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Analysis of chromosomes in peripheral blood lymphocytes of styrene-exposed workers
Mutation Research, 206 (1988) 167-169 Elsevier
167
MTR 01315
Analysis of chromosomes in peripheral blood lymphocytes of styrene-exposed workers Angelika Jablonickfi, Jarmila Karelov~, Helena Polfikovfi and Maria Vargovfi Research Institute of Preventive Medicine, 833 O1 Bratislava (Czechoslovakia)
(Received 9 November 1987) (Revision received 16 February 1988) (Accepted 29 March 1988)
Keywords: Styrene-exposed workers; Chromosomes; Peripheral blood lymphocytes; Lymphocytes, peripheral blood; Chromosomal aberrations
Summary A group of 11 workers exposed to an average of 253 mg. m -3 styrene had no elevation of the frequencies of chromosomal aberrations in their peripheral lymphocytes when compared to 11 healthy persons.
Styrene and its polymers a n d / o r metabolites are widely used in chemical industries as well as in the processing of various materials of common use. Almost the entire population is exposed to these agents, and this has prompted a vivid interest in checking the possible genetic impact of the exposure. Both styrene and styrene oxide are mutagenic in various in vivo and in vitro test systems (IARC, 1979). Studies suggesting genetic effects in workers occupationally exposed to styrene during its production and processing are of special interest (Meretoja et al., 1977, 1978; Meretoja and Vainio, 1979; Fleig and Theis, 1978; Hoegstedt et al., 1979; Andersson et al., 1980). Damage to chromosomes was found in workers occupationally exposed to high but not to low doses of styrene (Thiess et al., 1980; Watanabe et al., 1983; Hansteen et al., 1984; Pohiovfi and Srfim, 1985).
Correspondence: Dr. A. Jablonickfi, Research Institute of Preventive Medicine, 833 01 Bratislava (Czechoslovakia).
We analyzed the chromosomes of peripheral lymphocytes of workers in a laminated plastic shop, where the average styrene concentrations exceeded the Czechoslovak MAC-P values of 200 mg- m -3. Materials and methods
The exposed group consisted of 11 women aged 30-55 years, with average styrene exposure of 10 years. They were almost continuously exposed to styrene vapours (inhalation) or occasionally had direct skin contact with styrene during 8 working hours. The women manually laminated various kinds of sport utensils, boats and containers. The styrene concentrations in the working area ranged from 118 to 582 mg. m -3. The control group included 11 adult women of comparable age, social habits, living and working environment, except that they were not exposed to styrene. Histories of viral and other diseases during the previous 3 months were recorded together with data concerning contacts with other factors
0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
168
known to induce chromosomal aberrations (e.g. intake of drugs and alcohol, smoking habits, X-ray examination or rtg. therapy). All subjects with some illness or taking medication in the preceding period were excluded from this study. There were only two weak smokers in both analysis groups. Cytogenetic analysis was performed using short-term cultures of peripheral blood lymphocytes according to Hungerford (1965). BEM (Basal Eagle Medium, Institute of Virology, Slovak Acad. Sci., Bratislava) supplemented with 20% newborn calf serum (Bioveta, Ivanovice/Hanr), L-glutamine (I~ISOL, Prague), antibiotics (100 units/ml penicillin and 100/~g/ml streptomycin) and phytohaemagglutinin (PHA-15, Wellcome) was used as growth medium. The