INT . J . RADIAT. BIOL ., 1992, VOL . 61, NO . 3, 335-343

Chromosomal aberrations in human lymphocytes induced in vitroby very low doses of X-rays

D. C. LLOYDa*, A. A. EDWARDSa, A. LEONARD b, G. L . DEKNUDTb,L . VERSCHAEVE b , A. T. NATARAJAN F. DARROUDIc, G. OBE d , F. PALITTIe,C. TANZARELLAe and E. J. TAWN f

(Received 22 May 1991; revision received 22 August 1991 ; accepted 1 September 1991)

Abstract. This paper presents results of a collaborativeexperiment between six laboratories which examined the yieldsof unstable chromosomal aberrations in human lymphocytesinduced in vitro by X-rays over the dose range 0-300 mGy . Thework included data points of nominal doses of 0, 3, 5, 6, 10, 20,30, 50 and 300 mGy. Cells from 24 donors were examined and atotal of about 300000 metaphases were scored . The work wasundertaken to determine the limits of sensitivity of the systemtaking into account variations in scoring data due to inter-donor sample and inter-laboratory effects. Despite the existenceof these effects, aberration yields significantly in excess of con-trol values were seen at doses > 20 mGy and these were consis-tent with a linear extrapolation from higher doses . Below20 mGy the observed dicentric yields were generally lower thanbackground, but not significantly so . Excess acentric aber-rations, on the other hand, and centric rings, were higher thanthe controls but the increase was usually not significant . It isconcluded that the statistical uncertainties are such that below20 mGy this technique cannot distinguish between a linear or athreshold model .

1. Introduction

There is much interest and debate on the relation-ship between biological effect and absorbed radia-tion at low doses, but informative data on thisimportant issue are scarce and difficult to obtain .The usual approach is to extrapolate linearly to zerodose from higher dose data . Some authors havequestioned this linearity, e .g. Mole et al . (1983), whoexamined myeloid leukaemia induced in mice, buttheir lowest dose was 250 mGy. During the past 10years some data on the induction of chromosomeaberrations in human lymphocytes have been pub-

*Author for correspondence .aNational Radiological Protection Board, Chilton, Oxford-

shire, OX 11 ORQ UK .bCEN/SCK Mol, Belgium .`State University of Leiden, The Netherlands .'University of Essen, Germany .`University of Rome, Italy.fBritish Nuclear Fuels plc, Sellafield, UK .

0020-7616/92 $3 .00 © 1992 Taylor & Francis Ltd

lished which further question this assumed linearity(see Pohl-Ruling 1990 for a recent review) .An early indication came from Pohl-Ruling and

Fischer (1979), who measured chromosomal aber-rations in the lymphocytes of spa workers exposed toenhanced natural levels of external y-radiation andinternal radiations from inhaled radon decay pro-ducts. An attempt was made to estimate the averagedose to blood from both causes for each individual . Aplot of the yield of total aberrations against dose-rateindicated a linear relationship between effect anddose up to dose-rates of 2 mGy per 6 months, fol-lowed by a plateau at higher dose rates . Furtheranalysis of this work (Pohl-Ruling 1990) shows adecrease of aberration yield at dose rates aboveabout 10 mGy/year. There are several problems inconnection with this work (Edwards et al. 1989) .First, the estimation of a-particle doses to bloodrequires a series of assumptions leading to uncertain-ties that were acknowledged by the authors . Second,the dose to the lymphocytes was protracted on a timescale of years, and it is not known how this affects theobserved yield of aberrations in vivo . These problemsmay be avoided by measuring dose-response rela-tionships in vitro with well-defined physicaldosimetry .A number of in vitro studies at doses less than

