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Localization of breakpoints in the karyotype of workers professionally exposed to benzene
Mutation Research, 224 (1989) 235-240 Elsevier
235
MUTGEN 01474
Localization of breakpoints in the karyotype of workers professionally exposed to benzene M a r i a S~siadek a, J b z e f Jagielski a a n d R o m a n S m o l i k b " Department of Pathophysiology, Medical Academy of Wroctaw, 50-368 Wroctaw (Poland) and b Department of Internal and Occupational Medicine, Medical Academy of Wroctaw, 50-367 Wroctaw (Poland) (Received 19 July 1988) (Revision received 24 April 1989) (Accepted 1 May 1989)
Keywords: Benzene; Chromosome aberrations; Distribution of breakpoints
Summary Cytogenetic analysis was carried out on peripheral blood lymphocytes of 33 workers professionally exposed to benzene for 10-26 years. In the examined group structural chromosome aberrations were found in 147 metaphases (4.7%). Statistical analysis has shown that the distribution of breakpoints was not random. Chromosomes 2, 4 and 9 are almost twice as susceptible to breaks, while chromosomes 1 and 2 are almost twice as susceptible to gaps, as would be expected based on a random distribution of damage among chromosomes.
Benzene and its derivatives are important substances widely used in industry. Many workers are professionally exposed to the agents for a long time. Chronic benzene intoxication modulates immune response, granulocyte enzyme systems, leads to thrombocytopenia, leukopenia, anemia, even pancytopenia and, in extreme cases, to neoplastic diseases (Aksoy et al., 1972, 1976; Cronkite, 1987; Dean, 1978; Moszczyfiski et al., 1983; Smolik et al., 1973). The frequency of neoplasia in workers professionally exposed to benzene and its derivatives is significantly higher than in the unexposed population (Honeycombe, 1978; Rinsky et al., 1987; Yin et al., 1978). In groups of workers professionally exposed to benzene chromosome
Correspondence: Dr. M. S~siadek, Department of Pathophysiology, Medical Academy of Wrodaw, Marcinkowskiego 1, 50-368 Wroclaw (Poland).
aberrations, both structural and numerical, were found in 2.25-5.5% of analyzed metaphases. However, in patients with leukemia caused by benzene, a much higher percentage of chromosome aberrations was found (4-55%). The correlation between time of exposure to benzene, age, sex and percentage of damaged metaphases has not yet been determined (Aksoy et al., 1976; Clare et al., 1984; Sosiadek, 1984). Cytogenetic effects have been observed both in laboratory animals and in cultured cells treated with benzene (Dean, 1978; Morimoto and Wolff, 1980; Morimoto, 1983; Morimoto et al., 1983). Although the metabolism and toxicity of benzene in the organism are known, the mechanism of neoplastic cell transformation induced by benzene has not yet been ascertained. Activation of tyrosine-kinase groups of oncogenes may play a key role in the neoplastic transformation of cells (Cronkite, 1987; Kalf, 1987; Synder et al., 1987).
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
17 17 16
15 15 15 14 13 13
13 12 12 12 12 11 11 11 3
3 3
14 15 16
17 18 19 20 21 22
23 24 25 26 27 28 29 30 31
32 33
-
-
-
-
-
-
-
-
-
-
-
40 30
49 40 46 49 35 40 40 40 33
48 34 48 49 43 47
42 43 37
48
46
41 48 47
46 42 47
48 47 41
a N u m b e r of cigarettes p e r d a y x years of smoking.