cultures were incubated after adding 0.5 ml of heparinized whole-blood samples for 50-52 h at 37°C. Colchicine (0.5 /~g/ml, Fluka A.G.) was added for the last 2 h of the incubation. Cells were harvested by replacing the growth medium with hypotonic solution (0.075 M KC1) and fixed twice in methanol-glacial acetic acid (3 : 1). The slides were stained with Giemsa. The styrene metabolites, mandelic acid (MA) and phenylglyoxylic acid (PGA) corrected for creatinine, were determined in the urine samples of exposed workers taken on the same day as the blood for the cytogenetic analysis (Bardodrj et al., 1961; Bardodrj and Bardodrjovfi, 1970). Both groups of persons were examined within one week. The cytogenetic analysis involved examination of 100 metaphases of each proband; chromatid breaks (B'), chromosome breaks (B"), where the units of breakage are both chromatids at the same locus, and chromatid exchanges (E', an exchange
between two or more chromosomes) were recorded. Gaps were not considered as aberrations. Cells with breaks excluding gaps a n d / o r exchanges were classified as aberrant (AB.C.). No difference was made between first- and second-division cells in our analysis. According to our experience the proportion of first- and second-division cells is about 82% and 18% in 50-52 h cultures. The results were interpreted according to Srfim and Kuleshov (1980). If there is no difference in the frequencies of AB.C. between exposed and control groups and the level of AB.C. does not exceed 2%, there was no increased genetic risk in the occupationally exposed group. The value of the mitotic index (MI), as a number of dividing cells per 1000 cells in lymphocyte culture after metaphase arrest by colchicine was analysed in each proband. Inter-group differences were statistically evaluated using the Student's t-test and the test of significance of the difference between two relative values was performed with the technique of inverse trigonometric functions (Reisenauer, 1970). Results and discussion
There was no increase of the frequencies of aberrant cells in styrene exposed workers as compared to controls (Table 1). No differences were found with respect to the types of chromosomal aberrations. Both groups showed similar cell proliferation as indicated by the mitotic indices (MI). The concentrations of MA and PGA measured in the urine of workers at the end of a shift ranged between 214-711/xl/mmole creatinine and 50-175
TABLE 1 STRUCTURAL CHANGES IN PERIPHERAL BLOOD LYMPHOCYTES IN STYRENE-EXPOSED WORKERS Groups of persons analyzed
Number of women examined
Number of metaphases
Exposed workers
11
1100
Matching control
11
1100
B'
B"
E'
G
%AB.C.
B/C
MI~
9
5
0
3
1.27
0.012
19
11
7
0
6
1.36
0.016
22
B', chromatid breaks; B", chromosome breaks; E', chromatid exchanges; G, gaps; %AB.C., percentage portion of aberrant cells; B/C, breaks per cell; MI t~, mitotic index.
169 /tl/mmole creatinine, respectively. These values were closely correlated with the styrene exposure levels. O u r results s h o w t h a t t h e e x p o s u r e c o n d i t i o n s d e s c r i b e d d i d n o t l e a d to c h r o m o s o m a l a b e r r a t i o n s in t h e p e r i p h e r a l l y m p h o c y t e s o f e x p o s e d workers.
Acknowledgements W e are g r a t e f u l to D r . J. G r u n t f o r t h e statistical analysis o f e x p e r i m e n t a l d a t a , D r . S t a l n i k a n d Dr. Pinta for monitoring styrene concentrations a n d d a t a o n M A a n d P G A levels, a n d to Ms. M . P r a v d o v h f o r t e c h n i c a l assistance.
