Higher frequency of dicentrics and micronuclei in peripheral blood lymphocytes of cancer patients

Mutation Research 425 Ž1999. 1–8

Higher frequency of dicentrics and micronuclei in peripheral blood lymphocytes of cancer patients P. Venkatachalam a , Solomon F.D. Paul a , Mary N. Mohankumar a , B. Karthikeya Prabhu a , N. Gajendiran a , A. Kathiresan b, R.K. Jeevanram a

a,)

Health and Safety DiÕision, SHINE Group, Indira Gandhi Centre for Atomic Research, Kalpakkam 603 102 (TN), India b Tamilnad Hospitals, Cheran Nagar, Perumbakkam, Chennai 603 102 (TN), India Received 26 August 1998; revised 27 October 1998; accepted 11 November 1998

Abstract The presence of dicentric chromosome ŽDC. and micronuclei ŽMN. frequency in the peripheral blood lymphocytes of 25 cancer patients prior to chemo and radiotherapy and 21 healthy volunteers were studied. The overall DC and MN showed significantly higher frequency compared to those obtained in normal healthy volunteers Ž p - 0.0001.. However, among 25 patients only 15 showed a higher frequency of DC aberration, nine patients showed the presence of minutes ŽM. and seven patients showed chromatid breaks ŽChB.. The reasons for the higher frequency of aberration observed in these cancer patients are discussed in this paper. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Cancer; Chromosome; Dicentrics; Micronucleus; Virus; Lymphocyte

1. Introduction Chromosome rearrangements occurring over a period of time due to the exposure of various mutagens from environment and life style are known to cause cancer. The relationship between specific chromosomal rearrangements and the type of cancer has been studied extensively. These studies show translocation, deletion, gain or loss of chromosomes in tumour cells. The karyotype of various tumours have been reported in recent years w1,2x. However, in patients with Bloom’s syndrome, Fanconi’s anaemia and ataxia telangiectasia, the frequency of sponta) Corresponding author. Tel.: q91-4114-40352, 40362; Fax: q91-4114-40235; E-mail: [email protected]

neous chromosomal aberrations such as chromatid breaks ŽChB., gaps, dicentrics ŽDC. and sister chromatid exchanges ŽSCE. were shown to be higher than those of normal healthy individuals in peripheral blood lymphocytes w3x. Similarly higher incidence of breaks and SCE were reported in childhood leukaemia w4x and in chronic myeloid leukaemia ŽCML. patient’s w5x. Antoine et al. w6x have also reported an increased DC frequency in the peripheral blood lymphocytes of cervical cancer patients. On the contrary Diener et al. w7x and Kleinerman et al. w8x did not find any increase in chromosome aberrations in peripheral blood of cervical cancer patients and in Morbus Hodgkin’s disease. The limited but controversial reports available on the frequency of chromosome aberrations in periph-

0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 8 . 0 0 2 3 8 - 3

P. Venkatachalam et al.r Mutation Research 425 (1999) 1–8

2

eral blood lymphocytes of cancer patients prompted us to carry out a systematic study to measure the frequency of chromosomal aberrations such as DC and micronuclei ŽMN. in peripheral blood of cancer patients prior to the initiation of chemo or radiotherapy.

2. Materials and methods

average of 51.36 years. In this study among seven male patients two were smokers and among 18 female patients one had the habit of chewing tobacco. With respect to the nature of cancer, 48% had cancer of cervix, 8% had cancer of breast and oesophagus and the remaining 34% had cancer of thyroid, rectum, bladder. All the 21 of normal healthy volunteers were males and non-smokers. Their ages are given in Table 3.

