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The effect of smoking on the frequencies of asymmetrical and symmetrical chromosome exchanges in human lymphocytes
Mutation Research, 224 (1989) 151-156
151
Elsevier MUTGEN 01465
The effect of smoking on the frequencies of asymmetrical and symmetrical chromosome exchanges in human lymphocytes E. Janet Tawn and C. Louise Cartmell Geoffrey Schofield Cytogenetics Laboratory, BNF plc, Sellafield, Cumbria CA20 1PG (Great Britain)
(Received 2 February 1989) (Accepted 23 March 1989)
Keywords: Smoking; Chromosome exchanges, asymmetrical and symmetrical; Lymphocytes, human
Summary Cytogenetic analysis was performed on blood lymphocyte cultures from 12 moderate smokers (15-20 cigarettes/day) and 12 non-smokers. An increase in dicentrics was observed in the smokers using block-stained material but this was not significant. Analysis of banded material, enabling both symmetrical and asymmetrical aberrations to be scored, revealed a significant increase in total aberrations thus emphasizing the need to identify all chromosome rearrangements when examining the effects of low-level chronic exposures to clastogens.
The determination of chromosome aberration frequencies in short-term cultures of peripheral blood lymphocytes is a well established technique for assessing exposures to radiation, both as a biological dosimeter in individuals and as a population monitor for occupational exposure. One of the problems in interpreting the significance of aberration levels in radiation workers is the contribution made by exposure to other clastogenic agents encountered during normal life. The most widespread of these is smoking. Cigarette smoke, its condensate and their various fractions have all been shown to be mutagenic in a variety of biological test systems (DeMarini, 1983). Studies on h u m a n lymphocyte chromosomes have demonstrated an increase in sister-chromatid exchange
Correspondence: Dr. E. Janet Tawn, Geoffrey Schofield Cytogenetics Laboratory, BNF plc, Sellafield, Cumbria CA20 1PG (Great Britain).
frequencies in smokers compared to non-smokers but the effect on chromosome aberration frequencies has proved more difficult to establish (for review see Obe et al., 1984). Previous studies have been confined to scoring for asymmetrical aberrations, particularly dicentrics, using a block-staining technique. It is this technique that is customarily used for routine radiation dosimetry work. However in studies of chronic clastogenic exposure it is more appropriate to score both asymmetrical aberrations and the longer-lived symmetrical aberrations. This is more time consuming and requires a sophisticated staining technique which produces characteristic bands on the chromosomes. Each chromosome can then be individually identified. The present study is a continuation of earlier work in this laboratory on background levels of chromosome aberrations (Tawn, 1987). That report examined other factors as well as smoking and was the first to include data on banding analysis.
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
1
(cigarettes/
(years)
20
15
40
33
33
33
29
48
39
39
30
34
3
4
5
6
7
8
9
10
11
12
1
2
.
-
.
.
1
-
. .
.
-
-
-
.
-
.
1
-
1
-
.
.
trics
rings)
Acen-
Dicentrics
cells
( + centric
non-banded
.
.
1
-
-
-
.
-
-
-
.
.
b E q u i v a l e n t to 1 i n v e r s i o n plus 1 t r a n s l o c a t i o n .
.
Dicentrics
a Equivalent to I interstitial deletion plus 1 dicentric.
15
20
20
20
15
15
20
15
15
20
30
40
1
2
day)
Smoking
Age
.
.
1
1
1
1
1
.
.
.
.
.
locations
Trans-
.
.
.
Inver-
.
-
1
2
-
-
-
2
.
sions
.
.
.
.
--
-
-
-
1
-
1
deletions
Terminal
Com-
-
1b
-
-
1 a
-
-
-
plex
13
24
23
22
21
20
19
18
17
16
15
14
36
30
38
38
50
28
34
31
34
36
41
28
(years)
Age
No.
ABERRATIONS
in 100 banded cells
CHROMOSOME
Aberrations
AND
Non-smokers
SMOKING
Aberrations in 500
ON AGE,
No.
DATA
Smokers
INDIVIDUAL
TABLE
Aberrations
1
.
(1)
.
.
-
.
-
-
.
.
.
rings)
.
.
.
.
.
.
.
( + centric
Dicentrics
non-banded
2
3
-
1
-
.
.
.
.
.
.
trics
.
Acen-
cells
in 500
.
