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The inhibitory effect of the fungicides captan and captafol on eukaryotic topoisomerases in vitro and lack of recombinagenic activity in the wing spot test of Drosophila melanogaster
Mutation Research 518 (2002) 205–213
The inhibitory effect of the fungicides captan and captafol on eukaryotic topoisomerases in vitro and lack of recombinagenic activity in the wing spot test of Drosophila melanogaster Iwonna Rahden-Staron∗ Department of Biochemistry, Medical University of Warsaw, ul. Banacha 1, 02-097 Warszawa, Poland Received 27 November 2001; received in revised form 12 April 2002; accepted 22 April 2002
Abstract In studies on the mechanisms of mutagenic and carcinogenic action of captan and captafol-related chloroalkylthiocarboximide fungicides, two effects were tested: (i) the effect of both compounds on the activity of eukaryotic topoisomerases I and II in vitro, and (ii) their mutagenic and recombinagenic activity in the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster. Only captafol inhibited the activity of topoisomerase I (10–20% inhibition of activity in the range of 10–100 M). In contrast, both chemicals decreased the activity of topoisomerase II already at 1 M concentration (50 and 20% inhibition of activity by captafol and captan, respectively). Genotoxicity was tested in vivo by administrating both compounds by acute (3 h) and chronic feeding (48 h) of 3-day-old larvae. In acute feeding, captan and captafol demonstrated positive results only for small single and total spots in 10–100 mM exposure concentration range. Both chemicals were inconclusive for large single spots, as well as for twin spots. In chronic treatment, captan showed positive results only for small single and total spots at 2.5 and 5 mM concentrations. Captafol gave inconclusive results over all concentrations tested. The results of the acute treatment experiments which have been performed at very high doses (50% toxicity at higher doses) indicate very weak overall mutagenic activity of both test fungicides. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Captan; Captafol; Genotoxicity; SMART; Drosophila melanogaster; Topoisomerases I and II
1. Introduction There is a continuous debate concerning the possible role of many pesticides in chronic human health conditions [1]. Pesticides are thought to play a role in carcinogenesis, neurotoxicity, and reproductive and developmental processes. Among many pesticides there are groups of fungicides, e.g. captan Abbreviations: SMART, somatic mutation and recombination test; SCE, sister chromatid exchange; GSH, glutathione ∗ Tel.: +48-22-572-06-93; fax: +48-22-572-06-79. E-mail address: [email protected] (I. Rahden-Staron).
and captafol, two related chloroalkylthiocarboximide fungicides, used to control diseases of many fruits, and ornamental crops. They are also used as fungicides in paints, plastics, and leather. Thus, their use might cause long-term exposure of humans. Current regulatory policy to reduce cancer risks is based on the idea that chemicals which induce tumours in rodent cancer bioassays are potential human carcinogens. The US EPA categorised captan and captafol as group B2 (probable human) carcinogens, based upon findings of an increased incidence of malignant, or combined malignant and benign tumours in multiple experiments involving different strains of mice and rats [2,3]. In
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humans, a correlation has been observed between captan use and both leukaemia and prostate cancer [4]. Both compounds exhibited mutagenic activity in a variety of in vitro short-term tests for gene mutation, DNA repair, and chromosomal aberrations in prokaryotic and eukaryotic cells [5–7]. Both induced sister chromatid exchanges (SCE) and chromosomal aberrations [8]. In the studies performed under in vivo conditions, both captan and captafol were considered as non-genotoxic compounds [1], and captan as a non-genotoxic mouse-specific carcinogen [9]. The toxicity and carcinogenic activity of captafol has been better documented and is more pronounced than that of captan (for review see [5,10]). Since captan and captafol are present in the environment [11–13], there is a need for more data on mutagenicity in order to assess their potential hazards to human health. With very rare exceptions, pesticides do not react with DNA directly and the mechanisms of their carcinogenicity are, in general, similar to those of other non-genotoxic (epigenetic) carcinogens, e.g. promotion of spontaneous initiation, oxidative stress, cytotoxicity with sustained cell proliferation and others. One of the possibilities to get more valuable additional data is to monitor recombinagenic activity of chemical compounds, due to its possible connection with carcinogenicity [14,15]. It is important to understand whether or not indirect mechanisms, such as cytotoxicity and inhibition of DNA synthesis, induced by their exposure, are operating. SCE correlate with the chemicals’ cytotoxicity and may be induced by inhibition of the activities of topoisomerases [16–18]. Topoisomerases are nuclear enzymes capable of changing the topology of DNA and their activity is required during DNA replication, transcription, and homologous recombination [19–22]. Since inhibition of the activities of topoisomerases leads to an increase in the number of recombination events [23,24], it was worth checking whether the known ability of captafol and captan to induce SCE [8] is connected with their effect on topoisomerase activity [25,26]. To check the possible mechanism of carcinogenic action of captan and captafol, the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster was applied. The extensive knowledge of the genetics of D. melanogaster and
the long experimental experience with this organism have made it uniquely useful in mutation research and genetic toxicology. Cells of D. melanogaster possess xenobiotic metabolising systems similar to those in mammalian liver [27–29]. The development of SMART has provided a sensitive, rapid, and cheap assay to investigate the mutagenic and recombinagenic properties of chemicals [30–33].
