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Genotoxic evaluation of the herbicide trifluralin on human lymphocytes exposed in vitro
Genetic Toxicology
ELSEVIER
Mutation Research 371 (1996) 15-21
Genotoxic evaluation of the herbicide trifluralin on human lymphocytes exposed in vitro G. Ribas, J. Surrallrs 1, E. Carbonell, N. Xamena, A. Creus, R. Marcos * Grup de Mutag~nesi, Unitat de Gen~tica, Departament de Gen~tica i de Microbiologia, Edifici Cn, Universitat Autbnoma de Barcelona, 08193 Bellaterra, Spain Received 22 March 1996; revised 18 June 1996; accepted 19 June 1996
Abstract
The herbicide trifluralin was evaluated for genotoxicity in cultured human peripheral blood lymphocytes. Sister-chromatid exchanges (SCE), chromosome aberrations (CA) and micronuclei (MN) were scored as genetic endpoints. To detect eventual metabolic modification in the genotoxicity of this herbicide, the cultures for SCE and MN demonstration were also treated with $9 fraction. From our results we can conclude that trifluralin was able to exert a weak cytotoxic effect, reducing both the proliferative rate index (PRI) and the cytokinesis block proliferation index (CBPI), and also to induce a slight but statistically significant increase in the frequency of SCE. Under our conditions of testing, no genotoxic effects of trifluralin were observed in the CA and MN assays. Keywords: Trifluralin; Herbicide; Human lymphocyte; Cytogenetic assays; Genotoxicity
1. Introduction
Human exposure to pesticide chemicals has been associated with a variety of diseases, including cancer [1,2]. Because large amounts of pesticides are released into the environment daily, at increasing levels, they are of important concern not only from their potential toxicity but also from their eventual genetic and related effects. Thus, in the last years, many studies have stressed the potential genotoxic
* Corresponding author. Tel.: 34 3-5812052; Fax: 34 3-5812387. Present address: Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, 2300 RA Leiden, The Netherlands.
hazard that the exposure to pesticides can pose to humans [2]. Among pesticides, herbicides constitute an important proportion ( ~ 65%) of their production [3]. The wide use of herbicides, together with the fact that they usually are applied directly onto the soil, guarantee their spreading in the environment. Trifluralin, a dinitroaniline, is a pre-emergence soil-incorporated herbicide currently used on more than 50 crops, that acts entering the seedling in the hypocotyl region and disrupting cell division [4]. Until now, the genotoxic potential of trifluralin has been investigated by several researchers in assays measuring different endpoints and almost all yielded negative or inconclusive results [5-10]. Nevertheless, some tests have shown evidence of genotoxicity mainly in various plant species [11-13],
0165-1218/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 2 1 8 ( 9 6 ) 0 0 0 7 9 - 1
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G. Ribas et al. / Mutation Research 371 (1996) 15-21
when assaying for chromosome aberrations, and conflicting evidence was obtained in Drosophila melanogaster [14,15], in mouse bone marrow cells [16,17] and in cultured human lymphocytes [2]. Taking into account that in several studies the purity of the trifluralin used was not reported and, in some cases, the material tested contained only a low percentage of trifluralin, we decided to conduct further genotoxicity studies in cultured human lymphocytes to provide additional data and to extend the information available on the genotoxic effects of this herbicide. In our previous preliminary experiments with human lymphocytes [18], trifluralin gave negative results in the induction of chromosome aberrations (CA) and micronuclei (MN), and appeared as a poor inducer of sister chromatid exchanges (SCE). Last year we reported that trifluralin increases the ratio of excision repairable DNA lesions converted to micronuclei [19] and also produces significant increases in the length of the comet tail when evaluated by the single-cell gel electrophoresis assay [20]. We report here our recent results obtained with cultured human lymphocytes in the CA, MN and SCE assays after trifluralin treatment. Since the metabolism of this chemical could infuence its genotoxic activity, the assays for SCE and MN demonstration were performed with and without $9 fraction.
2. Materials and methods
2.1. Chemicals Trifluralin, 2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl) benzenamine (CAS No. 1582-09-08, 99.4% purity), was purchased from Dr. Ehrenstorfer's Laboratory (Augsburg, Germany). Ethyl methanesulphonate (EMS, CAS No. 62-50-0), cyclophosphamide (CP, CAS No. 6055-19-2) and mitomycin C (MMC, CAS No. 50-07-7) were obtained from Sigma Chemicals (St. Louis, MO, USA). $9 animal fraction was from Iffa Credo (L'Arbresle, France) and the different cofactors used to obtain the $9 mix, such as glucose-6-P and NADP, were from Sigma. Trifluralin and EMS were dissolved in dimethyl sulphoxide (DMSO, CAS No. 67-68-5, Panreac, Barcelona, Spain), at the final volume in culture of 1% of the
total. The other chemicals (CP and MMC) were dissolved in bidistilled water to the concentrations used, just prior to treatment.
2.2. Lymphocyte cultures Lymphocyte cultures were set up by adding 0.5 ml of heparinized whole blood, from young nonsmoking healthy donors, to 4.5 ml of RPMI 1640 chromosome medium supplemented with 16% heatinactivated fetal calf serum, antibiotics (penicillin and streptomycin) and glutamine (all obtained from Gibco, Eragny, France). Lymphocytes were stimulated by 4% phytohemagglutinin (PHA, Gibco). For sister chromatid exchanges (SCE) demonstration, the cultures were incubated at 37°C for 72 h and 5-bromodeoxyuridine (BrdU) at 15 ixg/ml was added 24 h after the initiation of cultures. The test compounds were added together with the BrdU. Throughout all cultures were maintained in the dark to minimize photolysis of BrdU. For chromosome aberrations (CA) the cultures were incubated for 54 h at 37°C, and the treatment with the chemicals was given 24 h after the initiation of cultures. For the micronuclei (MN) study, the cultures were incubated at 37°C for 72 h and, at 44h after their beginning, cytochalasin-B (Cyt-B, Sigma) at a final concentration of 6 p~g/ml was added, together with the chemicals to test, to arrest cytokinesis. In the three assays the treatment continued up to harvest and, for CA and SCE, at 2 h prior to harvesting, 0.6 i~g/ml of Colcemid (Gibco) was added to arrest the cells at metaphase. The cultures treated for 2 h with $9 mix (4 ml of chromosome medium with 0.5 ml of $9) and those without the $9 mix, were set up without heat inactivated fetal calf serum. After the treatment, cultures were washed twice with PBS and reconstituted in the same way as those cultures treated for 48 h. The cells were collected by centrifugation, and for CA and SCE, resuspended in a pre-warmed hypotonic solution (KC1 0.075 M) for 20 min and then fixed in acetic acid/methanol (1:3, v / v ) . For MN, the cells were washed once in RPMI 1640 medium and then, a mild hypotonic treatment (2-3 min in KC1 0.075 M at room temperature) was carried out. Next, the cells were centrifuged, and an acetic
G. Ribas et al. / Mutation Research 371 (1996) 15-21
acid/methanol (1:5, v / v ) solution was gently added. This fixation step was repeated twice and the resulting cells were resuspended in a small volume of fixative solution [21]. Air-dried preparations were made and the slides were stained with Giemsa (CA and MN) or fluorescence plus Giemsa (SCE) [22].
