- Email: [email protected]
Screening human populations for chromosome aberrations
Mutation Research, 143 (1985) Elsevier
155~160
155
MRLett. 0708
Screening human populations for chromosome aberrations* Amos Norman, Doris Bass and Denise Roe Department of Radiation Oncology, Laboratory of Biomedical and Environmental Sciences, and Jonsson Comprehensive Cancer Center, 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 relatively high frequencies of chromosome aberrations we have examined factors that influence the MNC in a learning set of blood samples obtained from 28 adults. The presence of cells with chromosome aberrations among approximately 170 metaphase cells per sample was the most important factor. Controlling for the effect of chromosome aberrations we found that age had a significant effect on MNC, but that donor sex, the mitotic index, the per cent of metaphase cells in the second or third division or the frequency of abnormal anaphase cells did not. Using logistic regression analysis we found that MNC was an excellent predictor of the presence of cells with chromosome aberrations among both the learning set and a test set of 17 additional blood samples.
The assay of micronuclei in peripheral blood lymphocytes provides a relatively simple and inexpensive measure of chromosome damage in human populations. However, its usefulness for identifying people with relatively high frequencies of chromosome aberrations is in doubt, partly because 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 of California and by UCLA Cancer Core Grant CA 16042. Please send all correspondence to: Amos Norman, Laboratory of Biomedical and Environmental Sciences, 900 Veteran Avenue, Los Angeles, CA 90024 (U.S.A.) (213) 825-5971.
have undertaken this study, therefore, in order to discover the factors that significantly influence the MNC and thus to evaluate MNC as predictors of chromosome aberrations. Materials and methods We selected 28 adults for a learning group and 17 for a test group. We included a number of people who had been exposed to ionizing radiations or to other clastogens as part of their work and 2 patients who were receiving radiation therapy for cancer. The assignment to the two groups was random except that the two patients were assigned to the test group. One subject from whom blood samples 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)
156
From each o f the 45 blood sa mples 3 sets of 2 cultures were established by addi ng 0.3 ml of whole blood to 5 ml of medium co ntaining RPMl1640 with Hepes (Gibco) plu s 15070 newborn calf seru m plu s 1% PHA. Set A , primarily for the assay of chromosome aberrations, contained also 2 x 10- 5 M bromodeoxyuridine (BUdR) and was incubated for 54 h , the last 2 h with colcemid . Set B, primarily fo r the assay o f the frequency of first, seco nd 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 the dete rmination of MNC was incubated for 76 h without either BUdR or colcemid . All incubations were at 38° C in the dark. At the end o f the culture per iod th e cells were collected by centrifugation, resuspended for 5 min in 0.075 M KCI and fixed in suspensio n with Carn oy' s. The cells were then dropped on microscope slides and air-dried . Slide s from 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 C were stained with 1% crystal violet or 4% Gurr' s Giemsa. All slides were coded ; the y were removed at random fo r scoring. Chromosome aberrations were sco red in a min imum of 100 fir st and 100 seco nd division metaphase cells in slides from sets A and B fo r the 28 blood samples in the learning set. The slides for the test set were analyzed for chromosome aberrati on s in appro ximately 100 fir st division metaphases. Slides from sets Band C were analyzed for MNC in 4500 lymphocytes per culture. Slides from Set C were also analyzed for abnormal anaphase cells - primarily cells with lagging chro mosomes, bridges, or tripolar mitoses. The mito tic index was also sco red by counting metaphase cells in a total of 2000 lymphocyte s. Cells with small, dense nucle i and cells with irregularly sha ped nuclei were excluded fro m all assays since th ese were pre sumably dead or dying cells. The MNC were the avera ges of th e counts obtained from the two cultures in Set C. T he natural logarithm of the MNC (LMN) was found to be approximatel y normally distributed, so it was used fo r tests o f significa nce of the effect of each
measured factor on MN C. In carryi ng ou t these tests, th e number of chromosome a berr atio ns and th e freq uency of cells with abnormal anaphases were each characterized as either none (0% ) or so me (> 0%) , becau se o f th e lar ge number of donors who had zero s for each . The effect of each of the measured factors on LMN was first tested . Th e effects of presence or ab sence of aberrations, presence or a bsence of abnormal anaphase cells and sex were tested using two sa mple independent r-test s, while the effects of kin etics (per cent of second and third division metaphase cells), mitotic index and age were tested using regre ssion analysis. Since the mo st important factor influencing LMN was the presence or ab sen ce o f ab errations, the effect of each of the factors was tested aft er controlling fo r th e effect