17
19 19 17
9 10 11
17
20 19 19
6 7 8
12
22 22 21
13
22
3 4 5
40 40
45
45
40 42 45
45 40 45
48 50 45
43 40
2
C
E
-
C 48 47
23
E
1
M M
M F M F M M F M M
F F F F M M
F F M
F
F
F M F
M F F
M M M
M F
E
Sex
F F
F
F
F M F
M F F
M M F
M M
C
300 180
450 400 400 500 400 300 180 200 200
400 250 300 400 300 350
350 400 300
300
350
350 400 400
400 180 300
300 250 250
400 400
E
300 400
250
180
300 400 400
400 200 200
400 300 300
400 400
C
C i g a r e t t e index
A g e (years)
Subject
Period of exposure (years)
OF CHROMOSOME
A G E , SEX, P E R I O D O F E X P O S U R E , C I G A R E T T E I N D E X a A N D P E R C E N T A G E E X P O S E D T O B E N Z E N E (E) A N D I N C O N T R O L S (C)
TABLE 1
100 100
100 100 100 100 100 100 100 30 100
100 100 30 100 100 100
100 100 100
100
100
100 100 100
100 100 30
100 100 100
100 100
E
100 100
100
100
100 100 100
100 100 100
100 100 100
100 100
C
PROFESSIONALLY
0 0
0 4 9 0 14 4 7 0 3
4 1 0 6 11 6
4 7 4
2
4
8 0 4
1 7 0
6 3 13
8 7
E
C
P e r c e n t a g e of c h r o m o some aberrations
IN W O R K E R S
N u m b e r of a n a l y z e d metaphases
ABERRATIONS
237
The aim of this work was to analyze the chromosomal localization of aberrations in the karyotypes of workers exposed to benzene and its derivatives. Material and methods
The studies were carried out on peripheral blood bcmphocytes of 33 workers occupationally exposed
to benzene and its derivatives for 10-23 years. During this period the concentration ~f benzene was below the m a x i m u m allowable concentration (MAC, 0.1 mg/1). Thirty-one of the workers did not show any clinical and hematological symptoms of chronic benzene intoxication. In 2 cases pancytopenia was observed.
I1
i-I !.l-l i-- -ii
i i-
--ii I1 -it -i!
-lit
1
-iib
i'i -it
1t--|-;(--i
-irl--i:i -"
l--i
.~ "
iii -
tt ',-
"-
.~--
-~i
Fig. 1. Karyotypes of a worker professionally exposed to benzene for 18 years, 46,XX(ctb(3)(p13)). Breakpoints are marked by arrows.
238
The control group consisted of 15 patients of approximately the same age, who had no contact with benzene or other chemical a n d / o r physical agents. Patients in both investigated groups were interviewed about infectious diseases, drugs and exposure to X-rays during the last 3 months before cytogenetic examination. All patients in both groups were smokers. None of them used alcohol heavily. Blood samples were obtained by venipuncture. Lymphocytes were cultured for 72 h according to the method of Moorhead et al. (1960). Cytogenetic analysis was performed using G T G - b a n d e d preparations (Seabright et al., 1975). Chromosome aberrations were classified according to ISCN (1985). Statistical analysis was based on the X2 test.
Results
In the group of 33 workers exposed to benzene and its derivatives 3090 metaphases were analyzed, while in the control group 1500 metaphases were studied. In the examined group structural chromosome aberrations were found in 147 metaphases (4.7%), in contrast with the control group, where aberrations were only found in 25 (1.68%) of analyzed metaphases (Table 1). Among the aberrations detected, there was no evidence, with one exception, of clonal origin. In one patient, a 43-year-old woman occupationally exposed to benzene for 18 years, a clonal aberration, probably at the fragile site ctb(3)(p13), occurred in 4% of analyzed metaphases (Fig. 1)
TABLE 2 OBSE RVED A N D E X P E C T E D N U M B E R S OF BREAKS A N D GAPS IN T H E EXPOSED G R O U P Chro-
II
mosome Rumber 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
Breaks
G a ps (O - E) 2
8.44 8.02 6.83 6.30 6.08 5.90 5.36 5.12 4.93 4.80 4.59 4.61 4.66 3.74 3.56 3.46 3.36 3.25 2.93 2.67 2.56 1.90 2.04 2.15
O
E
6 13 a 7 15 a 6 6 3 3 9a 2 2 2 1 3 2 2 1 0 0 0 0 1 3 0
7.26 6.9 5.87 5.42 5.23 5.07 4.61 4.24 4.13 3.94 3.96 4.01 3.22 3.06 3.84 2.89 2.8 5.25 2.3 2.20 1.63 1.75 4.40 1.85
E 0.21 5.39 0,22 16,9 0.11 0,17 0,56 0,36 5.74 0,95 0,97 1 1,53 0.002 0,88 0,27 1,16 5,52 2,3 2,20 1,63 0,32 0,45 1,85
O E 0.82 1.88 1.19 2.7 1.14 1.18 0.65 0.70 2.18 0.51 0.5 0.5 0.31 0.98 0.52 0.69 0.36 0 0 0.45 0 0.57 0.68 0
(O - E) 2 O
E
9a 16 a 6 6 3 3 3 3 3 2 2 2 0 0 0 0 0 0 1 0 0 0 0 1
5.15 4.89 4.17 3.84 3.71 3.6 3.27 3.01 2.93 2.8 2.8 2.8 2.8 2.17 2.11 2.05 1.98 1.79 1.63 1.56 1.16 1.24 3.12 1.31
E 2.88 25.2 0.8 1.21 0.13 0.1 0.022 0 0 0.23 0.23 0.23 2.8 2.17 2.11 2.05 1.98 1.79 0.24 1.56 1.16 1.24 3.12 0.07
O E 1.74 3.27 1.44 1.56 0.81 0.83 0.92 0.99 1.02 0.71 0.71 0.71 0 0 0 0 0 0 0.61 0 0 0 0 0.76
X 2 12.3, df 6, P = 0.45i a results are significant at p = 0.01; X 2 9.4, df 1, other results are not significant. II, % chromosome length (ISCN, 1985); O, observed number of aberrations; E, expected number of aberrations.