2.3. CA, SCE and MN analysis A total of 100 well spread metaphases in the CA assay, 50 in the SCE assay, and 1000 binucleated cells with well preserved cytoplasm in the MN assay, were examined when possible, for each experimental concentration and donor on coded slides. In the CA studies, aberrations such as gaps (achromatid lesions without dislocations in the chromatid) and breaks (lesions with some distortion in the continuity of the chromatid) were recorded. In addition, exchanges and fragments were also scored. In the SCE study, 100 metaphases per point were also analyzed to determine the proportion of cells that undergoes one, two and three or more divisions. The proliferative rate index (PRI) was calculated according to the formula PRI = (M1 + 2M2 + 3M3)/N, where M1, M2 and M3 indicate those metaphases corresponding to first, second and third or subsequent divisions, and N the total number of metaphases scored [23]. In the MN study, a minimum of 500 lymphocytes were scored to evaluate the percentage of cells with 1, 2, 3, 4 or more than 4 nuclei. A cytokinesis block proliferation index (CBPI) was calculated according to [19] as follows: CBPI = (MI + 2MII + 3(MIII + M I V ) / N , where MI to MIV represent the number of cells with one to four nuclei, respectively, and N is the number of cells scored. For the statistical evaluation of the SCE data, and following the recommendation of [24], we used the analysis of variance. For our experimental data the ~/(y + 1) transformation was appropriate to stabilise the variance. No replicate differences were found and when a significant F value was obtained ( p < 0.05), the Newman-Keuls test was applied to test the differences of each dose level to the control value. For the statistical evaluation of the other results, we used the Fisher's exact test for CA and micronucleated cells, and the chi-square test for PRI and CBPI. For all tests the level of significance was set at 0.05.
17
3. Results and discussion Tables 1 and 2 show the frequencies of SCE and the PRI values obtained after treatment with the herbicide trifluralin. In each table, the data obtained from the different experimental conditions are shown: treatments lasting 48 h without microsomal fraction, and treatments for 2 h without and with $9 microsomal fraction, respectively. The positive controls used were MMC (0.2 IxM) in the experiments without microsomal activation and CP (3 I~M) in the experiments with $9. Trifluralin treatments lasting for 48 h appear to induce a slight but statistically significant increase in SCE frequency in cultures set up with blood from two different donors at the highest concentration tested (50 p~g/ml). This weak genotoxic effect is accompanied by a slight reduction in cell proliferation when measured by the PRI. In the treatments for 2 h without microsomal activation, there is only a significant increase in SCE
Table 1 Frequencies of SCE and proliferative rate indices (PRI) in cultured human lymphocytes treated with the herbicide trifluralin: Donor A Treatment
Conc.(ixg/ml)
S C E / c e l l + SE
PRI
48 h ( - $9)
control 5 10 25 50 M M C (0.2 poM)
6.75 + 0.38 7.58+0.55 8.62+0.58 7.96 ___0.51 9.27 + 0.44 * 30.78+1.05 *
2.41 2.40 2.37 2.39 2.25 2.15 *
2 h ( - $9)
control 10 25 50 100 200 M M C (0.2 IxM)
6.98 + 0.32 6.82+0.46 9.05 + 0 . 4 2 * 6.26 + 0.44 7.76 + 0.46 8.22 _+0.48 10.48_+ 0.47 *
2.54 2.46 2.38 2.36 2.43 1.90 * 2.38
2 h (+$9)
control 10 25 50 100 200 CP (3 p.M)
7.12+0.30 7.02+0.38 7.37 + 0.47 6.06 + 0.37 7.32_+0.47 9.07 + 0.41 * 2 0 . 3 0 + 1.11 *
2.14 2.17 2.20 2.26 2.28 1.67 * 2.23
50 metaphases were scored for each concentration. p < 0.05 (Newman-Keuls test for SCE; chi-square test for PRI).
G. Ribas et al. /Mutation Research 371 (1996) 15-21
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Table 2 Frequencies of SCE and proliferative rate indices (PRI) in cultured human lymphocytes treated with the herbicide trifluralin: Donor B Treatment
Conc. (p~g/ml)
S C E / c e l l _+SE
PRI
48 h ( - $ 9 )
control 5 10 25 50 MMC (0.2 ~ M )
9.43_+0.61 11.08_+0.73 10.82_+0.58 10.74_+0.67 13.46_+0.72 * 36.02_+ 1.06 *
2.59 2.56 2.41 2.57 2.36 * 2.37
2h(-$9)
control 10 25 50 100 200 MMC (0.2 IxM)
7.55 _+0.31 9.26_+0.65 7.84-+0.52 7.35 5:0.56 8.79 _+0.49 9.19_+0.61 17.56_+0.81 *
2.13 1.54 1.72 * 1.82 * 1.92 1.40 * 2.10
2 h(+S9)
control 10 25 50 100 200 CP (10 I~M)
8.58 _+0.36 8.16+0.41 7.38+0.37 8.51 _+0.59 8.26_+0.55 9.93 _+0.60 * 25.86+_ 1.23 *
2.01 2.02 1.81 2.72 1.88 1.46 2.10
50 metaphases were scored for each concentration. * p < 0.05 (Newman-Keuls test for SCE; chi-square test for PRI).