References Andersson, H.C., E.A. Tranberg, A.H. Uggla and G. Zetterberg (1980) Chromosomal aberrations and sister-chromatid exchanges in lymphocytes of men occupationally exposed to styrene in a plastic-boat factory, Mutation Res., 73, 387-401. Bardod~j, Z., and E. BardodSjov~t (1970) Biotransformation of ethylbenzene, styrene and a-methylstyrene in man, Am. Ind. Hyg. Ass., 31,206. BardodSj, Z., E. Bardod~jovh and B. Mhlek (1961) Exposure test for styrene, (~s. Hyg., 6, 546 (in Czech). Fleig, I., and A.M. Thiess (1978) Mutagenicity study of workers employed in the styrene and polystyrene processing and manufacturing industry, Scand. J. Work. Environ. Health, 4, 254-258. Hansteen, I.-L., O. Jelmert, T. Torgrimsen and B. Forsund (1984) Low human exposure to styrene in relation to chromosome breaks, gaps and sister chromatid exchanges, Hereditas, 100, 87-91. Hoegstedt, B., K. Hedner, E. Mark-Vendel, F. Mitelman, A. Schuetz and S. Skerfving (1979) Increased frequency of
chromosome aberrations in workers exposed to styrene, Scand. J. Work Environ. Health, 5, 333-335. Hungerford, D.A. (1965) Leucocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KC1, Stain Technol., 40, 333-338. IARC (1979) Some monomers, plastics and synthetic elastomers and acrolein, IARC Monographs, Vol. 19, Lyon, pp. 231-274. Meretoja, T., and H. Vainio (1979) The use of human lymphocyte tests in the evaluation of potential mutagens: Clastogenic activity of styrene in occupational exposure, in: K. Berg (Ed.), Genetic Damage in Man Caused by Environmental Agents, Academic Press, New York, pp. 213-225. Meretoja, T., H. Vainio, M. Sorsa and H. H~konen (1977) Occupational styrene exposure and chromosomal aberrations, Mutation Res., 56, 193-197. Meretoja, T., H. JLrventaus, M. Sorsa and H. Vainio (1978) Chromosome aberrations in lymphocytes of workers exposed to styrene, Scand. J. Work. Environ. Health, 4, 259-264. Pohlovh, H., and R. Srhrn (1985) Cytogenetic analysis of peripheral blood lymphocytes of workers occupationally exposed to styrene, J. Hyg. Epidemiol. Microbiol. Immunol., 29, 155-162. Reisenauer, R. (1970) Methods of mathematical statistics and their application, SNTL, Prague, 239 pp. (in Czech). Srhrn, R.J., and N.P. Kuleshov (1980) Monitoring the occupational exposure to mutagens by the cytogenetic analysis of human peripheral lymphocytes in vivo, Arch. Toxicol., Suppl. 4, 11-18. Thiess, A.M., H. Schwelger and I. Fleig (1980) Chromosome investigations in lymphocytes of workers employed in areas in which styrene-containing unsaturated polyester resins are manufactured, Am. J. Med., 1, 205-210. Watanabe, T., A. Endo, M. Kumai and M. Ikeda (1983) Chromosome aberrations and sister chromatid exchanges in styrene-exposed workers with reference to their smoking habits, Environ. Mutagen., 5, 299-309.
167
MTR 01315
Analysis of chromosomes in peripheral blood lymphocytes of styrene-exposed workers Angelika Jablonickfi, Jarmila Karelov~, Helena Polfikovfi and Maria Vargovfi Research Institute of Preventive Medicine, 833 O1 Bratislava (Czechoslovakia)
(Received 9 November 1987) (Revision received 16 February 1988) (Accepted 29 March 1988)
Keywords: Styrene-exposed workers; Chromosomes; Peripheral blood lymphocytes; Lymphocytes, peripheral blood; Chromosomal aberrations
Summary A group of 11 workers exposed to an average of 253 mg. m -3 styrene had no elevation of the frequencies of chromosomal aberrations in their peripheral lymphocytes when compared to 11 healthy persons.
Styrene and its polymers a n d / o r metabolites are widely used in chemical industries as well as in the processing of various materials of common use. Almost the entire population is exposed to these agents, and this has prompted a vivid interest in checking the possible genetic impact of the exposure. Both styrene and styrene oxide are mutagenic in various in vivo and in vitro test systems (IARC, 1979). Studies suggesting genetic effects in workers occupationally exposed to styrene during its production and processing are of special interest (Meretoja et al., 1977, 1978; Meretoja and Vainio, 1979; Fleig and Theis, 1978; Hoegstedt et al., 1979; Andersson et al., 1980). Damage to chromosomes was found in workers occupationally exposed to high but not to low doses of styrene (Thiess et al., 1980; Watanabe et al., 1983; Hansteen et al., 1984; Pohiovfi and Srfim, 1985).
Correspondence: Dr. A. Jablonickfi, Research Institute of Preventive Medicine, 833 01 Bratislava (Czechoslovakia).
