- Email: [email protected]
Ageing in vivo does not influence micronucleus induction in human lymphocytes by x-irradiation
Mechanisms of Ageing and Development, 39 (1987) 113-119 Elsevier Scientific Publishers Ireland Ltd,
113
AGEING I N VIVO DOES NOT INFLUENCE MICRONUCLEUS INDUCTION IN HUMAN LYMPHOCYTES BY X-IRRADIATION
MICHAEL FENECH and ALEXANDER A. MORLEY Department of Haematoiogy, Flinders University and Medical Centre, Bedford Park, South Australia 5042 (Australia) (Received August 18th, 1986)
SUMMARY To test the hypothesis that the age-related decline in the stability of the genome is the consequence of an increasing deficiency in DNA repair we compared the extent of chromosome damage, after X.irradiation, in lymphocytes from healthy young and old individuals, using the expression of micronuclei as the end-point. Micronuclei have been shown to increase in number with age and they were enumerated using a recently described and improved technique which involves measurement of micronuclei in cells that were blocked from performing cytokinesis. The level of X-ray-induced micronuelei in cytokinesis.blocked cells, after exposure to 75 cGy and 150 cGy, was measured by subtracting the base-line micronucleus frequency in the control unirradiated cultures from the observed micronucleus frequency in the irradiated cultures. There was no difference between the results for the young and old subjects thus indicating that cells from the aged subjects do not exhibit increased chromosomal instability following X-irradiation. These results suggest that repair of those DNA lesions that lead to chromosome breakage does not decline with age. Key words: Cytokinesis-block micronucleus assay; Ageing; Human lymphocytes; Xirradiation
INTRODUCTION In vivo ageing may be the result of the accumulation of damage to important molecules such as DNA. Evidence for increase of DNA damage with age is provided both by those studies in which measurements of point mutation frequency are made [1,2] and studies which use classical cytogenetic techniques to measure gross chromosomal abnormalities [3,4]. An increasingly used alternative to classical cytogenetic techniques is the measurement of micronuclei (MN) in dividing cells. Micronuclei originate from acentric chromosome fragments or whole chromosomes that are not included in the daughter
0047-6374/87/$03.50 Printed and Published in Ireland
© 1987 ElsevierScientificPublishers Ireland Ltd.
114 nuclei after nuclear division and they provide an estimate of both chromosome breaks and whole chromosome loss. We have developed an improved micronucleus method, the cytokinesis-block (CB) method, in which micronuclei can be scored only in the those cells that have completed one nuclear division following stimulation by mitogen. The latter cells are recognised by their binucleate appearance (Fig. 1) after inhibition of cytokinesis by cytochalasin-B [5-7].With this method we have shown that the frequency of spontaneously expressed MN increases exponentially with age and this observation provided further evidence for the deterioration of the genome with in vivo ageing [5]. DNA repair systems are responsible for maintaining the integrity of DNA following spontaneous or mutagen-induced damage to DNA and it is possible that the age-related decline in the stability of the genome could be the consequence of increasing deficiency in DNA repair with age. To investigate this possibility we used the CB technique to determine if lymphocytes from elderly individuals developed more chromosomal damage after X-irradiation than did lymphocytes from young individuals. MATERIALS AND METHODS Cells and tissue culture
Blood was obtained from I 1 healthy young (mean age + I S.E. = 22.9 -+ 0.6) and 15 healthy elderly (mean age -+ 1 S.E. = 70.1 -+ 1.5) subjects who were non-smokers.
Fig. 1. Cytokinesis-blockedcells × 400 magnification(micronucleus,MN).
115 Lymphocytes were separated from whole blood on sodium metrizoate/Ficoll gradients (Nyegaard & Co. AS-Oslo), washed twice in Hank's balanced salt solution and resuspended in McCoy's modified medium 5A containing 15% heat inactivated foetal calf serum. The lymphocytes were cultured in l-ml aliquots in plastic tissue culture tubes (Sterilin, U.K.) at a concentration of 0.5 X 106. The lymphocytes were stimulated with phytohaemagglutinin (5 tzg/ml Burroughs Wellcome reagent-grade) to transform and divide and cultured at 37°C in a humidified atmosphere containing 10% CO2.