in one donor (A) at the concentration of 25 Ixg/ml. Nevertheless, it should be pointed out that the SCE frequencies observed in both donors at the highest concentration tested (200 ixg/ml) are on the borderline of significance. The results from the experiments for 2 h in the presence of $9 fraction showed a slight but significant increase in the SCE level in both donors and at the highest concentration tested. Also with this concentration, a significant reduction of cell proliferation was observed in the cultures from both donors, without and with metabolic activation. In conclusion, our results indicate that the herbicide trifluralin is able to exert weak cytotoxic and genotoxic effects. From the results reported in the literature, in vivo treatments with trifluralin for SCE induction in bone marrow of Chinese hamsters did not indicate any significant increase [9]; while in a study carried out without an extemal source of metabolic activation, a weak increase in the SCE level was obtained in cultured human lymphocytes [25]. The results corresponding to the induction of chromosome structural aberrations after treatment with trifluralin are shown in Table 3. The positive control used in this assay was EMS (1 mM). These data do not reveal significant clastogenic effects of
Table 3 Chromosome structural aberrations in cultured human lymphocytes treated with the herbicide trifluralin: Donors A and B Donor
Conc.
Chromatid-type aberrations
Chromosome-type aberrations
Total aberrations
(l,z g / m l )
g
c
c/c
T
G
C
C/C
T
( - gaps)
( + gaps)
No. cells with aberrations
A
control 1 5 25 50 EMS (1 mM)
3 4 0 1 1 10 *
0 0 4 1 4 19 *
0 0 0 0 0 3
0 0 4 1 4 25 *
0 0 0 0 0 0
1 2 1 0 2 1
0 0 0 0 0 1
1 2 1 0 2 3
1 2 5 1 6 28 *
4 6 5 2 7 37 ~
l 2 4 1 6 21 *
B
control 1 5 25 50 EMS (1 mM)
5 5 8* 9 * 2 13 *
4 2 1 0 4 13 *
0 1 0 0 1 3
4 4 1 0 6 19 *
1 1 0 0 0 0
1 1 0 4 l 9 "
0 0 0 0 1 0
1 1 0 4 3 9 *
5 5 1 4 9 28 *
11 11 9 13 * 11 41 *
5 4 1 3 7 19 *
100 metaphases were scored for each concentration; g, G: gaps; c, C: breaks; c / c , C / C : exchanges; T: total breaks without gaps. W e calculated the total of aberrations by assuming that one exchange implies two breaks. * p < 0.05 (Fisher's exact test)
G. Ribas et al. /Mutation Research 371 (1996) 15-21
this herbicide, because only a weak induction of chromatid gaps was found in one donor (B). Our negative findings agree with those obtained in Chinese hamster ovary (CHO) cells with and without metabolic activation [6,9], although they are in contrast with the weakly positive results obtained by [26] in human lymphocytes and by [27] in germ cells of male mice. Nevertheless, these last results have not been considered sufficiently relevant since they were obtained with a commercial product containing only 26% trifluralin, and with no indication of the other components or impurities. In this context it may be recalled that some positive evidences of genotoxicity and carcinogenicity obtained in the past with trifluralin can be attributed to the contamination with nitrosamines. Tables 4 - 6 show the results corresponding to the micronucleus assay in cytokinesis blocked cells. The positive controls used in these experiments were
Table 5 Micronuclei in human cultured lymphocytes treated with the herbicide trifluralin: Donor B Treatment
Conc.
BN cells CBPI
(Ixg/ml)
(%)
control 5 10 25 50 M M C (0.2 IxM)
55.4 55.4 53.4 60.8 60.0 51.8 52.0
2.36 2.40 2.25 2.19 * 2.00 * 1.53 * 2.30
20 13 11 8 14 13 62 *
2 h (-$9)
control 25 50 100 200 E M S (15 m M )
63.6 66.4 70.2 63.6 36.4 21.8
1.87 1.85 1.81 1.73 1.37 * 1.23 *
14 7 7 14 11 86 *
2 h (+$9)
control 25 50
56.6 59.0 59.6 52.2 27.4 47.4
1.90 2.12 2.10 1.79 * 1.27 * 1.92
16 10 13 12 32 * 47 *
48 h ( - $ 9 )
100
100 Table 4 Micronuclei in human cultured lymphocytes treated with the herbicide trifluralin: Donor A Treatment 48 h ( - $ 9 )
Conc.
BN cells CBPI
(l~g/ml)
(%)
control 5 10 25 50
54.2 57.4 56.6 68.0 70.2 65.8 50.2
2.52 2.53 2.42 2.18 * 2.15 * 1.76 * 2.47
55.4 52.2 51.2 28.8 3.4 19.8
1.83 1.62 1.59 1.30 1.03 1.23
* * * * *
17 t 111 *
61.2 59.2 61.2 48.2 32.8 56.6
1.83 2.07 1.93 1.51 * 1.34 * 1.70 ~
11 8 12 20 32 * 45 *
100 M M C (0.2 p,M) 2 h (-$9)
control 25 50 100 200 EMS (15 m M )
2 h (+$9)
control 25 50 100 200 CP (0.1 m M )
Total B N M N 15 13 10 24 12 28 * 63 * 16 7
11
1000 binucleated cells were examined for each concentration; BN, binucleated; BNMN, binucleated cells with one or more micronuclei; t, toxicity. * p < 0.05 (Fisher's exact test for BNMN cells; chi-square test for CBPI).
19
200 CP (0.1 m M )
Total B N M N
1000 binucleated cells were examined for each concentration; BN, binucleated; BNMN, binucleated cells with one or more micronuclei; t, toxicity. * p < 0.05 (Fisher's exact test for BNMN cells; chi-square test for CBPI).
