Mutation Research, 143 (1985) 155~160

Elsevier

MRLett. 0708

Screening human populations for chromosome aberrations*

Amos Norman, Doris Bass and Denise Roe

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Department of Radiation Oncology, Laboratory of Biomedical and Environmental Sciences, and Jonsson Comprehensive CancerCenter, University of California, Los Angeles, CA 90024 (U.S.A.)

(Accepted II March 1985)

Summary

In order to determine the usefulness of micro nuclear counts (MNC) for identifying people with relativelyhigh frequencies of chromosome aberrations we have examined factors that influence the MNC in a learningset of blood samples obtained from 28 adults. The presence of cells with chromosome aberrations amongapproximately 170 metaphase cells per sample was the most important factor. Controlling for the effect ofchromosome aberrations we found that age had a significant effect on MNC, but that donor sex, the mitoticindex, the per cent of metaphase cells in the second or third division or the frequency of abnormal anaphasecells did not. Using logistic regression analysis we found that MNC was an excellent predictor of thepresence of cells with chromosome aberrations among both the learning set and a test set of 17 additionalblood samples.

The assay of micronuclei in peripheral bloodlymphocytes provides a relatively simple and inex­pensive measure of chromosome damage in humanpopulations. However, its usefulness for identify­ing people with relatively high frequencies ofchromosome aberrations is in doubt, partlybecause of the dependence of micro nuclear counts(MNC) on the degree of lymphocyte proliferation[6] and partly because of unsettled questions con­cerning the origin of the micronuclei [4,7]. We

* This work was supported in part by Contract 82EROO038 bet­ween the U.S. Department of Energy and the University ofCalifornia and by UCLA Cancer Core Grant CA 16042.

Please send all correspondence to: Amos Norman, Laboratoryof Biomedical and Environmental Sciences, 900 VeteranAvenue, Los Angeles, CA 90024 (U.S.A.) (213) 825-5971.

have undertaken this study, therefore, in order todiscover the factors that significantly influence theMNC and thus to evaluate MNC as predictors ofchromosome aberrations.

Materials and methods

We selected 28 adults for a learning group and17 for a test group. We included a number of peo­ple who had been exposed to ionizing radiations orto other clastogens as part of their work and 2 pa­tients who were receiving radiation therapy forcancer. The assignment to the two groups was ran­dom except that the two patients were assigned tothe test group. One subject from whom bloodsamples were obtained about a year apart was in­cluded in both the learning group (first sample)and the test group (second sample).

0165-7992/85/$ 03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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From each of the 45 blood samples 3 sets of 2cultures were established by adding 0.3 ml ofwhole blood to 5 ml of medium containingRPMl1640 with Hepes (Gibco) plu s 15070 newborncalf seru m plus 1% PHA. Set A , primarily for theassay of chromosome aberrations, contained also 2x 10- 5 M bromodeoxyuridine (BUdR) and was

incubated for 54 h, the last 2 h with colcemid . SetB, primarily for the assay of the frequency of first,

second and third divi sion met aphases also contain­ed 2 x 10- 5 M BUdR and was incubated for 76 h,the last 2 h with colcimid. Set C, primarily for thedete rmination of MNC was incubated for 76 hwithout either BUdR or colcemid . All incubationswere at 38°C in the dark. At the end of the cultureper iod th e cells were collected by centrifugation,resuspended for 5 min in 0.075 M KCI and fixed insuspension with Carnoy' s. The cells were thendropped on microscope slides and air-dried . Slidesfrom sets A and B were sta ined by the FPG techni­qu e to allo w identifica tion of the first , second or

th ird division metapha se cells . Slides fro m Set Cwere stained with 1% crystal violet or 4% Gurr' sGiemsa. All slides were coded; the y were removedat random for scoring.

