Chromosome changes in lymphocytes after occupational exposure to toluene

Mutation Research, 102 (1982) 439-445

439

Elsevier BiomedicalPress

Chromosome changes in lymphocytes after occupational exposure to toluene M. Bauchinger 1 E. Schmid l, j. Dresp l, j. Kolin-Gerresheim ~, R. Hauf 2 and E. Suhr 2 t Division ofRadiobiology, Cytogenetics, GSF, 8042 Mi~nchen-Neuherber~ Post Oberschleissheim; and 2 Institute of Practical Occupational Medicine, 7800 Freiburg/Breisgau (West Germany)

(Received30 March 1982) (Accepted 18 May 1982)

Summary Cytogenetic analyses were carried out in peripheral lymphocytes from 20 male workers exposed only to toluene in a rotogravure plant for more than 16 years. As compared with a group of 24 unexposed controls, significantly higher yields of chromatid breaks, chromatid exchanges and gaps were observed. The number of SCEs was significantly increased in smoking and non-smoking toluene-exposed workers compared with the corresponding control groups.

Up to now, four chromosome studies of peripheral lymphocytes of workers exposed only to toluene or to a mixture of toluene and other organic solvents have been published (Forni et al., 1971; Funes-Cravioto et al., 1977; M~iki-Paakkanen et al., 1980; Haglund et al., 1980). Because the results are contradictory, further data should be established. The Institute of Practical Occupational Medicine in Freiburg (Federal Republic of Germany) carried out a study on the health hazards of 100 workers at a rotogravure plant with a toluene exposure of at least 10 years. For the present study, 20 individuals exposed for more than 16 years were selected from these workers for the analysis of chromosome aberrations and sister-chromatid exchanges (SCE).

Material and methods Chromosomes were analysed in peripheral lymphocytes of 20 males working on a rotogravure machine. Toluene with a benzene concentration of < 0.3% is used as a rapidly evaporating solvent for printing inks. The continuously measured toluene 0165-1218/82/0000-0000/$02.75 © ElsevierBiomedicalPress

OF CHROMOSOME

ABNORMALITIES±

S.E. A N D

S C E s - + S.E. I N L Y M P H O C Y T E S

OF TOLUENE-EXPOSED

WORKERS

Age

60

46

8

9

44.2

±7.0

36

44

19

20

1-20

32

18

Toluene

37

51

15

17

52

14

16

41

48

13

51

42

7

12

41

6

37

40

5

42

40

4

11

41

3

10

53

49

1

---+S . D .

(years)

2

No.

+

-

+

+

+

--

+

--

+

--

+

--

+

+

--

--

-

+

+

+

Smoking

S cells

±0.0024

0.0248

0.027

0.040

0.023

0.043

--+0.13

0.90

0.3

1.3

0.7

1.0

0.3

0

0.033

0.027

2.3

1.3

1.0~

0.3

0.3

1.0

2.0

1.0

1.0

0.7

0.7

0.3

1.0

1.3

(%)

0.030

0.013

0.030

0.013

0.003

0.030

0.027

0.043

0.023

0.007

0.023

0.023

0.017

0.020

p e r cell

Gaps

-+0.0010

0.0036

-

0.007

-

-

0.020

0.010

0.003

-

-

0.003

0.007

0.003

0.003

0.007

-

-

0.003

0.007

±0.0005

0.0015

0.003

-

-

0.007

0.003

0.003

-

-

0.003

-

0.003

0.007

-

-

-

-

-

-

Chromatid exchanges

Chromatid breaks

A b e r r a t i o n s p e r cell

±0.0008

0.0035

-

0.003

0.003

0.003

-

0.007

-

0.007

0.003

-

0.007

0.007

-

0.007

-

0.007

0.003

0.007

0.007

fragments

Acentric

-+0.0003

0.0005

-

0.003

0.003

-

-

-

-

-

-

-

0.003

-

-

-

-

-

-

-

Dicentrics

9.62±0.37

13.02 --+ 0.59

9.22 ± 0.43

13.72 ± 0.58

9.76 ± 0.52

9.46 ± 0.58

9.34 ± 0.58

10.78 ± 0.59

7.64±0.50

9.04±0.50

8.12±0.44

9.66 ± 0.49

9.46 ± 0.57

8.56 ± 0.38

11.40--+ 0.59

7.50 ± 0.41

8.70 ± 0.53

8.44±0.42

8.74 ± 0.42

8.84 ± 0.46

10.96 ± 0.59

p e r cell --+ S.E.

SCE

300 c e l l s w e r e a n a l y s e d f o r s t r u c t u r a l c h r o m o s o m e c h a n g e s , a n d 50 c e l l s f o r S C E i n e a c h s u b j e c t . S cells, cells w i t h s t r u c t u r a l c h r o m o s o m e a b e r r a t i o n s .