2.1. Study population

2.2. Sample collection

The blood samples were obtained from a total number of 46 persons. Among these, 25 were cancer patients unexposed to any chemo or radiotherapy and the remaining 21 were from normal healthy volunteers. Details such as age, gender and type of the cancer of the 25 patients are given in Table 1. Age of the cancer patients varied between 22 and 75 with an

Blood samples of cancer patients were collected from Tamilnad Hospitals, Chennai, Tamilnadu, India and Government Arignar Anna Cancer Hospital, Kancheepuram, Tamilnadu, India. About 3 ml blood from each patient was collected in a sterile heparinised vial. The blood samples were then transported to the laboratory in an ice bath. These samples were brought back to room temperature prior to setting up of the culture.

Table 1 Type of cancer, age and gender of patients S. no. Code. no.

Age Smokerr Gender Type of Žyears. non-smoker cancer

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

63 46 52 52 68 22 55 55 47 49 55 57 56 35 45 50 54 52 41 55 50 50 50 75 50

TNH-1 TNH-2 TNH-4 TNH-5 TNH-6 TNH-7 TNH-9 TNH-10 TNH-13 TNH-15 TNH-16 TNH-17 TNH-18 TNH-19 TNH-20 TNH-21 TNH-22 KAN-1 KAN-2 KAN-3 KAN-5 KAN-6 KAN-7 KAN-8 KAN-9

NS NS NS NS NS NS NS NS NS S NS NS NS NS NS NS S NS NS NS NS NS NS T NS

F F F M M M F F F M F M F F F F M F F F F F F F F

Thyroid Breast Neck Pyriformfossa Lung NHL Breast Cervix Cervix Oesophagus Oesophagus Rectum Endometrium Cervix Cervix Cervix Bladder Cervix Cervix Cervix Cervix Cervix Cervix Check Cervix

TNH, Tamilnad Hospital; KAN, Kancheepuram; F, female; M, male; S, smoker; NS, non-smoker; T, tobacco chewing.

2.3. Culture for DC chromosome aberration The method for DC chromosome aberration assay is described elsewhere w9x. Briefly, the method is given below. To 0.5 ml of the blood sample, 5 ml RPMI-1640 ŽSigma. culture medium supplemented with 7.5% NaHCO 3 , 20% fetal calf serum, 200 mM L-glutamine, penicillin 100 unitsrml and streptomycin 100 mgrml, was added. A total of 200 ml of phytohaemagglutinin-M ŽPHA-M, Gibco, BRL. was added to the culture to initiate cell division. At 46 h, the cells were blocked at metaphase by adding colcemid to a final concentration 0.1 mgrml. The culture was incubated for another 2 h. The cells were harvested by centrifuging the sample at 400 = g and the cell pellet obtained was given hypotonic treatment with 0.45% KCl for 20 min at 378C. The cells were washed with a mixture of methanol and acetic acid Ž3:1, Carnoy’s fixative. for three times. After the first wash, the cells were suspended in a mixture of methanol and acetic acid Ž1:1. for 10 min and centrifuged. After a final wash with Carnoy’s fixative, cell pellet was suspended in a known volume of fixative Ž0.5 ml.. The cells were then cast by allowing three drops of the cell suspension to fall on a pre-cooled slide from a height of 10 cm. The slide

P. Venkatachalam et al.r Mutation Research 425 (1999) 1–8

was air dried and stained with 10% Giemsa solution ŽpH 6.8., mounted with DPX and scored for DC using light microscope ŽNikon Optiphot-2, Japan. under 10 = 100x oil immersion.

3

from a height of 1 cm, air dried and stained with Giemsa. Cells with two daughter nuclei, surrounded by cytoplasm were scored for the presence of MN according to the modified criteria of Countryman and Heddle w11x.

2.4. Culture for MN

2.5. Statistical analysis

The method for cytokinesis-blocked MN assay has been described by Fenech and Morely w10x. The procedure is given briefly below. The culture was set up the same way mentioned for DC aberration till the addition of PHA-M. Cytochalasin-B ŽSigma. at a final concentration of 3 mgr5 ml culture was added at 44 h. The cells were incubated for another 28 h at 378C. The cells were then harvested, given hypotonic treatment and washed with Carnoy’s fixative as mentioned for DC aberration. Three drops of cell suspension were allowed to fall on to a clear cooled slide

The DC, excess acentrics ŽAC., ChB, minutes ŽM. and MN frequency obtained in cancer patients were compared with those obtained from normal healthy volunteers using Welch approximation unpaired ‘t’-test. 3. Results The aberrations such as DC, AC, ChB, M and MN scored in cancer patients and healthy volunteers are given in Tables 2 and 3, respectively. Table 4

Table 2 Frequency of various chromosomal aberration and MN observed in peripheral blood of cancer patients Code no.