500 mGy of low-LET radiation have been made,with between 1000 and 3000 cells scored per dosepoint. Generally these fit well to the linear model,although Luchnik and Sevan'kaev (1976) reported aplateau in the dicentric response to y-rays at dosesbetween 100 and 300 mGy. Repetition of this experi-ment by Lloyd et al. (1986) gave no indication of aplateau in this region . Kucerova et al . (1972) pro-duced some dicentric data for X-rays which mightindicate a response with a threshold at about150 mGy, although the authors themselves chose tointerpret the data as linear up to 500 mGy . Lloydet al . (1986) found a linear response in the low-dose

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336

region for X-rays down to 50 mGy for both dicen-trics and total aberrations. Wagner et al. (1983)found a linear-quadratic response using doses50-500 mGy of 220 kVp X-rays . The dose-squaredcontribution was small in the quoted dose range .

In vitro investigations at doses less than 100 mGyrequire a large number of cells to be examined,which would exceed the scoring capacity of a singlelaboratory. On this basis the International AtomicEnergy Agency (IAEA) coordinated a collaborativeproject involving several laboratories (Pohl-Rulinget al . 1983) . Two donors and eight X-ray doses(200 kVp, 1 mm Cu filtration) from zero to 300 mGy,six of which were no more than 50 mGy, were used .The scoring was shared between 10 laboratories andcomparisons showed some evidence of inter-laboratory variation . Despite this, results werepooled and the authors reported that the yields ofboth dicentrics and total aberrations were signifi-cantly below the control at 4 mGy, and were belowthe control at doses up to 20 mGy for dicentrics andup to 50 mGy for deletions . The authors interpretedthis as evidence for stimulation of repair mechanismsat doses below a few tens of mGy . Edwards et al .(1989), commenting on these data, pointed out thatthe dicentric yield at zero dose of 2 .8 per 1000 cellswas much higher than the commonly cited value ofabout 1 per 1000 cells (e .g . Bender et al . 1988) . It wasalso higher than the control value of 1 . 8 per 1000cells from a similar experiment by a similar groupusing neutrons (Pohl-Ruling et al . 1986), in whichone of the two donors was common to both experi-ments. It was concluded that the apparent loss ofaberrations at 4 mGy was principally caused by anexcessively high control measurement and was there-fore probably spurious .Because of the equivocal results of the IAEA-

sponsored experiment, the Commission of the Euro-pean Communities (CEC) supported a joint studyusing X-rays . Some results and a preliminary analy-sis have already been published (Lloyd et al. 1988,CEC 1991) . Further results and a more completeanalysis are presented here .

2. Materials and methods

Blood from all donors was irradiated at 37°C withX-rays of defined quality (169keV ISO wide series)(ISO 1979) in one laboratory . This was obtainedfrom an X-ray set operated at 250 kVp with a half-value layer of 4 .3 mm Cu. Because of the numbers ofsamples to be irradiated and processed the exposuresspanned 4 months . The doses quoted in this paperare averages. The 95% confidence limits on the

D. C. Lloyd et al .

stated doses, arising from day-to-day reproducibi-lity, are ±4% . The exposure times ranged from 1 to7 min . For each donor and dose 10 replicate 48hwhole-blood cultures (Eagle's MEM + 10% foetalcalf serum + Pha + BudR ; colcemid at 45 h) were setup and fixed according to published procedures(Lloyd et al . 1982) . After the fixation, cells fromreplicate cultures were pooled in order that replicateslides could be dispensed from a common stock .Sample slides of lymphocyte metaphases for eachpoint were stained with fluorescence plus Giemsa toensure the material contained an acceptably small(< 10%) number of second division cells . The aver-age value was 3 . 1 %. Unstained and coded replicateslides were distributed to the participating labora-tories where they were stained with conventionalGiemsa and scored for chromosome and chromatidaberrations. Results were collated and analysed todetect donor and laboratory variations and a dose-response relationship was determined . Initially fourdonors were used with nominal doses of 0, 3, 6, 10,20, 30, 50 and 300 mGy. In a second experiment,nominal doses of 0, 5, 30 and 300 mGy were usedwith 20 donors .

