- Email: [email protected]
Induction of chromosome aberrations and sister-chromatid exchanges by caprolactam in vitro
Mutation Research, 224 (1989) 333-337
333
Elsevier MUTGEN 02655
Induction of chromosome aberrations and sister-chromatid exchanges by caprolactam in vitro Hannu Norppa and Hilkka Jirventaus Department of lndustrial Hygiene and Toxicology, Institute of Occupational Health, Topeliuksenkatu 41 a A, SF~00250 Helsinki (Finland)
Keywords." Caprolactam; Chromosome aberrations
Summary Caprolactam (CAP) induced chromosome aberrations in whole-blood cultures of h u m a n lymphocytes at 50 m M without metabolic activation (24-h treatment) and at 200 m M in the presence of rat liver $9 mix (1-h treatment). CAP also produced a dose-dependent increase in polyploid cells, the effect being statistically significant at 25 and 50 m M without $9 mix and at 100 and 200 m M with $9 mix. Without metabolic activation, there was an increase in hypodiploid cells at 50 m M and hyperdiploid cells at 12.5 mM. In Chinese hamster ovary cells, CAP produced a marginal elevation of sister-chromatid exchanges at 125 m M in the presence of $9 mix (4-h treatment). The results show that CAP is able to induce cytogenetic changes in vitro at very high toxic concentrations.
Caprolactam (CAP) was included as a noncarcinogenic negative control chemical in the recent collaborative study on in vitro short-term tests of the International Programme on Chemical Safety (IPCS) (Ashby et al., 1985). Although it showed weak, unconfirmed positive effects at high doses in certain tests with fungi and mammalian cells, the only systems considered clearly positive were somatic mutations in Drosophila and chromosome aberrations in human lymphocytes. The purpose of the present study was to re-examine CAP for the induction of structural and numerical chromosome aberrations in human lymphocytes and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells.
Correspondence: Hannu Norppa, Department of Industrial Hygiene and Toxicology, Institute of Occupational Health, Topeliuksenkatu 41 a A, SF-00250 Helsinki (Finland).
Materials and methods H u m a n whole-blood lymphocyte cultures were established from venous blood sample of a 33year-old male donor as described earlier (Jantunen et al., 1985), with the exception that sterilized disposable 10-ml glass culture tubes with screw caps (Coming Ltd., Stone, U.K.) were used, and 0.2 ml of blood was added to 3.5 ml of culture medium. To prepare the treatment solutions, CAP (¢caprolactam; 99%, EGA-Chemie; Steinheim/A1buch, F.R.G.) was dissolved in R P M I 1640 medium (Grand Island Biological Co., Glasgow, U.K.) at concentrations ranging from 11.9 to 380.2 m g / m l . These solutions were added at a volume of 0.5 ml to the 3.7-ml (final concentrations in Table 1) lymphocyte cultures (2 for each treatment) that had been established 48 h earlier. Control cultures received an equal volume of R P M I 1640 medium.
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
334 Positive control cultures were treated with cyclophosphamide (CP; L~i~ike/Farmos-Yhtymfi Oy, Turku, Finland) prepared as earlier reported (M~iki-Paakkanen and Norppa, 1987; N o r p p a et al., 1985). A further dilution (10 mM, in R P M I 1640 medium) was added to 4.2-ml cultures at a volume of 42 /zl (final concentration 0.1 mM). Without metabolic activation, the treatment lasted until harvest, i.e., 24 h. For treatment with $9 mix, the culture medium was replaced for 1 h with 3.7 ml of medium containing rat liver $9 mix (for details, see M~iki-Paakkanen and Norppa, 1987). After the treatment and cell wash, the cells were suspended in 4 ml of complete growth medium (Jantunen et al., 1986; M~ki-Paakkanen and Norppa, 1987) and were further incubated (37 ° C) for 24 h. Cell harvest, hypotonic treatment, fixation, slide preparation, and Giemsa staining were performed as reported previously (M~iki-Paakkanen et al., 1980), except that Colcemid was present in the cultures for the last 2 h. The same lymphocyte slides were separately analyzed for structural chromosome aberrations (see Jantunen et al., 1986), numerical chromosome aberrations, and mitotic index. Cells with less than 46 chromosomes were considered hypodiploid, those with 47-59 chromosomes hyperdiploid, and those with 60 or more chromosomes polyploid. Duplicate cultures of C H O cells were established for each treatment as described earlier (Hyt0nen et al., 1983). CAP treatment solutions, prepared in McCoy's Medium 5A (Grand Island Biological Co.), ranged from 8.8 to 565.8 m g / m l (final concentrations in Table 3) and were added for 4 h, 24 h after culture initiation, to the cultures at a volume of 1 ml. $9 mix treatment medium was similar to that used for human lymphocytes, except that McCoy's Medium 5A was used instead of R P M I 1640. Control cultures were treated with 1 ml of the medium. Cyclophosphamide ( N o r p p a et al., 1985), at a final concentration of 0.01 mM, was used as a positive control. After the treatment, the cells were rinsed twice with 4 ml of phosphate-buffered saline (PBS) and were allowed to recover in complete medium for 24 h. Cell harvest, preparation of slides, and fluorescence-plus-Giemsa staining were performed according to previously described procedures
(Hyt¢Snen et al., 1983: Mfiki-Paakkanen et al., 1980). SCEs and proliferation indices were analyzed as earlier described ( N o r p p a et al., 1985).