CS

DC

AC

M

ChB

DCr cell

CB cells

MN

MNr cell

TNH-1 TNH-2 TNH-4 TNH-5 TNH-6 TNH-7 TNH-9 TNH-10 TNH-13 TNH-15 TNH-16 TNH-17 TNH-18 TNH-19 TNH-20 TNH-21 TNH-22 KAN-1 KAN-2 KAN-3 KAN-5 KAN-6 KAN-7 KAN-8 KAN-9 Total

136 106 196 141 161 190 250 125 100 200 100 100 100 126 106 150 100 200 125 150 150 150 100 100 100 3462

1 0 0 3 0 17 3 0 0 1 3 0 1 0 0 1 2 0 2 4 5 0 1 5 1 50

1 1 2 4 3 16 2 – – 2 2 – 1 – – – – – 2 – 2 1 – 2 3 44

1 – 1 – – 3 – 2 2 1 – – – 2 – 1 – 1 – 2 – 1 – – – 17

– 1 – 3 2 3 3 – 4 2 1 2 1 2 2 1 – 2 – 3 – 2 1 5 2 42

0.0074 ND ND 0.0213 ND 0.0895 0.0120 ND ND 0.0050 0.0300 ND 0.0100 ND ND 0.0067 0.0200 ND 0.0160 0.0267 0.0333 ND 0.0100 0.0500 0.0100

1000 1000 1000 1000 1000 1000 1000 1000 500 1000 1000 1000 1000 CF 1000 CF 200 1000 1000 1000 1000 1000 1000 1000 1000 21,700

39 45 33 19 15 20 36 28 17 29 26 18 10 – 15 – 16 17 18 21 15 9 39 123 12 620

0.039 0.045 0.033 0.019 0.015 0.020 0.036 0.028 0.034 0.029 0.026 0.018 0.010 – 0.015 – 0.080 0.017 0.018 0.021 0.015 0.009 0.039 0.123 0.012

CS, Metaphase cells scored; DC, dicentric chromosome; AC, excess acentrics; M, minutes; ChB, chromatid breaks; MN, micronuclei; ND, not detected; CF, culture failed; CB cells, cytokinesis blocked binucleated cells.

P. Venkatachalam et al.r Mutation Research 425 (1999) 1–8

4

Table 3 Frequency of various chromosomal aberration and MN observed in peripheral blood of male healthy volunteers S. no.

Age

CS

DC

AC

M

ChB

DCr cell

CB cells

MN

MNr cell

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Total

46 28 38 45 35 43 32 39 37 29 34 31 30 29 28 29 33 35 37 29 28

239 727 200 372 171 295 203 239 200 192 101 628 406 250 250 250 250 250 250 250 400 6123

– 1 1 1 – 1 1 – – – – 1 – – – – – 1 – – – 7

1 – – 3 1 1 – 5 – 1 1 – 2 1 – – 2 1 – – 1 20

– 2 1 – – 1 – – – 1 – 1 – 1 1 1 – – 1 – – 10

– – – – – – – 4 – – 1 – – 1 – 1 2 – – 1 3 13

ND 0.0014 0.005 0.0027 ND 0.0034 0.0049 ND ND ND ND 0.0016 ND ND ND ND ND 0.004 ND ND ND

1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 21,000

11 13 3 21 15 10 20 19 8 10 5 3 9 9 17 14 22 14 7 12 13 255

0.011 0.013 0.003 0.021 0.015 0.010 0.020 0.019 0.008 0.010 0.005 0.003 0.009 0.009 0.017 0.014 0.022 0.014 0.007 0.012 0.013