2. Materials and methods 2.1. Chemicals Captan and captafol were purchased from OrganikaAzot (Jaworzno, Poland). The purity of the test compounds was 99.8%. Camptothecin and etoposide were purchased from Sigma. Solutions of captan and captafol were made independently, either with 5% ethanol and 5% Tween-80 (Serva, Heidelberg, FRG) in water (chronic feeding), or with 2 vol.% DMSO (Fluka, AG) (acute feeding). Etoposide was dissolved in 5% ethanol and 5% Tween-80 in water. Camptothecin was dissolved in 2 vol.% DMSO. The structural formulae of the chemicals tested, their Chemical Abstract Services (CAS) registry numbers, and molecular weight are shown in Table 1. 2.2. Topoisomerases activity The activity of topoisomerase I was assayed in a nuclear extract from mouse lymphoma L5178Y cells. Nuclei were isolated according to Pommier et al. [34] and extracted with 0.35 M NaCl according to Estley et al. [35]. The topoisomerase I assay measured the relaxation of supercoiled pBR322 by the extract according to Liu [36]. The topoisomerase II assay determined the conversion of topologically knotted phage P4 DNA to the unknotted topoisomer and was performed according to Liu et al. [37]. Captan and captafol were mixed with the extract in the assay cocktail and pre-incubated for 10 min at 30 ◦ C before addition of the substrate. The results of the assays were quantified by densitometric scanning of negatives of photographed electrophoresis gels. One unit of enzyme activity relaxed 50% of the substrate DNA after 30 min at 30 ◦ C. DNA electrophoresis was performed on 0.8%
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Table 1 Compounds tested in Drosophila wing spot test Common name
Trade name
Chemical name
Captan
Orthocide merpan pillarcap
Captafol
Difolatan folcid
Chemical structure
Molecular weight
CAS no.
N-[(trichloromethyl)-thio]-4cyclohexene-1,2-dicarboximide
301
133-06-2
N-[(1,1,2,2-tetrachloroethyl) thio]-4-cyclo-hexene1,2-dicarboximide
349
2425-06-1
Health effects of captan: mutagen, suspect carcinogen, teratogen, LD50 (oral, rat) 9 g/kg. Health effects of captafol: dermatitis, suspect teratogen, respiratory sensitisation (asthma), mutagen, suspect carcinogen, LD50 (oral, rat) 6200 mg/kg.
agarose; 2 mM EDTA; 40 mM Tris–acetate; pH 7.8 at 1 V/cm according to Sambrook et al. [38]. 2.3. Somatic mutation and recombination test (SMART) Compounds were tested at 25 ◦ C in the wing-spot assay carried out according to Graf et al. [33] and Graf [39]. For the mwh/flr3 cross, females from an y; mwh jv stock were mated to males from flr3 /TM3, Ser stock. Both strains were received from Prof. F.W. Würgler, Institute of Toxicology, Swiss Federal Institute of Technology and University of Zürich (Switzerland). For genetic symbols and description, see [40]. F1 larvae from cross mwh/flr3 were collected 72 ± 4 h after 8 h of egg-laying and placed in plastic vials containing Drosophila instant medium (formula 4-24, Carolina Biological Supply Co., Burlington, NC, USA) with the test compound. Solubility problems were encountered with captan and captafol, which had to be tested as a suspension, either in a mixture of 5% Tween and 5% ethanol in water in chronic feeding, or in 2% DMSO only in acute feeding. Despite the better solubility of both compounds in 2% DMSO compared to 5% Tween, 5% ethanol in water, they were tested in 2% DMSO only in acute feeding (3 h); 2% DMSO has high toxicity in longer treatment.
An amount of 5 ml of a suspension of the compound was added to 1.5 g dry instant medium. The larvae were fed on this medium for the rest of their development (approximately 48 h) (chronic feeding). In some experiments, acute feeding [33] was used. In this procedure, captan or captafol in 2% DMSO was mixed with cellulose powder (1 ml/200 mg powder) and the larvae were placed on top of a gauze-covered bed of this wet powder in a bottle. Feeding through the gauze was allowed to occur for 3 h and then the larvae were transferred into another bottle and cultured on a reagent-free medium to obtain adult flies (acute feeding). The concentration ranged from 10 to 100 mM for acute feeding, and from 0.25 to 10 mM for chronic feeding of both captan and captafol. Negative solvent controls 2% DMSO or 5% Tween-80/5% ethanol/water controls were always conducted in parallel. An amount of 0.01 mM camptothecin, (inhibitor of topoisomerase I) and 1 mM etoposide (VP16) (inhibitor of topoisomerase II) were used as positive controls. After metamorphosis, the hatched adults were collected from the treatment vials and stored in 70% ethanol. Subsequently, wings were analysed according to standard procedures [33]. Three different types of spots (single spots of the mwh or the flr phenotype, twin spots with adjacent mwh and flr areas) were recorded separately in the marker transheterozygous wings. As cell genetics show, wing spot test
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assays detect several genetic endpoints. Twin spots are exclusively produced by mitotic recombination, while mwh single spots may in addition also be due to other mechanisms, such as gene mutation and deletion. The flr single spots on the other hand, arise from mutation or deletion and a few are due to mitotic double cross-over. 2.4. Statistical evaluation For a comparison of the induction frequencies only mwh clones in mwh single spots and in twin spots were taken into consideration [41]. For statistical testing, the decision procedure of Frei and Würgler [42] was applied. As customary for the evaluation of wing spot data [33], the following classes among the total spots are distinguished: small single spots (1–2 cells in size), large single spots (3 cells or larger), and twin spots. This classification is biologically meaningful [42]. For the calculation, the Kastenbaum–Bowman test was used with P = 0.05. Based on the number of wings analysed, the number of mwh clones and the number of cells scored in each wing (24,400), it is possible to calculate the clone formation frequency per cell cycle in 105 cells (see also: [42,43]).