MMC (0.2 p.M), EMS (15 mM) and CP (0.1 mM). These tables indicate the percentage of binucleated (BN) cells, the values of the cytokinesis block proliferation index (CBPI), and the number of cells presenting one or more micronuclei after scoring 1000 BN cells. Taking into account the simplicity and the sensitivity of the MN assay [28,29], it can be used to complement the results from SCE and CA assays. Due to the well known interindividual variability of the baseline frequency of MN, we have performed the MN experiments with blood from three donors. With reference to the CBPI there is a dose-related decrease. Although a significant increase in the number of BNMN cells was observed in four cases at the highest concentration of trifluralin tested, the overall evaluation of the results obtained without and with metabolic activation indicates a lack of response. These findings generally agree with those obtained in the CA assay, and indicate that in cultured human
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G. Ribas et al. / Mutation Research 371 (1996) 15-21
Table 6 Micronuclei in human cultured lymphocytes treated with the herbicide trifluralin: Donor C Treatment
Conc. (txg/ml)
BN cells CBPI (%)
Total BNMN
48 h ( - $9) control 5 l0 25 50 MMC (0.2 p~M)
65.6 68.2 70.6 74.0 76.6 77.6
2.48 1l 2.44 4 2.29 5 2.19 * 4 2.02 * 4 2.06 * 25
2 h ( - $9)
control 25 50 100 200 EMS (15 mM)
65.0 59.8 61.6 45.2 22.6 5.2
2.04 6 2.03 20 1.87 l0 1.50 * 11 1.23 ~ 26 1.05 * 43
control 25 50 100 200 CP (0.1 mM)
56.6 52.2 67.0 59.4 23.8 50.0
2.10 14 1.93 8 2.07 7 1.84 * 9 1.25 * 18 1.85 * 29
2 h(+S9)
1000 binucleated cells were examined for each concentration; BN, binucleated; BNMN, binucleated cells with one or more micronuclei; t, toxicity. * p < 0.05 (Fisher's exact test for BNMN cells; chi-square test for CBPI). l y m p h o c y t e s , trifluralin does n o t s h o w c l a s t o g e n i c effects. T a k i n g into a c c o u n t that the m i c r o n u c l e u s test is also able to d e t e c t a n e u g e n i c activity, o u r n e g a t i v e results are in c o n t r a s t w i t h the f i n d i n g s o f [14] a n d [15,30] in D r o s o p h i l a , w h o r e p o r t e d trifluralin to b e an a n e u p l o i d y i n d u c i n g c o m p o u n d a n d also w i t h t h o s e f r o m [26] in h u m a n l y m p h o c y t e s , in w h i c h c h r o m o s o m e a b e r r a t i o n s h a v e b e e n attributed to i n t e r f e r e n c e s w i t h the mitotic spindle. In c o n c l u s i o n , o u r results i n d i c a t e that the herbicide trifluralin is able to e x e r t w e a k c y t o t o x i c a n d g e n o t o x i c effects in c u l t u r e d h u m a n l y m p h o c y t e s a n d t h a t the S C E a s s a y a p p e a r s to b e m o r e s e n s i t i v e t h a n C A a n d M N assays in the d e t e c t i o n o f such activity.
Acknowledgements T h i s i n v e s t i g a t i o n was s u p p o r t e d in part b y the Spanish Ministry of Education and Science (Grant
No. S A F 9 4 - 0 6 9 7 , C I C Y T ) , the G e n e r a l i t a t o f C a t a l u n y a ( G R Q 9 3 - 2 0 2 3 , C I R I T ) a n d the E u r o p e a n C o m m u n i t i e s ( C T 9 1 - 0 1 6 1 , S T E P P r o g r a m ) . G. R i b a s a n d J. Surrall6s were s u p p o r t e d d u r i n g this w o r k b y f e l l o w s h i p s f r o m the U n i v e r s i t a t A u t b n o m a de B a r c e l o n a ( U A B ) a n d the S p a n i s h M i n i s t r y o f E d u c a t i o n a n d S c i e n c e (FPI), respectively. W e also t h a n k G. U m b e r t for h e r e x p e r t t e c h n i c a l h e l p in the p r e p a ration a n d scoring o f s a m p l e s a n d M. M c C a r t h y for h e r secretarial assistance.
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G. Ribas et al. /Mutation Research 371 (1996) 15-21 [12] Kabarity, A. and A. Nahas (1979) Induction of polyploidy and C-tumours after treating Allium cepa root tips with the herbicide 'Treflan', Biol. Plant, 21, 253-258. [13] Grigorenko, N.V., V.F. Fasilchenko, Y.G. Merezhinski, V.V. Morgun, V.F. Logvinenko and S.V. Sharmankin (1986) Cytogenetic activity of a herbicide treflan, and its metabolites as applied to maize (Russ.), Tsitol. Genet., 20, 294-298. [14] Bryant, M.L. and M.R. Murnik (1979) Mutagenicity of the herbicide trifluralin in Drosophila melanogaster, Genetics, 91, Suppl. 2. [15] Foureman, P.A. (1981) Identification of aneuploidy inducing chemicals in Drosophila, Environ, Mutagen., 3, 319. [16] Neh~z, M., A. P~ldy, A. Selypes and G. Berencsi (1980) Experiments on the mutagenic effects of two pesticides, DNOC and trifluralin, Mutation Res., 74, 202-203. [17] Pilinskaya, M.S.A. (1987) Evaluation of the cytogenetic effect of the herbicide treflan and of a number of its metabolites on mammalian somatic cells (Russ.), Tsitol. Genet., 21, 131-135. [18] Torres, C., G. Ribas, J. Surrall~s, N. Xamena, A. Creus and R. Marcos (1993) Genotoxicity of five herbicides in Drosophila melanogaster and in peripheral blood human lymphocytes, 23rd Annual Meeting of the European Environmental Mutagen Society, Abstracts Book, p. 216. [19] Surrall~s, J., N. Xamena, A. Creus and R. Marcos (1995) The suitability of the micronucleus assay in human lymphocytes as a new biomarker of excision repair, Mutation Res., 342, 43-59. [20] Ribas, G., G. Frenzilli, R. Barale and R. Marcos (1995) Herbicide-induced DNA damage in human lymphocytes evaluated by the single-cell gel electrophoresis (SCGE) assay, Mutation Res., 344, 41-54. [21] Surrall~s, J., E. Carbonell, R. Marcos, F. Degrassi, A. Antoccia and C. Tanzarella (1992) A collaborative study on the improvement of the micronucleus test in cultured human lymphocytes, Mutagenesis, 6, 407-410.