239 (Smeets et al., 1986). This w o m a n did not show any clinical or hematological symptoms of chronic benzene intoxication. The chromosomal distribution of the 147 chromosome aberrations and the statistical analysis are presented in Table 2. Chromosomes 2, 4 and 9 are almost twice as susceptible to breaks, while chromosomes 1 and 2 are almost twice as susceptible to gaps as expected. Chromosome 18 appears to be underrepresented for the presence of aberrations. In the control group 25 breakpoints were distributed randomly on 23 chromosome pairs. On chromosomes 1, 2, 3, 5, 7 and 10 two breakpoints were found.
Discussion In the present study, workers occupationally exposed to benzene and its derivatives exhibited a significantly increased frequency of metaphase cells with structural chromosome aberrations over that observed in a control population (4.7% versus 1.7%). The percentage of damaged cells in the benzene-exposed workers is higher than that recorded in earlier studies by Aksoy et al. (1976) and by Clare et al. (1984). This difference m a y be due to the level of benzene to which the workers in the present study were exposed (although the concentration of benzene was below the MAC) or to differences in ascertainment. The other possibility is that in the present study the scoring for chromosomal aberrations was performed not in 48-h cultures, as it is accepted now, but in 72-h cultures, so real damage could be underestimated. The percentage of cells with aberrations in the control group is similar to that described in the literature ( G u n d y et al., 1983; Mattei et al., 1979; Tawn, 1987). A statistical analysis of the chromosomal distribution of the breakpoints involved in the aberrations indicated that while in control individuals the distribution was random, it was n o n - r a n d o m in the cells of individuals exposed to benzene. These results for the benzene-exposed workers agree with earlier reports that the interchromosomal distribution of breakpoints in cells exposed in vitro to various mutagens is n o n - r a n d o m (Buckton, 1976; Honeycombe, 1978; Meyne et al., 1979; Mondello et al., 1984). However, earlier
investigators also reported that the interchromosomal distribution of breakpoints among control cells was non-random. Chromosomes 3, 7 and 16 were preferentially broken in those studies (Marlhens et al., 1986; Mattei et al., 1979). The apparently r a n d o m distribution of the breakpoints of chromosome aberrations observed in our control population m a y be due to the small number of aberrations observed. In workers exposed to benzene, the observed frequency of breakpoints in chromosomes 1, 2, 3 and 9 was twice as high as expected, based on relative chromosome length. Chromosomes 1, 3 and 9 also exhibit an increased sensitivity to the clastogenic activity of methotrexate (Mondello et al., 1984), cis-platinum(II) diamminedichloride, triethylenemelamine (Meyne et al., 1979) and busulfan (Honeycombe, 1978). This consistent sensitivity suggests an inherent fragility to D N A - d a m a g i n g agents or a preferential lack of repair in those regions. Of interest is the observation that the frequency of breakpoints in chromosome 5 was not higher than expected despite the fact that this chromosome is usually involved in the etiology of secondary leukemias (Van der Berghe et al., 1985). While this study demonstrates that the increased frequency of chromosomal aberrations in cells of workers occupationally exposed to benzene is non-randomly distributed among chromosomes, it is not yet possible to determine whether this n o n - r a n d o m distribution is involved in the etiology of the tumors induced by benzene. To evaluate this relationship, it would be necessary to determine also the distribution of chromosomal alterations occurring among cancers present in workers occupationally exposed to benzene.