3. Results 3.1. The effect of fungicides on topoisomerases I and II activity The effect of captan and captafol on eukaryotic topoisomerases was studied in vitro using nuclear extracts from mouse lymphoma cells. Reactions specific to topoisomerases I and II were relaxation of pBR322 in the absence of ATP and unknotting of phage P4 DNA, respectively. Captan had no effect on the relaxing activity of topoisomerase I up to a concentration level of 100 M. Captafol, however, inhibited 10–20% of the topoisomerase I activity in the range of 10–100 M. In contrast, both captan and captafol clearly inhibited the unknotting activity of topoisomerase II; 50% of inhibition was observed at 1 M captafol and 5 M captan (Figs. 1a and b, 2a and b). Camptothecin and etoposide used as positive controls in the SMART assay inhibited 50% of topoi-
Fig. 1. (a) Effect of captan (lanes 1–4) and captafol (lanes 6–9) on topoisomerase I from mouse lymphoma cells. The concentrations of compounds was: lane 1, 6–0 M; lane 2, 7–1 M; lane 3, 8–10 M; lane 4, 9–100 M; lane 5, 10–control pBR322 (SC). (b) Inhibitory effect of captan and captafol on topoisomerase I activity: % of inhibition of topoisomerase I activity by captan (䊉) and captafol (䉱).
somerase I and topoisomerase II activities at 5 and 10 M, respectively (data not shown). 3.2. Genotoxicity testing of the fungicides with the wing spot test The data of the wing analysis collected with the two compounds are shown in Table 2. Controls were pooled since there were no significant differences between them. Pooled controls used as carriers in the experiments gave spontaneous spot frequencies very similar to those found in other laboratories [44]. As concurrent controls showed only a few spots, data from the pooled large controls were used for control corrections in the determination of clone induction frequencies. For significance testing, however, the spot scores in treated groups were
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Fig. 2. (a) Effect of captan (lanes 1–4) and captafol (lanes 6–9) on topoisomerase II from mouse lymphoma cells. The concentrations of compounds was: lane 1, 6–0 M; lane 2, 7–1 M; lane 3, 8–10 M; lane 4, 9–100 M; lane 5, 10–control P4 DNA. (b) Inhibitory effect of captan and captafol on topoisomerase II activity: % of inhibition of topoisomerase II activity by captan (䊊) and captafol (䉭).
always compared with the corresponding concurrent controls. According to Graf [39] and Graf et al. [45], the optimal strategy in genotoxicity screening is to start with chronic exposure of 3-day-old larvae for 48 h, that is until pupation. In preliminary experiments of the present work, with younger (2-day-old) larvae, neither captafol, nor captan have induced twin spots and increased frequency of large single spots. The frequency of small single spots was considerably lower as compared with the frequency in 3-day-old larvae (data not shown). That data was in agreement with Graf [39] who showed that practically no twin spots are found in very young and in very old larvae. Based on these results 3-day-old larvae were used for all further experiments. Solubility problems were encountered with both compounds which had to be tested as a suspension under sub-optimal conditions (see Section 2). According
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to Frei and Würgler [42], a positive diagnosis of the test requires that the results of at least two of the three categories should be positive, i.e. when frequencies of total and small single spots per wing are two times, and for large and twin spots five times higher than the spontaneous mutation rate. In acute feeding experiments with captan and captafol in the exposure concentration range from 10 to 100 mM, the cross mwh/flr3 reveals clear positive results for both fungicides, but only for small single and total spots. However, results were inconclusive for large single spots, as well as for twin spots. Effects of both fungicides tested were different in chronic treatment compared with in acute feeding. Positive results were observed for small single and total spots only for captan at two concentrations: 2.5 and 5 mM. No dose-related induction for these spot categories was observed for both acute and chronic feeding (Table 2). Results for captafol were inconclusive or negative in chronic treatment over the whole concentration range. The observed toxicity in both treatments was higher for captafol than for captan. About 50% of the flies did not reach the adult stage in acute treatment at 25 mM captan and 10 mM captafol, and in chronic feeding at 5 mM for captan and the 1–1.5 mM exposure level for captafol. The analysis of the SMART assay data from acute and chronic treatments shows the lack of twin spots, which are produced by mitotic recombination exclusively [33]. To compare the in vivo test effects of both fungicides which inhibit in vitro topoisomerase II, 0.01 mM camptothecin and 1 mM etoposide, (DNA topoisomerases I and II inhibitors, respectively), were used as positive controls [34,46]. These recombinagenic and mutagenic compounds result in stimulation of recombination processes [47], and an increase in SCE frequency [48]. Mutations introduced by these agents result in gene deletion and rearrangements [16]. The flies survival at 0.01 mM camptothecin and 1 mM etoposide was similar to the controls. Solubility problems were encountered with etoposide, which had to be tested as a suspension. Data on induction potency of the control inhibitors observed in the present work for all types of spots (Table 2), as well as lack of toxicity at their tested doses were in agreement with the previous results of Frei and Würgler [44] who showed that induced clones are of 88 and 59% of recombinational origin for camptothecin and etoposide, respectively.