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[22] Perry, P.E. and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (Lond.), 251, 156-158. [23] Lamberti, L., P. Bigatti Ponzetto and G. Ardito (1983) Cell kinetics and sister chromatid exchange frequency in human lymphocytes, Mutation Res., 120, 193-199. [24] Cooke, D., J. Allen, M.G. Clare, C.J. Dor~ and L. Henderson (1989) Statistical methods for sister chromatid exchange experiments, in: D.J. Kirkland (Ed.), Statistical Evaluation on Mutagenicity Test Data, Cambridge University Press, Cambridge, pp. 155-183. [25] Ghiazza, G.F., G. Zavarise, M. Lanero and G. Ferraro (1984) SCE (sister chromatid exchange) induced in chromosomes of human lymphocytes by trifluralin, atrazine and simazine (ltal.). Boll. Soc. It. Biol. Sper., 60, 2149-2153. [26] Donna, A., P.G. Betta, F. Gagliardi, G.F. Ghiazza, M. Gallareto and V. Gabutto (1981) Preliminary experimental contribution to the study of possible carcinogenic activity of two herbicides containing atrazine-simazine and trifluralin as active principles, Pathologica, 73, 707-721. [27] Neh~z, M., A. Selypes, A. Pfildy, E. Mazzag, G. Berensci and R. J~rmay (1982) The effects of five weeks treatment with dinitro-o-cresol or trifluralin-containing pesticides in the germ cells of male mice, J. Appl. Toxicol., 2, 179-180. [28] Fenech, M. and A.A. Morley (1985) Measurement of micronuclei in lymphocytes, Mutation Res., 147, 29-36. [29] Fenech, M. (1993) The cytokinesis-block micronucleus tecnhique: A detailed description of the method and its application to genotoxicity studies in human populations, Mutation Res., 285, 35-44. [30] Foureman, P.A. (1988) The TX; Y test for the detection of nondisjunction and chromosome breakage in Drosophila melanogaster. II. Results of female exposures, Mutation Res., 203, 309-316.
ELSEVIER
Mutation Research 371 (1996) 15-21
Genotoxic evaluation of the herbicide trifluralin on human lymphocytes exposed in vitro G. Ribas, J. Surrallrs 1, E. Carbonell, N. Xamena, A. Creus, R. Marcos * Grup de Mutag~nesi, Unitat de Gen~tica, Departament de Gen~tica i de Microbiologia, Edifici Cn, Universitat Autbnoma de Barcelona, 08193 Bellaterra, Spain Received 22 March 1996; revised 18 June 1996; accepted 19 June 1996
Abstract
The herbicide trifluralin was evaluated for genotoxicity in cultured human peripheral blood lymphocytes. Sister-chromatid exchanges (SCE), chromosome aberrations (CA) and micronuclei (MN) were scored as genetic endpoints. To detect eventual metabolic modification in the genotoxicity of this herbicide, the cultures for SCE and MN demonstration were also treated with $9 fraction. From our results we can conclude that trifluralin was able to exert a weak cytotoxic effect, reducing both the proliferative rate index (PRI) and the cytokinesis block proliferation index (CBPI), and also to induce a slight but statistically significant increase in the frequency of SCE. Under our conditions of testing, no genotoxic effects of trifluralin were observed in the CA and MN assays. Keywords: Trifluralin; Herbicide; Human lymphocyte; Cytogenetic assays; Genotoxicity
1. Introduction
Human exposure to pesticide chemicals has been associated with a variety of diseases, including cancer [1,2]. Because large amounts of pesticides are released into the environment daily, at increasing levels, they are of important concern not only from their potential toxicity but also from their eventual genetic and related effects. Thus, in the last years, many studies have stressed the potential genotoxic
* Corresponding author. Tel.: 34 3-5812052; Fax: 34 3-5812387. Present address: Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, 2300 RA Leiden, The Netherlands.
hazard that the exposure to pesticides can pose to humans [2]. Among pesticides, herbicides constitute an important proportion ( ~ 65%) of their production [3]. The wide use of herbicides, together with the fact that they usually are applied directly onto the soil, guarantee their spreading in the environment. Trifluralin, a dinitroaniline, is a pre-emergence soil-incorporated herbicide currently used on more than 50 crops, that acts entering the seedling in the hypocotyl region and disrupting cell division [4]. Until now, the genotoxic potential of trifluralin has been investigated by several researchers in assays measuring different endpoints and almost all yielded negative or inconclusive results [5-10]. Nevertheless, some tests have shown evidence of genotoxicity mainly in various plant species [11-13],
0165-1218/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 2 1 8 ( 9 6 ) 0 0 0 7 9 - 1
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G. Ribas et al. / Mutation Research 371 (1996) 15-21
when assaying for chromosome aberrations, and conflicting evidence was obtained in Drosophila melanogaster [14,15], in mouse bone marrow cells [16,17] and in cultured human lymphocytes [2]. Taking into account that in several studies the purity of the trifluralin used was not reported and, in some cases, the material tested contained only a low percentage of trifluralin, we decided to conduct further genotoxicity studies in cultured human lymphocytes to provide additional data and to extend the information available on the genotoxic effects of this herbicide. In our previous preliminary experiments with human lymphocytes [18], trifluralin gave negative results in the induction of chromosome aberrations (CA) and micronuclei (MN), and appeared as a poor inducer of sister chromatid exchanges (SCE). Last year we reported that trifluralin increases the ratio of excision repairable DNA lesions converted to micronuclei [19] and also produces significant increases in the length of the comet tail when evaluated by the single-cell gel electrophoresis assay [20]. We report here our recent results obtained with cultured human lymphocytes in the CA, MN and SCE assays after trifluralin treatment. Since the metabolism of this chemical could infuence its genotoxic activity, the assays for SCE and MN demonstration were performed with and without $9 fraction.