Results and discussion
CAP significantly increased lymphocytes with chromatid-type aberrations and the total number of aberrant cells (_+gaps) and aberrations/100 cells at the highest concentrations available, with (200 raM) and without (50 raM) $9 mix (Table 1). These toxic doses also increased cells with chromosome-type aberrations and gaps with and without $9 mix, respectively. The number of aberrations was particularly high with metabolic activation, because several cells with multiple and complex chromatid-type aberrations were found. Although linear regression analysis would indicate a dose-dependent effect in m a n y of the aberration categories of Table 1, this evidence is not very convincing, as only one concentration both with and without $9 mix gave a result clearly deviating from the controls. As expected, CP was clastogenic with $9 mix, but not without it. A dose-dependent increase could be seen in the number of polyploid cells (mainly tetraploids or nearly tetraploids) both with and without $9 mix and in the total number of cells with deviating chromosome n u m b e r without $9 mix (Table 2). These frequencies were significantly increased at 25 and 50 m M without $9 mix and (polyploids only) at 100 and 200 m M with $9 mix. None of the treatments, with the exception of a sporadic finding at 12.5 m M without $9 mix, was able to significantly increase the number of hyperdiploid cells (Tables 1 and 2). Hypodiploidy, a parameter considered to be more sensitive to artifacts than hyperdiploidy, was elevated at 50 m M without $9 mix. When hyperdiploids and hypodiploids were pooled, a statistically significant effect could be observed without $9 mix at 50 raM. Exclusion of hypodiploids from the total number of cells with numerical chromosome changes yielded a significant increase at all concentrations tested without $9 mix and at the 2 highest ones available with $9 mix, with no obvious dose response.
335 TABLE 1 CHROMOSOME ABERRATIONS IN H U M A N WHOLE-BLOOD LYMPHOCYTE C U L T U R E S TREATED WITH CAPROLACTAM (CAP), 48 h AFTER C U L T U R E INITIATION, W I T H O U T (24-h TREATMENT) OR W I T H (1-h TREATMENT) RAT LIVER $9 MIX 200 metaphases/treatment (100 from each duplicate culture) were analyzed for chromosome aberrations and 2000 cells/treatment
(1000 from each duplicate culture) for mitotic index. Treatment
Mitotic
Cells with aberrations a (%)
(mM)
index a
Chromatid
Chromosome
(%)
type
type
Aberrations Gaps
Total gaps excluded
gaps included
per 100 cells, gaps excluded a
Without $9 mix
Control(medium)
10.1_+1.4
1.5_+0.5
0.0
0.0
1.5_+0.5
1.5_+0.5
1.5_+ 0.5
0.1
8.0_+0.8
0.5+0.5
0.0
0.5_+0.5
0.5_+0.5
1.0 +0.0
0.5_+ 0.5
CAP 12.5 25 50 100
7.2_+0.4 4.8_+0.7 3.5_+0.2 0.0
1.5+1.5 1.0+0.0 7.5_+0.5"*
1.0-+ 0.0 1.0_+ 0.0 1.0+0.0
0.5_+0.5 1.0_+0.0 3.0+0.0"
2.5_+1.5 2.0_+0.0 8.5_+0.5"*
6.3_+0.2
0.0
0.5_+0.5
0.0
0.5_+0.5
Cyclophosphamide
3.0_+1.0 3.0_+0.0 11.5-+1.5"**
2.5_+ 1.5 2.0_+ 0.0 13.0-+ 1 . 0 " * *
With $9 mix
Control(medium)
0.5_+0.5
0.5_+ 0.5
Cyclophosphamide 0.1
3.9_+0.2
13.5_+0.5 * * *
7.0_+2.0 * * *
0.0
20.5_+2.0 * * *
20.5_+2.5 * * *
22.5_+ 3.5 * * *
CAP 25 50 100 b 200 400
5.8_+0.1 4.9_+0.9 4.1-+0.3 2.2_+0.1 0.0
1.0+_1.0 1.0_+1.0 1.0-+1.0 5.1_+0.1 **
1.0-+1.0 1.5_+0.5 1.0-+1.0 8.3_+0.3 * * *
0.0 0.5_+0.5 0.0 0.8_+0.8
2.0-+2.0 2.5_+0.5 2.0_+2.0 13.4+0.3 * * *
2.0-+2.0 3.0_+1.0 3.0_+2.0 14.3-+1.2 * * *
2.0_+ 2.0 2.5-+ 0.5 2.5_+ 2.5 135.2_+37.2 * * *
a Mean of duplicate cultures ± SE. b 158 cells analyzed.
