377
Mutation Research, 51 (1978) 377--381 © Elsevier/North-Holland Biomedical Press
MUTAGENICITY TESTS WITH STYRENE OXIDE IN MAMMALS
L. FABRY a, A. LI~ONARD a and M. ROBERFROID b a Mammalian Genetics Laboratory, Department ofRadiobiology, C.E.N.-S.C.K., B-2400 Mol (Belgium), and b Laboratory of Biotoxicology, University of Louvain, Pharmacy School, UCL 7369 Brussels B-1200 (Belgium)
(Received 12 January 1978) (Revision received 6 April 1978) (Accepted 7 April 1978)
Summary The capability of styrene oxide to induce chromosome damage in vivo has been tested in the male mouse by examination of bone-marrow cells, by scoring micronuclei in polychromatic erythrocytes, by observation of meiotic chromosomes from treated males and by the dominant-lethality test. Furthermore, studies have been performed on short-term cultures of human lymphocytes in vitro. Whereas an increase in the yield of chromatid and chromosomal aberrations was observed after exposure in vitro, only negative results were obtained in the tests in vivo. One has, therefore, to conclude that styrene oxide is potentially capable of breaking mammalian chromosomes but that an exposure to an acute dose in vivo does not produce visible damage in somatic cells or in male germ cells.
Introduction Styrene and particularly its metabolite styrene oxide have been implicated as possible mutagenic agents. Indeed, these compounds have given positive results in mutagenicity tests on Salmonella typhimurium [2,12,14], yeast and Chinese hamster cells [10]. Moreover, Meretoja et al. [11] observed chromosomal aberrations in peripheral lymphocytes from 10 male workers exposed chronically to styrene in three plants manufacturing polyester plastic products. Such .workers, however, may also have been exposed to mutagenic agents other than styrene. In the present investigation we have, therefore, assayed the capability of styrene oxide to induce chromosomal damage using four mammalian test systems in vivo and one in vitro. The two tests on somatic cells in vivo were the induction of chromosomal aberrations in bone-marrow cells and the production of micronuclei in polychromatic erythrocytes. The two tests on
378 g e r m cells in vivo were the spermatocyte test on the treated m a l e s a n d the
dominant lethality induced in the post-meiotic male germ cells. Studies were also performed in short-term cultured human lymphocytes in vitro. Material and methods The tests in vivo were made on adult BALB/c male mice receiving a single i.p. injection of 2 5 0 mg styrene oxide per kg. The dose was chosen on the basis of the work of Leibman [6] on toxicity of styrene and of preliminary experiments on styrene oxide toxicity. According to Leibman the LD50 of styrene in rats is about 2.5 g/kg after i.p. injection, the acute toxicity of styrene oxide being about four times that of styrene. In our o w n experiments on the toxicity of styrene oxide all the animals given 500 mg/kg died within a few hours, whereas most of the mice treated with 250 or 125 mg/kg remained alive for several days. This styrene oxide (purity 97%) was obtained from Aldrich Europ (Janssen Pharmaceutica, Belgium) and was dissolved in paraffin oil. TABLE
1
CHROMOSOMAL
Treatment
Control 0.1 m M 0.5 m M
ABERRATIONS
Cells analysed
100 200 200
IN H U M A N
Total number of anomalies
1 13 18
LYMPHOCYTES
AFTER
EXPOSURE
IN V I T R O
Anomalies Chromatid gaps 1 5 10
Chromatid breaks . 4 3
Chromatid exchanges .
.