of aberrat ion s. Th e effects of kinetics, mitot ic index and age were analyzed using analysis o f cov arian ce; th e effect s of pre sence or ab sence of abnormal anaph ase cells and of sex analyzed using analysis of vari ance. One-tailed statistical tests were used . The cho ice of an appropriat e MNC cut-off for identifying peopl e with susp ected chromosome aberration s was determined using logistic regressio n a na lysis [1]. Thi s analysis models the probabilit y th at a particular person will ha ve at least one chro moso me aberration , ba sed on their characteri stics (factors). From th ese probabilities a cut-point can de derived to separate the people with a high chance of aberrations from those with a low chance of aberrations. Since multivariate normality o f the factors is not required in logistic regression ana lysis, the MNC were used. Result s
Fig. 1 sho ws the MNC distributions in the 28 learning and 17 test samples. Those samples in which cells with chromosome aberrations are found ar e speci fically indicated . As can be seen the M NC dist ributi ons vary o ver an orde r of ma gnitude among donors. Clearly, the chro mosome aberrations ar e found primarily among sa mples with large MN C. The frequency o f cells with chro-
157
CELLS WITH MN PER 4 500 CELLS 30 0
• 20 0
• 100 80 MN 60
•• •• ••
•
. 0 0
40 30
•go
20
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:.. 0
o• 000 0 0 0 0 · 0 0 000 0 0 0
o
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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 samp les without aberrati on s. the closed circles are fo r samples cont aini ng on e or more cells with chro mosome aberrations. In the learn ing set 9000 cells were assayed for MN; in the test set 4500 cells were assayed .
mosome aberrations in the 29 samples from the 28 donor s in the learning group was 0.46 per 100 firs t division metaphases (14 cells with aberrations per 3072 cells) and 0 .24 per 100 second division metaphases (9 cells with aberrations per 3 825 cells). For convenience we have co mbined the scores of first and second division metaphase cells by assuming that 100 second division cells were equal to 50 first division cells. The frequency of aberration s among the 28 samples in the learning set is shown in Table 1. The average frequency of cells with chromosome aberrations per 100 cells is 0.43 (21 cells with chromosome aberrations in 4844 cells) . The mean LMN of the 10 people with chromoso me aberrations in eithe r the fir st or second metaphase was 3.890 (standard error = 0.098), while that of the 18 people with no aberrations was 3.266 (standard error = 0.081) . Thi s difference is highly significa nt (p<0.0001) when analyzed using a two-sample r-test (one-tailed test).
The correlation between LMN and frequency of cells in second metaphase was 0.08 , while the correlation between LMN and frequency of cells in third metaphase was - 0.23 . Neither of the se cor relations are statistically signifi cant (p = 0.34 and p = 0.88 respectivel y, one-tailed tests). The correlation between LMN and mitotic index was 0.39 which is statistically significant (p = 0.02, onetailed test). There was a total of 37 abnormal anaphase cells among the 1085 scored in the 28 donors. The mean LMN of 19 donors with one or mo re abnormal anaphase cells was 3.637 (standa rd error = 0.086) while that of 9 donors with no abnormal anaphase cells was 3.175 (standard error = 0. 146). T his difference is statisticall y 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-tailed test) . The mean LMN of the 17 females was 3.585 (standard error = 0.109), while that for the 11 ma les was 3.341 (standard error = 0.126). This difference is not statistically significant (p = 0.08) when tested using a two-sample r-test (one-tai led test) . The mo st important factor influencing MNC is clearly the pre sence or ab sence of chromosome aberrations. After controlling for the effect of aberrations on LMN we found that the per cent of metaphase cells in second division was not significant (p = 0.77), the per cent of metaphase cells in third division was not significant (p = 0.82) , the mitotic inde x was no t significant (p = 0.13), the frequenc y of abnormal anaphases was not significant (p = 0.39) , and the effect o f sex was not significa nt (p = 0.30) . However, the ef fect of age on LMN remained significant (p = 0.02) . From thi s analysis the most import ant factor influencing the LMN after controlling for the effect of abe rrations was age. However , as ca n be seen in Fig. 2, the plot of LMN vs. age is srrikingly different when the data for sa mples with or without chromosome aberrations are considered separately. The LMN for the 18 people witho ut aberrations showed a statistically significant regre ssion on age (r = 0.57, p = 0.007) . Moreover this group show-
158
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Age Fig. 2. LMN vs. age. Th e open circles are for samples with no aberration s, the closed circles are for samples with aberrations. Th e regression lines are shown for the to tal set of point s and for th e ope n and closed circles alone. The slope o f the regression lines is significant ly great er than zero for the total and for the open set , but not for the closed circles.