CS, Metaphase cells scored; DC, dicentric chromosome; AC, excess acentrics; M, minutes; ChB, chromatid breaks; MN, micronuclei; ND, not detected; CB cells, cytokinesis blocked binucleated cells.

compares the average frequencies of these aberrations observed in cancer patients to those obtained in healthy volunteers. The increase in the aberration frequencies in cancer patients compared to healthy volunteers were highly significant except for M, where it was only significantly higher. Among these aberrations DC frequency was nearly 13 times higher compared to healthy volunteers as other aberrations were only 2 to 5 times higher in cancer patients. The average DC frequency in patients with cervical canTable 4 Comparison of various chromosomal aberrations and MN observed between healthy individuals and cancer patients Type of aberration

Healthy individuals

Cancer patients

p-value

Dicentricsrcell Acentricsrcell Ch. breakrcell Minutesrcell MNrcell

0.0011"0.0004 0.0033"0.0007 0.0021"0.0006 0.0016"0.0005 0.0121"0.0008

0.0144"0.0020 0.0127"0.0019 0.0121"0.0019 0.0049"0.0009 0.0289"0.0018

- 0.0001 - 0.0001 - 0.0001 - 0.01 - 0.0001

cer Ž0.0113 " 0.0026. alone showed a significant increase compared to that of healthy volunteers Ž p 0.005, Table 5.. The DC frequency Ž0.0174 " 0.0031. observed in patients with cancer other than the cervix, also showed a significant increase compared to that of healthy volunteers Ž p - 0.0001.. In one patient ŽTNH-7. remarkably high DC frequency was observed Ž0.089rcell.. In this patient among 17 DC, a single cell alone had 10 DC Žone pentacentric, one tricentric, four DC, Fig. 1.. The average DC frequency Ž0.0101 " 0.0017. after excluding the result obtained from this sample, was significantly Table 5 Frequency of DC in healthy individuals compared to that of cervical cancer and patients other than cervical cancer Subjects studied

Metaphase Dicentrics scored frequency

Healthy individuals 6123 Cervical cancer patients 1682 Other cancer patients 1880

p-value

0.0011"0.0004 0.0113"0.0026 - 0.005 0.0174"0.0031 - 0.0001

P. Venkatachalam et al.r Mutation Research 425 (1999) 1–8

5

Fig. 1. Multiple chromosome aberration observed in a lymphocyte of a non-hodgkins lymphoma patient prior to therapy.

higher compared to that of healthy volunteers Ž p 0.0001.. The MN frequency observed in cancer patients was also compared to that of age-matched healthy individuals. The MN frequency in age-matched control was obtained using the equation Y s 0.00029 X q 0.00171, where Y is the frequency of MN and X is the age of the donor. This equation was generated from the results obtained in this laboratory w12x. The

frequency of MN in cancer patients was significantly higher than that of age-matched controls Ž p 0.0001.. 4. Discussion Cytogenetic methods have been used extensively for monitoring base-line chromosome aberration frequency in population unexposed w13,14x, and ex-