Chromosome gaps
Chromosome fragments
. 3 1
-1
1 3
TABLE 2 C H R O M O S O M E A N O M A L I E S IN BONE-MARROW CELLS AT D I F F E R E N T I N T E R V A L S OF TIME A F T E R T R E A T M E N T W I T H 2 5 0 m g S T Y R E N E O X I D E P E R kg F o u r m i c e w e r e e x a m i n e d for e a c h t i m e interval. Interval (days)
Cells a n a l y s e d
AnomaLies
1 2 6 13
157 200 200 200
0 1 chromatid break 0 0
TABLE 3 RESULTS OF THE MICRONUCLEUS TEST Treatment
Controls Styrene oxide, 250 mg/kg
Animals examined
10 7
Cells analysed
10 0 0 0 7 000
Cells w i t h m i c r o n u c l e i Total
%
27 30
0.27 0.43
379
Animals receiving paraffin oil only were used as controls. The induction of chromosomal aberrations in the somatic cells was studied in bone-marrow cells [9], 1, 2, 6 and 13 days after treatment. Polychromatic erythrocytes from 8-week-old mice were examined for micronuclei [13] 30 h after injection. To test the ability of styrene oxide to induce chromosomal aberrations in male post-meiotic germ cells, each male was caged, after injection, with three virgin females from the same strain which were replaced after 7 and 14 days. The males were then kept for another two months, killed and their dividing spermatocytes examined for the presence of reciprocal translocations induced in the pre-meiotic cells [5]. The females were dissected 17 days after mating had started, and pre- and post-implantation losses were determined by conventional methods [1]. We restricted the dominant-lethality test to post-meiotic stages i.e. to weeks 1--3 after administration of styrene oxide, because no chemical has yet been shown exclusively to induce pre-meiotic dominant-lethal mutations in mice [1]. Furthermore, mitomycin C, the only chemical up to now that has been found [3] to act preferentially on meiotic stages (4th week), is also effective on spermatids (3rd week). Studies in vitro were carried out with human lymphocytes from one donor. Whole-blood samples (0.5 ml) were incubated [8] at 37°C for 72 h in 5 ml Ham's F-10 medium supplemented with bovine serum (9%), phytohaemagglutinin, streptomycin and penicillin [5]. Since concentrations of 1 mM of styrene oxide were found to diminish cell viability markedly, final concentra, tions of 0.1 and 0.5 mM of polystyrene oxide were used, the compound being added 24 h after initiation of the cultures [4]. TABLE 4 R E S U L T S O F T H E D I S S E C T I O N O F F E M A L E S M A T E D W I T H M A L E S T R E A T E D i.p. Observation
1st w e e k Controls
2nd w e e k Treated
Controls
3rd w e e k Treated
Controls
Treated
Total females
51
51
51
50
51
51
Pregnant females Total %
29 56.8
24 47.8
34 66.6
23 46.0
28 54.9
33 64.7
Corpora lutea Total Per female
252 8.7
220 9.2
297 8.7
203 8.8
243 8.7
301 9.1
Implantations Total Per female
229 7.9
194 8.1
264 7.8
185 8.0
210 7.5
253 7.7
Live e m b r y o s Total Per female
191 6.6
171 7.1
215 6.3
147 6.4
173 6.2
208 6.3
Dead embryos Total Per female
38 1.3
23 0.9
49 1.4
38 1.6
37 1.3
45 1.4
T o t a l l o s s (%)
24.2
22.8
27.6
27.6
28.8
30.9
9.2
11.8
11.1
8.9
13.6
15.9
15.1
10.5
16.5
18.7
15.2
14.9
Pre-implantation loss (%) P o s t - i m p l a n t a t i o n l o s s (%)
380 TABLE 5 R E S U L T S O F T H E E X A M I N A T I O N O F T H E S P E R M A T O C Y T E S I F R O M C O N T R O L AND T R E A T E D MALES Treatment
Animals examined
Cells analysed
Anomalies
Control Styrene oxide, 250 mg]kg
10 7
1000 1400
0 0