2. Materials and methods
2.1. Chemicals Trifluralin, 2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl) benzenamine (CAS No. 1582-09-08, 99.4% purity), was purchased from Dr. Ehrenstorfer's Laboratory (Augsburg, Germany). Ethyl methanesulphonate (EMS, CAS No. 62-50-0), cyclophosphamide (CP, CAS No. 6055-19-2) and mitomycin C (MMC, CAS No. 50-07-7) were obtained from Sigma Chemicals (St. Louis, MO, USA). $9 animal fraction was from Iffa Credo (L'Arbresle, France) and the different cofactors used to obtain the $9 mix, such as glucose-6-P and NADP, were from Sigma. Trifluralin and EMS were dissolved in dimethyl sulphoxide (DMSO, CAS No. 67-68-5, Panreac, Barcelona, Spain), at the final volume in culture of 1% of the
total. The other chemicals (CP and MMC) were dissolved in bidistilled water to the concentrations used, just prior to treatment.
2.2. Lymphocyte cultures Lymphocyte cultures were set up by adding 0.5 ml of heparinized whole blood, from young nonsmoking healthy donors, to 4.5 ml of RPMI 1640 chromosome medium supplemented with 16% heatinactivated fetal calf serum, antibiotics (penicillin and streptomycin) and glutamine (all obtained from Gibco, Eragny, France). Lymphocytes were stimulated by 4% phytohemagglutinin (PHA, Gibco). For sister chromatid exchanges (SCE) demonstration, the cultures were incubated at 37°C for 72 h and 5-bromodeoxyuridine (BrdU) at 15 ixg/ml was added 24 h after the initiation of cultures. The test compounds were added together with the BrdU. Throughout all cultures were maintained in the dark to minimize photolysis of BrdU. For chromosome aberrations (CA) the cultures were incubated for 54 h at 37°C, and the treatment with the chemicals was given 24 h after the initiation of cultures. For the micronuclei (MN) study, the cultures were incubated at 37°C for 72 h and, at 44h after their beginning, cytochalasin-B (Cyt-B, Sigma) at a final concentration of 6 p~g/ml was added, together with the chemicals to test, to arrest cytokinesis. In the three assays the treatment continued up to harvest and, for CA and SCE, at 2 h prior to harvesting, 0.6 i~g/ml of Colcemid (Gibco) was added to arrest the cells at metaphase. The cultures treated for 2 h with $9 mix (4 ml of chromosome medium with 0.5 ml of $9) and those without the $9 mix, were set up without heat inactivated fetal calf serum. After the treatment, cultures were washed twice with PBS and reconstituted in the same way as those cultures treated for 48 h. The cells were collected by centrifugation, and for CA and SCE, resuspended in a pre-warmed hypotonic solution (KC1 0.075 M) for 20 min and then fixed in acetic acid/methanol (1:3, v / v ) . For MN, the cells were washed once in RPMI 1640 medium and then, a mild hypotonic treatment (2-3 min in KC1 0.075 M at room temperature) was carried out. Next, the cells were centrifuged, and an acetic
G. Ribas et al. / Mutation Research 371 (1996) 15-21
acid/methanol (1:5, v / v ) solution was gently added. This fixation step was repeated twice and the resulting cells were resuspended in a small volume of fixative solution [21]. Air-dried preparations were made and the slides were stained with Giemsa (CA and MN) or fluorescence plus Giemsa (SCE) [22].
2.3. CA, SCE and MN analysis A total of 100 well spread metaphases in the CA assay, 50 in the SCE assay, and 1000 binucleated cells with well preserved cytoplasm in the MN assay, were examined when possible, for each experimental concentration and donor on coded slides. In the CA studies, aberrations such as gaps (achromatid lesions without dislocations in the chromatid) and breaks (lesions with some distortion in the continuity of the chromatid) were recorded. In addition, exchanges and fragments were also scored. In the SCE study, 100 metaphases per point were also analyzed to determine the proportion of cells that undergoes one, two and three or more divisions. The proliferative rate index (PRI) was calculated according to the formula PRI = (M1 + 2M2 + 3M3)/N, where M1, M2 and M3 indicate those metaphases corresponding to first, second and third or subsequent divisions, and N the total number of metaphases scored [23]. In the MN study, a minimum of 500 lymphocytes were scored to evaluate the percentage of cells with 1, 2, 3, 4 or more than 4 nuclei. A cytokinesis block proliferation index (CBPI) was calculated according to [19] as follows: CBPI = (MI + 2MII + 3(MIII + M I V ) / N , where MI to MIV represent the number of cells with one to four nuclei, respectively, and N is the number of cells scored. For the statistical evaluation of the SCE data, and following the recommendation of [24], we used the analysis of variance. For our experimental data the ~/(y + 1) transformation was appropriate to stabilise the variance. No replicate differences were found and when a significant F value was obtained ( p < 0.05), the Newman-Keuls test was applied to test the differences of each dose level to the control value. For the statistical evaluation of the other results, we used the Fisher's exact test for CA and micronucleated cells, and the chi-square test for PRI and CBPI. For all tests the level of significance was set at 0.05.
17
3. Results and discussion Tables 1 and 2 show the frequencies of SCE and the PRI values obtained after treatment with the herbicide trifluralin. In each table, the data obtained from the different experimental conditions are shown: treatments lasting 48 h without microsomal fraction, and treatments for 2 h without and with $9 microsomal fraction, respectively. The positive controls used were MMC (0.2 IxM) in the experiments without microsomal activation and CP (3 I~M) in the experiments with $9. Trifluralin treatments lasting for 48 h appear to induce a slight but statistically significant increase in SCE frequency in cultures set up with blood from two different donors at the highest concentration tested (50 p~g/ml). This weak genotoxic effect is accompanied by a slight reduction in cell proliferation when measured by the PRI. In the treatments for 2 h without microsomal activation, there is only a significant increase in SCE
Table 1 Frequencies of SCE and proliferative rate indices (PRI) in cultured human lymphocytes treated with the herbicide trifluralin: Donor A Treatment
Conc.(ixg/ml)
S C E / c e l l + SE
PRI
48 h ( - $9)
control 5 10 25 50 M M C (0.2 poM)
6.75 + 0.38 7.58+0.55 8.62+0.58 7.96 ___0.51 9.27 + 0.44 * 30.78+1.05 *
2.41 2.40 2.37 2.39 2.25 2.15 *
2 h ( - $9)
control 10 25 50 100 200 M M C (0.2 IxM)
6.98 + 0.32 6.82+0.46 9.05 + 0 . 4 2 * 6.26 + 0.44 7.76 + 0.46 8.22 _+0.48 10.48_+ 0.47 *
2.54 2.46 2.38 2.36 2.43 1.90 * 2.38
2 h (+$9)
control 10 25 50 100 200 CP (3 p.M)
7.12+0.30 7.02+0.38 7.37 + 0.47 6.06 + 0.37 7.32_+0.47 9.07 + 0.41 * 2 0 . 3 0 + 1.11 *
2.14 2.17 2.20 2.26 2.28 1.67 * 2.23
50 metaphases were scored for each concentration. p < 0.05 (Newman-Keuls test for SCE; chi-square test for PRI).