References Aksoy, M., K. Dincol et al. (1972) Details of blood changes in 32 patients with pancytopenia associated with long-term exposure to benzene, Br. J. Industr. Med., 29, 56-64. Aksoy, M., S. Erdem et al. (1976) Combination of genetic factors and chronic exposure in the aetiology of leukemia, Hum. Hered., 26, 149-153. Buckton, K.E. (1976) Identification with G and R banding of the position of breakage points induced in human chromosomes by in vitro X-irradiation, Int. J. Radiat., 29, 475-488.
240 Clare, M., A. Yardley-Jones et al. (1984) Chromosome analysis from peripheral blood lymphocytes of workers after an acute exposure to benzene, Br. J. Industr. Med., 41,249-253. Cronkite, E.P. (1987) Chemical leukemogenesis: benzene as a model, Semin. Hematol., 24, 2-11. Dean, B.J. (1978) Genetic toxicology of benzene, toluene, xylene and phenols, Mutation Res., 47, 75-97. Gundy, S., and L.P. Varga (1983) Chromosomal aberrations in healthy persons, Mutation Res., 120, 187-191. Honeycombe, J.R. (1978) The effects of busulphan on the chromosomes of normal human lymphocytes, Mutation Res., 57, 35-49. ISCN (1985) An International System for Human Cytogenetic Nomenclature, S. Karger, New York. Kalf, G.F. (1987) Recent advances in the metabolism and toxicity of benzene, CRC Crit. Rev. Toxicol., 18, 141-159. Marlhens, F., W. A1 Ackhar et al. (1986) The rate of chromosome breakage is age dependent in lymphocytes of adult controls, Mutation Res., 73, 290-297. Mattei, M.G., J. Ayme et al. (1979) Distribution of spontaneous chromosome breaks in man, Cytogenet. Cell Genet., 23, 95-102. Meyne, J., LH. Lockhart and F.H. Arrighi (1979) Nonrandom distribution of chromosomal aberrations induced by three chemicals, Mutation Res., 63, 201-209. Mondello, C., R. Giorgi and F. Nuzzo (1984) Chromosomal effects of methotrexate on cultured human lymphocytes, Mutation Res., 139, 67-70. Moorhead, P.S., P. Nowell et al. (1960) Chromosome preparation of leukocyte cultures from human peripheral blood, Exp. Cell Res., 20, 613-616. Morimoto, K. (1983) Induction of sister chromatid exchanges and cell division delays in human lymphocytes by microsomal activation of benzene, Cancer Res., 43, 1330-1334.
Morimoto, K., and S. Wolff (1980) Increase of sister chromatid exchanges and perturbations of cell division kinetics in human lymphocytes by benzene metabolites, Cancer Res., 40, 1189-1194. Morimoto, K., S. Wolff and K. Koizumi (1983) Induction of sister chromatid exchanges in human lymphocytes by microsomal activation of benzene metabolites, Mutation Res., 119, 355-360. Moszczyfiski, P., and J. Lisiewicz (1983) Occupational exposure to benzene, toluene and xylene and the T lymphocyte functions, J. Clin. Hematol. Oncol., 13, 37-41. Rinsky, R.A., M.S. Alexander et al. (1987) Benzene and leukemia, N. Engl. J. Med., 316, 1044-1050. S~tsiadek, M. (1984) Zmiany chromosomalne obserwowane u pracownik6w przewlekle naraZonych na benzen i jego pochodne, Pol. Tyg. Lek., 39, 40-41. Seabright, M., P. Cooke and M. Wheeler (1975) Variation in trypsin banding at different stages of contraction in human chromosomes and the definition, by measurement of the 'average' karyotype, Humangenetik, 29, 35-40. Smeets, D., J. Scheres and T. Hustinx (1986) The most common fragile site in man is 3p14, Hum. Genet., 74, 330. Smolik, R., K. Grzybek-Hryncewicz and A. Lange (1973) Serum complement level in workers exposed to benzene, toluene and xylene, Int. Arch. Arbeitsmed., 31,234-247. Snyder, R., L. Jowa et al. (1987) Formation of reactive metabolites from benzene, Arch. Toxicol., 60, 61-64. Tawn, E.J. (1987) The frequency of chromosome aberrations in a control population, Mutation Res., 182, 303-308. Van der Berghe H., K. Vermaelen et al. (1985) The 5q anomaly, Cancer Genet. Cytogenet., 17, 189-255. Yin, S.N., F.D. Tain et al. (1987) Leukaemia in benzene workers: a retrospective cohort study, Br. J. lndustr. Med., 44, 124-128.