X-irradiation Freshly isolated lymphocytes were exposed to X-rays at room temperature immediately before addition of phytohaemagglutinin. X-rays were delivered by a Phillips RT 100 at a rate of 400 cGy/min at 6 mA, 100 kV and 1.7 mm A1 filtration. The dose rate was measured using a Siemens dosimeter. Cytokinesis-block micronucleus assay Cytochalasin-B (Cyt-B, Sigma) was made up as a stock solution in dimethyl sulphoxide at a concentration of 2 mg/ml, divided into small portions and stored at --40°(2. The stock solution of Cyt-B was thawed, diluted in medium and added 44 h after the commencement of the culture to give a final concentration of 3/ag/ml. The culture was stopped at 72 h and a minimum of two cytocentrifuge preparations of cells on slides' were made per sample. The slides were stained with Jenner-Giemsa and micronuclei were scored in binucleate CB cells using XIO00 magnification. In this study CB cells were scored until 45 micronuclei were observed and the MN frequency determined. Consequently the number of CB cells scored was inversely related to the MN frequency and varied between 1000 and 9000 CB ceils for the unirradiated control cultures and between 300 and 900 CB cells for the irradiated cultures. This method has the advantage that the precision of the estimate is approximately the same both for cultures with few micronuclei and cultures with many micronuclei. Published criteria were applied for the identification of micronuclei [8]. Data analysis The Mann-Whitney U-test was used to compare micronucleus frequencies of young and old subjects for both control and irradiated cultures. RESULTS The numbers of MN in the individuals studied are plotted together with current data from our laboratory to show the correlation between MN frequency and age (Fig. 2). The results in the present study correspond well with our earlier observations which had indicated that there is an exponential increase in base-line MN frequency with age [5]. The mean for the values obtained for the elderly subjects was four times that observed for the young subjects (Table I), but there was considerably more scatter in the results for the aged individuals.
116 N : 96
120' (j
Z 0 •
60
•
•,
40
oo
20
0
2"o
"
4"o
"
do AGE (years)
Fig. 2. The effect of age on base-line MN frequencies obtained by the CB method. Circles represent current baseqine data. Triangles represent baseqine data for subjects studied. The number of CB cells scored varied between 1000 and 9000 depending on the bin frequency. To compare the response of the lymphocytes from the two groups to X-irradiation the level of induced micronuclei after exposure to 75 cGy and 150 cGy was measured for each individual by subtracting the base-line frequency in the unirradiated control cultures from the frequency observed for the irradiated cultures. The results obtained (Table I and Fig. 3) indicate that there was no difference between young and old individuals in the number of MN induced by X-rays. This suggested that lymphocytes from old subjects are as proficient as lymphocytes from young subjects in the repair of those DNA lesions that lead to chromosome breakage. DISCUSSION AND CONCLUSION The CB micronucleus method provides a precise measure of chromosome damage because, unlike conventional micronucleus assays, it enables micronuclei to be scored
117
TABLE I BASE-LINE AND X-RAY INDUCED MICRONUCLEUS FREQUENCIES (MEAN ± 1 S.E.) IN YOUNG AND ELDERLY SUBJECTS.
MN/I O00 CB cells Base4ine
Induced after 75 cGy
Induced after 150 cGy
Young
10.9 ±1.1 (iV= 11)
82.1 ±6.5 (iV = 11)
203.5 +15.3 (iV = 9)
Old
44.3 ± 5.3 a
82.2 ± 5.7 b
222.0 -L-_8.4 c
( N = 15)
(N= 15)
( N = 13)
ap < 0.01.
bp > 0.I0. cP > 0.I0.
(n ,.J
36
0
32,
,.I W m
L)
28' OlD
¢
24,
~"
20,
i
16.
~
12,
z
8
4
!