-
.
-
-
1
.
.
.
.
.
trics
.
Dicen-
.
.
1
1
1
2
.
.
.
.
.
.
.
.
.
.
locations
. -
-
-
1
sions
Inver-
in 100 banded
Trans-
Aberrations
-
-
-
-
1
deletions
Terminal
cells
to
153
Methods The study group comprised 12 smokers and 12 age-matched non-smokers, all with no known exposure to other clastogenic agents. It was not possible to obtain complete individual data on previous smoking history for the smokers. The men were selected retrospectively from new entrants to the site. At the time of their entry into the company it was standard practice to prepare slides for chromosome analysis from individuals going into certain working environments. These were not fully analysed but retained so that a baseline level would be available if the individual was ever involved in a possible radiation over-exposure. Peripheral blood lymphocytes were cultured for 48 h in Eagle's M E M containing 15% foetal bovine serum and stimulated with phytohaemagglutinin. Bromodeoxyuridine was present throughout culture at a concentration of 7.2 /~M. Colcemid was added 4 h prior to harvesting. For each individual a total of 500 first division cells were examined for asymmetrical aberrations from fluorescence-plus-Giemsa-stained slides, and 100 cells from G-banded slides were fully analysed for all observable asymmetrical and symmetrical aberrations. The analysis was confined to chromosome-type aberrations as these are of prime importance when considering aberration levels in lymphocytes of radiation-exposed individuals. Individual data are presented in Table 1. Results and discussion
Block-stained analysis for asymmetrical aberrations Chromosome analysis for asymmetrical aberrations revealed a 4-fold increase in dicentrics in the
smokers compared to the non-smokers but this did not reach statistical significance (Table 2). The acentric levels were similar for the two groups. Most reports have concentrated on dicentric frequency since acentrics are a heterogeneous group of aberrations and frequencies can vary considerably. The effect of smoking on dicentric levels has been difficult to evaluate. Large numbers of cells are needed to demonstrate significant changes in frequencies of an aberration with such a low level of occurrence. In addition such variables as the relative number and type of cigarettes, duration of smoking, and inhalation practices will often differ between studies. Interlaboratory differences in culturing techniques and scoring criteria, and the selection procedure to rule out exposure to other clastogens, can also be contributory factors to the variation between different reports. The studies by Obe and his colleagues (Obe and Herha, 1978; Obe et al., 1982, 1984) are the most extensive. This work demonstrated a doubling of dicentric frequency from 0.32 x 10-3/cell in non-smokers to 0.65 × 10 -3 in smokers. An even higher level of 2.34 x 10 -3 was found in their group of heavy smokers. Similar values were reported by Littlefield and Joiner (1986) in which the level of dicentrics was 0.33 x 10 -3 in the non-smokers rising to 3.00 x 10 -3 in heavy smokers of 20 years' duration. A previous study in our laboratory (Tawn, 1987) showed a doubling in dicentric frequency from 0.45 × 10-3 in the nonsmokers to 0.91 × 10 -3 in the smokers. The increase only became significant when the heavy smokers alone were considered, the level then being 2.80 × 10-3 compared to 0.34 x 10-3 in the remainder. Vijayalaxmi and Evans (1982) also reported a doubling in dicentric frequency in a
TABLE 2 ASYMMETRICAL ABERRATION FREQUENCIES FROM BLOCK-STAINED ANALYSIS
N u m b e r of individuals N u m b e r of cells analysed M e a n age (years) Dicentries/cell + S.E. × 1 0 - 3 Acentrics/cell + S.E. x 1 0 - 3
Total
Non-smokers
Smokers
24 12000 35.5 0.42 + 0.18 0.92 + 0.28
12 6 000 35.3 0.17 + 0.17 1,00 + 0.41
12 6 000 35.7 0.67 + 0.33 0.83 + 0.37
154
Aberrations determined by banding
TABLE 3 ABERRATION TYPES D E T E R M I N E D BY B A N D I N G
Total ceils Terminal deletions Translocations Inversions Dicentrics Complex a
Smokers
Non-smokers
1200 3 6 5 1 2
1200 1 5 1 1 -
a Exchanges involving 3 chromosomes. For frequency analysis, considered as a number of exchange events: (1) interstitial deletion plus dicentric, (2) inversion plus translocation.