Results and discussion The observations compiled in Tables 1--5 indicate a certain divergence between the tests in vitro and in vivo. A significant increase (P < 0.05) in the total number of anomalies was found after treatment of human l y m p h o c y t e s with 0.5 mM of styrene oxide b u t this appeared to be due, mainly, to an increase in chromatid gaps. On the other hand, if we except the positive findings of Loprieno et al. [10] who used a host-mediated assay, all tests on somatic cells in vivo (chromosomal aberrations in bone-marrow cells and micronuclei in erythrocytes) as well as on germ cells (translocations in pre-meiotic male germ cells and dominant lethality in postmeiotic ones) yielded negative results (Chi-square test). From this it appears that styrene oxide is capable of producing genetic damage in cells in vitro, as is also indicated by the results on Salmonella typhimurium [2,12,14], yeast and Chinese hamster cells [10], b u t could be less effective in vivo. The effects in vivo depend on uptake, distribution and excretion, and they may be modified by metabolic reactions. The effective concentration reaching the cells is, therefore, considerably higher in a culture system as shown by marked toxicity at 1 mM and so the differences observed between the results of the tests in vitro and in vivo could readily be accounted for by this. The data on chronically exposed men [11] are n o t necessarily contradictory to our results since exposure had continued much longer and these workers may have been exposed to other compounds. Acknowledgement Part of this investigation was supported by grants from the " F o n d s de la Recherche Fondamentale Collective" and by a grant to L.F. from the Belgian Nuclear Energy Study Center (C.E.N.--S.C.K.), Mol, Belgium. References 1 B a t e m a n , A.J., a n d S.S. Epstein, D o m i n a n t lethal m u t a t i o n s in m a m m a l s , in: A. H o l l a e n d e r (Ed.), C h e m i c a l M u t a g e n s , Vol. 2, P l e n u m , N e w Y o r k , 1 9 7 1 . 2 De Meester, C., F . P o n c e l e t , M. R o b e r f r o i d , J. R o n d e l e t and M. Mercier, M u t a g e n i c i t y of styrene and s t y r e n e o x i d e , M u t a t i o n Res., 56 ( 1 9 7 7 ) 1 4 7 - - 1 5 2 . 3 Ehling, U.H., C o m p a r i s o n o f r a d i a t i o n - and c h e m i c a l l y - i n d u c e d d o m i n a n t lethal m u t a t i o n s in male m i c e , M u t a t i o n Res., 11 ( 1 9 7 1 ) 3 5 - - 4 4 . 4 Evans, H.J., C y t o l o g i c a l m e t h o d s for d e t e c t i n g c h e m i c a l m u t a g e n s , in: A. H o l l a e n d e r (Ed.), Chemical M u t a g e n s , Vol. 4, P l e n u m , New Y o r k , 1 9 7 6 .
381 5 Ham, R.G., An i m p r o v e d n u t r i e n t s o l u t i o n for diploid Chinese h a m s t e r a nd h u m a n cell lines, Exptl. Cell Res., 29 (1963) 515. 6 Leibman , K.C., Metabolism and t o x i c i t y of styrene, Envizonm. Health Persp., 11 (1975) 115--119. 7 I.~onsxd, A., Observations on meiotic c h r o m o s o m e s of the male mous e as a t e s t of the p o t e n t i a l m u t a g e n i c i t y of chemicals in m a m m a l s , in: A. H o l l a e n d e r (Ed.), Chemical Mutagens, VoL 3, Plenum, New York, 1973. 8 L~onard, A., G.B. Gerber, D.G. Papworth, G. Decat, E.D. I ~ o n a r d and Gh. D e k n u d t , The radiosensitivities of l y m p h o c y t e s from pig, sheep, goat and cow, Mut a t i on Res., 36 (1976) 319--332. 9 L~onard, A., and E.D. I ~ o n a r d , Cytogenetic effects of m y l e r a n in vivo on bone-marrow cells of male mice, M u t a t i o n Res., 56 (1977) 329--333. 10 Loprieno, N., A. A b b o n d a n d o l o , R. Barale, S. Baroncelli, S. Bonatti, G. Bronzetti, A. Cammelini, C. Corsi, G. Corti, D. Fiezaa, C. Leporini, A. Mazzaccaro, R. Nieri, D. Rosellini and A.M. Rossi, Mutagenicity of industrial c o m p o u n d s styrene and its possible metaboHte styrene oxide, Mut a t i on Res., 40 (1976) 317--324. 11 Meretoja, T., H. Valnio0 M. Sorsa and H. H~/rk6nen, Occupational styrene e xpos ure and c h r o m o s o m a l aberrations, M u t a t i o n Res., 56 (1977) 193--197. 12 Mlluy, P., and A.J. Garro, Mutagenic activity of styrene oxide (1,2-epoxyethylbenzene), a pre s ume d styrene metaboHte, M u t a t i o n Res., 40 (1976) 15--18. 13 Schmid, W., The m i c r o n u c l e u s test for cytogenetic analysis, in: A. Hollaender (Ed.), Chemical Mutagens, VoL 4, Plenum, New York, 1976. 14 Vainio, H., R. P////kkSncn, K. R S n n h o i m , V. Raunio and P. Pelkonen, A s t u d y on the mutagenic activity of styrene and styrene oxide, Scand. J. Work Env. Health, 3 (1976) 147--151.