Probabilities calculated according to Fisher's exact (1-tailed) or binomic test (aberrations/100 cells): * p < 0.05; * * p < 0.01; * * * p < 0.001.
In CHO cells, a marginal increase in SCEs was obtained at the highest concentration of CAP available (125 mM) in the presence of $9 mix (Table 3). This dose induced a clear decrease in proliferation index and was too toxic for analysis without $9 mix. CP had a strong effect on SCEs in the presence of metabolic activation. The present results agree qualitatively with the IPCS collaborative study (Ashby et al., 1985): CAP induced structural chromosome aberrations in human lymphocytes. In the IPCS study, however, Howard et al. (1985) observed (dose-dependent) chromosome aberration induction at clearly
lower doses than we did, despite their shorter treatment times. Another discrepancy is that they did not see polyploidy induction. On the other hand, Ishidate and Sofuni (1985) did observe polyploidy, along with chromosome aberrations (including multi-aberrant cells), in Chinese hamster lung cells at CAP concentrations comparable to our positive ones. Also another study (Priston and Dean, 1985) in rat liver cells, although interpreted negative, indicated a statistically significant increase of chromosome aberrations (gaps included, in comparison with concurrent controls) at the highest dose of CAP tested (8.8 mM), with a
336 TABLE 2 N U M E R I C A L C H R O M O S O M E C H A N G E S 1N H U M A N W H O L E - B L O O D L Y M P H O C Y T E C U L T U R E S T R E A T E D WITH C A P R O L A C T A M (CAP), 48 h A F T E R C U L T U R E I N I T I A T I O N , W I T H O U T (24-h T R E A T M E N T ) O R WITH (1-h TREATMENT) RAT LIVER $9 MIX 200 metaphases (100 from each duplicate culture) were analyzed per treatment. Treatment
Cells with deviating chromosome number (%, mean of duplicates _+SE)
(mM)
Polyploid
Hyperdiploid
Hypodiploid
Hyper- and
Total
hypo-diploids combined
hypodiploids
hypodiploids
excluded
included
Without $9 mix Control (medium)
0.0
1.0 _+1.0
6.0 + 1.0
7.0 _+2.0
1.0 + 1.0
7.0 _+2.0
Cyclophosphamide 0.1
0.0
4.0 _+1.0
3.0 _+ 1.0
7.0 _+0.0
4.0 _+1.0
7.0 + 0.0
CAP 12.5 25 50 100
1.5_+0.5 2.5_+2.5 * 4.0-+3.0 ** (No metaphases)
5.5+2.5 ** 2.5_+0.5 2.5_+0.5
4.5_+1.5 7.5_+0.5 11.5_+3.5 *
10.0_+1.0 10.0_+0.0 14.0+3.0 *
With $9 mix Control(medium)
2.0_+0.0
2.0+1.0
7.0_+2.0
Cyclophosphamide 0.1
1.5_+1.5
5.5_+0.5
CAP 25 50 100 200 400
1.5_+0.5 2.0 _+1.0 6.5_+0.5 * 8.0_+4.0 * * (No metaphases)
0.5_+0.5 3.0 + 1.0 3.0_+0.0 1.0_+ 0.0
a
7 . 0 + 2 . 0 ** 5.0+_2.0 * 6.5_+2.5 **
11.5_+0.5 12.5_+2.5 * 18.0_+6.0 * * *
9.0_+1.0
4.0+1.0
lh0+l.0
8.0_+2.0
13.5_+2.5
7.0_+1.0
15.0_+l.0
6.0_+1.0 5.0 _+ 1.0 6.0_+3.0 7.3 _+0.7
6.5_+1.5 8.0 _+2.0 9.0_+3.0 8.9_+0.1
2.0_+0.0 5.0 _+0.0 9.5_+0.5 * 9.1 +4.1 *
8.0_+1.0 10.0 + 1.0 15.5+_2.5 16.4_+3.4
" 191 ceils analyzed. Probabilities calculated according to Fisher's exact test (1-tailed): * p < 0.05; * * p < 0.01; * * * p < 0.001. TABLE 3 S I S T E R - C H R O M A T I D E X C H A N G E S (SCEs) IN C H I N E S E H A M S T E R O V A R Y (CHO) CELLS T R E A T E D W I T H C A P R O L A C T A M (CAP) FOR 4 h W I T H O U T OR WITH RAT LIVER $9 MIX For each treatment, 60 second-division metaphases (30 from each duplicate culture) were analyzed for SCEs and 200 metaphases (100 from each duplicate culture) for proliferation indices. Treatment
Without $9 mix "
(mM)
SCEs/cell
With $9 mix a Proliferation index
SCEs/cell
Proliferation index
Control
9.0 _+0.0
1.97 _+0.01
10.2 + 0.1
1.84 _+0.01
Cyclophosphamide 0.01
9.6 + 0.8
1.99 _+0.01
39.9 _+0.6
1.73 + 0.03
10.0 _+0.6 9.5 _+0.1 10.0 _+0.0 No result No result
1.97 _+0.02 1.94 _+0.02 1.87 + 0.01 0.04 _+0.00 0.00
10.6 _+0.2 10.0 _+0.3 10.7 + 0.4 11.9 _+0.0 * * No result
1.86 _+0.03 1.85 _+0.01 1.79 _+0.01 1.32 + 0.02 0.00
CAP 15.6 31.3 62.5 125 250
a Mean of duplicate cultures + SE. ** p < 0.01, t-test (1-tailed), 51 cells analyzed.