FREQUENCY

TABLE 1

441 concentration in the air of the rotary machine room was between 200 and 300 ppm. The maximal allowable concentration (MAC) for toluene in the Federal Republic of Germany is 200 ppm (750 mg/m3). Toluene is mainly absorbed by inhalation of vapours. Additional low amounts of liquid toluene are absorbed through the skin during the cleaning of hands. The measured concentrations of toluene in blood were between 0.001 and 0.01700. There was no exposure to other industrial chemicals. 11 workers were heavy smokers ( > 10 cigarettes per day), 1 was a moderate smoker ( < 10 cigarettes per day), 8 were non-smokers. From the same plant, a group of 24 workers (8 heavy smokers, 1 moderate smoker and 15 non-smokers) with similar age and social environment, but without exposure to toluene, served as controls. Some were machinists or fitters, others were employed in the store-rooms or post-office. In both groups, alcohol was consumed moderately during an 8-h work day, but the alcohol consumption was higher in the control group. All the 44 persons examined were healthy, and no one had previously received radiation or cytostatic therapy. Subjective complaints, such as poor appetite, nausea, vertigo, headache, rapid fatigue, nervousness, sleeplessness and alcohol intolerance, which are often attributed to chronic exposure to toluene, were not more frequently mentioned by rotogravure printers than by controls. From data of regular medical examinations during the preceding 10 years, there was no evidence for significant changes in the blood picture or for a toxic liver effect. Neurological examinations revealed no damage of the central nervous system (Suhr, 1975). For the chromosome analyses, standard cultures with 0.5 ml whole blood, 4 ml FI0 medium, 0.5 ml foetal calf serum, 0.13 ml PHA and 9.7 × 10 -6 M BrdU were used. Colcemid (0.1 /~g/ml) was present in the cultures for the final 3 h. Structural chromosome damage was analysed exclusively in first-division metaphases (M~) identified by uniformly stained sister chromatids from 44-h cultures. 300 cells with a complete chromosome complement were scored from each rotogravure printer and 500 cells from each control person. 50 'harlequin'-stained cells (M2) per individual were scored for SCEs from 54-h cultures. To account for the reduction of BrdU concentration with increasing sampling time owing to cell proliferation, an initial concentration of 3.3 × 10 -5 M was used (Kolin-Gerresheim and Bauchinger, 1981). A detailed description of F P G staining is given elsewhere (Apelt et al., 1981).

R ~ The results of the chromosome analysis of toluene-exposed workers are compiled in Table 1. The control data are given in Table2. For statistical comparison, single-person data were used. The Mann-Whitney rank U test (significance taken as P < 0.05) was applied to compare the chromosome changes of different groups. In the group of toluene-exposed workers a significantly higher number of cells with structural chromosome changes (S-cells) than in controls was observed ( P = 0.006). The damage was predominantly of the chromatid type with significantly

OF CHROMOSOME

ABNORMALITIES

-+ S.E. A N D

S C E s ÷ S.E. I N L Y M P H O C Y T E S

OF UNEXPOSED

CONTROLS

44

30

42

16

17

18

Gaps

S cells

0.019 -+0.003

± 10.5

0.012

0.014

0.012

0.012 0.036

0.006

0.014

0.034

±0.06

0.51

0.2

0.8

0.4

0.2 0.2

0.2

0.8

0.2

0.6

1.0

0.024 0.030

0.2

0.4

1.0

1.2

0.4

0.4

0.2

0.6

0.4

0.4

0.6

1.0

0.6

0.2

(%)

0.018

0.010

0.010

0.008

0.032

0.036

0.010

0.008

0.032

0.062

0.016

0.010

0.014

0.014

p e r cell

42.4

+

+

+

+

--

--

--

+

--

--

--

--

+

--

--

--

-

a O n e c e n t r i c r i n g is i n c l u d e d .

1-24

22

49

15

50

29

24

46

13

14

23

47

12

35

38

11

22

48

59

45

9

lO

29 30

51

47

7

8

19

--

44

20 21

+

46

5

6

+

--

57

29

+

--

3

37

Smoking

4

62

t

± S.D.

(years)

Age

2

No.