.1. •
$
0 i •
tt,
YOUNG
•
OLD
75 cGy
•
•
YOUNG
OLD
150 cGy
Fig. 3. The frequency of X-ray induced MN (i.e. observed minus base-line) in lymphocytes from young and elderly subjects. The number of CB cells scored varied between 300 and 900 depending on the MN frequency.
118
only in those cells that have completed one nuclear division. This method is thus more precise than the conventional micronucleus assay which is affected by the variable response to phytohaemagglutinin shown by lymphocytes from different individuals and even by lymphocytes from the same individual under varying culture conditions. The rapidity of the CB method makes it a more sensitive technique than chromosome analysis because thousands of CB cells can be scored. This sensitivity was demonstrated in our previous studies in which it was shown that in vitro exposures to 5 cGy were unequivocally detected by the CB method whereas standard metaphase analysis procedures do not enable the detection of exposures of less than 20 c Gy [5]. The CB micronucleus method is therefore superior to chromosome analysis for detection of small differences in the extent of chromosome damage induced by ionising radiation. The results presented in this paper show that there is no difference between lymphocytes of young and old subjects in the number of X-ray-induced MN. They therefore do not support the hypothesis that cells from aged subjects exhibit increased chromosomal instability following exposure to ionising radiation. This conclusion is in contrast with the results of Dutkowski et al. [9] who demonstrated by using conventional chromosome analysis that chromosomes in lymphocytes from elderly subjects express more damage after exposure and incorporation of tritiated thymidine for 24 h prior to analysis than do chromosomes from young subjects. A direct comparison with the results of Dutkowski et al. is difficult owing to the chronic nature of exposure in their experiments, the different quality of radiation used and the unknown effects at the molecular level of tritiated thymidine incorporated in DNA. Consequently the contrasting conclusions may simply be explained by a difference in cellular response with age to the different radiobiological effects of an acute dose of X-irradiation and chronic exposure to tritiated thymidine. It is currently held that chromosome breaks induced by ionising radiation are the result of double-strand breaks (DSBs) in DNA [10,11]. The results described therefore suggest that DSB repair does not decline with in vivo ageing. There is other evidence that ageing in vivo [12] or in vitro [13] does not alter the kinetics of DNA strand break repair after X-irradiation. In their study on the rat Neidmuller et al. [14] found no change in DSB repair with increasing age although there was a slight reduction in single strand break repair and a marked increase in the extent of excision repair. Kutlaca et al. [15] observed that lymphocytes from aged subjects are approximately twice as sensitive to the lethal effects of X-irradiation as lymphocytes from young individuals. DSBs are commonly regarded as the lesion principally responsible for lethality [16,17] but, since repair of DSBs does not seem to decline with age, it is likely that a class of lesion other than DSBs may be the important lethal lesion and that it is the repair of this other lesion that declines with age. REFERENCES 1 K.J. Trainor, D. Wigmore, A. Chrysostomou, J. Dempsey, R. Seshadri and A.A. Morley, Mutation frequency in human lymphocytes increases with age. Mech. Ageing Dev,, 27 (1984) 83-86.