6

P. Venkatachalam et al.r Mutation Research 425 (1999) 1–8

posed to low level radiation w15,16x. This technique has also been used to monitor population exposed to industrial chemicals w17,18x and chemotherapeutic agents w5,19,20x. With respect to the increase in DC, MN and translocation frequency in the peripheral blood lymphocytes of cancer patients compared to those of healthy volunteers, it was suspected that these patients had undergone radiotherapy or chemotherapy in the past. The medical records of these patients, however, had indicated that they were not given any of these treatments prior to the collection of blood samples. If the patients had undergone radiotherapy, the frequency of DC would have been much higher than that observed in the present study. It has been reported that chemotherapeutic agents like bleomycin w19x, cyclophosphamide w17x and chemicals like ethylene oxide w21x induce chromosome aberrations. However, these patients were not treated any of these drugs, as evidenced from medical records. Perhaps increase in chromosomal aberrations in blood lymphocytes of cancer patients could have been due to the exposure of certain mutagenic agents capable of inducing dicentric aberrations. Similar to our results, Antoine et al. w6x have shown significantly higher frequencies of DC in peripheral blood lymphocytes of patients with cervical cancer. Honeycomb w5x has shown AC and ChB in patients with CML. Aronson et al. w4x also have observed AC and ChB in the blood of children with leukaemia. At the same time Diener et al. w7x, Kleinerman et al. w8x, and Catena et al. w22x did not observe any increase in the frequency of DC in blood lymphocytes of Morbus Hodgkin lymphoma, cervical cancer and adenocarcinoma, respectively. In the present study, it has been observed that even though the overall DC frequency observed in cancer patients was higher compared to that of normal individuals, only 15 out of 25 cancer patients had shown a higher frequency of DC. To confirm the results further, the frequency of MN was measured. A correlation between DC and MN was carried out in patients who had detectable DC frequency. This was studied only in 13 out of 15 patients who had detectable DC frequency. The two patients who were not included for the study were TNH-7 and TNH-21. In the case of TNH-7 the number of DC was more because of the presence of single rogue cell and was

not due to the uniform increase in aberration frequency among metaphase cells scored. Since the increase in DC was due to the presence of single rogue cell, it was not included in the study as it was felt that this may not result in corresponding increase in MN. TNH-21 was not included as the culture for MN had failed. The correlation study carried out in the remaining 13 patients showed a ‘r ’ value of 0.55 ŽFig. 2.. It is not known whether a better correlation would have be seen if the number of metaphases and binucleated cells scored were large as the total number of DC observed in cancer patients was nearly 12 times higher than that of observed in normal subjects, whereas in the case of MN it was only 2.5 times higher in cancer patients compared to that of observed in normal subjects. With respect to MN frequency in cancer patients the comparison was made to that obtained in age matched healthy individuals as it is known that MN frequency increases with age. However, Fenech et al. w23x and Gatenberg

Fig. 2. Correlation between DC and MN observed in cancer patients Ž r s 0.55..

P. Venkatachalam et al.r Mutation Research 425 (1999) 1–8

et al. w24x have shown that frequency of MN in cancer patients was same as that of normal persons. The exact reason for the higher DC frequency observed in 15 out of 25 cancer patients compared to that of normal subjects is not known. However, one can think that it could be due to chromosome instability as it has been reported that there exist genomic instability at low frequency in peripheral blood lymphocytes in individuals affected by several types of cancer w25,26x. An increased frequency of structural chromosomal aberrations has also been reported in families with high incidence of cancer w27x. Similar to DC and MN, the frequency of acentrics, ChB and M are also higher in cancer patients which could either due to chromosome instability or due to some agents such as virus inducing these aberrations. The association between virus and cancer is well established. The presence of Hepatitis-B virus in primary hepatocellular carcinoma, Epstein Bar virus in Burkitt’s lymphoma, human T cell lymphoma virus in adult T cell lymphoma and leukaemia and human papilloma virus in cervical cancer indicate the involvement of these viruses in these cancers w2x. It is known that certain viruses have the capacity to induce a large number of DC, tricentrics and quadracentrics resulting in the formation of a rogue cell w28x. In the present study, also one patient ŽTNH-7. had a cell with multicentric aberration ŽRogue cell, Fig. 1. indicating the possibility on the interaction of such virus. Though it is well known that viruses play an important role in the induction of cancer and induce DC, the cytogenetic analysis was carried out to look for specific translocation in a given tumour cell w2x. Even if one looks for the presence of DC the chances of seeing such aberrations are remote, as they are unstable in nature and hence eliminated during the fast division of tumour cells. Whereas translocations remain in tumour cells as these aberrations are of stable in nature. Since there is a strong relationship between specific translocation and the type of cancer most efforts had gone to identify specific translocations in tumour cells and not for other aberrations in blood lymphocytes. It is believed that tumour triggering agents may interact with cells of the body including blood cells causing chromosome aberration. Thus, one may expect presence of DC in blood lymphocytes other than the organ which has turned malignant.