3. Results and discussion

In neither experiment did the data for chromatidtype aberrations (gaps, breaks and exchanges) showevidence for a dose response ; this was expected sincethe cells were irradiated in G o . Further discussion istherefore confined to unstable chromosome typeaberrations .

3 .1 . Experiment 1

Pooled data from each laboratory and each of thefour donors of the first experiment are shown inTable 1, where all scored cells are included so thatcomparisons can be made with Lloyd et al. (1988) .However, analysis in the present paper has beenrestricted to cells with 46 centromeres and withcomplete aberrations, namely those where the dicen-trics and rings are accompanied by their acentricfragments. There are a few minor differencesbetween the data in Table 1 and the data of Tables 1in Lloyd et al . (1988) and CEC (1991) . These aredue to a later re-evaluation of some slides and scoresheets . Table 2 divides the data to illustrate the inter-laboratory variations . Data using doses from 0 to19 . 3 mGy have been combined because the yieldsare dominated by the control level of aberrations .The 28 . 7 and 47 .7 mGy data have been combined

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because the yield of dicentrics due to irradiationapproximately equals that due to spontaneousevents. The 290 mGy data stand alone because theyield is predominantly radiation-induced . The samereasoning was used in presenting the data in Table 3,where the variations between donor samples areillustrated .

3 .1 .1 . Laboratory variations . In order to test theextent of inter-laboratory variation in scoring, weassumed that aberrations were distributed as thePoisson distribution and applied the Pearson x 2 test .The explicit form is given in equation (1) where y isthe mean yield, Ni the number of cells scored and 0 .the number of aberrations scored . For the six labor-atories assuming no laboratory difference, the quan-tity in equation (1) should be distributed as x2 on 5degrees of freedom .

x2=E(0i-Ni u) 2 I Ni µ

( 1 )i

Values for x 2 are shown in Table 4 . The 95%confidence limits of x 2 , that is the value which maybe exceeded by chance with a probability of 5%, is11 . 1 . Thus in Table 4 all three values show signifi-cant inter-laboratory variation for dicentrics andtwo show the same for acentrics . Inspection of Table2 shows that for dicentrics laboratory 3 tends toscore high. For acentrics laboratories 1 and 6 tendto score low whilst 2 and 5 are high . Centric ringswere not tested because the numbers in Table 2 aresmall. However, there is no obvious laboratoryvariation in any dose range .

3 .1 .2 . Donor sample variations. Although we refer tosample-to-sample variation as `donor sample varia-tion', we do not wish to imply that the cause is

Chromosome damage and low radiation dose

337

Table 1 . Experiment 1 : pooled results for four donors and six laboratories showing frequencies of chromosome aberrations

solely an intrinsic difference in sensitivity betweendonors. Other possibilities include intra-donoreffects with time and unknown experimental varia-tion. We have not tested for these as each donor wassampled only once in this study . Samples were notall processed on the same day, but strenuous effortswere made on each occasion to replicate the experi-mental conditions .

Table 5 shows an analysis to investigate variationin the samples from the four donors . The number ofdegrees of freedom is 3 and the 5% level of x 2 is 7 .8 .Thus for dicentrics there is significant variationbetween the samples at 0-19 .3 mGy - and at290 mGy. From Table 3 it is clear that the samplefrom donor D appears to have a high control levelof dicentrics and that from donor A is more sensi-tive, and B less sensitive, than from donors C and D .From Table 3, centric ring yields are perfectly inline with dicentric yields .