We analyzed the chromosomes of peripheral lymphocytes of workers in a laminated plastic shop, where the average styrene concentrations exceeded the Czechoslovak MAC-P values of 200 mg- m -3. Materials and methods
The exposed group consisted of 11 women aged 30-55 years, with average styrene exposure of 10 years. They were almost continuously exposed to styrene vapours (inhalation) or occasionally had direct skin contact with styrene during 8 working hours. The women manually laminated various kinds of sport utensils, boats and containers. The styrene concentrations in the working area ranged from 118 to 582 mg. m -3. The control group included 11 adult women of comparable age, social habits, living and working environment, except that they were not exposed to styrene. Histories of viral and other diseases during the previous 3 months were recorded together with data concerning contacts with other factors
0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
168
known to induce chromosomal aberrations (e.g. intake of drugs and alcohol, smoking habits, X-ray examination or rtg. therapy). All subjects with some illness or taking medication in the preceding period were excluded from this study. There were only two weak smokers in both analysis groups. Cytogenetic analysis was performed using short-term cultures of peripheral blood lymphocytes according to Hungerford (1965). BEM (Basal Eagle Medium, Institute of Virology, Slovak Acad. Sci., Bratislava) supplemented with 20% newborn calf serum (Bioveta, Ivanovice/Hanr), L-glutamine (I~ISOL, Prague), antibiotics (100 units/ml penicillin and 100/~g/ml streptomycin) and phytohaemagglutinin (PHA-15, Wellcome) was used as growth medium. The cultures were incubated after adding 0.5 ml of heparinized whole-blood samples for 50-52 h at 37°C. Colchicine (0.5 /~g/ml, Fluka A.G.) was added for the last 2 h of the incubation. Cells were harvested by replacing the growth medium with hypotonic solution (0.075 M KC1) and fixed twice in methanol-glacial acetic acid (3 : 1). The slides were stained with Giemsa. The styrene metabolites, mandelic acid (MA) and phenylglyoxylic acid (PGA) corrected for creatinine, were determined in the urine samples of exposed workers taken on the same day as the blood for the cytogenetic analysis (Bardodrj et al., 1961; Bardodrj and Bardodrjovfi, 1970). Both groups of persons were examined within one week. The cytogenetic analysis involved examination of 100 metaphases of each proband; chromatid breaks (B'), chromosome breaks (B"), where the units of breakage are both chromatids at the same locus, and chromatid exchanges (E', an exchange
between two or more chromosomes) were recorded. Gaps were not considered as aberrations. Cells with breaks excluding gaps a n d / o r exchanges were classified as aberrant (AB.C.). No difference was made between first- and second-division cells in our analysis. According to our experience the proportion of first- and second-division cells is about 82% and 18% in 50-52 h cultures. The results were interpreted according to Srfim and Kuleshov (1980). If there is no difference in the frequencies of AB.C. between exposed and control groups and the level of AB.C. does not exceed 2%, there was no increased genetic risk in the occupationally exposed group. The value of the mitotic index (MI), as a number of dividing cells per 1000 cells in lymphocyte culture after metaphase arrest by colchicine was analysed in each proband. Inter-group differences were statistically evaluated using the Student's t-test and the test of significance of the difference between two relative values was performed with the technique of inverse trigonometric functions (Reisenauer, 1970). Results and discussion
There was no increase of the frequencies of aberrant cells in styrene exposed workers as compared to controls (Table 1). No differences were found with respect to the types of chromosomal aberrations. Both groups showed similar cell proliferation as indicated by the mitotic indices (MI). The concentrations of MA and PGA measured in the urine of workers at the end of a shift ranged between 214-711/xl/mmole creatinine and 50-175
TABLE 1 STRUCTURAL CHANGES IN PERIPHERAL BLOOD LYMPHOCYTES IN STYRENE-EXPOSED WORKERS Groups of persons analyzed
Number of women examined
Number of metaphases
Exposed workers
11
1100
Matching control
11
1100
B'
B"
E'
G
%AB.C.
B/C
MI~
9
5
0
3
1.27
0.012
19
11
7
0
6
1.36
0.016
22
B', chromatid breaks; B", chromosome breaks; E', chromatid exchanges; G, gaps; %AB.C., percentage portion of aberrant cells; B/C, breaks per cell; MI t~, mitotic index.