7

5. Conclusion The study shows that there is an increase in the frequency of aberrations in the peripheral blood lymphocytes of cancer patients. It is believed that this increase may be due to either chromosome instability or some agents such as virus or the combination of both factors.

Acknowledgements Authors wish to thank Dr. S.M. Lee, Director, SHINE Group, Shri L.V. Krishnan, Former Director, Safety Research and Health Physics Group, Shri A.R. Sundararajan, Former Associate Director, Safety Research and Health Physics Group, Dr. A. Natarajan, Head, Health and Safety Division, IGCAR, Kalpakkam, for their encouragement in carrying out this work. The authors are grateful to Dr. M. Veluswamy, CMD, Tamilnad Hospitals, Chennai, for granting permission to irradiate the blood samples. Thanks are due to Dr. A. Vaitheeswaran and J. Thirulogachander, Medical Physicts, Tamilnad Hospitals for their help during calibration of dose and blood sample irradiation.

References w1x E. Solomon, J. Borrow, A.D. Goddard, Chromosome aberration and cancer, Science 214 Ž1991. 1153–1159. w2x A.A. Sandberg, S. Kakati, Chromosome alterations and oncogenes in human neoplasia, In: T. Sharma ŽEd.., Trends in Chromosome Research, Springer-Verlag, 1991, pp. 190– 204. w3x R.D Wegner, Cytogenetic characterisation of human disorders with increased spontaneous and induced chromosomal instability. In: R.C. Sabti, G. Obe ŽEds.., Eukaryotic Chromosomes, Narosa Publishing House, 1991, pp. 126–152. w4x M.M. Aronson, R.C. Miller, B.B. Hill, W.W. Nicholes, A.T. Meadowes, Acute and long term cytogenetic effects of treatment in childhood cancer. Sister chromatid exchanges and chromosome aberrations, Mutat. Res. 92 Ž1982. 291–307. w5x J. Honeycomb, The cytogenetic effects of busulphan therapy on the Ph1-positive cells and lymphocytes from patients with chronic myeloid leukaemia, Mutat. Res. 81 Ž1981. 81–102. w6x J.L. Antoine, G.B. Gerber, A. Leonard, F. Richard, A. Wambersie, Chromosome aberration in patients treated with telecobalt therapy for mammary carcinoma, Radiat. Res. 86 Ž1981. 171–177.