337 s u g g e s t i v e d o s e r e s p o n s e . I n o t h e r in v i t r o studies o n the i n d u c t i o n of c h r o m o s o m e a b e r r a t i o n s , p o l y p l o i d y , a n e u p l o i d y , or s p i n d l e d i s t u r b a n c e s C A P g a v e n e g a t i v e results (see A s h b y et al., 1985). T h e c o n c l u s i o n of t h e I P C S s t u d y o n S C E i n d u c t i o n b y C A P was n e g a t i v e ( A s h b y et al., 1985). A closer look, h o w e v e r , r e v e a l s a m a r g i n a l e f f e c t of C A P (in c o m p a r i s o n w i t h c o n c u r r e n t c o n t r o l s ) o n S C E s in all b u t 1 of the 6 s e p a r a t e p a p e r s - - in a c c o r d a n c e w i t h o u r p r e s e n t f i n d i n g . T h u s , a l t h o u g h t h e effect o f C A P o n S C E s is e x t r e m e l y w e a k a n d o c c u r s at t o x i c doses, it c a n b e seen in several d i f f e r e n t in v i t r o s y s t e m s w i t h or w i t h o u t m e t a b o l i c a c t i v a t i o n . I n c o n c l u s i o n , C A P is a b l e to i n d u c e s t r u c t u r a l and numerical chromosome aberrations and, m a r g i n a l l y , S C E s in v a r i o u s cell c u l t u r e s y s t e m s at t o x i c c o n c e n t r a t i o n s w h i c h , in m o s t cases, are v e r y h i g h in t e r m s of m o l a r or w e i g h t / v o l u m e c o n c e n t r a t i o n s . W h e t h e r this a c t i v i t y has a n y relev a n c e w i t h r e s p e c t to the s i t u a t i o n in v i v o r e m a i n s to b e d e t e r m i n e d ,
References Ashby, J., F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.) (1985) Evaluation of Short-Term Tests for Carcinogens, Progress in Mutation Research, Vol. 5, Elsevier, Amsterdam, 752 pp. Howard, C.A., T. Sheldon and C.R. Richardson (1985) Tests for the induction of chromosomal aberrations in human peripheral lymphocytes in culture, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term
Tests for Carcinogens, Progress in Mutation Research, Vol. 5, Elsevier, Amsterdam, pp. 457-467. HytSnen, S., I. Alfheim and M. Sorsa (1983) Effects of emissions from residential wood stoves on SCE induction in CHO cells, Mutation Res., 118, 69-75. Ishidate, M., and T. Sofuni (1985) The in vitro chromosomal aberration test using Chinese hamster lung (CHL) fibroblast cells in culture, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D, Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Progress in Mutation Research, Vol. 5, Elsevier, Amsterdam, pp. 427-432. Jantunen, K., J. MS_ki-Paakkanen and H. Norppa (1986) Induction of chromosome aberrations by styrene and vinylacetate in cultured human lymphocytes: dependence on erythrocytes, Mutation Res., 159, 109-116. Miiki-Paakkanen, J., and H. Norppa (1987) Chromosome aberrations and sister-chromatid exchanges induced by technical grade toluene diisocyanate and methylenediphenyl diisocyanate in cultured human lymphocytes, Toxicol. Lett., 36, 37-43, Maki-Paakkanen, J., K. Husgafvel-Pursiainen, P.-L. Kalliom~iki, J. Tuominen and M. Sorsa (1980) Toluene-exposed workers and chromosome aberrations, J. Toxicol. Environ. Health, 6, 775-781. Norppa, H., F. Tursi, P. Pfaffli, J. M~tki-Paakkanen and H. J~irventaus (1985) Chromosome damage induced by vinyl acetate through in vitro formation of acetaldehyde in human lymphocytes and Chinese hamster ovary cells, Cancer Res., 45, 4816-4821. Priston, R.A.J., and B.J. Dean (1985) Tests for the induction of chromosome aberrations, polyploidy and sister-chromatid exchanges in rat liver (RL4) cells, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Progress in Mutation Research, Vol. 5, Elsevier, Amsterdam, pp. 387-395.