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4. Discussion The wing test system detects a great variety of genotoxins belonging to different chemical classes, including compounds directly or indirectly interacting with DNA. The results presented in this work demonstrate the weak overall mutagenic activity of captan and captafol under in vivo conditions in somatic cells of D. melanogaster and the ability of both compounds to inhibit the activity of topoisomerase II in vitro. However, captafol was a much more potent inhibitor of topoisomerase II relative to captan. The activity of topoisomerase I was inhibited only by captafol, but to a much lower extent as compared to its inhibitory effect on topoisomerase II. The question, whether or not inhibition of topoisomerase(s) is a major event in captan and captafol-induced mutagenicity and carcinogenicity, is considered on the metabolism of both compounds in vivo and on the importance of the thresholds basis [49]. Since captafol and captan show in vitro an inhibitory effect, especially on one of two topoisomerases, the wing spot test of D. melanogaster, which detect genotoxic activity of other topoisomerase poisons [44], seemed to be a good predictor of genotoxic activity of test fungicides. Camptothecin and etoposide were used as positive controls in the SMART assay and gave clear positive results for their recombinagenic activity, in agreement with Frei and Würgler [44]. The analysis of the SMART data for captan and captafol from both treatments showed the lack of twin spots and induction potency for single spots only in acute treatment, but with no dose–effect relationship. Single spots are due to recombination as twin spots are; they can be also due to point mutations, deficiencies and non-disjunction events [33]. Because of that the observed lack of twin spots in the used inversion-free mwh/flr3 marker heterozygotes may not be a sufficient proof that excludes recombinagenic properties of the two test fungicides. Captafol and captan inhibit 50% of topoisomerase II at 1 and 5 M, and etoposide at 10 M, respectively. The very weak inhibitory effect on topoisomerase I was demonstrated only by captafol. Simultaneous comparison of the inhibitory effects on topoisomerase II of etoposide and both test fungicides compared with end-effects in the SMART assay, can lead to the prediction that under test conditions both fungicides
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will not demonstrate recombinagenic activity either due to a lack of such activity, insufficient uptake by tissues, or effective metabolism. The fact that the test fungicides did not stay dissolved during the assays could cause possible fluctuations in the uptake of chemicals by larvae. It may be the reason of an insufficient level in tissues to reach the threshold’s value to reveal fungicides’ biological activity. On the other hand, both captafol and captan may be metabolised by Drosophila cytochrome P-450 mono-oxygenase system [50] to reactive products so efficiently that they do not reach the threshold’s value. Both compounds are not sufficient to retain their mutagenic activity until the late third instar stage when mwh mutants are produced with a high frequency. Under such conditions only small single spots would be observed, because they are induced very late in larvae development. However, the results of the preliminary experiments, with younger 2-day-old larvae, also showed induction of only small single spots category. This supports the correctness of interpretation and points out that the absence of twin spots and large single spots is not due to the relatively late treatment of larvae. Positive responses were found in the acute treatment, but there was no dose–effect relationship. The acute feeding experiments were performed at very high doses, whereby 50% and higher toxicity was observed. In vitro studies showed that mechanism-based toxicity of both test fungicides was related to their main metabolite tetrahydrophtalimide (THPI) which is later degraded to other toxic compounds [51]. This toxicity could also be related to a decrease in non-protein sulfhydryl groups, mainly of GSH [5,52] which, in turn, might influence the integrity and functions of the mitotic spindle [53]. The lack of a dose–effect relationship and an overall very weak mutagenicity may be a proof of a non-genotoxic mechanism of the two test fungicides (and/or their metabolites). They can interact with proteins inducing various types of lesions in the chromosomes of the imaginal disk cells which can give rise of mutations. The results discussed above suggest rather a multifactorial origin of captan- and captafol-induced chromosomal aberration and SCE [8]. The specific effect of inhibition of topoisomerase II does not seem to be a major event in captan and captafol mutagenicity and carcinogenicity.
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Acknowledgements The author is grateful to Dr. U. Graf for his keen interest and critical commentary regarding the manuscript; to Prof. Dr. F.E. Würgler, Dr. U. Graf, and H. Frei for the opportunity to learn SMART technique in their laboratory during the training course on Somatic Genotoxicity Assays with Drosophila of the European Environmental Mutagen Society held at the Institute of Toxicology, Swiss Federal Institute of Technology and University of Zürich, Schwerzenbach, Switzerland. This course was supported by a grant provided by the International Union Against Cancer (International Cancer Research Exchange Technology Transfer (ICRETT) Grant). The work was supported by funds from the Medical University of Warsaw, Poland (1WK/N/99). References [1] L. Swirsky Gold, T.H. Slone, B.N. Ames, N.B. Manley, Pesticide residues in food and cancer risk: a critical analysis, in: R. Krieger (Ed.), Handbook of Pesticide Toxicology, 2nd Edition, Academic Press, New York, 2001, pp. 799–843. [2] J.A. Quest, P.A. Fenner-Crisp, W. Burnam, M. Copley, K.L. Dearfield, K.L. Hamernik, D.S. Saunders, R.J. Whiting, R. Engler, Evaluation of the carcinogenic potential of pesticides. Part 4. Chloroalkylthiodicarboximide compounds with fungicidal activity, Regul. Toxicol. Pharmacol. 