3.1 .3 . Dose response. Despite the laboratory anddonor sample variations the pooled aberration dataof Table 1 should give an average dose responsebecause very nearly equal numbers of cells havebeen scored by each laboratory on each donor foreach dose. Pooling cannot therefore introduce syste-matic bias. In Table 6 are shown the results offitting the data in Table 1 by the iteratively re-weighted least-squares method, using Poissonweights, to the quadratic model of dose response(Y=C+aD+fD2) and to a linear fit (Y=C+aD),the latter over the dose range 0-47 .7 mGy. The fitof the data to these simple models is very good(x2 = 5 . 2 on 5 degrees of freedom) and it is thereforemost likely that the reduction in dicentric yield seenin Table 1 at the three lowest doses is not ofbiological origin but may be an artefact of the

DIC =dicentrics, CR=centric rings, AF=excess acentric fragments, rings and minutes . Multiply damaged cells : 'indicates that thedata include a cell with 2 DIC ; "two cells each with 2 DIC ; ba cell with 3 DIC; `a cell with a tricentric ; da cell with 2 CR .

Cells scoredDIC CR

AFDose(mGy) Complete

Withincompleteaberrations

Withfragments

Withoutfragments

Withfragments

Withoutfragments

0 11969 4 20 4 0 0 353 . 13 11927 5 13` 3 0 2 475 .80 11 719 6 14" 6 1 0 469 . 65 11992 2 13 2 1 0 4519 . 3 11 .980 6 23 - 4 2 2 5628-8 11952 6 29' 6 4 0 5847 . 7 11544 0 28' 0 0 0 52

290 11921 15 152 aab 14 5d 2 153

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Superscripts as in Table 1 .

higher-than-expected frequency of spontaneouslyarising aberrations .

3.1 .4. Highly damaged cells . An unusual feature ofthis experiment was the relatively high number ofmultiply damaged cells; i .e. containing more than

Superscripts as in Table 1 .

one exchange type aberration, but they were notimmensely damaged `rogue' cells as described byAwa and Neel (1986) . Their distribution betweendoses, laboratories and donors is indicated by thesuperscripts in Tables 1-3 . None of these cells wereobserved in the zero dose material. However Benderet al . (1990) recently reported a similar finding in avery large survey of background aberration levels incontrol subjects. Despite these observations theanalyses in the present paper have used Poissonstatistics for calculating uncertainties on datapoints. However, omitting these 11 cells from theanalysis does not alter the overall conclusion of thestudy. On Poisson statistics alone, only one multiplydamaged cell, at 290 mGy, would have beenexpected. No trend with laboratory is obvious(Table 2) since at least one such cell was found byeach. However, there is some indication in Table 3that the cells were found predominantly in thesamples from donors A and D, and it is interestingto note that these were the samples identified inparagraph 3 .1 .2 above as having higher aberrationyields .

3 .2. Experiment 2

In this study fewer doses were used and, in each of20 donors at each dose, all laboratories scored 500complete cells except for 280 mGy where 200 cellswere deemed sufficient . The results are shown inTables 7 and 8 . The laboratories numbered 1-6 are

338 D. C. Lloyd et al .

Table 2 . Data of Table 1 rearranged to show the scoring Table 4 . Values of xZ for dicentrics and excess acentricresults from each laboratory fragments to test laboratory variation in the data of Table 2

Doserange(mGy)

Labora- Completetory

cells DIC CR AF

Dose range (mGy)

DIC

AF

0-19.3

16.8

6028.8-47 . 7

12.5

371

100000-19 .3 10 2 0 9 290

13.9

6.02

10 054 8 2 583

10000 268 1 564

9 986 9 1 295

9547 17° 0 576

10 000 13 as 0 20

28 . 8-47 . 7 1

4 000 11 as 0 82

4031 5 1 29 Table 5 . Values of x 2 for dicentrics and excess acentric

3

4000 12 0 18 fragments to test the data in Table 3 for variations between the

4

3 998 8 0 15 samples from the different donors

5

3 467 16 1 336

4 000 5 2 7 Dose range (mGy)

DIC

AF

290 1

2000 22 1 23 0-19 . 3

9.4

4.52

1999 17 2 d 35 28.8-47 . 7

6.9

4.53

1999 41 1 23 290

17.3

4.44

1991 228 0 215

1932 22 0 296

2000 28 ab 1 22

Table 3 . Data of Table 1 rearranged to show the aberrationsscored for each donor