Chromosome aberrations were scored in amin imum of 100 first and 100 seco nd divisionmetaphase cells in slides from sets A and B for the28 blood samples in the learning set. The slides forthe test set were analyzed for chromosome aberra­tion s in appro ximately 100 fir st division meta­phases. Slides from sets Band C were analyzed forMNC in 4500 lymphocytes per culture. Slides fromSet C were also analyzed for abnormal anaphasecells - primarily cells with lagging chro mosomes,bridges, or tripolar mitoses. The mito tic index wasalso sco red by counting metaphase cells in a totalof 2000 lymphocyte s. Cells with small, dense nucle iand cells with irregularly shaped nuclei were ex­cluded from all assays since th ese were presumablydead or dying cells.

The MNC were the avera ges of the counts ob­tained from the two cultures in Set C. The naturallogarithm of the MNC (LMN) was found to be ap­proximately normally distributed, so it was usedfor tests o f significance of the effect of each

measured factor on MN C. In carryi ng ou t thesetests, th e number of chromosome aberrations andth e frequency of cells with abnormal anaphaseswere each characterized as either none (0% ) orsome (> 0%) , becau se of the lar ge number ofdonors who had zero s for each .

The effect of each of the measured factors onLMN was first tested . Th e effects of presence orab sence of aberrations, presence or absence of ab­normal anaphase cells and sex were tested using

two sample independent r-test s, while the effects ofkinetics (per cent of second and third divisionmetaphase cells), mitotic index and age were testedusing regre ssion analysis. Since the mo st importantfactor influencing LMN was the presence orabsence of aberrations, the effect of each of thefactors was tested after controlling fo r th e effect ofaberrat ion s. Th e effects of kinetics, mitot ic indexand age were analyzed using analysis o f covari­an ce; the effect s of presence or ab sence of abnor­mal anaph ase cells and of sex analyzed using ana­

lysis of vari ance. One-tailed statistical tests wereused .

The cho ice of an appropriat e MNC cut-off foridentifying peopl e with suspected chromosomeaberration s was determined using logistic regres­sion analysis [1]. Thi s analysis models the pro­bability th at a particular person will have at leastone chro mosome aberration , based on theircharacteri stics (factors). From th ese probabilities acut-point can de derived to separate the peoplewith a high chance of aberrations from those witha low chance of aberrations. Since multivariatenormality of the factors is not required in logisticregression ana lysis, the MNC were used.

Result s

Fig. 1 shows the MNC distributions in the 28learning and 17 test samples. Those samples inwhich cells with chromosome aberrations arefound are speci fically indicated . As can be seen theMNC dist ributions vary over an orde r of mag­nitude among donors. Clearly, the chromosomeaberrations ar e found primarily among sampleswith large MNC. The frequency o f cells with chro-

CELLS WITH MN PER 4 500 CELLS

30 0

•20 0

•

100

80 •••MN •• •••

60 • • . 0. 0 0 :..

40 •o 000 0

30 • 0 0 0go · 0 0

000 0

20 0

0

o 0

1O'-------L.-----,---,------'-----,------,---c-.Test Set (17) Learn ing Set (28)

Fig. I . Cells with MN per 4500 lymphocytes. The open circles

ar e fo r the samples without aberrations. the closed circles are

fo r samples cont aini ng one or more cells with chro mosome

aberrations. In the learning set 9000 cells were assayed for MN;

in the test set 4500 cells were assayed .

mosome aberrations in the 29 samples from the 28donors in the learning group was 0.46 per 100 firs tdivision metaphases (14 cells with aberrations per3072 cells) and 0.24 per 100 second divisionmetaphases (9 cells with aberrations per 3 825cells). For convenience we have combined thescores of first and second division metaphase cellsby assuming that 100 second division cells wereequal to 50 first division cells. The frequency ofaberrations among the 28 samples in the learningset is shown in Table 1. The average frequency ofcells with chromosome aberrations per 100 cells is0.43 (21 cells with chromosome aberrations in

4844 cells) .The mean LMN of the 10 people with chromo­

some aberrations in eithe r the first or second

metaphase was 3.890 (standard error = 0.098),while that of the 18 people with no aberrations was3.266 (standard error = 0.081) . Thi s difference ishighly significant (p<0.0001) when analyzed usinga two-sample r-test (one-tailed test).