Mutation Research, 51 (1978) 377--381 © Elsevier/North-Holland Biomedical Press
MUTAGENICITY TESTS WITH STYRENE OXIDE IN MAMMALS
L. FABRY a, A. LI~ONARD a and M. ROBERFROID b a Mammalian Genetics Laboratory, Department ofRadiobiology, C.E.N.-S.C.K., B-2400 Mol (Belgium), and b Laboratory of Biotoxicology, University of Louvain, Pharmacy School, UCL 7369 Brussels B-1200 (Belgium)
(Received 12 January 1978) (Revision received 6 April 1978) (Accepted 7 April 1978)
Summary The capability of styrene oxide to induce chromosome damage in vivo has been tested in the male mouse by examination of bone-marrow cells, by scoring micronuclei in polychromatic erythrocytes, by observation of meiotic chromosomes from treated males and by the dominant-lethality test. Furthermore, studies have been performed on short-term cultures of human lymphocytes in vitro. Whereas an increase in the yield of chromatid and chromosomal aberrations was observed after exposure in vitro, only negative results were obtained in the tests in vivo. One has, therefore, to conclude that styrene oxide is potentially capable of breaking mammalian chromosomes but that an exposure to an acute dose in vivo does not produce visible damage in somatic cells or in male germ cells.
Introduction Styrene and particularly its metabolite styrene oxide have been implicated as possible mutagenic agents. Indeed, these compounds have given positive results in mutagenicity tests on Salmonella typhimurium [2,12,14], yeast and Chinese hamster cells [10]. Moreover, Meretoja et al. [11] observed chromosomal aberrations in peripheral lymphocytes from 10 male workers exposed chronically to styrene in three plants manufacturing polyester plastic products. Such .workers, however, may also have been exposed to mutagenic agents other than styrene. In the present investigation we have, therefore, assayed the capability of styrene oxide to induce chromosomal damage using four mammalian test systems in vivo and one in vitro. The two tests on somatic cells in vivo were the induction of chromosomal aberrations in bone-marrow cells and the production of micronuclei in polychromatic erythrocytes. The two tests on
378 g e r m cells in vivo were the spermatocyte test on the treated m a l e s a n d the
dominant lethality induced in the post-meiotic male germ cells. Studies were also performed in short-term cultured human lymphocytes in vitro. Material and methods The tests in vivo were made on adult BALB/c male mice receiving a single i.p. injection of 2 5 0 mg styrene oxide per kg. The dose was chosen on the basis of the work of Leibman [6] on toxicity of styrene and of preliminary experiments on styrene oxide toxicity. According to Leibman the LD50 of styrene in rats is about 2.5 g/kg after i.p. injection, the acute toxicity of styrene oxide being about four times that of styrene. In our o w n experiments on the toxicity of styrene oxide all the animals given 500 mg/kg died within a few hours, whereas most of the mice treated with 250 or 125 mg/kg remained alive for several days. This styrene oxide (purity 97%) was obtained from Aldrich Europ (Janssen Pharmaceutica, Belgium) and was dissolved in paraffin oil. TABLE
1
CHROMOSOMAL
Treatment
Control 0.1 m M 0.5 m M
ABERRATIONS
Cells analysed
100 200 200
IN H U M A N
Total number of anomalies
1 13 18
LYMPHOCYTES
AFTER
EXPOSURE
IN V I T R O
Anomalies Chromatid gaps 1 5 10
Chromatid breaks . 4 3
Chromatid exchanges .
.
