckhart, 1979; Kellerer, 1987). Deeper insight into the processes revolved was gamed first by the concept of the 'hit' phenomenon, which led to a quantified picture of the relevant features of the energy deposition. Essentrolly this was a formalism developed to explain the shapes of dose-effect curves and their statm-

tics. The concept was subsequently refined and reformulated in terms of 'target' phenomena, meaning that the hit process did not in every instance lead to a significant end result, but only if the hit were associated with a specific target The target theory served to bridge the gap between the formalism of the hit theory and the real world m the form of molecular sizes and biologically important structures. The targets could be visualized as enzyme molecules, virus particles, genes, and chromosome threads. (Douglas Lea (1956) estimated molecular weights of viruses and genes on the basis of target calculations ) Following the demonstration m m a n y different experimental systems of target-s~ze estimates being highly dependent upon exposure conditions and adjunct elements, the target theory has been modified to encompass indirect effects as well (Thoday and Read, 1947; Alper and Howard-Flanders, 1960; Elkmd, 1987). Today, the target itself is a vague and moving concept, and theory has retreated from the specific and physxcally well defined object of the initml phase. Instead, we are m reality back in the ' h i t ' formalism, the target concept being no longer central in the analysis of dose-effect curves and their dynamics. In fact, one might rather speak of an 'event' formalism, since the ' h i t ' concept seems less suitable for the identification of the complex set of processes leading from the initml energy absorpuon to the stable endpoint underlying the dose-effect measure to be analyzed (Magee and Chatterjee, 1987; Hickson and Harris, 1988). The present model of low-dose effect interpretation leads to an inhomogeneous p~cture of radmt~on effects and their management. It seems we are faced w~th two sets of conditions. On the one hand, conventional experimental radiation exposure, where energy ~mpact and insults far exceed anything cells and organisms have experienced and survwed d u n n g their evolution, and which is totally related to man-made conditions, d~rectly or indirectly. And on the other hand, exposures and results at a level of energy and event frequency which have been part of the environmental challenges to be met by hwng orgamsms since life arose on earth, and to which it has adapted by way of a natural version of the trmge policy for casualty evaluation and handhng:

203 by preventton or repair of damage, i.e, restitution, or by modification and adaptation to damage, i.e., mutation, or by sequestration and elimination of damage, t.e., death. The analytical f o r m a h s m - target, hit or event - m a y well serve as a descriptive tool for p h e n o m ena in the high dose range, where physical p a r a m eters connected with the total insult are of a different order of m a g m t u d e than those met with m normal b a c k g r o u n d range conditions. In lowdose situations, the naturally developed response apparatus of the organism will react and cope with the insult according to the three processes described by the model. It follows that in the low dose range, the effect of one insult will not be independent of previous Insults, and so s u m m a u o n of effects of insults cannot always be made. Further, the acuvation of the response mechantsms will be related to the form of the insult (magnitude, rate, energy density), which implies that the conditional response will lead to unpredictable reactions under given circumstances. High-dose experiments will provide reformation which is adequate and relevant for high-dose exposures (e.g., radiation therapy, or some types of accident), but their interpretation in the low dose range is difficult and possibly inappropriate (PohlRifling, 1989). The conclusion must then be that the linear (or other) interpolation between highdose expertmental results and the ' s p o n t a n e o u s ' rate cannot m principle be supported by scientific arguments or logic. Regulatory needs have been a driving force for m u c h of radiation biology. The present interpretation does not provide an acceptable basis for a regulatory philosophy. In order to resolve the dilemma, it appears necessary to accept the complementary character of the sltuauon: one c a n n o t concurrently have a scientifically defendable interpretation a n d an admimstratlvely useful description based on simple physical assumptions. The resolution comes in the recogmtion of the dichoto m y of the situation: on the one h a n d there is the necessity of a regulatory set of rules, which has to be acceptable simply on the basis of everyday life, just as traffic regulations are. (Traffic lights and speed limits are workable and largely respected regulatory mstitutlons, with no proper scientific justification.) A n d on the other h a n d there is the

n o r m a l scientific investigation, where results are accepted as nature's proper answers to questions properly asked, and interpreted on the basis of first principles. The present model would serve primarily to deepen our understanding of cellular and organismal physiology and evolution. Occasionally, it m a y also provide useful input into rtsk analysis or epldemiological understanding. But basically it would act as a scientific corrective to health physics in the same sense as systematic b o t a n y contributes to agriculture. The p r o b l e m has been discussed in a wider context by R o w e (1986). His somewhat resigned, but also provocative c o n c h i d m g point m a y be relevant m the present context: ' I n any given risk analysis, the uncertainties m a y be so great as to requtre value j u d g e m e n t s to resolve the uncertainty. If value diversity is also very high, then the analysis can only show the h n m s of knowledge. Outside these hmits the political process for dealing with value diversity must be used to resolve the issues at hand.'

Acknowledgements T h a n k s are due to J o h n O r m e r o d and Erling Seeberg for helpful discussions. Ms J.J. Etde has patiently typed and retyped m a n y versions of the manuscript.

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