17 (1993) 19–34. [3] H. Tsuda, K. Matsumoto, H. Ogino, M. Ito, I. Hirono, M. Nagao, K. Sato, R. Cabral, H. Bartsch, Demonstration of initiation potential of carcinogens by induction of preneoplastic glutathione S-transferase P-form-positive liver cell foci: possible in vivo assay system for environmental carcinogens, Jpn. J. Cancer Res. 84 (1993) 230–236. [4] P.K. Mills, Correlation analysis of pesticide use data and cancer incidence rates in California counties, Arch. Environ. Health 53 (1998) 410–413. [5] I. Rahden-Staron, M. Szumilo, P. Ziemkiewicz, The effects of captan and captafol on different bacterial strains and on c-mitosis in V-79 Chinese hamster fibroblasts, Acta Biochim. Polon. 41 (1994) 45–55. [6] T.C. Hour, L. Chen, J.K. Lin, Comparative investigation on the mutagenicities of organophosphate, phtalimide, pyrethroid and carbamate insecticides by the Ames and lactam tests, Mutagenesis 13 (1998) 157–166. [7] C. Lu, R.M. Pfeil, C.P. Rice, Determination of mutational spectrum of the pesticide, captan, with an improved set of Escherichia coli LacZ mutants, Mutat. Res. 343 (1995) 219–227. [8] H. Tezuka, N. Ando, R. Suzuki, M. Terahata, M. Moriya, Y. Shirazu, Sister-chromatid exchanges and chromosomal
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wing somatic mutation and recombination test in Drosophila melanogaster, Experientia 51 (1995) 168–173. D.L. Lindsey, G.G. Zimm (Eds.), The Genome of Drosophila melanogaster, Academic Press, San Diego, CA, 1992, p. 1133. H. Frei, J. Clements, D. Howe, F.E. Würgler, The genotoxicity of the anti-cancer drug mitoxantrone in somatic and germ cells of Drosophila melanogaster, Mutat. Res. 279 (1992) 21–33. H. Frei, F.E. Würgler, Statistical methods to decide whether mutagenicity test data from Drosophila assays indicate a positive, negative, or inconclusive result, Mutat. Res. 203 (1988) 258–297. H. Frei, F.E. Würgler, Optimal experimental design and sample size for statistical evaluation of data from somatic mutation and recombination tests (SMART) in Drosophila, Mutat. Res. 334 (1995) 247–258. H. Frei, F.E. Würgler, Induction of somatic mutation and recombination by four inhibitors of eukaryotic topoisomerases assayed in the wing spot test of Drosophila melanogaster, Mutagenesis 11 (1996) 315–325. U. Graf, H. Frei, A. Kägi, A.J. Katz, F.E. Würgler, Thirty compounds tested in the Drosophila wing spot test, Mutat. Res. 222 (1989) 359–373. D.A. Burden, N. Osheroff, Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme, Biochim. Biophys. Acta 1400 (1998) 139–154. Y. Aratani, T. Andoh, H. Koyama, Effects of DNA topoisomerase inhibitors on non-homologous and homologous recombination in mammalian cells, Mutat. Res. 362 (1996) 181–191. A. Mukherjee, S. Sen, K. Agarwal, Ciproflaxin: mammalian DNA topoisomerase type II poison in vivo, Mutat. Res. 301 (1993) 87–92. D.J. Kirkland, L. Müller, Interpretation of the biological relevance of genotoxicity test results: the importance of thresholds, Mutat. Res. 464 (2000) 137–147. I. Rahden-Staron, H. Czeczot, M. Szumilo, Induction of rat liver cytochrome CYP 1A and CYP 2B by different fungicides, nitrofurans, and quercetin, Mutat. Res. 498 (2001) 57–66. R.I. Krieger, T. Thongsinthusakt, Captan metabolism in humans yields two biomarkers, tetrahydrophtalimide (THPI) and thiazolidine-2-thione-4-carboxylic acid (TTCA) in urine, Drug Chem. Toxicol. 16 (1993) 207– 225. M. Moriya, K. Kato, Y. Shirazu, Effect of cysteine and liver metabolism activation system on the activities of mutagenic pesticides, Mutat. Res. 57 (1978) 259–263. A. Önfelt, Spindle disturbances in mammalian cells. Part I. Changes in the quantity of the sulfhydryl groups in relation to survival and c-mitosis in V79 Chinese hamster cells after treatment with colcemid, diamine, carbaryl and methyl mercury, Chem. Biol. Interact. 46 (1983) 201– 217.
The inhibitory effect of the fungicides captan and captafol on eukaryotic topoisomerases in vitro and lack of recombinagenic activity in the wing spot test of Drosophila melanogaster Iwonna Rahden-Staron∗ Department of Biochemistry, Medical University of Warsaw, ul. Banacha 1, 02-097 Warszawa, Poland Received 27 November 2001; received in revised form 12 April 2002; accepted 22 April 2002
Abstract In studies on the mechanisms of mutagenic and carcinogenic action of captan and captafol-related chloroalkylthiocarboximide fungicides, two effects were tested: (i) the effect of both compounds on the activity of eukaryotic topoisomerases I and II in vitro, and (ii) their mutagenic and recombinagenic activity in the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster. Only captafol inhibited the activity of topoisomerase I (10–20% inhibition of activity in the range of 10–100 M). In contrast, both chemicals decreased the activity of topoisomerase II already at 1 M concentration (50 and 20% inhibition of activity by captafol and captan, respectively). Genotoxicity was tested in vivo by administrating both compounds by acute (3 h) and chronic feeding (48 h) of 3-day-old larvae. In acute feeding, captan and captafol demonstrated positive results only for small single and total spots in 10–100 mM exposure concentration range. Both chemicals were inconclusive for large single spots, as well as for twin spots. In chronic treatment, captan showed positive results only for small single and total spots at 2.5 and 5 mM concentrations. Captafol gave inconclusive results over all concentrations tested. The results of the acute treatment experiments which have been performed at very high doses (50% toxicity at higher doses) indicate very weak overall mutagenic activity of both test fungicides. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Captan; Captafol; Genotoxicity; SMART; Drosophila melanogaster; Topoisomerases I and II
1. Introduction There is a continuous debate concerning the possible role of many pesticides in chronic human health conditions [1]. Pesticides are thought to play a role in carcinogenesis, neurotoxicity, and reproductive and developmental processes. Among many pesticides there are groups of fungicides, e.g. captan Abbreviations: SMART, somatic mutation and recombination test; SCE, sister chromatid exchange; GSH, glutathione ∗ Tel.: +48-22-572-06-93; fax: +48-22-572-06-79. E-mail address: [email protected] (I. Rahden-Staron).