333
Elsevier MUTGEN 02655
Induction of chromosome aberrations and sister-chromatid exchanges by caprolactam in vitro Hannu Norppa and Hilkka Jirventaus Department of lndustrial Hygiene and Toxicology, Institute of Occupational Health, Topeliuksenkatu 41 a A, SF~00250 Helsinki (Finland)
Keywords." Caprolactam; Chromosome aberrations
Summary Caprolactam (CAP) induced chromosome aberrations in whole-blood cultures of h u m a n lymphocytes at 50 m M without metabolic activation (24-h treatment) and at 200 m M in the presence of rat liver $9 mix (1-h treatment). CAP also produced a dose-dependent increase in polyploid cells, the effect being statistically significant at 25 and 50 m M without $9 mix and at 100 and 200 m M with $9 mix. Without metabolic activation, there was an increase in hypodiploid cells at 50 m M and hyperdiploid cells at 12.5 mM. In Chinese hamster ovary cells, CAP produced a marginal elevation of sister-chromatid exchanges at 125 m M in the presence of $9 mix (4-h treatment). The results show that CAP is able to induce cytogenetic changes in vitro at very high toxic concentrations.
Caprolactam (CAP) was included as a noncarcinogenic negative control chemical in the recent collaborative study on in vitro short-term tests of the International Programme on Chemical Safety (IPCS) (Ashby et al., 1985). Although it showed weak, unconfirmed positive effects at high doses in certain tests with fungi and mammalian cells, the only systems considered clearly positive were somatic mutations in Drosophila and chromosome aberrations in human lymphocytes. The purpose of the present study was to re-examine CAP for the induction of structural and numerical chromosome aberrations in human lymphocytes and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells.
Correspondence: Hannu Norppa, Department of Industrial Hygiene and Toxicology, Institute of Occupational Health, Topeliuksenkatu 41 a A, SF-00250 Helsinki (Finland).
Materials and methods H u m a n whole-blood lymphocyte cultures were established from venous blood sample of a 33year-old male donor as described earlier (Jantunen et al., 1985), with the exception that sterilized disposable 10-ml glass culture tubes with screw caps (Coming Ltd., Stone, U.K.) were used, and 0.2 ml of blood was added to 3.5 ml of culture medium. To prepare the treatment solutions, CAP (¢caprolactam; 99%, EGA-Chemie; Steinheim/A1buch, F.R.G.) was dissolved in R P M I 1640 medium (Grand Island Biological Co., Glasgow, U.K.) at concentrations ranging from 11.9 to 380.2 m g / m l . These solutions were added at a volume of 0.5 ml to the 3.7-ml (final concentrations in Table 1) lymphocyte cultures (2 for each treatment) that had been established 48 h earlier. Control cultures received an equal volume of R P M I 1640 medium.
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
334 Positive control cultures were treated with cyclophosphamide (CP; L~i~ike/Farmos-Yhtymfi Oy, Turku, Finland) prepared as earlier reported (M~iki-Paakkanen and Norppa, 1987; N o r p p a et al., 1985). A further dilution (10 mM, in R P M I 1640 medium) was added to 4.2-ml cultures at a volume of 42 /zl (final concentration 0.1 mM). Without metabolic activation, the treatment lasted until harvest, i.e., 24 h. For treatment with $9 mix, the culture medium was replaced for 1 h with 3.7 ml of medium containing rat liver $9 mix (for details, see M~iki-Paakkanen and Norppa, 1987). After the treatment and cell wash, the cells were suspended in 4 ml of complete growth medium (Jantunen et al., 1986; M~ki-Paakkanen and Norppa, 1987) and were further incubated (37 ° C) for 24 h. Cell harvest, hypotonic treatment, fixation, slide preparation, and Giemsa staining were performed as reported previously (M~iki-Paakkanen et al., 1980), except that Colcemid was present in the cultures for the last 2 h. The same lymphocyte slides were separately analyzed for structural chromosome aberrations (see Jantunen et al., 1986), numerical chromosome aberrations, and mitotic index. Cells with less than 46 chromosomes were considered hypodiploid, those with 47-59 chromosomes hyperdiploid, and those with 60 or more chromosomes polyploid. Duplicate cultures of C H O cells were established for each treatment as described earlier (Hyt0nen et al., 1983). CAP treatment solutions, prepared in McCoy's Medium 5A (Grand Island Biological Co.), ranged from 8.8 to 565.8 m g / m l (final concentrations in Table 3) and were added for 4 h, 24 h after culture initiation, to the cultures at a volume of 1 ml. $9 mix treatment medium was similar to that used for human lymphocytes, except that McCoy's Medium 5A was used instead of R P M I 1640. Control cultures were treated with 1 ml of the medium. Cyclophosphamide ( N o r p p a et al., 1985), at a final concentration of 0.01 mM, was used as a positive control. After the treatment, the cells were rinsed twice with 4 ml of phosphate-buffered saline (PBS) and were allowed to recover in complete medium for 24 h. Cell harvest, preparation of slides, and fluorescence-plus-Giemsa staining were performed according to previously described procedures
(Hyt¢Snen et al., 1983: Mfiki-Paakkanen et al., 1980). SCEs and proliferation indices were analyzed as earlier described ( N o r p p a et al., 1985).