Chromosome gaps
Chromosome fragments
. 3 1
-1
1 3
TABLE 2 C H R O M O S O M E A N O M A L I E S IN BONE-MARROW CELLS AT D I F F E R E N T I N T E R V A L S OF TIME A F T E R T R E A T M E N T W I T H 2 5 0 m g S T Y R E N E O X I D E P E R kg F o u r m i c e w e r e e x a m i n e d for e a c h t i m e interval. Interval (days)
Cells a n a l y s e d
AnomaLies
1 2 6 13
157 200 200 200
0 1 chromatid break 0 0
TABLE 3 RESULTS OF THE MICRONUCLEUS TEST Treatment
Controls Styrene oxide, 250 mg/kg
Animals examined
10 7
Cells analysed
10 0 0 0 7 000
Cells w i t h m i c r o n u c l e i Total
%
27 30
0.27 0.43
379
Animals receiving paraffin oil only were used as controls. The induction of chromosomal aberrations in the somatic cells was studied in bone-marrow cells [9], 1, 2, 6 and 13 days after treatment. Polychromatic erythrocytes from 8-week-old mice were examined for micronuclei [13] 30 h after injection. To test the ability of styrene oxide to induce chromosomal aberrations in male post-meiotic germ cells, each male was caged, after injection, with three virgin females from the same strain which were replaced after 7 and 14 days. The males were then kept for another two months, killed and their dividing spermatocytes examined for the presence of reciprocal translocations induced in the pre-meiotic cells [5]. The females were dissected 17 days after mating had started, and pre- and post-implantation losses were determined by conventional methods [1]. We restricted the dominant-lethality test to post-meiotic stages i.e. to weeks 1--3 after administration of styrene oxide, because no chemical has yet been shown exclusively to induce pre-meiotic dominant-lethal mutations in mice [1]. Furthermore, mitomycin C, the only chemical up to now that has been found [3] to act preferentially on meiotic stages (4th week), is also effective on spermatids (3rd week). Studies in vitro were carried out with human lymphocytes from one donor. Whole-blood samples (0.5 ml) were incubated [8] at 37°C for 72 h in 5 ml Ham's F-10 medium supplemented with bovine serum (9%), phytohaemagglutinin, streptomycin and penicillin [5]. Since concentrations of 1 mM of styrene oxide were found to diminish cell viability markedly, final concentra, tions of 0.1 and 0.5 mM of polystyrene oxide were used, the compound being added 24 h after initiation of the cultures [4]. TABLE 4 R E S U L T S O F T H E D I S S E C T I O N O F F E M A L E S M A T E D W I T H M A L E S T R E A T E D i.p. Observation
1st w e e k Controls
2nd w e e k Treated
Controls
3rd w e e k Treated
Controls
Treated
Total females
51
51
51
50
51
51
Pregnant females Total %
29 56.8
24 47.8
34 66.6
23 46.0
28 54.9
33 64.7
Corpora lutea Total Per female
252 8.7
220 9.2
297 8.7
203 8.8
243 8.7
301 9.1
Implantations Total Per female
229 7.9
194 8.1
264 7.8
185 8.0
210 7.5
253 7.7
Live e m b r y o s Total Per female
191 6.6
171 7.1
215 6.3
147 6.4
173 6.2
208 6.3
Dead embryos Total Per female
38 1.3
23 0.9
49 1.4
38 1.6
37 1.3
45 1.4
T o t a l l o s s (%)
24.2
22.8
27.6
27.6
28.8
30.9
9.2
11.8
11.1
8.9
13.6
15.9
15.1
10.5
16.5
18.7
15.2
14.9
Pre-implantation loss (%) P o s t - i m p l a n t a t i o n l o s s (%)
380 TABLE 5 R E S U L T S O F T H E E X A M I N A T I O N O F T H E S P E R M A T O C Y T E S I F R O M C O N T R O L AND T R E A T E D MALES Treatment
Animals examined
Cells analysed
Anomalies
Control Styrene oxide, 250 mg]kg
10 7
1000 1400
0 0