119 2 Vijaylaxmi and HJ. Evans, Measurement of spontaneous and X-irradiation induced 6-thioguanineresistant human blood lymphocytes using a T-cell cloning technique. Mutat. Res., 125 (1984) 87-94. 3 T.A. Lezhava, Heterochromatisation as a key factor in ageing. Mech. Ageing Dev., 28 (1984) 279-287. 4 E.L. Schneider, Cytogenetics of ageing. In E.L. Schneider (ed.), The Genetics o f Ageing, Plenum, New York, 1979, pp. 27-52. 5 M. Fenech and A.A. Morley, Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation. Mutat. Res., 161 (1986) 193-198. 6 M. Fenech and A.A. Motley, Solutions to the kinetic problem in the micronucleus assay. Cytobios, 42 (1985) 233-246. 7 M. Fenech and A.A. Morley, Measurement of micronuclei in human lymphocytes. Murat. Res., 147 (1985) 29-36. 8 J.A. Heddle, B.L. Constance, E.F. Saunders and D. Benz, Sensitivity to five mutagens in Fanconi's anaemia as measured by the micronucleus method, Cancer Res., 38 (1978) 2983-2988. 9 R.T. Dutkowski, R, Lesh, L. Staiano-Coico, H. Thaler, G. Darlington and M.E. Weksler, Increased chromosomal instability in lymphocytes from elderly humans. Murat. Res., 149 (1985) 505-512. 10 H.J. Evans, Molecular mechanisms in the induction of chromosome aberrarions. In D.Scott, B.A. Bridges and F.H. Sobels (eds.), Progress in Genetic Toxicology, Elsevier/North Holland, 1977, pp. 57-74. 11 K.E. Chadwick and H.P. Leenhouts, The Molecular Theory o f Radiation Biology Springer-Verlag New York, 1981, pp. 51-62. 12 D.R. Turner, V.C. Grfffith and A.A. Morley, Ageing in vivo does not alter the kinetics of DNA strand break repair. Mech. Ageing Dev., 19 (1982) 325-331. 13 F. Suzuki, E. Watanabe and M. Horikawa, Repair of X-ray damage in ageing human diploid cells. Exp. Cell Res., 127 (1980) 299-307. 14 H. Neidmuller, G. Hofecker and M. Skalicky, Changes of DNA repair mechanisms during the ageing of the rat. Mech. AgeingDev., 29 (1985) 221-238. 15 R. Kutlaca, R. Seshadti and A.A. Morley, Effect of age on sensitivity of human lymphocytes to radiation, a brief note.Mech. AgeingDev., 19 (1982) 97-101. 16 P.E. Bryant, Enzymatic restriction of mammalian cell DNA: evidence for double-strand breaks as potentially lethal lesions. Int. Z Radiat. Biol., 48H) (1985) 55-60. 17 I.R. Radford, The level of induced DNA double-strand breakage correlates with cell killing after X-irradiation. Int. J. Radiat. Biol., 48(1) (1985) 45-54.
113
AGEING I N VIVO DOES NOT INFLUENCE MICRONUCLEUS INDUCTION IN HUMAN LYMPHOCYTES BY X-IRRADIATION
MICHAEL FENECH and ALEXANDER A. MORLEY Department of Haematoiogy, Flinders University and Medical Centre, Bedford Park, South Australia 5042 (Australia) (Received August 18th, 1986)
SUMMARY To test the hypothesis that the age-related decline in the stability of the genome is the consequence of an increasing deficiency in DNA repair we compared the extent of chromosome damage, after X.irradiation, in lymphocytes from healthy young and old individuals, using the expression of micronuclei as the end-point. Micronuclei have been shown to increase in number with age and they were enumerated using a recently described and improved technique which involves measurement of micronuclei in cells that were blocked from performing cytokinesis. The level of X-ray-induced micronuelei in cytokinesis.blocked cells, after exposure to 75 cGy and 150 cGy, was measured by subtracting the base-line micronucleus frequency in the control unirradiated cultures from the observed micronucleus frequency in the irradiated cultures. There was no difference between the results for the young and old subjects thus indicating that cells from the aged subjects do not exhibit increased chromosomal instability following X-irradiation. These results suggest that repair of those DNA lesions that lead to chromosome breakage does not decline with age. Key words: Cytokinesis-block micronucleus assay; Ageing; Human lymphocytes; Xirradiation
INTRODUCTION In vivo ageing may be the result of the accumulation of damage to important molecules such as DNA. Evidence for increase of DNA damage with age is provided both by those studies in which measurements of point mutation frequency are made [1,2] and studies which use classical cytogenetic techniques to measure gross chromosomal abnormalities [3,4]. An increasingly used alternative to classical cytogenetic techniques is the measurement of micronuclei (MN) in dividing cells. Micronuclei originate from acentric chromosome fragments or whole chromosomes that are not included in the daughter
0047-6374/87/$03.50 Printed and Published in Ireland
© 1987 ElsevierScientificPublishers Ireland Ltd.