Results and discussion
CAP significantly increased lymphocytes with chromatid-type aberrations and the total number of aberrant cells (_+gaps) and aberrations/100 cells at the highest concentrations available, with (200 raM) and without (50 raM) $9 mix (Table 1). These toxic doses also increased cells with chromosome-type aberrations and gaps with and without $9 mix, respectively. The number of aberrations was particularly high with metabolic activation, because several cells with multiple and complex chromatid-type aberrations were found. Although linear regression analysis would indicate a dose-dependent effect in m a n y of the aberration categories of Table 1, this evidence is not very convincing, as only one concentration both with and without $9 mix gave a result clearly deviating from the controls. As expected, CP was clastogenic with $9 mix, but not without it. A dose-dependent increase could be seen in the number of polyploid cells (mainly tetraploids or nearly tetraploids) both with and without $9 mix and in the total number of cells with deviating chromosome n u m b e r without $9 mix (Table 2). These frequencies were significantly increased at 25 and 50 m M without $9 mix and (polyploids only) at 100 and 200 m M with $9 mix. None of the treatments, with the exception of a sporadic finding at 12.5 m M without $9 mix, was able to significantly increase the number of hyperdiploid cells (Tables 1 and 2). Hypodiploidy, a parameter considered to be more sensitive to artifacts than hyperdiploidy, was elevated at 50 m M without $9 mix. When hyperdiploids and hypodiploids were pooled, a statistically significant effect could be observed without $9 mix at 50 raM. Exclusion of hypodiploids from the total number of cells with numerical chromosome changes yielded a significant increase at all concentrations tested without $9 mix and at the 2 highest ones available with $9 mix, with no obvious dose response.
335 TABLE 1 CHROMOSOME ABERRATIONS IN H U M A N WHOLE-BLOOD LYMPHOCYTE C U L T U R E S TREATED WITH CAPROLACTAM (CAP), 48 h AFTER C U L T U R E INITIATION, W I T H O U T (24-h TREATMENT) OR W I T H (1-h TREATMENT) RAT LIVER $9 MIX 200 metaphases/treatment (100 from each duplicate culture) were analyzed for chromosome aberrations and 2000 cells/treatment
(1000 from each duplicate culture) for mitotic index. Treatment
Mitotic
Cells with aberrations a (%)
(mM)
index a
Chromatid
Chromosome
(%)
type
type
Aberrations Gaps
Total gaps excluded
gaps included
per 100 cells, gaps excluded a
Without $9 mix
Control(medium)
10.1_+1.4
1.5_+0.5
0.0
0.0
1.5_+0.5
1.5_+0.5
1.5_+ 0.5
0.1
8.0_+0.8
0.5+0.5
0.0
0.5_+0.5
0.5_+0.5
1.0 +0.0
0.5_+ 0.5
CAP 12.5 25 50 100
7.2_+0.4 4.8_+0.7 3.5_+0.2 0.0
1.5+1.5 1.0+0.0 7.5_+0.5"*
1.0-+ 0.0 1.0_+ 0.0 1.0+0.0
0.5_+0.5 1.0_+0.0 3.0+0.0"
2.5_+1.5 2.0_+0.0 8.5_+0.5"*
6.3_+0.2
0.0
0.5_+0.5
0.0
0.5_+0.5
Cyclophosphamide
3.0_+1.0 3.0_+0.0 11.5-+1.5"**
2.5_+ 1.5 2.0_+ 0.0 13.0-+ 1 . 0 " * *
With $9 mix
Control(medium)
0.5_+0.5
0.5_+ 0.5
Cyclophosphamide 0.1
3.9_+0.2
13.5_+0.5 * * *
7.0_+2.0 * * *
0.0
20.5_+2.0 * * *
20.5_+2.5 * * *
22.5_+ 3.5 * * *
CAP 25 50 100 b 200 400
5.8_+0.1 4.9_+0.9 4.1-+0.3 2.2_+0.1 0.0
1.0+_1.0 1.0_+1.0 1.0-+1.0 5.1_+0.1 **
1.0-+1.0 1.5_+0.5 1.0-+1.0 8.3_+0.3 * * *
0.0 0.5_+0.5 0.0 0.8_+0.8
2.0-+2.0 2.5_+0.5 2.0_+2.0 13.4+0.3 * * *
2.0-+2.0 3.0_+1.0 3.0_+2.0 14.3-+1.2 * * *
2.0_+ 2.0 2.5-+ 0.5 2.5_+ 2.5 135.2_+37.2 * * *
a Mean of duplicate cultures ± SE. b 158 cells analyzed.