group of moderate smokers compared to nonsmokers although their values of 4.2 × 10 -3 and 1.4 x 10- 3 respectively were both somewhat higher than other reports. However a similar non-smoking level of 1.4 × 10-3 for dicentrics was found in a study of nurses which rose to 2.3 × 10-3 in the smokers (Stucker et al., 1986). In the present work a rigid selection procedure was adopted in order to eliminate other variables such as occupational and medical exposure to possible clastogens. This may account for the comparatively low values for dicentric frequency of 0.17 × 10 -3 for non-smokers and 0.67 × 10 -3 for smokers. In a recent control population study Galloway et al. (1986) demonstrated a smoking effect particularly for chromatid aberrations although this was not confirmed in 2 further large studies (Anderson et al., 1988; Bender et al., 1988). Unfortunately these 3 reports fail to give full details of the different aberration types.
(a) Frequencies of aberration types. Tables 3 and 4 set out the different aberration types and their frequencies and for ease of comparison Table 4 also includes data from the previous study from this laboratory. All 3 categories of chromosome aberration i.e. asymmetrical exchanges, symmetrical exchanges and terminal deletions showed higher levels in smokers compared to non-smokers. When the 3 groups were combined this difference became significant (X 2 = 4.48, df= 1, P = 0.05)." Levels of dicentrics and acentrics (terminal + interstitial deletions) were higher than those found in the nonbanding part of the study and confirm observations in this laboratory that better ascertainment is possible with banding. In our previous study on background levels of chromosome aberrations, banding analysis revealed significant increases in symmetrical aberrations and total chromosome aberrations in smokers compared to non-smokers. In that work the two groups were not matched and there were 3 times as many non-smokers as smokers. The levels of symmetrical and asymmetrical exchanges in the non-smokers are similar in the two studies but there is some difference in values for the smokers. This could be due to differences in the smoking profiles of the two groups. Also the errors attached to the frequencies in the smokers of the previous study were quite large due to the limited number of cells analysed. It is, however, notable that both studies showed considerable increases in symmetrical aberrations in smokers compared to non-smokers. Asymmetrical and symmetrical ex-
TABLE 4 ABERRATION F R E Q U E N C I E S D E T E R M I N E D BY B A N D I N G Present study Non-smokers Asymmetrical exchange aberrations/ cell+ S.E. × 10 -3 Symmetrical exchange aberrations/ cell + S.E. x 10- 3 Terminal deletions/cell + S.E. × 10- 3 Total aberrations/cell + S.E. × 10 3
Tawn (1987) Smokers
Non-smokers
Smokers
0.83 + 0.83
2.50 + 1.44
1.05 5:0.74
1.54 + 1.54
5.00 + 2.04 0.83 + 0.83 6.67:5:2.36
10.83 + 3.00 2.50 + 1.44 15.83 + 3.63
5.26 + 1.67 2.10 + 1.05 8.41 -t- 2.10
16.92 + 5.10 6.15 + 3.08 24.61:5:6.15
155 TABLE 5 ABERRATIONS INVOLVING EXCLUSIVELY CHROMOSOMES 7 AND 14 Individual Non-smokers 2 5 12 Smokers 13 15 20 21
inv(14)(qllq32) t(7;14)(p22;qll) t(7;14)(q34;q11) inv(14)(qllq32) t(7;14)(p13;q11) inv(7)(p13q32) inv(14)(qllq32) t(7;14Xpl3;qll) inv(7)(q21q36)
changes are formed in equal proportions (Savage, 1979) but cells carrying asymmetrical aberrations will be lost more quickly from the peripheral blood. The inclusion of symmetrical aberrations in the cytogenetic analysis therefore provides a better measure of a chronic clastogenic exposure such as that resulting from cigarette smoking.
(b) Exchanges involving exclusively chromosomes 7 and 14. The non-random occurrence of translocations and inversions involving exclusively chromosomes 7 and 14 in cultures of peripheral blood lymphocytes has been widely reported (for review see Tawn, 1988). Nine such exchanges were seen in this study (Table 5), 3 in the non-smokers and 6 in the smokers. The origin and significance of these 9earrangements remain unclear but they are not thought to be increased by exposure to clastogens. A recent report by Prieur et al. (1988) revealed a higher incidence in newborns than adults which led to the suggestion that such rearrangements are generated before birth and may be related to the rearrangements of immunoglobulin family genes. Removal of these 9 exchanges from the analysis still leaves an excess of aberrations in the smokers. Conclusions
The men in this study smoked 15-20 cigarettes/day and can be considered as moderate smokers. Determination of a smoking effect by
looking at asymmetrical chromosome aberrations in such a group is always going to be difficult, requiring large numbers of cells to be examined. However analysis of banded material, which also enables symmetrical aberrations to be scored, confirms that smoking does have a detectable clastogenic effect even in relatively small studies such as this.