ed, after adjusting for age that the mitotic index wa 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 donors without aberrations were significant ly related to LMN . The resulting equation wa s LMN = 2.0819 + (0.0218) Age + (0.0064) MI (I)
cla ssify the 28 people in the learning set we see that 9 of 10 with aberrations are correctly identified. We al so see that 17 of the 18 people without aberrations are also correctly cla ssified . In the test set 10 of the II people with aberrations are properly classified , and 4 of the 6 without aberrations are al so properly cla ssified . A more general approach to finding a cutoff is to use a log istic regression model [I]. This gives a prediction o f the probability of find ing 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, proportion o f metaphases in seco nd or th ird division, and abno rmal anaphases were not significa nt (p>0 .1O). Using thi s model we obtained the predictions sho wn in Table I. On the ba sis of the table it was decided to use as a cuto ff a probability o f 0.44 for findi ng at least one aberration. For the normal people this corresponds to MNC of 48; for suspect people this corresponds to MNC of 27. Applying thi s criterion to the 28 people in the learning gr o up lead s to the sa me result as before: 9 out of 10 people with aberrations correctly clas sified , and 17 out of 18 without aberrations correctly cla ssified. Applying it to the test sam ple leads to an improved classification : all 11 people with aberrations were correctly identified. Discussion
(The standard errors of the regression coefficients for age and mitotic index were 0.0063 .and 0.0032, respectively). The results o f the analysis of the 10 people with aberrations sho wed, however , no significa nt 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 chromosome aberrations we can sim ply use the data plotted in Fig. I . By in spection MNC of 38 or greater are clearly associated st ro ngly with the presence of chromoso me aberrations. Usin g th is criterion to
An impor tant reason for screening populations for ch romosome damage is to avoid the hard and expensive effo rt of assaying chromosome aberrations by conventional means in people who are not likel y to have significantly high aberration frequ encies. Our results demonstrate that the MNC are use ful fo r predicting high frequencies of aberrations. We defined 'high' as one or more cells with un stable chromosome aberratio ns per 100-200 metaphases. That is approxim ately double the average frequenc y of such cells in our learning population. Clea rly as th e cuto ff value is ra ised the
159 TABLE 1 OBSERVED CHROMOSOME ABERRATIONS PER COUNTED CELLS AND PREDICTED PROBABILITY OF AT LEAST ONE SUCH ABERRATION IN EACH SUBJECT FROM THE LOGISTIC REGRESSION MODEL No aberrations
Aberration s
Aberrations/ Subject cells
Predi ction
Aberrati on s/ Subject cells
Prediction
0/121 0/123 0/136 0/139
N N N N
0.052 0.015 0.008 0.114
11208 1/172 1/140 1/13 8
S N N S
0.999 0.035 0.589 0.930
0/ 145 0/ 164 0/ 168 0/172
N N S N
0.020 0.100 0.437 0.088
21180 2/164 3/222 21122
S S S S
0.850 0.920 0.996 0.953
0/179 0/183 0/188 0/188
N N N N
0.026 0.040 0.023 0.589
4/170 4/162
S S
0.976 0.939
0/194 0/196 01200
N N N
0.052 0.100 0.068
01216 01224 01230
N N N
0.052 0.004 0.023
The number of cells'are the sum of first division metaphase plus half the second division metaphase 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 the cutoff frequency is lower in people who we suspected, primarily fro m 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 normal group. 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 expo su re to significant levels of c1astogens than to score cells in the general populace. The average frequency of cells with chromosome aberrations in the learning group is about 4 per 1000 metaphases. This agrees reasonably well with