-+0.0005

0.0019

-

0.004

0.004

-

0.002

-

-

-

0.002

-

-

0.004

0.010

0.002

-

0.002

-

-

0.002

0.008

0.004

-+0.0002

0.0004

-

-

-+0.0004

0.0023

0.002

0.004

-

-

0.002

0.002

0.006

-

0.002

0.004

-

0.004

0.004

0.002

0.002

0.004

0.002

0.002

0.004

0.004

0.004

0.002

0.002

fragments

Acentric

-

-

-

-

0.002

0.004

-

-

-

-

-

-

0.002

-

-

-

-

-

-

exchanges

0.002

Chromatid

Chromatid breaks

A b e r r a t i o n s p e r cell

-+0.0002

0.0005

_

-

-

0.002

0.002

0.002

-

0.002

-

0.002 a

-

0.002

-

-

-

-

-

-

-

-

Dicentrics

8.18--+0.25

8.52 ± 0 . 4 4

8.88 ± 0.43

10.28~0.52

6.48 -+ 0.33

7.50 ± 0.40 6.38 ± 0 . 3 3

6.26 -+ 0.37

8.14 ± 0.50

10.06 -+ 0.51

6.78 -+ 0.43

7.02 -+ 0.33

8.16_+0.51

7.30±0.44

8.76 -4- 0.42

7.14 ± 0.47

9.12 --+0.46

10.32 ± 0.50

7.44 -+ 0.52

9.02 --+0.48

8.82 ± 0.59

9.22 ± 0.47

8.84 --+ 0.55

7.90 -+ 0.42 7.90 ± 0.45

-+ S.E.

p e r cell

SCE

5 0 0 cells were a n a l y s e d f o r s t r u c t u r a l c h r o m o s o m e c h a n g e s , a n d 50 c e l l s f o r S C E i n e a c h s u b j e c t . S cells, cells w i t h s t r u c t u r a l c h r o m o s o m e a b e r r a t i o n s .

FREQUENCY

TABLE 2

443 TABLE 3 STATISTICAL E V A L U A T I O N OF SCE DATA a

Controls, s. Controls, n.s. Toluene, s. Toluene, n.s.

N u m b e r of individuals

Number of cells

Mean SCE per cell+-S.E.

9 15 12 8

450 750 600 400

8.89 +-0.41 7.75 -4-0.25 10.33 -+ 0.49 8.55 +--0.27

a s., smokers; n.s., non-smokers.

increased yields of chromatid breaks (P = 0.014) and chromatid exchanges (P = 0.049). In contrast, the yields of acentrics and dicentrics were unchanged. Additionally, the yield of gaps was significantly increased (P = 0.048). For the statistical evaluation of SCE data, the subjects of either group were subdivided into smokers and non-smokers (Table 3). Such an analysis revealed significantly higher SCE values for non-smoking rotogravure workers compared with non-smoking controls (P=0.021). This was also true for smoking rotogravure workers compared with smoking controls (P = 0.032). In both groups, smokers had significantly higher SCE values than non-smokers (controls, P = 0.020; toluene exposed, P = 0.003).

Discussion

Conventional chromosome analysis from 24 workers (Forni et al., 1971) and from 32 workers (Maki-Paakkanen .et al., 1980) at rotogravure plants revealed no significant differences between the frequencies of chromosome changes in the toluene groups and in the control groups. Haglund et al. (1980) reported on chromosome analyses from paint-industry workers exposed to a mixture of organic solvents, mainly containing xylene or toluene. For the frequency of structural chromosome aberrations in 5 workers with the highest exposure (toluene concentration > 100 m g / m 3) and their matched controls, no differences were found. In contrast with these studies, Funes-Cravioto et al. (1977) found increased frequencies of chromatid and isochromatid breaks in 14 workers exposed only to toluene in a rotoprinting factory compared with controls. This result is similar to the observation of our study. The negative results published by Forni et al. (1971) hold for cells with chromosome-type changes that were classified according to Buckton et al. (1962) as C u cells and C s cells. If we use this classification for our data, both results are in line. However, in Forni's paper no information is available on the number of cells with chromatid-type aberrations (B cells), which is significantly increased in the present study. Published results of SCE analysis revealed no significant differences between exposed workers and controls (Funes-Cravioto et al., 1977; M~iki-Paakkanen et al., 1980; Haglund et al., 1980). Owing to the low number of subjects analysed - - only 4