114 nuclei after nuclear division and they provide an estimate of both chromosome breaks and whole chromosome loss. We have developed an improved micronucleus method, the cytokinesis-block (CB) method, in which micronuclei can be scored only in the those cells that have completed one nuclear division following stimulation by mitogen. The latter cells are recognised by their binucleate appearance (Fig. 1) after inhibition of cytokinesis by cytochalasin-B [5-7].With this method we have shown that the frequency of spontaneously expressed MN increases exponentially with age and this observation provided further evidence for the deterioration of the genome with in vivo ageing [5]. DNA repair systems are responsible for maintaining the integrity of DNA following spontaneous or mutagen-induced damage to DNA and it is possible that the age-related decline in the stability of the genome could be the consequence of increasing deficiency in DNA repair with age. To investigate this possibility we used the CB technique to determine if lymphocytes from elderly individuals developed more chromosomal damage after X-irradiation than did lymphocytes from young individuals. MATERIALS AND METHODS Cells and tissue culture
Blood was obtained from I 1 healthy young (mean age + I S.E. = 22.9 -+ 0.6) and 15 healthy elderly (mean age -+ 1 S.E. = 70.1 -+ 1.5) subjects who were non-smokers.
Fig. 1. Cytokinesis-blockedcells × 400 magnification(micronucleus,MN).
115 Lymphocytes were separated from whole blood on sodium metrizoate/Ficoll gradients (Nyegaard & Co. AS-Oslo), washed twice in Hank's balanced salt solution and resuspended in McCoy's modified medium 5A containing 15% heat inactivated foetal calf serum. The lymphocytes were cultured in l-ml aliquots in plastic tissue culture tubes (Sterilin, U.K.) at a concentration of 0.5 X 106. The lymphocytes were stimulated with phytohaemagglutinin (5 tzg/ml Burroughs Wellcome reagent-grade) to transform and divide and cultured at 37°C in a humidified atmosphere containing 10% CO2.
X-irradiation Freshly isolated lymphocytes were exposed to X-rays at room temperature immediately before addition of phytohaemagglutinin. X-rays were delivered by a Phillips RT 100 at a rate of 400 cGy/min at 6 mA, 100 kV and 1.7 mm A1 filtration. The dose rate was measured using a Siemens dosimeter. Cytokinesis-block micronucleus assay Cytochalasin-B (Cyt-B, Sigma) was made up as a stock solution in dimethyl sulphoxide at a concentration of 2 mg/ml, divided into small portions and stored at --40°(2. The stock solution of Cyt-B was thawed, diluted in medium and added 44 h after the commencement of the culture to give a final concentration of 3/ag/ml. The culture was stopped at 72 h and a minimum of two cytocentrifuge preparations of cells on slides' were made per sample. The slides were stained with Jenner-Giemsa and micronuclei were scored in binucleate CB cells using XIO00 magnification. In this study CB cells were scored until 45 micronuclei were observed and the MN frequency determined. Consequently the number of CB cells scored was inversely related to the MN frequency and varied between 1000 and 9000 CB ceils for the unirradiated control cultures and between 300 and 900 CB cells for the irradiated cultures. This method has the advantage that the precision of the estimate is approximately the same both for cultures with few micronuclei and cultures with many micronuclei. Published criteria were applied for the identification of micronuclei [8]. Data analysis The Mann-Whitney U-test was used to compare micronucleus frequencies of young and old subjects for both control and irradiated cultures. RESULTS The numbers of MN in the individuals studied are plotted together with current data from our laboratory to show the correlation between MN frequency and age (Fig. 2). The results in the present study correspond well with our earlier observations which had indicated that there is an exponential increase in base-line MN frequency with age [5]. The mean for the values obtained for the elderly subjects was four times that observed for the young subjects (Table I), but there was considerably more scatter in the results for the aged individuals.