ReferenCes Anderson, D., P.C. Jenkinson, R.S. Dewdney, A.J. Francis, P. Godbert and K.R. Butterworth (1988) Chromosome aberrations, mitogen-induced blastogenesis and proliferative rate index in peripheral lymphocytes from 106 control individuals of the U.K. population, Mutation Res., 204, 407-420. Bender, M.A, R.J. Preston, R.C. Leonard, B.E. Pyatt, P.C. Gooch and M.D. Shelby (1988) Chromosomal aberration and sister-chromatid exchange frequencies in peripheral blood lymphocytes of a large human population sample, Mutation Res., 204, 421-433. DeMarini, D.N. (1983) Genotoxicity of tobacco-smoke and tobacco smoke condensate, Mutation Res., 114, 59-89. Galloway, S.M., P.K. Berry, W.W. Nichols, S.R. Wolman, K.A. Soper, P.D. Stolley and P. Archer (1986) Chromosome aberrations in individuals occupationally exposed to ethylene oxide, and in a large control population, Mutation Res., 170, 55-74. Littlefield, L.G., and E.E. Joiner (1986) Analysis of chromosome aberrations in lymphocytes of long-term heavy smokers, Mutation Res., 170, 145-150. Obe, G., and J. Herha (1978) Chromosomal ~berrations in heavy smokers, Hum. Genet., 41,259-263. Obe, G., H.-J. Vogt, S. Madle, A. Fahning and W.-D. Heller (1982) Double-blind study on the effect of cigarette smoking on the chromosomes of human peripheral blood lymphocytes in vivo, Mutation Res., 92, 309-319. Obe, G., W.-D. Heller and H.-J. Vogt (1984) Mutagenic activity of cigarette smoke, in" G. Obe (Ed.), Mutations in Man, Springer, Berlin, pp. 223-246. Prieur, M., W. Al Achkar, A. Aurias, J. Conturier, A.M. Dutrillaux, B. DutriUaux, A. Flury-Herard, M. GerbaultSeureau, F. Hoffschir, E. Lamoliatte, D. Lefrancois, M. Lombard, M. Muleris, M. Ricoul, L. Sabatier and E. Viegas-Pequignot (1988) Acquired chromosome rearrangements in human lymphocytes: effects of ageing, Hum. Genet., 79, 147-150. Savage, J.R.K. (1979) Survival and transmission of syn~etrical chromosome aberrations, in: S. Okada, M. Imamura, T. Terashima and H. Yamaguchi (Eds.), Proc. 6th Int. Congr ~. Radiat. Res., Tokyo, Japanese Association for Radiation Research, Tokyo, pp. 519-524. Stucker, I., A. Hirsch, T. Doloy, I. Bastie-Sigeac and D. Hereon (1986) Urine mutagenicity, chromosomal abnormalities and sister chromatid exchanges in lymphocytes of
156 nurses handling cytostatic drugs, Int. Arch. Occup. Environ. Health, 57, 195-205. Tawn, E.J. (1987) The frequency of chromosome aberrations in a control population, Mutation Res., 182, 303-308. Tawn, E.J. (1988) The non-random occurrence of exchanges involving chromosomes 7 and 14 in human lymphocytes: a
prospective study of control individuals, Mutation Res., 199, 215-220. Vijayalaxmi, and H.J. Evans (1982) In vivo and in vitro effects of cigarette smoke on chromosomal damage and sisterchromatid exchange in human peripheral blood lymphocytes, Mutation Res., 92, 321-332.