444 in Funes-Cravioto's study and 5 in that of Haglund et al. - - a meaningful comparison with our SCE data is only feasible for the study of Maki-Paakkanen et al. In contrast with these authors, we found a significant increase of SCEs for smoking and non-smoking toluene-exposed workers. Up to now only one study exists of the cytogenetic effect in human lymphocytes after exposure to toluene in vitro. Though Gerner-Smidt and Friedrich (1978) described negative results for conventional and SCE analyses after treatment with 15.2/tg to 1.52 mg toluene per ml, no distinct conclusions can be drawn owing to the insufficient number of cells analysed (total 236) as stated by the authors themselves. It is evident from our findings that toluene or its metabolites (Dean, 1978) may induce a weak clastogenic effect in human lymphocytes in vivo. The observed chromosome changes were of the chromatid type (breaks and exchanges). Chromatid-type structural changes induced by chemical mutagens are often accompanied by gaps, which in the present study were significantly increased too. It is known that, with increasing culture times, chromatid breaks and gaps occur more frequently, i.e., one cannot reliably decide whether these changes are induced by a chemical or at least partly by the culture conditions. Since we have scored exclusively first-division cells for conventional chromosome analysis, such an influence is far less likely. However, on the other hand, a synergistic effect of toluene and BrdU cannot be fully excluded. The observed chromosomal changes can certainly not be attributed to the BrdU treatment. If we compare the yields of the relevant aberration types per cell (gaps, 0.019-+0.005; chromatid breaks, 0.002 -+ 0.0005; acentrics, 0.0021 -+ 0.0004) which we had previously observed in 12700 cells of control cultures without BrdU (Bauchinger et al., 1980), there are no recognizable differences as compared with the data of the present study (Table 2). A weak cytogenetic effect of toluene may only be detected if the following criteria are present simultaneously. A sufficiently large number of subjects, exposed to high toluene concentrations and a large number of cells, scored, in previously published studies only 100 Cells were analysed per individual. The negative results for conventional chromosome analysis of Forni et al. (1971), M~iki-Paakkanen et al. (1980), as well as of the latter for SCE analysis, may be mainly explained by the lower toluene exposure of the workers. As compared with 200-300 ppm in our study, the mean concentrations stated by Forni et al. were usually below and only sometimes just above the MAC of toluene (200 ppm), and were 7-112 ppm in the study of M~iki-Paakkanen et al. Such an explanation seems to be justified if one considers the positive results of Funes-Cravioto et al. (1977) from workers exposed to toluene in a concentration of 100-200 ppm with occasional rises up to 500-700 ppm.

Acknowledgements We thank Mrs. Bamberg, Mrs. Giesen and Mrs. Rocznik for their skilful technical assistance. Thanks are also due to the physicians of the rotogravure plant and to the occupational monitoring office for their co-operation and provision of the blood samples.

445

References Apelt, F., J. Kolin-Gerresheim and M. Bauchinger (1981) Azathioprine, a clastogene in human somatic cells? Analysis of chromosome damage and SCE in lymphocytes after exposure in vivo and in vitro, Mutation Res., 88, 61-72. Bauchinger, M., J. Kolin-Gerresheim, E. Schmid and J. Dresp (1980) Chromosome analyses of nuclearpower plant workers, Int. J. Radiation Biol., 38, 577-581. Buckton, K.E., P.A. Jacobs, W.M. Court Brown and R. Doll (1962) A study of the chromosome damage persisting after X-ray therapy for ankylosing spondylitis, Lancet, II, 676-682. Dean, B.J. (1978) Genetic toxicology of benzene, toluene, xylenes and phenols, Mutation Res., 47, 75-97. Forni, A., E. Pacifico and A. Limonta (1971) Chromosome studies in workers exposed to benzene or toluene or both, Arch. Environ. Health, 22, 373-378. Funes-Cravioto, F., B. Kolmodin-Hedman, J. Lindsten, M. Nordenskj61d, C. Zapata-Gayon, B. Lambert, E. Nordberg, R. Obin and A. Swensson (1977) Chromosome aberrations and sister chromatid exchange in workers in chemical laboratories and a rotoprinting factory and in children of women laboratory workers, Lancet, II, 322-325. Gerner-Smidt, P., and U. Friedrich (1978) The mutagenic effect of benzene, toluene and xylene studied by the SCE technique, Mutation Res., 58, 313-316. Haglund, U., J. Lundberg and L. Zech (1980) Chromosome aberrations and sister chromatid exchanges in Swedish paint industry workers, Scand. J. Work Environ. Health, 6, 291-298. Kolin-Gerresheim, J., and M. Bauchinger (1981) Dependence of the frequency of harlequin-stained cells on BrdU concentration in human lymphocyte cultures, Mutation Res., 91, 25 i-254. M~ki-Paakkanen, J., K. Husgafvel-Pursiainen, P.L. Kalliomaki, J. Tuominen and M. Sorsa (1980) Toluene exposed workers and chromosome aberrations, J. Toxicol. Environ. Health, 6, 775-781. Suhr, E. (1975) Vergleichende Untersuchungen tiber den Gesundheitszustand toluolexponierter Tiefdrucker, Thesis, Medical Faculty, Albert-Ludwigs-University Freiburg im Breisgau, Institut ftir Praktische Arbeitsmedizin, pp. 1-97.