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The correlation between LMN and frequency ofcells in second metaphase was 0.08 , while the cor­relation between LMN and frequency of cells inthird metaphase was - 0.23 . Neither of the se cor ­relations are statistically signifi cant (p = 0.34 andp = 0.88 respectivel y, one-tailed tests). The correla­tion between LMN and mitotic index was 0.39which is statistically significant (p = 0.02, one­

tailed test). There was a total of 37 abnormalanaphase cells among the 1085 scored in the 28

donors. The mean LMN of 19 donors with one ormo re abnormal anaphase cells was 3.637 (standa rderror = 0.086) while that of 9 donors with no ab­normal anaphase cells was 3.175 (standard error =

0. 146). This difference is statistically significant (p

= 0.004) when tested using a two sample r-test(one-tailed test) .

The correlation between LMN and age was 0.60,which is highly significant (p = 0.0004, one-tailedtest) . The mean LMN of the 17 females was 3.585(standard error = 0.109), while that for the 11ma les was 3.341 (standard error = 0.126). Thisdifference is not statistically significant (p = 0.08)when tested using a two-sample r-test (one-tai ledtest) .

The most important factor influencing MNC isclearly the presence or absence of chromosomeaberrations. After controlling for the effect ofaberrations on LMN we found that the per cent ofmetaphase cells in second division was not signifi­cant (p = 0.77), the per cent of metaphase cells inthird division was not significant (p = 0.82) , themitotic index was no t significant (p = 0.13), thefrequency of abnormal anaphases was not signifi­cant (p = 0.39) , and the effect o f sex was notsignificant (p = 0.30) . However, the ef fect of ageon LMN remained significant (p = 0.02) .

From this analysis the most import ant factor in­fluencing the LMN after controll ing for the effectof abe rrations was age. However , as can be seen inFig. 2, the plot of LMN vs. age is srrikingly dif­ferent when the data for samples with or withoutchromosome aberrations are considered separate­ly. The LMN for the 18 people without aberrationsshowed a statistically significant regre ssion on age(r = 0.57, p = 0.007) . Moreover this group show-

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LMN = 2.0819 + (0.0218) Age + (0.0064) MI(I)

Fig. 2. LMN vs. age. The open circles are for samples with noaberrations, the closed circles are for samples with aberrations.Th e regression lines are shown for the to tal set of point s and forthe ope n and closed circles alone. The slope of the regressionlines is significant ly great er than zero for the total and for theopen set , but not for the closed circles.

ed, after adjusting for age that the mitotic indexwa s a marginally significant factor (adjusted r =

0.68, p = 0.06). After adjusting for age and

mitotic index no other factors in the se donorswithout aberrations were significant ly related to

LMN . The resulting equation was

classify the 28 people in the learning set we see that9 of 10 with aberrations are correctly identified.We also see that 17 of the 18 people without aber­

rations are also correctly classified . In the test set10 of the II people with aberrations are properly

classified , and 4 of the 6 without aberrations are

also properly classified .

A more general approach to finding a cutoff is

to use a log ist ic regression model [I]. This gives aprediction of the probability of finding an aberra­tion, weighting all the significant fa ctors . Only 2

fa ctors were found significant: the MNC (p <0.000 I) and whether the subject was classified as'normal ' or 'suspect' (p = 0.03) . All the other fac­tors tested , including age, sex, mitotic index, pro­

portion o f metaphases in second or th ird division,and abnormal anaphases were not significant

(p>0 .1O). Using thi s model we obtained thepredictions shown in Table I.

On the ba sis of the table it was decided to use asa cutoff a probability of 0.44 for finding at leastone aberration. For the normal people this cor­responds to MNC of 48; for suspect people thiscorresponds to MNC of 27. Applying thi s criterion

to the 28 people in the learning group lead s to the

same result as before: 9 out of 10 people with aber­

rations correctly classified , and 17 out of 18without aberrations correctly classified. Applyingit to the test sample leads to an improved classifica­tion : all 11 people with aberrations were correctly

identified.