and captafol, two related chloroalkylthiocarboximide fungicides, used to control diseases of many fruits, and ornamental crops. They are also used as fungicides in paints, plastics, and leather. Thus, their use might cause long-term exposure of humans. Current regulatory policy to reduce cancer risks is based on the idea that chemicals which induce tumours in rodent cancer bioassays are potential human carcinogens. The US EPA categorised captan and captafol as group B2 (probable human) carcinogens, based upon findings of an increased incidence of malignant, or combined malignant and benign tumours in multiple experiments involving different strains of mice and rats [2,3]. In
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humans, a correlation has been observed between captan use and both leukaemia and prostate cancer [4]. Both compounds exhibited mutagenic activity in a variety of in vitro short-term tests for gene mutation, DNA repair, and chromosomal aberrations in prokaryotic and eukaryotic cells [5–7]. Both induced sister chromatid exchanges (SCE) and chromosomal aberrations [8]. In the studies performed under in vivo conditions, both captan and captafol were considered as non-genotoxic compounds [1], and captan as a non-genotoxic mouse-specific carcinogen [9]. The toxicity and carcinogenic activity of captafol has been better documented and is more pronounced than that of captan (for review see [5,10]). Since captan and captafol are present in the environment [11–13], there is a need for more data on mutagenicity in order to assess their potential hazards to human health. With very rare exceptions, pesticides do not react with DNA directly and the mechanisms of their carcinogenicity are, in general, similar to those of other non-genotoxic (epigenetic) carcinogens, e.g. promotion of spontaneous initiation, oxidative stress, cytotoxicity with sustained cell proliferation and others. One of the possibilities to get more valuable additional data is to monitor recombinagenic activity of chemical compounds, due to its possible connection with carcinogenicity [14,15]. It is important to understand whether or not indirect mechanisms, such as cytotoxicity and inhibition of DNA synthesis, induced by their exposure, are operating. SCE correlate with the chemicals’ cytotoxicity and may be induced by inhibition of the activities of topoisomerases [16–18]. Topoisomerases are nuclear enzymes capable of changing the topology of DNA and their activity is required during DNA replication, transcription, and homologous recombination [19–22]. Since inhibition of the activities of topoisomerases leads to an increase in the number of recombination events [23,24], it was worth checking whether the known ability of captafol and captan to induce SCE [8] is connected with their effect on topoisomerase activity [25,26]. To check the possible mechanism of carcinogenic action of captan and captafol, the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster was applied. The extensive knowledge of the genetics of D. melanogaster and
the long experimental experience with this organism have made it uniquely useful in mutation research and genetic toxicology. Cells of D. melanogaster possess xenobiotic metabolising systems similar to those in mammalian liver [27–29]. The development of SMART has provided a sensitive, rapid, and cheap assay to investigate the mutagenic and recombinagenic properties of chemicals [30–33].
2. Materials and methods 2.1. Chemicals Captan and captafol were purchased from OrganikaAzot (Jaworzno, Poland). The purity of the test compounds was 99.8%. Camptothecin and etoposide were purchased from Sigma. Solutions of captan and captafol were made independently, either with 5% ethanol and 5% Tween-80 (Serva, Heidelberg, FRG) in water (chronic feeding), or with 2 vol.% DMSO (Fluka, AG) (acute feeding). Etoposide was dissolved in 5% ethanol and 5% Tween-80 in water. Camptothecin was dissolved in 2 vol.% DMSO. The structural formulae of the chemicals tested, their Chemical Abstract Services (CAS) registry numbers, and molecular weight are shown in Table 1. 2.2. Topoisomerases activity The activity of topoisomerase I was assayed in a nuclear extract from mouse lymphoma L5178Y cells. Nuclei were isolated according to Pommier et al. [34] and extracted with 0.35 M NaCl according to Estley et al. [35]. The topoisomerase I assay measured the relaxation of supercoiled pBR322 by the extract according to Liu [36]. The topoisomerase II assay determined the conversion of topologically knotted phage P4 DNA to the unknotted topoisomer and was performed according to Liu et al. [37]. Captan and captafol were mixed with the extract in the assay cocktail and pre-incubated for 10 min at 30 ◦ C before addition of the substrate. The results of the assays were quantified by densitometric scanning of negatives of photographed electrophoresis gels. One unit of enzyme activity relaxed 50% of the substrate DNA after 30 min at 30 ◦ C. DNA electrophoresis was performed on 0.8%
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Table 1 Compounds tested in Drosophila wing spot test Common name
Trade name
Chemical name
Captan
Orthocide merpan pillarcap
Captafol
Difolatan folcid
Chemical structure
Molecular weight
CAS no.
N-[(trichloromethyl)-thio]-4cyclohexene-1,2-dicarboximide
301
133-06-2
N-[(1,1,2,2-tetrachloroethyl) thio]-4-cyclo-hexene1,2-dicarboximide
349
2425-06-1
Health effects of captan: mutagen, suspect carcinogen, teratogen, LD50 (oral, rat) 9 g/kg. Health effects of captafol: dermatitis, suspect teratogen, respiratory sensitisation (asthma), mutagen, suspect carcinogen, LD50 (oral, rat) 6200 mg/kg.
agarose; 2 mM EDTA; 40 mM Tris–acetate; pH 7.8 at 1 V/cm according to Sambrook et al. [38]. 2.3. Somatic mutation and recombination test (SMART) Compounds were tested at 25 ◦ C in the wing-spot assay carried out according to Graf et al. [33] and Graf [39]. For the mwh/flr3 cross, females from an y; mwh jv stock were mated to males from flr3 /TM3, Ser stock. Both strains were received from Prof. F.W. Würgler, Institute of Toxicology, Swiss Federal Institute of Technology and University of Zürich (Switzerland). For genetic symbols and description, see [40]. F1 larvae from cross mwh/flr3 were collected 72 ± 4 h after 8 h of egg-laying and placed in plastic vials containing Drosophila instant medium (formula 4-24, Carolina Biological Supply Co., Burlington, NC, USA) with the test compound. Solubility problems were encountered with captan and captafol, which had to be tested as a suspension, either in a mixture of 5% Tween and 5% ethanol in water in chronic feeding, or in 2% DMSO only in acute feeding. Despite the better solubility of both compounds in 2% DMSO compared to 5% Tween, 5% ethanol in water, they were tested in 2% DMSO only in acute feeding (3 h); 2% DMSO has high toxicity in longer treatment.