169 /tl/mmole creatinine, respectively. These values were closely correlated with the styrene exposure levels. O u r results s h o w t h a t t h e e x p o s u r e c o n d i t i o n s d e s c r i b e d d i d n o t l e a d to c h r o m o s o m a l a b e r r a t i o n s in t h e p e r i p h e r a l l y m p h o c y t e s o f e x p o s e d workers.
Acknowledgements W e are g r a t e f u l to D r . J. G r u n t f o r t h e statistical analysis o f e x p e r i m e n t a l d a t a , D r . S t a l n i k a n d Dr. Pinta for monitoring styrene concentrations a n d d a t a o n M A a n d P G A levels, a n d to Ms. M . P r a v d o v h f o r t e c h n i c a l assistance.
References Andersson, H.C., E.A. Tranberg, A.H. Uggla and G. Zetterberg (1980) Chromosomal aberrations and sister-chromatid exchanges in lymphocytes of men occupationally exposed to styrene in a plastic-boat factory, Mutation Res., 73, 387-401. Bardod~j, Z., and E. BardodSjov~t (1970) Biotransformation of ethylbenzene, styrene and a-methylstyrene in man, Am. Ind. Hyg. Ass., 31,206. BardodSj, Z., E. Bardod~jovh and B. Mhlek (1961) Exposure test for styrene, (~s. Hyg., 6, 546 (in Czech). Fleig, I., and A.M. Thiess (1978) Mutagenicity study of workers employed in the styrene and polystyrene processing and manufacturing industry, Scand. J. Work. Environ. Health, 4, 254-258. Hansteen, I.-L., O. Jelmert, T. Torgrimsen and B. Forsund (1984) Low human exposure to styrene in relation to chromosome breaks, gaps and sister chromatid exchanges, Hereditas, 100, 87-91. Hoegstedt, B., K. Hedner, E. Mark-Vendel, F. Mitelman, A. Schuetz and S. Skerfving (1979) Increased frequency of
chromosome aberrations in workers exposed to styrene, Scand. J. Work Environ. Health, 5, 333-335. Hungerford, D.A. (1965) Leucocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KC1, Stain Technol., 40, 333-338. IARC (1979) Some monomers, plastics and synthetic elastomers and acrolein, IARC Monographs, Vol. 19, Lyon, pp. 231-274. Meretoja, T., and H. Vainio (1979) The use of human lymphocyte tests in the evaluation of potential mutagens: Clastogenic activity of styrene in occupational exposure, in: K. Berg (Ed.), Genetic Damage in Man Caused by Environmental Agents, Academic Press, New York, pp. 213-225. Meretoja, T., H. Vainio, M. Sorsa and H. H~konen (1977) Occupational styrene exposure and chromosomal aberrations, Mutation Res., 56, 193-197. Meretoja, T., H. JLrventaus, M. Sorsa and H. Vainio (1978) Chromosome aberrations in lymphocytes of workers exposed to styrene, Scand. J. Work. Environ. Health, 4, 259-264. Pohlovh, H., and R. Srhrn (1985) Cytogenetic analysis of peripheral blood lymphocytes of workers occupationally exposed to styrene, J. Hyg. Epidemiol. Microbiol. Immunol., 29, 155-162. Reisenauer, R. (1970) Methods of mathematical statistics and their application, SNTL, Prague, 239 pp. (in Czech). Srhrn, R.J., and N.P. Kuleshov (1980) Monitoring the occupational exposure to mutagens by the cytogenetic analysis of human peripheral lymphocytes in vivo, Arch. Toxicol., Suppl. 4, 11-18. Thiess, A.M., H. Schwelger and I. Fleig (1980) Chromosome investigations in lymphocytes of workers employed in areas in which styrene-containing unsaturated polyester resins are manufactured, Am. J. Med., 1, 205-210. Watanabe, T., A. Endo, M. Kumai and M. Ikeda (1983) Chromosome aberrations and sister chromatid exchanges in styrene-exposed workers with reference to their smoking habits, Environ. Mutagen., 5, 299-309.