the estimate of 1-3 per 1000, depending on age, in a general Japanse population [8] and of 5-8 per 1000, depending on sex, in a British population [2]. The median MNC in th e learning group is about 7.7 per 1000 lymphocytes. This agrees reasonably well with values of 2-7 per 1000, depending on donor, found by Krepinsky and Heddle [4] and about 4 per 1000 reported by Hostedt [3]. We have found, using an improved technique [6] that on the average only about 50070 of the cells in our culture have divided at least once during the 76-h culture period . That means that the MNC are actually about 15 per 1000 proliferating cells. Obviously the frequency of cells with acentric chromosome fragments is too sma ll to give rise to so many cells with MN . There are at least 4 explanations for the discrepancy: (I) Nuclear debris is mistaken for MN - if
160
that were so, we would expect to find increased MNC after high radiation doses or long culture time s when more cells are dying; but the MNC actually decrease [6]. (2) Small acentric chromosome fragment s are not counted in metaphase, but the y become visible as MN because of additional DNA synthesis during culture and because of swelling in hypotonic medium [7]. (3) Acentric chromatid fra gments give rise to MN [4] - if that were so we expect some of the chromatid fragments to appear as chromosome fragments in the second division metaphase; 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% of anaphase cells are abnormal and fro m 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 o f lymphocyte proliferation in culture [4,6]. Our results indicate that neither mitotic index nor the relative frequencies of first, second and third division metaphase are useful measures of the contribution of this factor to MNC. We believe that the variabilit y 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 pre vious study [5] to be a significant factor in MNC. The MNC counts, on the average, are higher in women than in men in both studies . Although the differences are not statistically significant they may be real, reflecting the increased probability of X chromosome loss during cell division in women than in men in both studies. Although the difference s are not statistically significant they ma y be real, reflecting the increased probabilit y of X
chromosome loss during cell division in women [2]. Thus the degree of lymphocyte proliferation, age and sex can contribute to the variability of MNC. Despite this, our result s show that MNC is an excellent predictor of the pre sence of one or more cells with chromosome aberrations among one to two hundred metaphase cells. The MN assay appears useful, therefore, as a screen for selecting people for further studies of chromosome aberr ations.
References I Cox, D.R. , An alysis of Binary Data , C ha pma n and Hall, Lond on, 1977, pp . 14-29. 2 Ga lloway, S.M ., and K.E . Buckton , Aneuploidy and aging: ch rom osome stud ies on a random sample of the populat ion using Gvbandin g, Cytogenet , Cell Ge net., 20 (1978) 78- 95. 3 Hostedt , B., Micronuclei in lymph ocytes with preserved cyto plasm, A met hod for the assessment o f cytoge net ic damage in man, Mutation Res., 130 (1984) 6 1-6 5. 4 Krep insk y, A .B., and J .A . Hedd le, Micronucl ei as a rapid an d inexpensive measure of radiat ion-induced chro moso mal abe rr at ions , in: Ishihara and Sasak i (Ed s.), Rad iat ionIndu ced Chromosome Dam age in Man , Liss, New York , 1983, pp . 93- 109. 5 Norman, A ., S. Cochran, D. Bass a nd D. Roe, Effects of age, sex a nd medical X-ra ys on chr om osome dama ge, lnt. J . Radi at. Biol ., 46 (1984) 317-321. 6 Pincu, M., D. Bass and A . Norman , An improved micronuclear assay in lymphocytes, Mut at ion Res., 139 (1984) 61- 65. 7 Pin cu, M., H . Callisen and A. Norman, Micron uclear DNA distributions in human lymphocytes , Int. J. Radiat. BioI., 1985, in press. 8 Ton omu ra, A ., K. Kunikazu and F. Saito , Types and frequ encies of chromosome aberrations in perip heral lympho cytes of genera l populations, in: Ishihara a nd Sasa ki (Eds .), Radiation-Induced Chromosom e Dam age in Man , Liss, New York , 1983, pp . 605-616 .