116 N : 96
120' (j
Z 0 •
60
•
•,
40
oo
20
0
2"o
"
4"o
"
do AGE (years)
Fig. 2. The effect of age on base-line MN frequencies obtained by the CB method. Circles represent current baseqine data. Triangles represent baseqine data for subjects studied. The number of CB cells scored varied between 1000 and 9000 depending on the bin frequency. To compare the response of the lymphocytes from the two groups to X-irradiation the level of induced micronuclei after exposure to 75 cGy and 150 cGy was measured for each individual by subtracting the base-line frequency in the unirradiated control cultures from the frequency observed for the irradiated cultures. The results obtained (Table I and Fig. 3) indicate that there was no difference between young and old individuals in the number of MN induced by X-rays. This suggested that lymphocytes from old subjects are as proficient as lymphocytes from young subjects in the repair of those DNA lesions that lead to chromosome breakage. DISCUSSION AND CONCLUSION The CB micronucleus method provides a precise measure of chromosome damage because, unlike conventional micronucleus assays, it enables micronuclei to be scored
117
TABLE I BASE-LINE AND X-RAY INDUCED MICRONUCLEUS FREQUENCIES (MEAN ± 1 S.E.) IN YOUNG AND ELDERLY SUBJECTS.
MN/I O00 CB cells Base4ine
Induced after 75 cGy
Induced after 150 cGy
Young
10.9 ±1.1 (iV= 11)
82.1 ±6.5 (iV = 11)
203.5 +15.3 (iV = 9)
Old
44.3 ± 5.3 a
82.2 ± 5.7 b
222.0 -L-_8.4 c
( N = 15)
(N= 15)
( N = 13)
ap < 0.01.
bp > 0.I0. cP > 0.I0.
(n ,.J
36
0
32,
,.I W m
L)
28' OlD
¢
24,
~"
20,
i
16.
~
12,
z
8
4
!
.1. •
$
0 i •
tt,
YOUNG
•
OLD
75 cGy
•
•
YOUNG
OLD
150 cGy
Fig. 3. The frequency of X-ray induced MN (i.e. observed minus base-line) in lymphocytes from young and elderly subjects. The number of CB cells scored varied between 300 and 900 depending on the MN frequency.
118
only in those cells that have completed one nuclear division. This method is thus more precise than the conventional micronucleus assay which is affected by the variable response to phytohaemagglutinin shown by lymphocytes from different individuals and even by lymphocytes from the same individual under varying culture conditions. The rapidity of the CB method makes it a more sensitive technique than chromosome analysis because thousands of CB cells can be scored. This sensitivity was demonstrated in our previous studies in which it was shown that in vitro exposures to 5 cGy were unequivocally detected by the CB method whereas standard metaphase analysis procedures do not enable the detection of exposures of less than 20 c Gy [5]. The CB micronucleus method is therefore superior to chromosome analysis for detection of small differences in the extent of chromosome damage induced by ionising radiation. The results presented in this paper show that there is no difference between lymphocytes of young and old subjects in the number of X-ray-induced MN. They therefore do not support the hypothesis that cells from aged subjects exhibit increased chromosomal instability following exposure to ionising radiation. This conclusion is in contrast with the results of Dutkowski et al. [9] who demonstrated by using conventional chromosome analysis that chromosomes in lymphocytes from elderly subjects express more damage after exposure and incorporation of tritiated thymidine for 24 h prior to analysis than do chromosomes from young subjects. A direct comparison with the results of Dutkowski et al. is difficult owing to the chronic nature of exposure in their experiments, the different quality of radiation used and the unknown effects at the molecular level of tritiated thymidine incorporated in DNA. Consequently the contrasting conclusions may simply be explained by a difference in cellular response with age to the different radiobiological effects of an acute dose of X-irradiation and chronic exposure to tritiated thymidine. It is currently held that chromosome breaks induced by ionising radiation are the result of double-strand breaks (DSBs) in DNA [10,11]. The results described therefore suggest that DSB repair does not decline with in vivo ageing. There is other evidence that ageing in vivo [12] or in vitro [13] does not alter the kinetics of DNA strand break repair after X-irradiation. In their study on the rat Neidmuller et al. [14] found no change in DSB repair with increasing age although there was a slight reduction in single strand break repair and a marked increase in the extent of excision repair. Kutlaca et al. [15] observed that lymphocytes from aged subjects are approximately twice as sensitive to the lethal effects of X-irradiation as lymphocytes from young individuals. DSBs are commonly regarded as the lesion principally responsible for lethality [16,17] but, since repair of DSBs does not seem to decline with age, it is likely that a class of lesion other than DSBs may be the important lethal lesion and that it is the repair of this other lesion that declines with age. REFERENCES 1 K.J. Trainor, D. Wigmore, A. Chrysostomou, J. Dempsey, R. Seshadri and A.A. Morley, Mutation frequency in human lymphocytes increases with age. Mech. Ageing Dev,, 27 (1984) 83-86.