235
MUTGEN 01474
Localization of breakpoints in the karyotype of workers professionally exposed to benzene M a r i a S~siadek a, J b z e f Jagielski a a n d R o m a n S m o l i k b " Department of Pathophysiology, Medical Academy of Wroctaw, 50-368 Wroctaw (Poland) and b Department of Internal and Occupational Medicine, Medical Academy of Wroctaw, 50-367 Wroctaw (Poland) (Received 19 July 1988) (Revision received 24 April 1989) (Accepted 1 May 1989)
Keywords: Benzene; Chromosome aberrations; Distribution of breakpoints
Summary Cytogenetic analysis was carried out on peripheral blood lymphocytes of 33 workers professionally exposed to benzene for 10-26 years. In the examined group structural chromosome aberrations were found in 147 metaphases (4.7%). Statistical analysis has shown that the distribution of breakpoints was not random. Chromosomes 2, 4 and 9 are almost twice as susceptible to breaks, while chromosomes 1 and 2 are almost twice as susceptible to gaps, as would be expected based on a random distribution of damage among chromosomes.
Benzene and its derivatives are important substances widely used in industry. Many workers are professionally exposed to the agents for a long time. Chronic benzene intoxication modulates immune response, granulocyte enzyme systems, leads to thrombocytopenia, leukopenia, anemia, even pancytopenia and, in extreme cases, to neoplastic diseases (Aksoy et al., 1972, 1976; Cronkite, 1987; Dean, 1978; Moszczyfiski et al., 1983; Smolik et al., 1973). The frequency of neoplasia in workers professionally exposed to benzene and its derivatives is significantly higher than in the unexposed population (Honeycombe, 1978; Rinsky et al., 1987; Yin et al., 1978). In groups of workers professionally exposed to benzene chromosome
Correspondence: Dr. M. S~siadek, Department of Pathophysiology, Medical Academy of Wrodaw, Marcinkowskiego 1, 50-368 Wroclaw (Poland).
aberrations, both structural and numerical, were found in 2.25-5.5% of analyzed metaphases. However, in patients with leukemia caused by benzene, a much higher percentage of chromosome aberrations was found (4-55%). The correlation between time of exposure to benzene, age, sex and percentage of damaged metaphases has not yet been determined (Aksoy et al., 1976; Clare et al., 1984; Sosiadek, 1984). Cytogenetic effects have been observed both in laboratory animals and in cultured cells treated with benzene (Dean, 1978; Morimoto and Wolff, 1980; Morimoto, 1983; Morimoto et al., 1983). Although the metabolism and toxicity of benzene in the organism are known, the mechanism of neoplastic cell transformation induced by benzene has not yet been ascertained. Activation of tyrosine-kinase groups of oncogenes may play a key role in the neoplastic transformation of cells (Cronkite, 1987; Kalf, 1987; Synder et al., 1987).
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
17 17 16
15 15 15 14 13 13
13 12 12 12 12 11 11 11 3
3 3
14 15 16
17 18 19 20 21 22
23 24 25 26 27 28 29 30 31
32 33
-
-
-
-
-
-
-
-
-
-
-
40 30
49 40 46 49 35 40 40 40 33
48 34 48 49 43 47
42 43 37
48
46
41 48 47
46 42 47
48 47 41
a N u m b e r of cigarettes p e r d a y x years of smoking.