151
Elsevier MUTGEN 01465
The effect of smoking on the frequencies of asymmetrical and symmetrical chromosome exchanges in human lymphocytes E. Janet Tawn and C. Louise Cartmell Geoffrey Schofield Cytogenetics Laboratory, BNF plc, Sellafield, Cumbria CA20 1PG (Great Britain)
(Received 2 February 1989) (Accepted 23 March 1989)
Keywords: Smoking; Chromosome exchanges, asymmetrical and symmetrical; Lymphocytes, human
Summary Cytogenetic analysis was performed on blood lymphocyte cultures from 12 moderate smokers (15-20 cigarettes/day) and 12 non-smokers. An increase in dicentrics was observed in the smokers using block-stained material but this was not significant. Analysis of banded material, enabling both symmetrical and asymmetrical aberrations to be scored, revealed a significant increase in total aberrations thus emphasizing the need to identify all chromosome rearrangements when examining the effects of low-level chronic exposures to clastogens.
The determination of chromosome aberration frequencies in short-term cultures of peripheral blood lymphocytes is a well established technique for assessing exposures to radiation, both as a biological dosimeter in individuals and as a population monitor for occupational exposure. One of the problems in interpreting the significance of aberration levels in radiation workers is the contribution made by exposure to other clastogenic agents encountered during normal life. The most widespread of these is smoking. Cigarette smoke, its condensate and their various fractions have all been shown to be mutagenic in a variety of biological test systems (DeMarini, 1983). Studies on h u m a n lymphocyte chromosomes have demonstrated an increase in sister-chromatid exchange
Correspondence: Dr. E. Janet Tawn, Geoffrey Schofield Cytogenetics Laboratory, BNF plc, Sellafield, Cumbria CA20 1PG (Great Britain).
frequencies in smokers compared to non-smokers but the effect on chromosome aberration frequencies has proved more difficult to establish (for review see Obe et al., 1984). Previous studies have been confined to scoring for asymmetrical aberrations, particularly dicentrics, using a block-staining technique. It is this technique that is customarily used for routine radiation dosimetry work. However in studies of chronic clastogenic exposure it is more appropriate to score both asymmetrical aberrations and the longer-lived symmetrical aberrations. This is more time consuming and requires a sophisticated staining technique which produces characteristic bands on the chromosomes. Each chromosome can then be individually identified. The present study is a continuation of earlier work in this laboratory on background levels of chromosome aberrations (Tawn, 1987). That report examined other factors as well as smoking and was the first to include data on banding analysis.
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
1
(cigarettes/
(years)
20
15
40
33
33
33
29
48
39
39
30
34
3
4
5
6
7
8
9
10
11
12
1
2
.
-
.
.
1
-
. .
.
-
-
-
.
-
.
1
-
1
-
.
.
trics
rings)
Acen-
Dicentrics
cells
( + centric
non-banded
.
.
1
-
-
-
.
-
-
-
.
.
b E q u i v a l e n t to 1 i n v e r s i o n plus 1 t r a n s l o c a t i o n .
.
Dicentrics
a Equivalent to I interstitial deletion plus 1 dicentric.
15
20
20
20
15
15
20
15
15
20
30
40
1
2
day)
Smoking
Age
.
.
1
1
1
1
1
.
.
.
.
.
locations
Trans-
.
.
.
Inver-
.
-
1
2
-
-
-
2
.
sions
.
.
.
.
--
-
-
-
1
-
1
deletions
Terminal
Com-
-
1b
-
-
1 a
-
-
-
plex
13
24
23
22
21
20
19
18
17
16
15
14
36
30
38
38
50
28
34
31
34
36
41
28
(years)
Age
No.
ABERRATIONS
in 100 banded cells
CHROMOSOME
Aberrations
AND
Non-smokers
SMOKING
Aberrations in 500
ON AGE,
No.
DATA
Smokers
INDIVIDUAL
TABLE
Aberrations
1
.
(1)
.
.
-
.
-
-
.
.
.
rings)
.
.
.
.
.
.
.
( + centric
Dicentrics
non-banded
2
3
-
1
-
.
.
.
.
.
.
trics
.
Acen-
cells
in 500
.
-
.
-
-
1
.
.
.
.
.
trics
.
Dicen-
.
.
1
1
1
2
.
.
.
.
.
.
.
.
.