TolOI

72

o

60

ON• y

•

o

•48

Age36

oo

24

o

I

I •III

. : _ _-~YesI __ -0- • ..DNo----..,-.-- .. ...-"----:. ,/ .....

...-0 : ->0 1 ->0%

/0./ 1f 0 I

..-- Io I

II

4.0

4.4

2.4

2.8

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Discussion(The standard errors of the regression coefficientsfor age and mitotic index were 0.0063 .and 0.0032,

respectively). The results o f the analysis of the 10people with aberrations showed, however , no

significant effects of age or any other factors(mitotic index had the smallest p -valu e, p = 0.07, r

;, 0.49) .In order to determine an appropriate cutoff in

MNC for use in screening people for chromosomeaberrations we can simply use the data plotted inFig. I . By inspection MNC of 38 or greater areclearly associated stro ngly with the presence ofchromosome aberrations. Usin g th is criterion to

An impor tant reason for screening populations

for ch romosome damage is to avoid the hard andexpensive effort of assaying chromosome aberra­

tions by conventional means in people who are not

likel y to have significantly high aberration fre­

qu encies. Our results demonstrate that the MNCare use ful fo r predicting high frequencies of aber­rations. We defined 'high' as one or more cellswith unstable chromosome aberrations per100-200 metaphases. That is approxim ately doublethe average frequency of such cells in our learningpopulation. Clea rly as th e cuto ff value is ra ised the

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TABLE 1

OBSERVED CHROMOSOME ABERRATIONS PER COUNTED CELLS ANDPREDICTED PROBABILITY OF AT LEAST ONE SUCH ABERRATION IN EACHSUBJECT FROM THE LOGISTIC REGRESSION MODEL

No aberrations Aberrations

Aberrations/ Subject Predi ction Aberrations/ Subject Predictioncells cells

0/121 N 0.052 11208 S 0.9990/123 N 0.015 1/172 N 0.0350/136 N 0.008 1/140 N 0.5890/139 N 0.114 1/13 8 S 0.930

0/ 145 N 0.020 21180 S 0.8500/ 164 N 0.100 2/164 S 0.9200/168 S 0.437 3/222 S 0.9960/172 N 0.088 21122 S 0.953

0/179 N 0.026 4/170 S 0.9760/183 N 0.040 4/162 S 0.9390/188 N 0.0230/188 N 0.589

0/194 N 0.0520/196 N 0.10001200 N 0.068

01216 N 0.05201224 N 0.00401230 N 0.023

The number of cells'are the sum of first division metaphase plus half the second divisionmetaphase cells analyzed. Subjects Nand S, respectively, are normal and suspect.

number of samples that are candidates for conve n­tional chromosome analysis will drop . Most in­teresting, in th is respect , is our finding that thecutoff frequency is lower in people who we sus­pected, primarily from their employment record,to have a higher than normal aberration frequen­cy . This reflects the fact that the suspects did in­deed show a higher probability of high chromo­some aberration frequencies then the normalgroup. The adoption of the lower cutoff will lead,nevertheless, to more work; but it is common prac­tice to work harder to score chromosome aberra­tions in people suspected of exposure to significantlevels of c1astogens than to score cells in thegeneral populace.

The average frequency of cells with chromosomeaberrations in the learning group is about 4 per1000 metaphases. This agrees reasonably well with

the estimate of 1-3 per 1000, depending on age, ina general Japanse population [8] and of 5-8 per1000, depending on sex, in a British population [2].The median MNC in the learning group is about7.7 per 1000 lymphocytes. This agrees reasonablywell with values of 2-7 per 1000, depending ondonor, found by Krepinsky and Heddle [4] andabout 4 per 1000 reported by Hostedt [3]. We havefound, using an improved technique [6] that on theaverage only about 50070 of the cells in our culturehave divided at least once during the 76-h cultureperiod . That means that the MNC are actuallyabout 15 per 1000 proliferating cells . Obviously thefrequency of cells with acentric chromosomefragments is too sma ll to give rise to so many cellswith MN .