An amount of 5 ml of a suspension of the compound was added to 1.5 g dry instant medium. The larvae were fed on this medium for the rest of their development (approximately 48 h) (chronic feeding). In some experiments, acute feeding [33] was used. In this procedure, captan or captafol in 2% DMSO was mixed with cellulose powder (1 ml/200 mg powder) and the larvae were placed on top of a gauze-covered bed of this wet powder in a bottle. Feeding through the gauze was allowed to occur for 3 h and then the larvae were transferred into another bottle and cultured on a reagent-free medium to obtain adult flies (acute feeding). The concentration ranged from 10 to 100 mM for acute feeding, and from 0.25 to 10 mM for chronic feeding of both captan and captafol. Negative solvent controls 2% DMSO or 5% Tween-80/5% ethanol/water controls were always conducted in parallel. An amount of 0.01 mM camptothecin, (inhibitor of topoisomerase I) and 1 mM etoposide (VP16) (inhibitor of topoisomerase II) were used as positive controls. After metamorphosis, the hatched adults were collected from the treatment vials and stored in 70% ethanol. Subsequently, wings were analysed according to standard procedures [33]. Three different types of spots (single spots of the mwh or the flr phenotype, twin spots with adjacent mwh and flr areas) were recorded separately in the marker transheterozygous wings. As cell genetics show, wing spot test
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assays detect several genetic endpoints. Twin spots are exclusively produced by mitotic recombination, while mwh single spots may in addition also be due to other mechanisms, such as gene mutation and deletion. The flr single spots on the other hand, arise from mutation or deletion and a few are due to mitotic double cross-over. 2.4. Statistical evaluation For a comparison of the induction frequencies only mwh clones in mwh single spots and in twin spots were taken into consideration [41]. For statistical testing, the decision procedure of Frei and Würgler [42] was applied. As customary for the evaluation of wing spot data [33], the following classes among the total spots are distinguished: small single spots (1–2 cells in size), large single spots (3 cells or larger), and twin spots. This classification is biologically meaningful [42]. For the calculation, the Kastenbaum–Bowman test was used with P = 0.05. Based on the number of wings analysed, the number of mwh clones and the number of cells scored in each wing (24,400), it is possible to calculate the clone formation frequency per cell cycle in 105 cells (see also: [42,43]).
3. Results 3.1. The effect of fungicides on topoisomerases I and II activity The effect of captan and captafol on eukaryotic topoisomerases was studied in vitro using nuclear extracts from mouse lymphoma cells. Reactions specific to topoisomerases I and II were relaxation of pBR322 in the absence of ATP and unknotting of phage P4 DNA, respectively. Captan had no effect on the relaxing activity of topoisomerase I up to a concentration level of 100 M. Captafol, however, inhibited 10–20% of the topoisomerase I activity in the range of 10–100 M. In contrast, both captan and captafol clearly inhibited the unknotting activity of topoisomerase II; 50% of inhibition was observed at 1 M captafol and 5 M captan (Figs. 1a and b, 2a and b). Camptothecin and etoposide used as positive controls in the SMART assay inhibited 50% of topoi-
Fig. 1. (a) Effect of captan (lanes 1–4) and captafol (lanes 6–9) on topoisomerase I from mouse lymphoma cells. The concentrations of compounds was: lane 1, 6–0 M; lane 2, 7–1 M; lane 3, 8–10 M; lane 4, 9–100 M; lane 5, 10–control pBR322 (SC). (b) Inhibitory effect of captan and captafol on topoisomerase I activity: % of inhibition of topoisomerase I activity by captan (䊉) and captafol (䉱).
somerase I and topoisomerase II activities at 5 and 10 M, respectively (data not shown). 3.2. Genotoxicity testing of the fungicides with the wing spot test The data of the wing analysis collected with the two compounds are shown in Table 2. Controls were pooled since there were no significant differences between them. Pooled controls used as carriers in the experiments gave spontaneous spot frequencies very similar to those found in other laboratories [44]. As concurrent controls showed only a few spots, data from the pooled large controls were used for control corrections in the determination of clone induction frequencies. For significance testing, however, the spot scores in treated groups were
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Fig. 2. (a) Effect of captan (lanes 1–4) and captafol (lanes 6–9) on topoisomerase II from mouse lymphoma cells. The concentrations of compounds was: lane 1, 6–0 M; lane 2, 7–1 M; lane 3, 8–10 M; lane 4, 9–100 M; lane 5, 10–control P4 DNA. (b) Inhibitory effect of captan and captafol on topoisomerase II activity: % of inhibition of topoisomerase II activity by captan (䊊) and captafol (䉭).