Probabilities calculated according to Fisher's exact (1-tailed) or binomic test (aberrations/100 cells): * p < 0.05; * * p < 0.01; * * * p < 0.001.
In CHO cells, a marginal increase in SCEs was obtained at the highest concentration of CAP available (125 mM) in the presence of $9 mix (Table 3). This dose induced a clear decrease in proliferation index and was too toxic for analysis without $9 mix. CP had a strong effect on SCEs in the presence of metabolic activation. The present results agree qualitatively with the IPCS collaborative study (Ashby et al., 1985): CAP induced structural chromosome aberrations in human lymphocytes. In the IPCS study, however, Howard et al. (1985) observed (dose-dependent) chromosome aberration induction at clearly
lower doses than we did, despite their shorter treatment times. Another discrepancy is that they did not see polyploidy induction. On the other hand, Ishidate and Sofuni (1985) did observe polyploidy, along with chromosome aberrations (including multi-aberrant cells), in Chinese hamster lung cells at CAP concentrations comparable to our positive ones. Also another study (Priston and Dean, 1985) in rat liver cells, although interpreted negative, indicated a statistically significant increase of chromosome aberrations (gaps included, in comparison with concurrent controls) at the highest dose of CAP tested (8.8 mM), with a
336 TABLE 2 N U M E R I C A L C H R O M O S O M E C H A N G E S 1N H U M A N W H O L E - B L O O D L Y M P H O C Y T E C U L T U R E S T R E A T E D WITH C A P R O L A C T A M (CAP), 48 h A F T E R C U L T U R E I N I T I A T I O N , W I T H O U T (24-h T R E A T M E N T ) O R WITH (1-h TREATMENT) RAT LIVER $9 MIX 200 metaphases (100 from each duplicate culture) were analyzed per treatment. Treatment
Cells with deviating chromosome number (%, mean of duplicates _+SE)
(mM)
Polyploid
Hyperdiploid
Hypodiploid
Hyper- and
Total
hypo-diploids combined
hypodiploids
hypodiploids
excluded
included
Without $9 mix Control (medium)
0.0
1.0 _+1.0
6.0 + 1.0
7.0 _+2.0
1.0 + 1.0
7.0 _+2.0
Cyclophosphamide 0.1
0.0
4.0 _+1.0
3.0 _+ 1.0
7.0 _+0.0
4.0 _+1.0
7.0 + 0.0
CAP 12.5 25 50 100
1.5_+0.5 2.5_+2.5 * 4.0-+3.0 ** (No metaphases)
5.5+2.5 ** 2.5_+0.5 2.5_+0.5
4.5_+1.5 7.5_+0.5 11.5_+3.5 *
10.0_+1.0 10.0_+0.0 14.0+3.0 *
With $9 mix Control(medium)
2.0_+0.0
2.0+1.0
7.0_+2.0
Cyclophosphamide 0.1
1.5_+1.5
5.5_+0.5
CAP 25 50 100 200 400
1.5_+0.5 2.0 _+1.0 6.5_+0.5 * 8.0_+4.0 * * (No metaphases)
0.5_+0.5 3.0 + 1.0 3.0_+0.0 1.0_+ 0.0
a
7 . 0 + 2 . 0 ** 5.0+_2.0 * 6.5_+2.5 **
11.5_+0.5 12.5_+2.5 * 18.0_+6.0 * * *
9.0_+1.0
4.0+1.0
lh0+l.0
8.0_+2.0
13.5_+2.5
7.0_+1.0
15.0_+l.0
6.0_+1.0 5.0 _+ 1.0 6.0_+3.0 7.3 _+0.7
6.5_+1.5 8.0 _+2.0 9.0_+3.0 8.9_+0.1
2.0_+0.0 5.0 _+0.0 9.5_+0.5 * 9.1 +4.1 *
8.0_+1.0 10.0 + 1.0 15.5+_2.5 16.4_+3.4
" 191 ceils analyzed. Probabilities calculated according to Fisher's exact test (1-tailed): * p < 0.05; * * p < 0.01; * * * p < 0.001. TABLE 3 S I S T E R - C H R O M A T I D E X C H A N G E S (SCEs) IN C H I N E S E H A M S T E R O V A R Y (CHO) CELLS T R E A T E D W I T H C A P R O L A C T A M (CAP) FOR 4 h W I T H O U T OR WITH RAT LIVER $9 MIX For each treatment, 60 second-division metaphases (30 from each duplicate culture) were analyzed for SCEs and 200 metaphases (100 from each duplicate culture) for proliferation indices. Treatment
Without $9 mix "
(mM)
SCEs/cell
With $9 mix a Proliferation index
SCEs/cell
Proliferation index
Control
9.0 _+0.0
1.97 _+0.01
10.2 + 0.1
1.84 _+0.01
Cyclophosphamide 0.01
9.6 + 0.8
1.99 _+0.01
39.9 _+0.6
1.73 + 0.03
10.0 _+0.6 9.5 _+0.1 10.0 _+0.0 No result No result
1.97 _+0.02 1.94 _+0.02 1.87 + 0.01 0.04 _+0.00 0.00
10.6 _+0.2 10.0 _+0.3 10.7 + 0.4 11.9 _+0.0 * * No result
1.86 _+0.03 1.85 _+0.01 1.79 _+0.01 1.32 + 0.02 0.00
CAP 15.6 31.3 62.5 125 250
a Mean of duplicate cultures + SE. ** p < 0.01, t-test (1-tailed), 51 cells analyzed.