155~160
155
MRLett. 0708
Screening human populations for chromosome aberrations* Amos Norman, Doris Bass and Denise Roe Department of Radiation Oncology, Laboratory of Biomedical and Environmental Sciences, and Jonsson Comprehensive Cancer Center, 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 relatively high frequencies of chromosome aberrations we have examined factors that influence the MNC in a learning set of blood samples obtained from 28 adults. The presence of cells with chromosome aberrations among approximately 170 metaphase cells per sample was the most important factor. Controlling for the effect of chromosome aberrations we found that age had a significant effect on MNC, but that donor sex, the mitotic index, the per cent of metaphase cells in the second or third division or the frequency of abnormal anaphase cells did not. Using logistic regression analysis we found that MNC was an excellent predictor of the presence of cells with chromosome aberrations among both the learning set and a test set of 17 additional blood samples.
The assay of micronuclei in peripheral blood lymphocytes provides a relatively simple and inexpensive measure of chromosome damage in human populations. However, its usefulness for identifying people with relatively high frequencies of chromosome aberrations is in doubt, partly because 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 of California and by UCLA Cancer Core Grant CA 16042. Please send all correspondence to: Amos Norman, Laboratory of Biomedical and Environmental Sciences, 900 Veteran Avenue, Los Angeles, CA 90024 (U.S.A.) (213) 825-5971.
have undertaken this study, therefore, in order to discover the factors that significantly influence the MNC and thus to evaluate MNC as predictors of chromosome aberrations. Materials and methods We selected 28 adults for a learning group and 17 for a test group. We included a number of people who had been exposed to ionizing radiations or to other clastogens as part of their work and 2 patients who were receiving radiation therapy for cancer. The assignment to the two groups was random except that the two patients were assigned to the test group. One subject from whom blood samples 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)
156
From each o f the 45 blood sa mples 3 sets of 2 cultures were established by addi ng 0.3 ml of whole blood to 5 ml of medium co ntaining RPMl1640 with Hepes (Gibco) plu s 15070 newborn calf seru m plu s 1% PHA. Set A , primarily for the assay of chromosome aberrations, contained also 2 x 10- 5 M bromodeoxyuridine (BUdR) and was incubated for 54 h , the last 2 h with colcemid . Set B, primarily fo r the assay o f the frequency of first, seco nd 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 the dete rmination of MNC was incubated for 76 h without either BUdR or colcemid . All incubations were at 38° C in the dark. At the end o f the culture per iod th e cells were collected by centrifugation, resuspended for 5 min in 0.075 M KCI and fixed in suspensio n with Carn oy' s. The cells were then dropped on microscope slides and air-dried . Slide s from 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 C were stained with 1% crystal violet or 4% Gurr' s Giemsa. All slides were coded ; the y were removed at random fo r scoring. Chromosome aberrations were sco red in a min imum of 100 fir st and 100 seco nd division metaphase cells in slides from sets A and B fo r the 28 blood samples in the learning set. The slides for the test set were analyzed for chromosome aberrati on s in appro ximately 100 fir st division metaphases. Slides from sets Band C were analyzed for MNC in 4500 lymphocytes per culture. Slides from Set C were also analyzed for abnormal anaphase cells - primarily cells with lagging chro mosomes, bridges, or tripolar mitoses. The mito tic index was also sco red by counting metaphase cells in a total of 2000 lymphocyte s. Cells with small, dense nucle i and cells with irregularly sha ped nuclei were excluded fro m all assays since th ese were pre sumably dead or dying cells. The MNC were the avera ges of th e counts obtained from the two cultures in Set C. T he natural logarithm of the MNC (LMN) was found to be approximatel y normally distributed, so it was used fo r tests o f significa nce of the effect of each
measured factor on MN C. In carryi ng ou t these tests, th e number of chromosome a berr atio ns and th e freq uency of cells with abnormal anaphases were each characterized as either none (0% ) or so me (> 0%) , becau se o f th e lar ge number of donors who had zero s for each . The effect of each of the measured factors on LMN was first tested . Th e effects of presence or ab sence of aberrations, presence or a bsence of abnormal anaphase cells and sex were tested using two sa mple independent r-test s, while the effects of kin etics (per cent of second and third division metaphase cells), mitotic index and age were tested using regre ssion analysis. Since the mo st important factor influencing LMN was the presence or ab sen ce o f ab errations, the effect of each of the factors was tested aft er controlling fo r th e effect of