17
19 19 17
9 10 11
17
20 19 19
6 7 8
12
22 22 21
13
22
3 4 5
40 40
45
45
40 42 45
45 40 45
48 50 45
43 40
2
C
E
-
C 48 47
23
E
1
M M
M F M F M M F M M
F F F F M M
F F M
F
F
F M F
M F F
M M M
M F
E
Sex
F F
F
F
F M F
M F F
M M F
M M
C
300 180
450 400 400 500 400 300 180 200 200
400 250 300 400 300 350
350 400 300
300
350
350 400 400
400 180 300
300 250 250
400 400
E
300 400
250
180
300 400 400
400 200 200
400 300 300
400 400
C
C i g a r e t t e index
A g e (years)
Subject
Period of exposure (years)
OF CHROMOSOME
A G E , SEX, P E R I O D O F E X P O S U R E , C I G A R E T T E I N D E X a A N D P E R C E N T A G E E X P O S E D T O B E N Z E N E (E) A N D I N C O N T R O L S (C)
TABLE 1
100 100
100 100 100 100 100 100 100 30 100
100 100 30 100 100 100
100 100 100
100
100
100 100 100
100 100 30
100 100 100
100 100
E
100 100
100
100
100 100 100
100 100 100
100 100 100
100 100
C
PROFESSIONALLY
0 0
0 4 9 0 14 4 7 0 3
4 1 0 6 11 6
4 7 4
2
4
8 0 4
1 7 0
6 3 13
8 7
E
C
P e r c e n t a g e of c h r o m o some aberrations
IN W O R K E R S
N u m b e r of a n a l y z e d metaphases
ABERRATIONS
237
The aim of this work was to analyze the chromosomal localization of aberrations in the karyotypes of workers exposed to benzene and its derivatives. Material and methods
The studies were carried out on peripheral blood bcmphocytes of 33 workers occupationally exposed
to benzene and its derivatives for 10-23 years. During this period the concentration ~f benzene was below the m a x i m u m allowable concentration (MAC, 0.1 mg/1). Thirty-one of the workers did not show any clinical and hematological symptoms of chronic benzene intoxication. In 2 cases pancytopenia was observed.
I1
i-I !.l-l i-- -ii
i i-
--ii I1 -it -i!
-lit
1
-iib
i'i -it
1t--|-;(--i
-irl--i:i -"
l--i
.~ "
iii -
tt ',-
"-
.~--
-~i
Fig. 1. Karyotypes of a worker professionally exposed to benzene for 18 years, 46,XX(ctb(3)(p13)). Breakpoints are marked by arrows.
238
The control group consisted of 15 patients of approximately the same age, who had no contact with benzene or other chemical a n d / o r physical agents. Patients in both investigated groups were interviewed about infectious diseases, drugs and exposure to X-rays during the last 3 months before cytogenetic examination. All patients in both groups were smokers. None of them used alcohol heavily. Blood samples were obtained by venipuncture. Lymphocytes were cultured for 72 h according to the method of Moorhead et al. (1960). Cytogenetic analysis was performed using G T G - b a n d e d preparations (Seabright et al., 1975). Chromosome aberrations were classified according to ISCN (1985). Statistical analysis was based on the X2 test.
Results
In the group of 33 workers exposed to benzene and its derivatives 3090 metaphases were analyzed, while in the control group 1500 metaphases were studied. In the examined group structural chromosome aberrations were found in 147 metaphases (4.7%), in contrast with the control group, where aberrations were only found in 25 (1.68%) of analyzed metaphases (Table 1). Among the aberrations detected, there was no evidence, with one exception, of clonal origin. In one patient, a 43-year-old woman occupationally exposed to benzene for 18 years, a clonal aberration, probably at the fragile site ctb(3)(p13), occurred in 4% of analyzed metaphases (Fig. 1)
TABLE 2 OBSE RVED A N D E X P E C T E D N U M B E R S OF BREAKS A N D GAPS IN T H E EXPOSED G R O U P Chro-
II
mosome Rumber 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
Breaks
G a ps (O - E) 2
8.44 8.02 6.83 6.30 6.08 5.90 5.36 5.12 4.93 4.80 4.59 4.61 4.66 3.74 3.56 3.46 3.36 3.25 2.93 2.67 2.56 1.90 2.04 2.15
O
E
6 13 a 7 15 a 6 6 3 3 9a 2 2 2 1 3 2 2 1 0 0 0 0 1 3 0
7.26 6.9 5.87 5.42 5.23 5.07 4.61 4.24 4.13 3.94 3.96 4.01 3.22 3.06 3.84 2.89 2.8 5.25 2.3 2.20 1.63 1.75 4.40 1.85
E 0.21 5.39 0,22 16,9 0.11 0,17 0,56 0,36 5.74 0,95 0,97 1 1,53 0.002 0,88 0,27 1,16 5,52 2,3 2,20 1,63 0,32 0,45 1,85
O E 0.82 1.88 1.19 2.7 1.14 1.18 0.65 0.70 2.18 0.51 0.5 0.5 0.31 0.98 0.52 0.69 0.36 0 0 0.45 0 0.57 0.68 0
(O - E) 2 O
E
9a 16 a 6 6 3 3 3 3 3 2 2 2 0 0 0 0 0 0 1 0 0 0 0 1
5.15 4.89 4.17 3.84 3.71 3.6 3.27 3.01 2.93 2.8 2.8 2.8 2.8 2.17 2.11 2.05 1.98 1.79 1.63 1.56 1.16 1.24 3.12 1.31
E 2.88 25.2 0.8 1.21 0.13 0.1 0.022 0 0 0.23 0.23 0.23 2.8 2.17 2.11 2.05 1.98 1.79 0.24 1.56 1.16 1.24 3.12 0.07
O E 1.74 3.27 1.44 1.56 0.81 0.83 0.92 0.99 1.02 0.71 0.71 0.71 0 0 0 0 0 0 0.61 0 0 0 0 0.76
X 2 12.3, df 6, P = 0.45i a results are significant at p = 0.01; X 2 9.4, df 1, other results are not significant. II, % chromosome length (ISCN, 1985); O, observed number of aberrations; E, expected number of aberrations.