Doserange(mGy)

Donor Complete(sex, age,

cellssmoking)

scored DIC CR AF

0-19.3 A

14690 21 0 69(M, 21, NS)

B

14 993 17 2 52(M, 35, NS)

C

14 846 13 0 1 49(M, 41, NS)

D

15 058 328888 1 59

28. 8-47 . 7

(M, 42, NS)

A

5 500 21 1 33B

5 961 9 1 27C

6 006 12 2 0 20D

6 029 15 a 2 30

290 A

2997 58aa b 4 d 36B

2 998 22 1 48C

2 934 35 0 39D

2 992 37 0 30

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identical to those shown in Table 2 . A finer break-down of the data was also given in CEC (1991)although, as with the preliminary publication ofexperiment 1, there have been a few changes to thedata in the light of re-evaluation of some slides .

3.2 .1 . Laboratory variation . The values of x2 com-puted according to equation (1) are shown in Table7. It is clear from these data that there is a smallinter-laboratory variation in scoring for dicentrics,particularly at 280 mGy . This is due mainly to lownumbers of dicentrics found by laboratories 4 and 5 .These are thought to be low because the numbers ofdicentrics listed in Table 7 should be about doublethose in Table 2 since twice as many cells werescored. However, for laboratories 4 and 5 thesenumbers are approximately the same in both tables .For centric rings the only anomalous result is at28 .5 mGy where laboratory 2 scored a remarkablylarge number (eight) . In contrast inter-laboratoryvariation in the scoring of acentrics is marked at alldoses. In particular laboratory 2 has scored remark-ably high at three of the four doses but this is offsetby very low values from laboratory 1 . The samelaboratories are implicated in Table 2 althoughthere the disparity is not quite so marked .

3 .2.2 . Donor sample variation . Table 8 shows resultsof the Pearson x2 test for variation among thesamples from the 20 donors . The 95% confidencelimit on 19 degrees of freedom is 30 . 1 . For dicentricsthe tests indicate a significant variability at 5 mGybut not at zero dose . At these low doses with meansof 2 to 2 .5 dicentrics per donor the x2 test is notrecommended. However, the u-test of Papworth(1970) gave similar conclusions with regard to sig-nificance. At zero dose u=16 (p=0-44) and at5mGy u=2 .72 (p=0 .003) .

This result is expected because average aber-ration yields at these two doses are very close . Themain cause of this anomaly is the sample fromdonor G, where 7 dicentrics were found . However,for this sample the distribution between laboratorieswas unusual; laboratories 2 and 3 scored 4 and 3dicentrics respectively and the others zero . There isalso an indication in Table 8 of a high x 2 value for

Table 6. Control (C), linear (a) and quadratic (f) coefficients and errors obtained by fitting to the dicentric data of Table 1

Dose range

C

a

Ii(mGy)

(10-3 ± SE)

(10 -5 mGy - '±SE) (10 -$ mGy -2 ±SE)

x2

d.f.

0-47 . 7

1 .2+0.2

2.9+1.0

5.2

50-290

1 .2±0.2

2.7±1.2

4.4±4.2

5.2

5

d.f. =Degrees of freedom .

Chromosome damage and low radiation dose

339

dicentrics at 280 mGy indicating donor variability .Superficially, the sample from donor L seems to givea substantially higher yield of dicentrics than theothers, but the overall yield at this dose is low,suggesting that the low yields are more likely torepresent the anomalies . For centric rings, signifi-cant variation occurs only at 280 mGy where thesample from donor T gives an anomalously highresult. For acentrics a large variability appears atall doses .