aberrat ion s. Th e effects of kinetics, mitot ic index and age were analyzed using analysis o f cov arian ce; th e effect s of pre sence or ab sence of abnormal anaph ase cells and of sex analyzed using analysis of vari ance. One-tailed statistical tests were used . The cho ice of an appropriat e MNC cut-off for identifying peopl e with susp ected chromosome aberration s was determined using logistic regressio n a na lysis [1]. Thi s analysis models the probabilit y th at a particular person will ha ve at least one chro moso me aberration , ba sed on their characteri stics (factors). From th ese probabilities a cut-point can de derived to separate the people with a high chance of aberrations from those with a low chance of aberrations. Since multivariate normality o f the factors is not required in logistic regression ana lysis, the MNC were used. Result s
Fig. 1 sho ws the MNC distributions in the 28 learning and 17 test samples. Those samples in which cells with chromosome aberrations are found ar e speci fically indicated . As can be seen the M NC dist ributi ons vary o ver an orde r of ma gnitude among donors. Clearly, the chro mosome aberrations ar e found primarily among sa mples with large MN C. The frequency o f cells with chro-
157
CELLS WITH MN PER 4 500 CELLS 30 0
• 20 0
• 100 80 MN 60
•• •• ••
•
. 0 0
40 30
•go
20
• • •.
:.. 0
o• 000 0 0 0 0 · 0 0 000 0 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 samp les without aberrati on s. the closed circles are fo r samples cont aini ng on e or more cells with chro mosome aberrations. In the learn ing set 9000 cells were assayed for MN; in the test set 4500 cells were assayed .
mosome aberrations in the 29 samples from the 28 donor s in the learning group was 0.46 per 100 firs t division metaphases (14 cells with aberrations per 3072 cells) and 0 .24 per 100 second division metaphases (9 cells with aberrations per 3 825 cells). For convenience we have co mbined the scores of first and second division metaphase cells by assuming that 100 second division cells were equal to 50 first division cells. The frequency of aberration s among the 28 samples in the learning set is shown in Table 1. The average frequency of cells with chromosome aberrations per 100 cells is 0.43 (21 cells with chromosome aberrations in 4844 cells) . The mean LMN of the 10 people with chromoso me aberrations in eithe r the fir st or second metaphase was 3.890 (standard error = 0.098), while that of the 18 people with no aberrations was 3.266 (standard error = 0.081) . Thi s difference is highly significa nt (p<0.0001) when analyzed using a two-sample r-test (one-tailed test).
The correlation between LMN and frequency of cells in second metaphase was 0.08 , while the correlation between LMN and frequency of cells in third metaphase was - 0.23 . Neither of the se cor relations are statistically signifi cant (p = 0.34 and p = 0.88 respectivel y, one-tailed tests). The correlation between LMN and mitotic index was 0.39 which is statistically significant (p = 0.02, onetailed test). There was a total of 37 abnormal anaphase cells among the 1085 scored in the 28 donors. The mean LMN of 19 donors with one or mo re abnormal anaphase cells was 3.637 (standa rd error = 0.086) while that of 9 donors with no abnormal anaphase cells was 3.175 (standard error = 0. 146). T his difference is statisticall y 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-tailed test) . The mean LMN of the 17 females was 3.585 (standard error = 0.109), while that for the 11 ma les was 3.341 (standard error = 0.126). This difference is not statistically significant (p = 0.08) when tested using a two-sample r-test (one-tai led test) . The mo st important factor influencing MNC is clearly the pre sence or ab sence of chromosome aberrations. After controlling for the effect of aberrations on LMN we found that the per cent of metaphase cells in second division was not significant (p = 0.77), the per cent of metaphase cells in third division was not significant (p = 0.82) , the mitotic inde x was no t significant (p = 0.13), the frequenc y of abnormal anaphases was not significant (p = 0.39) , and the effect o f sex was not significa nt (p = 0.30) . However, the ef fect of age on LMN remained significant (p = 0.02) . From thi s analysis the most import ant factor influencing the LMN after controlling for the effect of abe rrations was age. However , as ca n be seen in Fig. 2, the plot of LMN vs. age is srrikingly different when the data for sa mples with or without chromosome aberrations are considered separately. The LMN for the 18 people witho ut aberrations showed a statistically significant regre ssion on age (r = 0.57, p = 0.007) . Moreover this group show-
158
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Age Fig. 2. LMN vs. age. Th e open circles are for samples with no aberration s, the closed circles are for samples with aberrations. Th e regression lines are shown for the to tal set of point s and for th e ope n and closed circles alone. The slope o f the regression lines is significant ly great er than zero for the total and for the open set , but not for the closed circles.