G. Ribas et al. /Mutation Research 371 (1996) 15-21
18
Table 2 Frequencies of SCE and proliferative rate indices (PRI) in cultured human lymphocytes treated with the herbicide trifluralin: Donor B Treatment
Conc. (p~g/ml)
S C E / c e l l _+SE
PRI
48 h ( - $ 9 )
control 5 10 25 50 MMC (0.2 ~ M )
9.43_+0.61 11.08_+0.73 10.82_+0.58 10.74_+0.67 13.46_+0.72 * 36.02_+ 1.06 *
2.59 2.56 2.41 2.57 2.36 * 2.37
2h(-$9)
control 10 25 50 100 200 MMC (0.2 IxM)
7.55 _+0.31 9.26_+0.65 7.84-+0.52 7.35 5:0.56 8.79 _+0.49 9.19_+0.61 17.56_+0.81 *
2.13 1.54 1.72 * 1.82 * 1.92 1.40 * 2.10
2 h(+S9)
control 10 25 50 100 200 CP (10 I~M)
8.58 _+0.36 8.16+0.41 7.38+0.37 8.51 _+0.59 8.26_+0.55 9.93 _+0.60 * 25.86+_ 1.23 *
2.01 2.02 1.81 2.72 1.88 1.46 2.10
50 metaphases were scored for each concentration. * p < 0.05 (Newman-Keuls test for SCE; chi-square test for PRI).
in one donor (A) at the concentration of 25 Ixg/ml. Nevertheless, it should be pointed out that the SCE frequencies observed in both donors at the highest concentration tested (200 ixg/ml) are on the borderline of significance. The results from the experiments for 2 h in the presence of $9 fraction showed a slight but significant increase in the SCE level in both donors and at the highest concentration tested. Also with this concentration, a significant reduction of cell proliferation was observed in the cultures from both donors, without and with metabolic activation. In conclusion, our results indicate that the herbicide trifluralin is able to exert weak cytotoxic and genotoxic effects. From the results reported in the literature, in vivo treatments with trifluralin for SCE induction in bone marrow of Chinese hamsters did not indicate any significant increase [9]; while in a study carried out without an extemal source of metabolic activation, a weak increase in the SCE level was obtained in cultured human lymphocytes [25]. The results corresponding to the induction of chromosome structural aberrations after treatment with trifluralin are shown in Table 3. The positive control used in this assay was EMS (1 mM). These data do not reveal significant clastogenic effects of
Table 3 Chromosome structural aberrations in cultured human lymphocytes treated with the herbicide trifluralin: Donors A and B Donor
Conc.
Chromatid-type aberrations
Chromosome-type aberrations
Total aberrations
(l,z g / m l )
g
c
c/c
T
G
C
C/C
T
( - gaps)
( + gaps)
No. cells with aberrations
A
control 1 5 25 50 EMS (1 mM)
3 4 0 1 1 10 *
0 0 4 1 4 19 *
0 0 0 0 0 3
0 0 4 1 4 25 *
0 0 0 0 0 0
1 2 1 0 2 1
0 0 0 0 0 1
1 2 1 0 2 3
1 2 5 1 6 28 *
4 6 5 2 7 37 ~
l 2 4 1 6 21 *
B
control 1 5 25 50 EMS (1 mM)
5 5 8* 9 * 2 13 *
4 2 1 0 4 13 *
0 1 0 0 1 3
4 4 1 0 6 19 *
1 1 0 0 0 0
1 1 0 4 l 9 "
0 0 0 0 1 0
1 1 0 4 3 9 *
5 5 1 4 9 28 *
11 11 9 13 * 11 41 *
5 4 1 3 7 19 *
100 metaphases were scored for each concentration; g, G: gaps; c, C: breaks; c / c , C / C : exchanges; T: total breaks without gaps. W e calculated the total of aberrations by assuming that one exchange implies two breaks. * p < 0.05 (Fisher's exact test)
G. Ribas et al. /Mutation Research 371 (1996) 15-21
this herbicide, because only a weak induction of chromatid gaps was found in one donor (B). Our negative findings agree with those obtained in Chinese hamster ovary (CHO) cells with and without metabolic activation [6,9], although they are in contrast with the weakly positive results obtained by [26] in human lymphocytes and by [27] in germ cells of male mice. Nevertheless, these last results have not been considered sufficiently relevant since they were obtained with a commercial product containing only 26% trifluralin, and with no indication of the other components or impurities. In this context it may be recalled that some positive evidences of genotoxicity and carcinogenicity obtained in the past with trifluralin can be attributed to the contamination with nitrosamines. Tables 4 - 6 show the results corresponding to the micronucleus assay in cytokinesis blocked cells. The positive controls used in these experiments were
Table 5 Micronuclei in human cultured lymphocytes treated with the herbicide trifluralin: Donor B Treatment
Conc.