3 .2 .3 . Dose-response . The pooled yields shown inTable 8 may be compared with those in Table 1 .The dicentric yield of 49 in 60 000 cells at zero dosein Table 8 is consistent with a nominal value of 1 in1000 and is significantly lower than the yield of 20in 11969 cells from Table 1 . At 28 .5 mGy theincreases above the respective control yields areconsistent for dicentrics. At 280 mGy the meanyields for the second experiment are about 30%lower than for the first . It is interesting to note thatthe yields of dicentrics obtained at about 300 mGyin both experiments described in this paper arelower than would be expected from many publishedacute X-ray curves (Lloyd and Edwards 1983) . Thereason for this could be that the energy of X-rays inboth experiments is higher (mean energy 169 keV)than the less filtered beams (- 90 keV average) thatare more often used in radiobiological studies . Theyield in the second experiment is, however, stilllower than would be expected from many y-raycurves (e.g . Lloyd and Edwards 1983) and this is ananomaly which gives more weight to the inferencedrawn earlier, that laboratories 4 and 5 scored lowat 280 mGy in the second experiment .

Although there are only four dose points in thisexperiment an attempt was made to fit dicentrics tothe linear and quadratic models as was done forexperiment 1 (Table 6) . The best linear fit was

For both equations the x2 values are 2 .6 on 1 degree

Y=(0 . 71±0 . 14)10-3 +(2 .5+1 . 0)10-5D (2)and for the quadratic model :

Y= (0 . 71 ±0 . 14)10-3 + (2 .4± 1 .2)10-5D+(1 . 1±4 . 3)10-8D2 (3)

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Table 7 . Experiment 2 : unstable aberrations scored by doseand laboratory : donors combined

The values for 0, 4 . 82 and 28 . 5 mGy are in 10 000 cells andfor 280 mGy in 4000 cells. Superscripts as in Table 1 .

of freedom. These fits are essentially consistent withthose in Table 6 .

3 .2 .4 . Highly damaged cells . In this experiment onlythree highly damaged cells were recorded, and asabout twice as many cells were scored comparedwith experiment 1 this is a significantly lower fre-quency than the 11 shown in Table 1 . They areagain indicated by superscripts in Tables 7 and 8but there are too few to draw any conclusionregarding relationships to dose, donor orlaboratory .

The second experiment in particular showsclearly that, in terms of yield, acentrics are morevariable than dicentrics, and for this reason we have

D. C. Lloyd et al .

not attempted to analyse a dose-response relation-ship. Such an analysis at these low doses is alsoconfounded by the relatively high control level ofthese aberrations . Even so there is still no convinc-ing evidence from either Tables 1 or 8 that yields ofacentrics show any anomalous behaviour with dose .This conclusion is in marked contrast with that ofPohl-Ruling et al . (1983) .

In the second experiment the yield of dicentricsat 4.82 mGy was lower than at zero dose, but notsignificantly so. However, taken together withexperiment I and with Pohl-Ruling et al . (1983),there are now six data points of irradiation withdoses between 3 and 10 mGy, all of which give alower dicentric yield than their respective controls .All three sets of dicentric data up to 50 mGy havebeen plotted for comparison in Figure 1 . The twodata points from Pohl-Ruling et al . have a commoncontrol which shows a yield much higher thanexpected from the literature . The three points fromthe first experiment reported here also have a com-mon control level which is a little higher than thatgenerally expected, whilst the single point from thesecond experiment has a control value close toexpectation . However, for centric rings the observedcontrol level is 1 in 60 000 cells compared with 10for a dose of 4 .82 mGy (Table 8) . Thus, if yields ofdicentrics and centric rings are summed then thereis no decrease at 4 .82 mGy compared with thecontrol . It is interesting to note that if centric ringswere the sole criterion for judging biological effectthen the results might lead to a conclusion that4 .82 mGy caused a 10-fold increase in biologicaleffect .

It should be noted that the quadratic fits fordicentrics in both experiments reported in thispaper (Table 6 and equation 3), and also Pohl-Riiling et al .'s fit to their data over the range0-300 mGy give positive values for the a-coefficientsdespite the low dose yields being lower than theircontrols. Had the best fits produced negative aterms this would have been more convincing evi-dence that a low dose dip in yields was a real effect .