always compared with the corresponding concurrent controls. According to Graf [39] and Graf et al. [45], the optimal strategy in genotoxicity screening is to start with chronic exposure of 3-day-old larvae for 48 h, that is until pupation. In preliminary experiments of the present work, with younger (2-day-old) larvae, neither captafol, nor captan have induced twin spots and increased frequency of large single spots. The frequency of small single spots was considerably lower as compared with the frequency in 3-day-old larvae (data not shown). That data was in agreement with Graf [39] who showed that practically no twin spots are found in very young and in very old larvae. Based on these results 3-day-old larvae were used for all further experiments. Solubility problems were encountered with both compounds which had to be tested as a suspension under sub-optimal conditions (see Section 2). According
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to Frei and Würgler [42], a positive diagnosis of the test requires that the results of at least two of the three categories should be positive, i.e. when frequencies of total and small single spots per wing are two times, and for large and twin spots five times higher than the spontaneous mutation rate. In acute feeding experiments with captan and captafol in the exposure concentration range from 10 to 100 mM, the cross mwh/flr3 reveals clear positive results for both fungicides, but only for small single and total spots. However, results were inconclusive for large single spots, as well as for twin spots. Effects of both fungicides tested were different in chronic treatment compared with in acute feeding. Positive results were observed for small single and total spots only for captan at two concentrations: 2.5 and 5 mM. No dose-related induction for these spot categories was observed for both acute and chronic feeding (Table 2). Results for captafol were inconclusive or negative in chronic treatment over the whole concentration range. The observed toxicity in both treatments was higher for captafol than for captan. About 50% of the flies did not reach the adult stage in acute treatment at 25 mM captan and 10 mM captafol, and in chronic feeding at 5 mM for captan and the 1–1.5 mM exposure level for captafol. The analysis of the SMART assay data from acute and chronic treatments shows the lack of twin spots, which are produced by mitotic recombination exclusively [33]. To compare the in vivo test effects of both fungicides which inhibit in vitro topoisomerase II, 0.01 mM camptothecin and 1 mM etoposide, (DNA topoisomerases I and II inhibitors, respectively), were used as positive controls [34,46]. These recombinagenic and mutagenic compounds result in stimulation of recombination processes [47], and an increase in SCE frequency [48]. Mutations introduced by these agents result in gene deletion and rearrangements [16]. The flies survival at 0.01 mM camptothecin and 1 mM etoposide was similar to the controls. Solubility problems were encountered with etoposide, which had to be tested as a suspension. Data on induction potency of the control inhibitors observed in the present work for all types of spots (Table 2), as well as lack of toxicity at their tested doses were in agreement with the previous results of Frei and Würgler [44] who showed that induced clones are of 88 and 59% of recombinational origin for camptothecin and etoposide, respectively.
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4. Discussion The wing test system detects a great variety of genotoxins belonging to different chemical classes, including compounds directly or indirectly interacting with DNA. The results presented in this work demonstrate the weak overall mutagenic activity of captan and captafol under in vivo conditions in somatic cells of D. melanogaster and the ability of both compounds to inhibit the activity of topoisomerase II in vitro. However, captafol was a much more potent inhibitor of topoisomerase II relative to captan. The activity of topoisomerase I was inhibited only by captafol, but to a much lower extent as compared to its inhibitory effect on topoisomerase II. The question, whether or not inhibition of topoisomerase(s) is a major event in captan and captafol-induced mutagenicity and carcinogenicity, is considered on the metabolism of both compounds in vivo and on the importance of the thresholds basis [49]. Since captafol and captan show in vitro an inhibitory effect, especially on one of two topoisomerases, the wing spot test of D. melanogaster, which detect genotoxic activity of other topoisomerase poisons [44], seemed to be a good predictor of genotoxic activity of test fungicides. Camptothecin and etoposide were used as positive controls in the SMART assay and gave clear positive results for their recombinagenic activity, in agreement with Frei and Würgler [44]. The analysis of the SMART data for captan and captafol from both treatments showed the lack of twin spots and induction potency for single spots only in acute treatment, but with no dose–effect relationship. Single spots are due to recombination as twin spots are; they can be also due to point mutations, deficiencies and non-disjunction events [33]. Because of that the observed lack of twin spots in the used inversion-free mwh/flr3 marker heterozygotes may not be a sufficient proof that excludes recombinagenic properties of the two test fungicides. Captafol and captan inhibit 50% of topoisomerase II at 1 and 5 M, and etoposide at 10 M, respectively. The very weak inhibitory effect on topoisomerase I was demonstrated only by captafol. Simultaneous comparison of the inhibitory effects on topoisomerase II of etoposide and both test fungicides compared with end-effects in the SMART assay, can lead to the prediction that under test conditions both fungicides
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will not demonstrate recombinagenic activity either due to a lack of such activity, insufficient uptake by tissues, or effective metabolism. The fact that the test fungicides did not stay dissolved during the assays could cause possible fluctuations in the uptake of chemicals by larvae. It may be the reason of an insufficient level in tissues to reach the threshold’s value to reveal fungicides’ biological activity. On the other hand, both captafol and captan may be metabolised by Drosophila cytochrome P-450 mono-oxygenase system [50] to reactive products so efficiently that they do not reach the threshold’s value. Both compounds are not sufficient to retain their mutagenic activity until the late third instar stage when mwh mutants are produced with a high frequency. Under such conditions only small single spots would be observed, because they are induced very late in larvae development. However, the results of the preliminary experiments, with younger 2-day-old larvae, also showed induction of only small single spots category. This supports the correctness of interpretation and points out that the absence of twin spots and large single spots is not due to the relatively late treatment of larvae. Positive responses were found in the acute treatment, but there was no dose–effect relationship. The acute feeding experiments were performed at very high doses, whereby 50% and higher toxicity was observed. In vitro studies showed that mechanism-based toxicity of both test fungicides was related to their main metabolite tetrahydrophtalimide (THPI) which is later degraded to other toxic compounds [51]. This toxicity could also be related to a decrease in non-protein sulfhydryl groups, mainly of GSH [5,52] which, in turn, might influence the integrity and functions of the mitotic spindle [53]. The lack of a dose–effect relationship and an overall very weak mutagenicity may be a proof of a non-genotoxic mechanism of the two test fungicides (and/or their metabolites). They can interact with proteins inducing various types of lesions in the chromosomes of the imaginal disk cells which can give rise of mutations. The results discussed above suggest rather a multifactorial origin of captan- and captafol-induced chromosomal aberration and SCE [8]. The specific effect of inhibition of topoisomerase II does not seem to be a major event in captan and captafol mutagenicity and carcinogenicity.
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