Results and discussion The observations compiled in Tables 1--5 indicate a certain divergence between the tests in vitro and in vivo. A significant increase (P < 0.05) in the total number of anomalies was found after treatment of human l y m p h o c y t e s with 0.5 mM of styrene oxide b u t this appeared to be due, mainly, to an increase in chromatid gaps. On the other hand, if we except the positive findings of Loprieno et al. [10] who used a host-mediated assay, all tests on somatic cells in vivo (chromosomal aberrations in bone-marrow cells and micronuclei in erythrocytes) as well as on germ cells (translocations in pre-meiotic male germ cells and dominant lethality in postmeiotic ones) yielded negative results (Chi-square test). From this it appears that styrene oxide is capable of producing genetic damage in cells in vitro, as is also indicated by the results on Salmonella typhimurium [2,12,14], yeast and Chinese hamster cells [10], b u t could be less effective in vivo. The effects in vivo depend on uptake, distribution and excretion, and they may be modified by metabolic reactions. The effective concentration reaching the cells is, therefore, considerably higher in a culture system as shown by marked toxicity at 1 mM and so the differences observed between the results of the tests in vitro and in vivo could readily be accounted for by this. The data on chronically exposed men [11] are n o t necessarily contradictory to our results since exposure had continued much longer and these workers may have been exposed to other compounds. Acknowledgement Part of this investigation was supported by grants from the " F o n d s de la Recherche Fondamentale Collective" and by a grant to L.F. from the Belgian Nuclear Energy Study Center (C.E.N.--S.C.K.), Mol, Belgium. References 1 B a t e m a n , A.J., a n d S.S. Epstein, D o m i n a n t lethal m u t a t i o n s in m a m m a l s , in: A. H o l l a e n d e r (Ed.), C h e m i c a l M u t a g e n s , Vol. 2, P l e n u m , N e w Y o r k , 1 9 7 1 . 2 De Meester, C., F . P o n c e l e t , M. R o b e r f r o i d , J. R o n d e l e t and M. Mercier, M u t a g e n i c i t y of styrene and s t y r e n e o x i d e , M u t a t i o n Res., 56 ( 1 9 7 7 ) 1 4 7 - - 1 5 2 . 3 Ehling, U.H., C o m p a r i s o n o f r a d i a t i o n - and c h e m i c a l l y - i n d u c e d d o m i n a n t lethal m u t a t i o n s in male m i c e , M u t a t i o n Res., 11 ( 1 9 7 1 ) 3 5 - - 4 4 . 4 Evans, H.J., C y t o l o g i c a l m e t h o d s for d e t e c t i n g c h e m i c a l m u t a g e n s , in: A. H o l l a e n d e r (Ed.), Chemical M u t a g e n s , Vol. 4, P l e n u m , New Y o r k , 1 9 7 6 .
381 5 Ham, R.G., An i m p r o v e d n u t r i e n t s o l u t i o n for diploid Chinese h a m s t e r a nd h u m a n cell lines, Exptl. Cell Res., 29 (1963) 515. 6 Leibman , K.C., Metabolism and t o x i c i t y of styrene, Envizonm. Health Persp., 11 (1975) 115--119. 7 I.~onsxd, A., Observations on meiotic c h r o m o s o m e s of the male mous e as a t e s t of the p o t e n t i a l m u t a g e n i c i t y of chemicals in m a m m a l s , in: A. H o l l a e n d e r (Ed.), Chemical Mutagens, VoL 3, Plenum, New York, 1973. 8 L~onard, A., G.B. Gerber, D.G. Papworth, G. Decat, E.D. I ~ o n a r d and Gh. D e k n u d t , The radiosensitivities of l y m p h o c y t e s from pig, sheep, goat and cow, Mut a t i on Res., 36 (1976) 319--332. 9 L~onard, A., and E.D. I ~ o n a r d , Cytogenetic effects of m y l e r a n in vivo on bone-marrow cells of male mice, M u t a t i o n Res., 56 (1977) 329--333. 10 Loprieno, N., A. A b b o n d a n d o l o , R. Barale, S. Baroncelli, S. Bonatti, G. Bronzetti, A. Cammelini, C. Corsi, G. Corti, D. Fiezaa, C. Leporini, A. Mazzaccaro, R. Nieri, D. Rosellini and A.M. Rossi, Mutagenicity of industrial c o m p o u n d s styrene and its possible metaboHte styrene oxide, Mut a t i on Res., 40 (1976) 317--324. 11 Meretoja, T., H. Valnio0 M. Sorsa and H. H~/rk6nen, Occupational styrene e xpos ure and c h r o m o s o m a l aberrations, M u t a t i o n Res., 56 (1977) 193--197. 12 Mlluy, P., and A.J. Garro, Mutagenic activity of styrene oxide (1,2-epoxyethylbenzene), a pre s ume d styrene metaboHte, M u t a t i o n Res., 40 (1976) 15--18. 13 Schmid, W., The m i c r o n u c l e u s test for cytogenetic analysis, in: A. Hollaender (Ed.), Chemical Mutagens, VoL 4, Plenum, New York, 1976. 14 Vainio, H., R. P////kkSncn, K. R S n n h o i m , V. Raunio and P. Pelkonen, A s t u d y on the mutagenic activity of styrene and styrene oxide, Scand. J. Work Env. Health, 3 (1976) 147--151.