8

P. Venkatachalam et al.r Mutation Research 425 (1999) 1–8

w7x A. Diener, G. Stephen, Th. Vogl, J. Lissner, The induction of chromosome aberrations during the course of radiation therapy for Morbus hodgkin, Radiat. Res. 114 Ž1988. 528–536. w8x R.A. Kleinerman, L.G. Littelfield, R.E. Tarone, S.G. Machado, M. Blettner, L.J. Peters, J.D. Boice, Chromosome aberrations in peripheral lymphocytes and radiation dose to active bone marrow in patients treated for cancer of the cervix, Radiat. Res. 119 Ž1989. 176–190. w9x IAEA Report, Biological dosimetry, Chromosome aberration analysis for dose assessment, In: Technical Report Series No. 260, IAEA, Vienna, 1986, pp. 1–69. w10x M.A. Fenech, A.A. Morely, Measurement of micronuclei in lymphocytes, Mutat. Res. 147 Ž1985. 29–36. w11x P.I. Countryman, J.A. Heddle, The production of MN from chromosome aberrations in irradiated culture of human lymphocytes, Mutat. Res. 41 Ž1976. 321–332. w12x S.F.D. Paul, Comparison of dose–response curves obtained for micronucleus and dicentric frequency in biological dosimetry, PhD thesis, Submitted to University of Madras, Chennai, 1996. w13x M.A. Bender, A.A. Awa, A.L. Brooks, H.J. Evans, P.G. Groer, L.G. Littlefield, C. Pereira, R.J. Preston, B.W. Wacholz, Current status of cytogenetic procedures to detect and quantify previous exposures to radiation, Mutat. Res. 196 Ž1988. 103–159. w14x D. Anderson, P.C. Jenkinson, R.S. Dewdney, J. Francis, P. Godbert, K.R. Butterworth, Chromosome aberrations, mitogen-induced blastogenesis and proliferative rate index in peripheral lymphocytes from 106 control individuals of the UK population, Mutat. Res. 204 Ž1988. 407–420. w15x D.C. Lloyd, R.J. Purrott, E.J. Reeder, The incidence of unstable chromosome aberrations in peripheral blood lymphocytes from unirradiated and occupationally exposed people, Mutat. Res. 72 Ž1980. 523–532. w16x P. Bigatti, L. Lamberti, G. Ardito, F. Armellino, Cytogenetic monitoring of hospital workers exposed to low-level ionising radiation, Mutat. Res. 204 Ž1988. 343–347. w17x M. Sorsa, L. Pyy, S. Salomaa, L. Nylund, J.W. Yager, Biological and environmental and occupational exposure to cyclophosphamide in industry and hospitals, Mutat. Res. 204 Ž1988. 465–479.

w18x G. De Jong, N.J. van Sittert, A.T. Natarajan, Cytogenetic monitoring of industrial populations potentially exposed to genotoxic chemicals and of control populations, Mutat. Res. 204 Ž1988. 451–464. w19x G. Obe, W. Matthiessen, D. Gobel, Chromosomal aberration in the peripheral lymphocytes of cancer patients treated with high-energy electrons and bleomycin, Mutat. Res. 81 Ž1981. 133–141. w20x F. Sarto, R. Tomanin, L. Giacomelli, A. Canova, F. Raimondi, C. Ghiotto, M.V. Fiorentino, Evaluation of chromosomal aberration in lymphocytes and micronuclei in lymphocytes, oral mucosa and hair root cells of patients under antiblastic therapy, Mutat. Res. 228 Ž1990. 157–169. w21x S.M. Galloway, P.K. Berry, W.W. Nichols, S.R. Wolman, K.A. Soper, P.D. Stolley, P. Archer, Chromosome aberrations in individuals occupationally exposed to ethylene oxide and in a large control population, Mutat. Res. 170 Ž1986. 55–74. w22x C. Catena, D. Conti, P. Parasacchi, P. Marenco, B. Bortolato, M. Boutturi, M. Leoni, M. Portaluri, E. Righ, Micronuclei in cytokinesis blocked lymphocytes may predict patient response to radiotherapy, Int. J. Radiat. Biol. 70 Ž1996. 301– 308. w23x M. Fenech, J. Denham, W. Francis, A. Morely, Micronuclei in cytokinesis blocked lymphocytes of cancer patients following fractionated partial-body radiotherapy, Int. J. Radiat. Biol. 57 Ž1990. 373–383. w24x H.W. Gantenberg, K. Wuttke, C. Streffer, W.U. Muller, Micronuclei in human lymphocytes irradiated in vitro or in vivo, Radiat. Res. 128 Ž1991. 276–281. w25x M.R. Thompson, R. McInnes, H. Willard, Genetics in Medicine, N.B., Philadelphia, USA, 1991. w26x P. Venole, B. Tedeschi, D. Caporossi, B. Nicoletti, A study on lymphocytes of neuroblastoma patients, Cancer Genet. Cytogenet. 36 Ž1988. 13–23. w27x P. Venole, B. Tedeschi, B. Nicoletti, Fragile site induction by aphidicolin may be increased neuroblastoma patients, Cancer Genet. Cytogenet. 36 Ž1988. 13–23. w28x V.J. Neel, A.A. Awa, The saga of Rogue lymphocytes, RERF Update 5 Ž1993. 7–9.