.
locations
. -
-
-
1
sions
Inver-
in 100 banded
Trans-
Aberrations
-
-
-
-
1
deletions
Terminal
cells
to
153
Methods The study group comprised 12 smokers and 12 age-matched non-smokers, all with no known exposure to other clastogenic agents. It was not possible to obtain complete individual data on previous smoking history for the smokers. The men were selected retrospectively from new entrants to the site. At the time of their entry into the company it was standard practice to prepare slides for chromosome analysis from individuals going into certain working environments. These were not fully analysed but retained so that a baseline level would be available if the individual was ever involved in a possible radiation over-exposure. Peripheral blood lymphocytes were cultured for 48 h in Eagle's M E M containing 15% foetal bovine serum and stimulated with phytohaemagglutinin. Bromodeoxyuridine was present throughout culture at a concentration of 7.2 /~M. Colcemid was added 4 h prior to harvesting. For each individual a total of 500 first division cells were examined for asymmetrical aberrations from fluorescence-plus-Giemsa-stained slides, and 100 cells from G-banded slides were fully analysed for all observable asymmetrical and symmetrical aberrations. The analysis was confined to chromosome-type aberrations as these are of prime importance when considering aberration levels in lymphocytes of radiation-exposed individuals. Individual data are presented in Table 1. Results and discussion
Block-stained analysis for asymmetrical aberrations Chromosome analysis for asymmetrical aberrations revealed a 4-fold increase in dicentrics in the
smokers compared to the non-smokers but this did not reach statistical significance (Table 2). The acentric levels were similar for the two groups. Most reports have concentrated on dicentric frequency since acentrics are a heterogeneous group of aberrations and frequencies can vary considerably. The effect of smoking on dicentric levels has been difficult to evaluate. Large numbers of cells are needed to demonstrate significant changes in frequencies of an aberration with such a low level of occurrence. In addition such variables as the relative number and type of cigarettes, duration of smoking, and inhalation practices will often differ between studies. Interlaboratory differences in culturing techniques and scoring criteria, and the selection procedure to rule out exposure to other clastogens, can also be contributory factors to the variation between different reports. The studies by Obe and his colleagues (Obe and Herha, 1978; Obe et al., 1982, 1984) are the most extensive. This work demonstrated a doubling of dicentric frequency from 0.32 x 10-3/cell in non-smokers to 0.65 × 10 -3 in smokers. An even higher level of 2.34 x 10 -3 was found in their group of heavy smokers. Similar values were reported by Littlefield and Joiner (1986) in which the level of dicentrics was 0.33 x 10 -3 in the non-smokers rising to 3.00 x 10 -3 in heavy smokers of 20 years' duration. A previous study in our laboratory (Tawn, 1987) showed a doubling in dicentric frequency from 0.45 × 10-3 in the nonsmokers to 0.91 × 10 -3 in the smokers. The increase only became significant when the heavy smokers alone were considered, the level then being 2.80 × 10-3 compared to 0.34 x 10-3 in the remainder. Vijayalaxmi and Evans (1982) also reported a doubling in dicentric frequency in a
TABLE 2 ASYMMETRICAL ABERRATION FREQUENCIES FROM BLOCK-STAINED ANALYSIS
N u m b e r of individuals N u m b e r of cells analysed M e a n age (years) Dicentries/cell + S.E. × 1 0 - 3 Acentrics/cell + S.E. x 1 0 - 3
Total
Non-smokers
Smokers
24 12000 35.5 0.42 + 0.18 0.92 + 0.28
12 6 000 35.3 0.17 + 0.17 1,00 + 0.41
12 6 000 35.7 0.67 + 0.33 0.83 + 0.37
154
Aberrations determined by banding
TABLE 3 ABERRATION TYPES D E T E R M I N E D BY B A N D I N G
Total ceils Terminal deletions Translocations Inversions Dicentrics Complex a
Smokers
Non-smokers
1200 3 6 5 1 2
1200 1 5 1 1 -
a Exchanges involving 3 chromosomes. For frequency analysis, considered as a number of exchange events: (1) interstitial deletion plus dicentric, (2) inversion plus translocation.
group of moderate smokers compared to nonsmokers although their values of 4.2 × 10 -3 and 1.4 x 10- 3 respectively were both somewhat higher than other reports. However a similar non-smoking level of 1.4 × 10-3 for dicentrics was found in a study of nurses which rose to 2.3 × 10-3 in the smokers (Stucker et al., 1986). In the present work a rigid selection procedure was adopted in order to eliminate other variables such as occupational and medical exposure to possible clastogens. This may account for the comparatively low values for dicentric frequency of 0.17 × 10 -3 for non-smokers and 0.67 × 10 -3 for smokers. In a recent control population study Galloway et al. (1986) demonstrated a smoking effect particularly for chromatid aberrations although this was not confirmed in 2 further large studies (Anderson et al., 1988; Bender et al., 1988). Unfortunately these 3 reports fail to give full details of the different aberration types.