ed, after adjusting for age that the mitotic index wa 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 donors without aberrations were significant ly related to LMN . The resulting equation wa s LMN = 2.0819 + (0.0218) Age + (0.0064) MI (I)
cla ssify the 28 people in the learning set we see that 9 of 10 with aberrations are correctly identified. We al so see that 17 of the 18 people without aberrations are also correctly cla ssified . In the test set 10 of the II people with aberrations are properly classified , and 4 of the 6 without aberrations are al so properly cla ssified . A more general approach to finding a cutoff is to use a log istic regression model [I]. This gives a prediction o f the probability of find ing 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, proportion o f metaphases in seco nd or th ird division, and abno rmal anaphases were not significa nt (p>0 .1O). Using thi s model we obtained the predictions sho wn in Table I. On the ba sis of the table it was decided to use as a cuto ff a probability o f 0.44 for findi ng at least one aberration. For the normal people this corresponds to MNC of 48; for suspect people this corresponds to MNC of 27. Applying thi s criterion to the 28 people in the learning gr o up lead s to the sa me result as before: 9 out of 10 people with aberrations correctly clas sified , and 17 out of 18 without aberrations correctly cla ssified. Applying it to the test sam ple leads to an improved classification : all 11 people with aberrations were correctly identified. Discussion
(The standard errors of the regression coefficients for age and mitotic index were 0.0063 .and 0.0032, respectively). The results o f the analysis of the 10 people with aberrations sho wed, however , no significa nt 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 chromosome aberrations we can sim ply use the data plotted in Fig. I . By in spection MNC of 38 or greater are clearly associated st ro ngly with the presence of chromoso me aberrations. Usin g th is criterion to
An impor tant reason for screening populations for ch romosome damage is to avoid the hard and expensive effo rt of assaying chromosome aberrations by conventional means in people who are not likel y to have significantly high aberration frequ encies. Our results demonstrate that the MNC are use ful fo r predicting high frequencies of aberrations. We defined 'high' as one or more cells with un stable chromosome aberratio ns per 100-200 metaphases. That is approxim ately double the average frequenc y of such cells in our learning population. Clea rly as th e cuto ff value is ra ised the
159 TABLE 1 OBSERVED CHROMOSOME ABERRATIONS PER COUNTED CELLS AND PREDICTED PROBABILITY OF AT LEAST ONE SUCH ABERRATION IN EACH SUBJECT FROM THE LOGISTIC REGRESSION MODEL No aberrations
Aberration s
Aberrations/ Subject cells
Predi ction
Aberrati on s/ Subject cells
Prediction
0/121 0/123 0/136 0/139
N N N N
0.052 0.015 0.008 0.114
11208 1/172 1/140 1/13 8
S N N S
0.999 0.035 0.589 0.930
0/ 145 0/ 164 0/ 168 0/172
N N S N
0.020 0.100 0.437 0.088
21180 2/164 3/222 21122
S S S S
0.850 0.920 0.996 0.953
0/179 0/183 0/188 0/188
N N N N
0.026 0.040 0.023 0.589
4/170 4/162
S S
0.976 0.939
0/194 0/196 01200
N N N
0.052 0.100 0.068
01216 01224 01230
N N N
0.052 0.004 0.023
The number of cells'are the sum of first division metaphase plus half the second division metaphase 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 the cutoff frequency is lower in people who we suspected, primarily fro m 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 normal group. 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 expo su re to significant levels of c1astogens than to score cells in the general populace. The average frequency of cells with chromosome aberrations in the learning group is about 4 per 1000 metaphases. This agrees reasonably well with