BN cells CBPI
(Ixg/ml)
(%)
control 5 10 25 50 M M C (0.2 IxM)
55.4 55.4 53.4 60.8 60.0 51.8 52.0
2.36 2.40 2.25 2.19 * 2.00 * 1.53 * 2.30
20 13 11 8 14 13 62 *
2 h (-$9)
control 25 50 100 200 E M S (15 m M )
63.6 66.4 70.2 63.6 36.4 21.8
1.87 1.85 1.81 1.73 1.37 * 1.23 *
14 7 7 14 11 86 *
2 h (+$9)
control 25 50
56.6 59.0 59.6 52.2 27.4 47.4
1.90 2.12 2.10 1.79 * 1.27 * 1.92
16 10 13 12 32 * 47 *
48 h ( - $ 9 )
100
100 Table 4 Micronuclei in human cultured lymphocytes treated with the herbicide trifluralin: Donor A Treatment 48 h ( - $ 9 )
Conc.
BN cells CBPI
(l~g/ml)
(%)
control 5 10 25 50
54.2 57.4 56.6 68.0 70.2 65.8 50.2
2.52 2.53 2.42 2.18 * 2.15 * 1.76 * 2.47
55.4 52.2 51.2 28.8 3.4 19.8
1.83 1.62 1.59 1.30 1.03 1.23
* * * * *
17 t 111 *
61.2 59.2 61.2 48.2 32.8 56.6
1.83 2.07 1.93 1.51 * 1.34 * 1.70 ~
11 8 12 20 32 * 45 *
100 M M C (0.2 p,M) 2 h (-$9)
control 25 50 100 200 EMS (15 m M )
2 h (+$9)
control 25 50 100 200 CP (0.1 m M )
Total B N M N 15 13 10 24 12 28 * 63 * 16 7
11
1000 binucleated cells were examined for each concentration; BN, binucleated; BNMN, binucleated cells with one or more micronuclei; t, toxicity. * p < 0.05 (Fisher's exact test for BNMN cells; chi-square test for CBPI).
19
200 CP (0.1 m M )
Total B N M N
1000 binucleated cells were examined for each concentration; BN, binucleated; BNMN, binucleated cells with one or more micronuclei; t, toxicity. * p < 0.05 (Fisher's exact test for BNMN cells; chi-square test for CBPI).
MMC (0.2 p.M), EMS (15 mM) and CP (0.1 mM). These tables indicate the percentage of binucleated (BN) cells, the values of the cytokinesis block proliferation index (CBPI), and the number of cells presenting one or more micronuclei after scoring 1000 BN cells. Taking into account the simplicity and the sensitivity of the MN assay [28,29], it can be used to complement the results from SCE and CA assays. Due to the well known interindividual variability of the baseline frequency of MN, we have performed the MN experiments with blood from three donors. With reference to the CBPI there is a dose-related decrease. Although a significant increase in the number of BNMN cells was observed in four cases at the highest concentration of trifluralin tested, the overall evaluation of the results obtained without and with metabolic activation indicates a lack of response. These findings generally agree with those obtained in the CA assay, and indicate that in cultured human
20
G. Ribas et al. / Mutation Research 371 (1996) 15-21
Table 6 Micronuclei in human cultured lymphocytes treated with the herbicide trifluralin: Donor C Treatment
Conc. (txg/ml)
BN cells CBPI (%)
Total BNMN
48 h ( - $9) control 5 l0 25 50 MMC (0.2 p~M)
65.6 68.2 70.6 74.0 76.6 77.6
2.48 1l 2.44 4 2.29 5 2.19 * 4 2.02 * 4 2.06 * 25
2 h ( - $9)
control 25 50 100 200 EMS (15 mM)
65.0 59.8 61.6 45.2 22.6 5.2
2.04 6 2.03 20 1.87 l0 1.50 * 11 1.23 ~ 26 1.05 * 43
control 25 50 100 200 CP (0.1 mM)
56.6 52.2 67.0 59.4 23.8 50.0
2.10 14 1.93 8 2.07 7 1.84 * 9 1.25 * 18 1.85 * 29
2 h(+S9)
1000 binucleated cells were examined for each concentration; BN, binucleated; BNMN, binucleated cells with one or more micronuclei; t, toxicity. * p < 0.05 (Fisher's exact test for BNMN cells; chi-square test for CBPI). l y m p h o c y t e s , trifluralin does n o t s h o w c l a s t o g e n i c effects. T a k i n g into a c c o u n t that the m i c r o n u c l e u s test is also able to d e t e c t a n e u g e n i c activity, o u r n e g a t i v e results are in c o n t r a s t w i t h the f i n d i n g s o f [14] a n d [15,30] in D r o s o p h i l a , w h o r e p o r t e d trifluralin to b e an a n e u p l o i d y i n d u c i n g c o m p o u n d a n d also w i t h t h o s e f r o m [26] in h u m a n l y m p h o c y t e s , in w h i c h c h r o m o s o m e a b e r r a t i o n s h a v e b e e n attributed to i n t e r f e r e n c e s w i t h the mitotic spindle. In c o n c l u s i o n , o u r results i n d i c a t e that the herbicide trifluralin is able to e x e r t w e a k c y t o t o x i c a n d g e n o t o x i c effects in c u l t u r e d h u m a n l y m p h o c y t e s a n d t h a t the S C E a s s a y a p p e a r s to b e m o r e s e n s i t i v e t h a n C A a n d M N assays in the d e t e c t i o n o f such activity.
Acknowledgements T h i s i n v e s t i g a t i o n was s u p p o r t e d in part b y the Spanish Ministry of Education and Science (Grant
No. S A F 9 4 - 0 6 9 7 , C I C Y T ) , the G e n e r a l i t a t o f C a t a l u n y a ( G R Q 9 3 - 2 0 2 3 , C I R I T ) a n d the E u r o p e a n C o m m u n i t i e s ( C T 9 1 - 0 1 6 1 , S T E P P r o g r a m ) . G. R i b a s a n d J. Surrall6s were s u p p o r t e d d u r i n g this w o r k b y f e l l o w s h i p s f r o m the U n i v e r s i t a t A u t b n o m a de B a r c e l o n a ( U A B ) a n d the S p a n i s h M i n i s t r y o f E d u c a t i o n a n d S c i e n c e (FPI), respectively. W e also t h a n k G. U m b e r t for h e r e x p e r t t e c h n i c a l h e l p in the p r e p a ration a n d scoring o f s a m p l e s a n d M. M c C a r t h y for h e r secretarial assistance.
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