239 (Smeets et al., 1986). This w o m a n did not show any clinical or hematological symptoms of chronic benzene intoxication. The chromosomal distribution of the 147 chromosome aberrations and the statistical analysis are presented in Table 2. Chromosomes 2, 4 and 9 are almost twice as susceptible to breaks, while chromosomes 1 and 2 are almost twice as susceptible to gaps as expected. Chromosome 18 appears to be underrepresented for the presence of aberrations. In the control group 25 breakpoints were distributed randomly on 23 chromosome pairs. On chromosomes 1, 2, 3, 5, 7 and 10 two breakpoints were found.
Discussion In the present study, workers occupationally exposed to benzene and its derivatives exhibited a significantly increased frequency of metaphase cells with structural chromosome aberrations over that observed in a control population (4.7% versus 1.7%). The percentage of damaged cells in the benzene-exposed workers is higher than that recorded in earlier studies by Aksoy et al. (1976) and by Clare et al. (1984). This difference m a y be due to the level of benzene to which the workers in the present study were exposed (although the concentration of benzene was below the MAC) or to differences in ascertainment. The other possibility is that in the present study the scoring for chromosomal aberrations was performed not in 48-h cultures, as it is accepted now, but in 72-h cultures, so real damage could be underestimated. The percentage of cells with aberrations in the control group is similar to that described in the literature ( G u n d y et al., 1983; Mattei et al., 1979; Tawn, 1987). A statistical analysis of the chromosomal distribution of the breakpoints involved in the aberrations indicated that while in control individuals the distribution was random, it was n o n - r a n d o m in the cells of individuals exposed to benzene. These results for the benzene-exposed workers agree with earlier reports that the interchromosomal distribution of breakpoints in cells exposed in vitro to various mutagens is n o n - r a n d o m (Buckton, 1976; Honeycombe, 1978; Meyne et al., 1979; Mondello et al., 1984). However, earlier
investigators also reported that the interchromosomal distribution of breakpoints among control cells was non-random. Chromosomes 3, 7 and 16 were preferentially broken in those studies (Marlhens et al., 1986; Mattei et al., 1979). The apparently r a n d o m distribution of the breakpoints of chromosome aberrations observed in our control population m a y be due to the small number of aberrations observed. In workers exposed to benzene, the observed frequency of breakpoints in chromosomes 1, 2, 3 and 9 was twice as high as expected, based on relative chromosome length. Chromosomes 1, 3 and 9 also exhibit an increased sensitivity to the clastogenic activity of methotrexate (Mondello et al., 1984), cis-platinum(II) diamminedichloride, triethylenemelamine (Meyne et al., 1979) and busulfan (Honeycombe, 1978). This consistent sensitivity suggests an inherent fragility to D N A - d a m a g i n g agents or a preferential lack of repair in those regions. Of interest is the observation that the frequency of breakpoints in chromosome 5 was not higher than expected despite the fact that this chromosome is usually involved in the etiology of secondary leukemias (Van der Berghe et al., 1985). While this study demonstrates that the increased frequency of chromosomal aberrations in cells of workers occupationally exposed to benzene is non-randomly distributed among chromosomes, it is not yet possible to determine whether this n o n - r a n d o m distribution is involved in the etiology of the tumors induced by benzene. To evaluate this relationship, it would be necessary to determine also the distribution of chromosomal alterations occurring among cancers present in workers occupationally exposed to benzene.
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