In a recent paper Pohl-Ruling et al . (1991) indi-cated that a very low in vivo exposure to radiationdue to fallout from the 1986 Chernobyl reactoraccident could be discerned by an approximately 6-fold elevation in aberration yields in blood samplestaken before and after the accident . The dosesinvolved were 0 .2-0 . 5 mGy in the first year in addi-tion to the usual 0 .9 mGy year -1 pre-accident back-ground dose. This would imply a remarkably highlycurved relationship between dose and effect. Thecase rests essentially on data from two persons inSalzburg sampled once before and three times after

Dose(mGy) Laboratory DIC CR AF

0 1 9 0 22 14 b 1 613 10 0 284 4 0 165 7 0 186 58 0 9

X2 8 . 2 97

4 . 82 1 4 1 142 11 4 543 11 0 164 4 0 175 5 2 536 6 3 29

x2 8 . 0 57

28 .5 1 11 1 82 17 8 873 23 8 2 394 11 1 175 17 0 346 9 0 26

x2 9 . 5 110

280 1 35 0 452 31 3 343 47 2 424 23 0 285 20 2 476 46 4 75

x2 19 . 0 29

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the accident. As cells from each time point werescored by different laboratories it is necessary toassume no inherent inter-laboratory variability. Afeature of the present paper is that a difference,greater than may be expected by chance, isreported in aberration yields obtained by laborator-ies that have scored identical metaphase prep-arations. This was not surprising to us, as inter-laboratory variability has been shown previously(e.g. Lloyd et al. 1987) in studies involving higherradiation doses . The study of Pohl-Ruling et al.(1991) showed a remarkably large ratio (1 . 1 :1 .0) ofrings to dicentrics in the first pre-exposure samples .Unexpectedly large ratios have been shownoccasionally in the present paper (Table 7,28 . 5 mGy) but these were not found by all labora-tories or at most doses. In addition when the rela-tively small number of cells per time point in thePohl-Ruling et al. (1991) study is considered weconclude that the reported elevated yield due todose from Chernobyl fallout should be viewed withcaution .

Chromosome damage and low radiation dose

Superscripts as in Table 1 .

4. Conclusions

An attempt has been made to investigate theshape of the dose-effect relationship at very lowdoses of X-rays for the induction of chromosomalaberrations in human lymphocytes . Even at a rela-tively high dose of about 300 mGy where nearly allof the scored aberrations are radiation-induced,there is evidence for laboratory variations and donorsample variations both of which can confound themeasured aberration yield . At doses of about50 mGy and below, where control levels of aber-rations contribute an increasing fraction, evidencefor significant variations in scoring between labora-tories and between donors may still be found. Theoften-quoted conclusion that the scoring of acentricsis more variable than that for dicentrics is supportedby the laboratory variations shown in Tables 4 and7 . Donor variations may, however, be less signifi-cant, since Table 8 indicates this trend while Table 5does not. Overall we suggest that more variableresults are obtained with acentrics rather than dicen-trics. Despite these variations, results for each donorand each laboratory have been combined but, in

Table 8 . Aberrations scored for each donor in 3000 cells for 0, 4 . 82 and 28 . 5 mGy and 1200 cells at 280 mGy; Laboratories combined