the estimate of 1-3 per 1000, depending on age, in a general Japanse population [8] and of 5-8 per 1000, depending on sex, in a British population [2]. The median MNC in th e learning group is about 7.7 per 1000 lymphocytes. This agrees reasonably well with values of 2-7 per 1000, depending on donor, found by Krepinsky and Heddle [4] and about 4 per 1000 reported by Hostedt [3]. We have found, using an improved technique [6] that on the average only about 50070 of the cells in our culture have divided at least once during the 76-h culture period . That means that the MNC are actually about 15 per 1000 proliferating cells. Obviously the frequency of cells with acentric chromosome fragments is too sma ll to give rise to so many cells with MN . There are at least 4 explanations for the discrepancy: (I) Nuclear debris is mistaken for MN - if
160
that were so, we would expect to find increased MNC after high radiation doses or long culture time s when more cells are dying; but the MNC actually decrease [6]. (2) Small acentric chromosome fragment s are not counted in metaphase, but the y become visible as MN because of additional DNA synthesis during culture and because of swelling in hypotonic medium [7]. (3) Acentric chromatid fra gments give rise to MN [4] - if that were so we expect some of the chromatid fragments to appear as chromosome fragments in the second division metaphase; 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% of anaphase cells are abnormal and fro m 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 o f lymphocyte proliferation in culture [4,6]. Our results indicate that neither mitotic index nor the relative frequencies of first, second and third division metaphase are useful measures of the contribution of this factor to MNC. We believe that the variabilit y 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 pre vious study [5] to be a significant factor in MNC. The MNC counts, on the average, are higher in women than in men in both studies . Although the differences are not statistically significant they may be real, reflecting the increased probability of X chromosome loss during cell division in women than in men in both studies. Although the difference s are not statistically significant they ma y be real, reflecting the increased probabilit y of X
chromosome loss during cell division in women [2]. Thus the degree of lymphocyte proliferation, age and sex can contribute to the variability of MNC. Despite this, our result s show that MNC is an excellent predictor of the pre sence of one or more cells with chromosome aberrations among one to two hundred metaphase cells. The MN assay appears useful, therefore, as a screen for selecting people for further studies of chromosome aberr ations.
References I Cox, D.R. , An alysis of Binary Data , C ha pma n and Hall, Lond on, 1977, pp . 14-29. 2 Ga lloway, S.M ., and K.E . Buckton , Aneuploidy and aging: ch rom osome stud ies on a random sample of the populat ion using Gvbandin g, Cytogenet , Cell Ge net., 20 (1978) 78- 95. 3 Hostedt , B., Micronuclei in lymph ocytes with preserved cyto plasm, A met hod for the assessment o f cytoge net ic damage in man, Mutation Res., 130 (1984) 6 1-6 5. 4 Krep insk y, A .B., and J .A . Hedd le, Micronucl ei as a rapid an d inexpensive measure of radiat ion-induced chro moso mal abe rr at ions , in: Ishihara and Sasak i (Ed s.), Rad iat ionIndu ced Chromosome Dam age in Man , Liss, New York , 1983, pp . 93- 109. 5 Norman, A ., S. Cochran, D. Bass a nd D. Roe, Effects of age, sex a nd medical X-ra ys on chr om osome dama ge, lnt. J . Radi at. Biol ., 46 (1984) 317-321. 6 Pincu, M., D. Bass and A . Norman , An improved micronuclear assay in lymphocytes, Mut at ion Res., 139 (1984) 61- 65. 7 Pin cu, M., H . Callisen and A. Norman, Micron uclear DNA distributions in human lymphocytes , Int. J. Radiat. BioI., 1985, in press. 8 Ton omu ra, A ., K. Kunikazu and F. Saito , Types and frequ encies of chromosome aberrations in perip heral lympho cytes of genera l populations, in: Ishihara a nd Sasa ki (Eds .), Radiation-Induced Chromosom e Dam age in Man , Liss, New York , 1983, pp . 605-616 .