There are at least 4 explanations for the discre­pancy: (I) Nuclear debris is mistaken for MN - if

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that were so, we would expect to find increasedMNC after high radiation doses or long culturetime s when more cells are dying; but the MNC ac­tually decrease [6]. (2) Small acentric chromosomefragments are not counted in metaphase, but the ybecome visible as MN because of additional DNAsynthesis during culture and because of swelling inhypotonic medium [7]. (3) Acentric chromatidfra gments give rise to MN [4] - if that were so weexpect some of the chromatid fragments to appearas chromosome fragments in the second divisionmetaphase; but we don't find evidence for this. (4)The MN arise from spindle defects - this is sup­ported by our finding here that some 2% ofanaphase cells are abnormal and from the meas­urements of micronuclear DNA distributions [7].More work is required to establish the relative im­portance of these mechanisms.

The variability of MNC within a human popula­tion is due in part to differences in degree of lym­phocyte proliferation in culture [4,6]. Our resultsindicate that neither mitotic index nor the relativefrequencies of first, second and third divisionmetaphase are useful measures of the contributionof this factor to MNC. We believe that the varia­bility due to this factor can be reduced by confin­ing the assay to the proliferating lymphocytes [6].Age has been shown both in thi s and in a previousstudy [5] to be a significant factor in MNC. TheMNC counts, on the average, are higher in womenthan in men in both studies . Although the dif­ferences are not statistically significant they maybe real, reflecting the increased probability of Xchromosome loss during cell division in womenthan in men in both studies. Although the dif­ference s are not statistically significant they maybe real, reflecting the increased probability of X

chromosome loss during cell division in women[2]. Thus the degree of lymphocyte proliferation,age and sex can contribute to the variability ofMNC. Despite this, our result s show that MNC isan excellent predictor of the presence of one ormore cells with chromosome aberrations amongone to two hundred metaphase cells. The MNassay appears useful, therefore, as a screen forselecting people for further studies of chromosomeaberrations.

References

I Cox, D.R. , Analysis of Binary Data , Chapma n and Hall,

London, 1977, pp . 14-29.2 Ga lloway, S.M ., and K.E . Buckton , Aneuploidy and aging:

ch romosome studies on a random sample of the populat ionusing Gvbanding, Cytogenet , Cell Genet., 20 (1978) 78- 95.

3 Hostedt , B., Micronuclei in lymphocytes with preserved

cytoplasm, A met hod for the assessment of cytoge net ic

damage in man, Mutation Res., 130 (1984) 61-6 5.4 Krep insky, A.B., and J .A . Hedd le, Micronucl ei as a rapid

and inexpensive measure of radiat ion-induced chromosomal

abe rrat ions , in: Ishihara and Sasak i (Ed s.) , Rad iat ion­

Indu ced Chromosome Damage in Man , Liss, New York ,

1983, pp . 93- 109.5 Norman, A ., S. Cochran, D. Bass and D. Roe, Effects of age,

sex and medical X-ra ys on chrom osome dama ge, lnt. J .

Radiat. Biol . , 46 (1984) 317-321.6 Pincu, M., D. Bass and A. Norman , An improved micro­

nuclear assay in lymphocytes, Mut at ion Res., 139 (1984)61- 65.

7 Pincu, M., H . Callisen and A. Norman, Micron uclear DNAdistributions in human lymphocytes , Int. J. Radiat. BioI.,

1985, in press.

8 Ton omu ra, A., K. Kunikazu and F. Saito , Types and fre­quencies of chromosome aberrations in perip heral lym­

phocytes of genera l populations, in: Ishihara and Sasaki

(Eds .), Radiation-Induced Chromosom e Damage in Man ,

Liss, New York , 1983, pp . 605-616 .