341

Donor 0 mGy 4 .82 mGy 28.5 mGy 280 mGy

DIC CR AFDIC CR AF DIC CR AF(sex, age,smoking) DIC CR AF

E (M, 29, NS) 3 0 4 5 1 6 4 0 7 6 0 8F (F, 25, NS) 1 0 10 0 0 17 10 0 12 10 0 14G (M, 39, NS) 2 0 4 7 1 14 5 0 16 10 0 14H (M, 26, S) 3 0 17 1 1 4 9 0 12 16 1 23I (M, 31, NS) 4 0 12 2 0 9 7 1 13 12 0 10J (F, 19, NS) 6b 0 6 1 2 14 6 2 9 15 2 15K (M, 25, S) 5 0 9 2 1 17 7 0 23 11 1 22L (F, 28, NS) 5a 0 3 0 0 8 2 0 15 22 0 19M (F, 58, NS) 2 0 14 5 0 12 4 2 10 11 0 18N (F, 36, NS) 2 0 6 2 0 13 5 0 6 5 0 9O (F, 25, NS) 1 0 6 4 0 7 3 0 10 11 0 28P (M, 35, NS) 3 0 4 2 2 5 2 1 5 8 0 8Q (M, 23, NS) 2 0 4 1 0 9 2 0 7 8 0 7R (M, 30, NS) 2 0 4 2 1 4 5a 0 8 7 0 17S (M, 21,NS) 0 1 5 1 0 5 3 0 9 5 2 18T (F, 31, NS) 3 0 4 1 0 12 1 0 8 9 4 7U (F, 31, NS) 2 0 7 0 0 10 3 3 10 10 0 12V (F, 26, NS) 1 0 9 1 0 5 4 1 15 6 1 14W (F, 27, NS) 0 0 2 4 0 9 4 2 13 8 0 4X (M, 37, NS) 2 0 4 0 1 3 2 0 3 12 0 4

Total 49 1 134 41 10 183 88 12 211 202 11 271X2 20 . 0 44 . 7 35 . 6 44 . 4 25 . 2 37 . 3 31 .6 60 . 4d .f. 19 19 19 19 19 19 19 19p 0 . 39 0 . 0008 0 . 012 0 . 0008 0 . 15 0.0073 0.035 <0-0001

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D. C. Lloyd et al .

averaging, equal weight was given to each donor

Acknowledgementand each laboratory in order that variations shouldnot produce bias . Under these circumstances fourdoses of less than 10 mGy all produced observeddicentric yields lower than their corresponding con-trols. In Table 1, we believe the control level ishigher than normal, although no one donor sampleyielded exceptionally high spontaneous frequencies(8, 5, 3 and 4 dicentrics in approximately 3000 cells) .No such criticism may be levelled at the control datain the experiment shown in Table 8 . It is importantto note that if the yield of centric rings is combinedwith that of dicentrics then the low-dose anomalydisappears .

The studies reported here sound a number ofcautionary notes . The statistical variations of smallnumbers, believed to be distributed as the Poissondistribution, can lead to spurious conclusions . This isparticularly the case if the presence of multiplydamaged cells means that uncertainties on datapoints are somewhat greater than Poisson (Benderet al . 1990) . This work has shown conclusively thatlinearity of biological effect can be demonstratedwith this system down to doses of about 20 mGy.Below that, statistical variations connected withsmall numbers preclude any definitive statement .Thus the question of whether linearity extends downto zero dose, or whether there is a plateau (thres-hold) indicative of inducible repair remains open .Certainly there is no evidence for a super-linearresponse at very low doses, and there is nothing inthe present work which is incompatible with theprudent radiological protection assumption of alinear initial slope . The problems discussed hereinvolved scoring large numbers of cells ( - 300 000),making it very unlikely that the true response atdoses less than 20 mGy will ever be measureddirectly with these techniques .

0 .5

0.4

Sa`

0 .3

0 .z~

u 0.1I

0

10

20

30

40

50Dose, mGy

Figure 1 . Dicentric yields as a function of dose : •, Pohl-Ruling et al., 1983 ; x, this work, experiment 1 ; O, thiswork, experiment 2 .

We wish to acknowledge Mr D . R. McClure of theUK National Radiological Protection Board forirradiating the blood samples and performingmeticulous physical dosimetry. Five of the collabor-ating laboratories (Chilton, Essen, Leiden, Mol andRome) were partly supported by grants from theCommission of the European Communities . We areindebted to Dr K. H . Chadwick of CEC for hisencouragement and constructive comments duringthe course of this project .

References

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