(a) Frequencies of aberration types. Tables 3 and 4 set out the different aberration types and their frequencies and for ease of comparison Table 4 also includes data from the previous study from this laboratory. All 3 categories of chromosome aberration i.e. asymmetrical exchanges, symmetrical exchanges and terminal deletions showed higher levels in smokers compared to non-smokers. When the 3 groups were combined this difference became significant (X 2 = 4.48, df= 1, P = 0.05)." Levels of dicentrics and acentrics (terminal + interstitial deletions) were higher than those found in the nonbanding part of the study and confirm observations in this laboratory that better ascertainment is possible with banding. In our previous study on background levels of chromosome aberrations, banding analysis revealed significant increases in symmetrical aberrations and total chromosome aberrations in smokers compared to non-smokers. In that work the two groups were not matched and there were 3 times as many non-smokers as smokers. The levels of symmetrical and asymmetrical exchanges in the non-smokers are similar in the two studies but there is some difference in values for the smokers. This could be due to differences in the smoking profiles of the two groups. Also the errors attached to the frequencies in the smokers of the previous study were quite large due to the limited number of cells analysed. It is, however, notable that both studies showed considerable increases in symmetrical aberrations in smokers compared to non-smokers. Asymmetrical and symmetrical ex-
TABLE 4 ABERRATION F R E Q U E N C I E S D E T E R M I N E D BY B A N D I N G Present study Non-smokers Asymmetrical exchange aberrations/ cell+ S.E. × 10 -3 Symmetrical exchange aberrations/ cell + S.E. x 10- 3 Terminal deletions/cell + S.E. × 10- 3 Total aberrations/cell + S.E. × 10 3
Tawn (1987) Smokers
Non-smokers
Smokers
0.83 + 0.83
2.50 + 1.44
1.05 5:0.74
1.54 + 1.54
5.00 + 2.04 0.83 + 0.83 6.67:5:2.36
10.83 + 3.00 2.50 + 1.44 15.83 + 3.63
5.26 + 1.67 2.10 + 1.05 8.41 -t- 2.10
16.92 + 5.10 6.15 + 3.08 24.61:5:6.15
155 TABLE 5 ABERRATIONS INVOLVING EXCLUSIVELY CHROMOSOMES 7 AND 14 Individual Non-smokers 2 5 12 Smokers 13 15 20 21
inv(14)(qllq32) t(7;14)(p22;qll) t(7;14)(q34;q11) inv(14)(qllq32) t(7;14)(p13;q11) inv(7)(p13q32) inv(14)(qllq32) t(7;14Xpl3;qll) inv(7)(q21q36)
changes are formed in equal proportions (Savage, 1979) but cells carrying asymmetrical aberrations will be lost more quickly from the peripheral blood. The inclusion of symmetrical aberrations in the cytogenetic analysis therefore provides a better measure of a chronic clastogenic exposure such as that resulting from cigarette smoking.
(b) Exchanges involving exclusively chromosomes 7 and 14. The non-random occurrence of translocations and inversions involving exclusively chromosomes 7 and 14 in cultures of peripheral blood lymphocytes has been widely reported (for review see Tawn, 1988). Nine such exchanges were seen in this study (Table 5), 3 in the non-smokers and 6 in the smokers. The origin and significance of these 9earrangements remain unclear but they are not thought to be increased by exposure to clastogens. A recent report by Prieur et al. (1988) revealed a higher incidence in newborns than adults which led to the suggestion that such rearrangements are generated before birth and may be related to the rearrangements of immunoglobulin family genes. Removal of these 9 exchanges from the analysis still leaves an excess of aberrations in the smokers. Conclusions
The men in this study smoked 15-20 cigarettes/day and can be considered as moderate smokers. Determination of a smoking effect by
looking at asymmetrical chromosome aberrations in such a group is always going to be difficult, requiring large numbers of cells to be examined. However analysis of banded material, which also enables symmetrical aberrations to be scored, confirms that smoking does have a detectable clastogenic effect even in relatively small studies such as this.
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