119 2 Vijaylaxmi and HJ. Evans, Measurement of spontaneous and X-irradiation induced 6-thioguanineresistant human blood lymphocytes using a T-cell cloning technique. Mutat. Res., 125 (1984) 87-94. 3 T.A. Lezhava, Heterochromatisation as a key factor in ageing. Mech. Ageing Dev., 28 (1984) 279-287. 4 E.L. Schneider, Cytogenetics of ageing. In E.L. Schneider (ed.), The Genetics o f Ageing, Plenum, New York, 1979, pp. 27-52. 5 M. Fenech and A.A. Morley, Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation. Mutat. Res., 161 (1986) 193-198. 6 M. Fenech and A.A. Motley, Solutions to the kinetic problem in the micronucleus assay. Cytobios, 42 (1985) 233-246. 7 M. Fenech and A.A. Morley, Measurement of micronuclei in human lymphocytes. Murat. Res., 147 (1985) 29-36. 8 J.A. Heddle, B.L. Constance, E.F. Saunders and D. Benz, Sensitivity to five mutagens in Fanconi's anaemia as measured by the micronucleus method, Cancer Res., 38 (1978) 2983-2988. 9 R.T. Dutkowski, R, Lesh, L. Staiano-Coico, H. Thaler, G. Darlington and M.E. Weksler, Increased chromosomal instability in lymphocytes from elderly humans. Murat. Res., 149 (1985) 505-512. 10 H.J. Evans, Molecular mechanisms in the induction of chromosome aberrarions. In D.Scott, B.A. Bridges and F.H. Sobels (eds.), Progress in Genetic Toxicology, Elsevier/North Holland, 1977, pp. 57-74. 11 K.E. Chadwick and H.P. Leenhouts, The Molecular Theory o f Radiation Biology Springer-Verlag New York, 1981, pp. 51-62. 12 D.R. Turner, V.C. Grfffith and A.A. Morley, Ageing in vivo does not alter the kinetics of DNA strand break repair. Mech. Ageing Dev., 19 (1982) 325-331. 13 F. Suzuki, E. Watanabe and M. Horikawa, Repair of X-ray damage in ageing human diploid cells. Exp. Cell Res., 127 (1980) 299-307. 14 H. Neidmuller, G. Hofecker and M. Skalicky, Changes of DNA repair mechanisms during the ageing of the rat. Mech. AgeingDev., 29 (1985) 221-238. 15 R. Kutlaca, R. Seshadti and A.A. Morley, Effect of age on sensitivity of human lymphocytes to radiation, a brief note.Mech. AgeingDev., 19 (1982) 97-101. 16 P.E. Bryant, Enzymatic restriction of mammalian cell DNA: evidence for double-strand breaks as potentially lethal lesions. Int. Z Radiat. Biol., 48H) (1985) 55-60. 17 I.R. Radford, The level of induced DNA double-strand breakage correlates with cell killing after X-irradiation. Int. J. Radiat. Biol., 48(1) (1985) 45-54.