337 s u g g e s t i v e d o s e r e s p o n s e . I n o t h e r in v i t r o studies o n the i n d u c t i o n of c h r o m o s o m e a b e r r a t i o n s , p o l y p l o i d y , a n e u p l o i d y , or s p i n d l e d i s t u r b a n c e s C A P g a v e n e g a t i v e results (see A s h b y et al., 1985). T h e c o n c l u s i o n of t h e I P C S s t u d y o n S C E i n d u c t i o n b y C A P was n e g a t i v e ( A s h b y et al., 1985). A closer look, h o w e v e r , r e v e a l s a m a r g i n a l e f f e c t of C A P (in c o m p a r i s o n w i t h c o n c u r r e n t c o n t r o l s ) o n S C E s in all b u t 1 of the 6 s e p a r a t e p a p e r s - - in a c c o r d a n c e w i t h o u r p r e s e n t f i n d i n g . T h u s , a l t h o u g h t h e effect o f C A P o n S C E s is e x t r e m e l y w e a k a n d o c c u r s at t o x i c doses, it c a n b e seen in several d i f f e r e n t in v i t r o s y s t e m s w i t h or w i t h o u t m e t a b o l i c a c t i v a t i o n . I n c o n c l u s i o n , C A P is a b l e to i n d u c e s t r u c t u r a l and numerical chromosome aberrations and, m a r g i n a l l y , S C E s in v a r i o u s cell c u l t u r e s y s t e m s at t o x i c c o n c e n t r a t i o n s w h i c h , in m o s t cases, are v e r y h i g h in t e r m s of m o l a r or w e i g h t / v o l u m e c o n c e n t r a t i o n s . W h e t h e r this a c t i v i t y has a n y relev a n c e w i t h r e s p e c t to the s i t u a t i o n in v i v o r e m a i n s to b e d e t e r m i n e d ,
References Ashby, J., F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.) (1985) Evaluation of Short-Term Tests for Carcinogens, Progress in Mutation Research, Vol. 5, Elsevier, Amsterdam, 752 pp. Howard, C.A., T. Sheldon and C.R. Richardson (1985) Tests for the induction of chromosomal aberrations in human peripheral lymphocytes in culture, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term
Tests for Carcinogens, Progress in Mutation Research, Vol. 5, Elsevier, Amsterdam, pp. 457-467. HytSnen, S., I. Alfheim and M. Sorsa (1983) Effects of emissions from residential wood stoves on SCE induction in CHO cells, Mutation Res., 118, 69-75. Ishidate, M., and T. Sofuni (1985) The in vitro chromosomal aberration test using Chinese hamster lung (CHL) fibroblast cells in culture, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D, Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Progress in Mutation Research, Vol. 5, Elsevier, Amsterdam, pp. 427-432. Jantunen, K., J. MS_ki-Paakkanen and H. Norppa (1986) Induction of chromosome aberrations by styrene and vinylacetate in cultured human lymphocytes: dependence on erythrocytes, Mutation Res., 159, 109-116. Miiki-Paakkanen, J., and H. Norppa (1987) Chromosome aberrations and sister-chromatid exchanges induced by technical grade toluene diisocyanate and methylenediphenyl diisocyanate in cultured human lymphocytes, Toxicol. Lett., 36, 37-43, Maki-Paakkanen, J., K. Husgafvel-Pursiainen, P.-L. Kalliom~iki, J. Tuominen and M. Sorsa (1980) Toluene-exposed workers and chromosome aberrations, J. Toxicol. Environ. Health, 6, 775-781. Norppa, H., F. Tursi, P. Pfaffli, J. M~tki-Paakkanen and H. J~irventaus (1985) Chromosome damage induced by vinyl acetate through in vitro formation of acetaldehyde in human lymphocytes and Chinese hamster ovary cells, Cancer Res., 45, 4816-4821. Priston, R.A.J., and B.J. Dean (1985) Tests for the induction of chromosome aberrations, polyploidy and sister-chromatid exchanges in rat liver (RL4) cells, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Progress in Mutation Research, Vol. 5, Elsevier, Amsterdam, pp. 387-395.