Micronucleus induction in erythrocytes of the Hyla pulchella tadpoles (Amphibia: Hylidae) exposed to insecticide endosulfan

Mutation Research 587 (2005) 67–72

Micronucleus induction in erythrocytes of the Hyla pulchella tadpoles (Amphibia: Hylidae) exposed to insecticide endosulfan Rafael C. Lajmanovich a,∗ , Mariana Cabagna b , Paola M. Peltzer a , Gabriela A. Stringhini c , Andr´es M. Attademo c a

c

National Council for Scientific and Technical Research (CONICET), Faculty of Biochemistry and Biological Sciences, FBCB-UNL, Pje. El Pozo s/n (3000), Santa Fe, Argentina b Cathedra of Normal Morphology, Faculty of Biochemistry and Biological Sciences, FBCB-UNL, Pje. El Pozo s/n (3000), Santa Fe, Argentina High School of Health, Faculty of Biochemistry and Biological Sciences, EES-FBCB-UNL (3001), Santa Fe, Argentina Received 10 June 2005; received in revised form 28 July 2005; accepted 3 August 2005 Available online 16 September 2005

Abstract Endosulfan is a synthetic chlorinated and environmental genotoxic pesticide used worldwide for crop production. We used the micronucleus test in erythrocytes of Hyla pulchella tadpoles in order to develop an experimental model for detecting genotoxic effects of the synthetic chlorinated cyclodiene endosulfan. The frequency of micronuclei was examined in blood smears obtained from tadpoles exposed in vivo to three different concentrations 2.5, 5, and 10 ␮g/l of the compound and fixed at two sampling times 48 and 96 h. As a positive control larvae were exposed to 40 mg/l of cyclophosphamide. Results obtained here demonstrated the genotoxic effects of the commercial formulation endosulfan in the experimental model assayed. © 2005 Elsevier B.V. All rights reserved. Keywords: Amphibians; Endosulfan; Micronucleus test; Tadpoles erythrocyte

1. Introduction Endosulfan is a synthetic chlorinated and environmental genotoxic pesticide used worldwide for crop production [1]. Commercial endosulfan is composed ∗ Corresponding author. Tel.: +54 342 4740152; fax: +54 342 4750394. E-mail address: [email protected] (R.C. Lajmanovich).

1383-5718/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2005.08.001

of two isomers ␣-endosulfan and ␤-endosulfan. It was introduced into the earth’s environment in 1956 as a general use organochlorine insecticide, and it was applied to protect crops such as grains, tea, fruits, and vegetables from a variety of insects [2]. In Argentina, endosulfan is legally and widely used [3], and its residuals have been detected in wild fauna [4] and river waters where it produced death of fishes [5]. Large amounts of this insecticide can be found in surface water near areas of application [6]. The breakdown of endosulfan

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in water is faster (5 weeks) under neutral conditions (pH 7) than at acidic conditions or basic conditions (5 months) [7]. Organochlorines characteristically exhibit low water solubility, high lipid solubility, environmental persistence and have a target toxic action on the nervous system of organisms [8]. Endosulfan is highly toxic to fish and aquatic invertebrates [9]. In amphibians it also exhibited a wide range of toxicities [10]. Although some studies reported a mutagenic effect (reverse mutations, cross overs, and mitotic gene conversions) for technical-grade endosulfan in Saccharomyces cerevisiae [11], other studies not found this effect using Escherichia coli and Salmonella typhimurium [12]. However, the data regarding its genotoxicity, especially that of its two isomers, are insufficient [13]. Several tests have been developed with aquatic animals, which can be used to assess the genotoxicity of chemicals, using such endpoints as chromosomal aberrations, sister-chromatid exchanges and micronuclei (MN) induction [14]. The MN assay has been used as a measure of genotoxicity in amphibians under laboratory and field conditions [15–17] and has shown potential for in situ monitoring of water quality [18,19]. MN are small fragments of intracytoplasmic chromatin which arise from chromosomes breaks or whole chromosomes, after the action of clastogenic substances or spindle-poisons [17]. Therefore, MN frequencies have been considered reliable indexes of both chromosome breakage and chromosome loss [20,21]. The present study evaluates the induction of commercial formulation endosulfan to cause genetic damage by the MN test in erythrocytes of Hyla pulchella tadpoles. This work forms part of a project to investigate adverse impact from the massive exposure to pesticides on native populations of anurans in Argentina [22,23].

tion of 40 ppm (mg/l). All test solutions were freshly prepared before each experiment. 2.2. Tadpoles H. pulchella tadpoles were selected to carry out the present study. This species has an extensive distribution on neotropical region [24], and it is frequently found both on natural and altered sites (agricultural land and urban territories) [25]. In addition, it is relatively easy to handle and acclimate to laboratory conditions. All the tadpoles used in the experiment were collected from temporary ponds of the Paran´a River floodplain (31◦ 43 S; 60◦ 34 W Argentina). Prometamorphic larvae (from stages 26 up to 36) [26] were used for the bioassay. The average total size (snout-tail) was 18 ± 2 mm and average weight was 0.05 ± 0.02 g. Before the experiment, tadpoles were acclimatized in glass tanks (12.5 cm diameter and 13.5 cm high) with 1 l artificial pond water (APW) [27], at 22 ± 2 ◦ C for 7 days, and 12 h–12 h light–dark. 2.3. Experimental design The 96-h sub-lethal tests were conducted according to USEPA Standard Methods [28], with 10 larvae per treatment group. In the sub-lethal test, endosulfan forms an emulsion with APW and as such was applied in three different concentrations: 2.5, 5, and 10 ␮g/l. Negative controls were conducted in APW during the same period. CP was used as a positive control, at a concentration of 40 ppm (mg/l). All test solutions were prepared in triplicate immediately before each experiment. The water, containing the compound and the food was changed, every 2 days. The MN frequency in each group was measured after 48 and 96 h. 2.4. The micronucleus test in tadpoles

2. Materials and methods 2.1. Chemicals Endosulfan (CAS No. 115-29-7) commercial grade, tradename Piastra, was obtained from Agarcros, Argentina. The purity of the endosulfan as indicated by the manufacturer is 35% (w/w) and ‘inert’ ingredients are 65%. Cyclophosphamide (CP) (CAS no. 50-18-0, Filaxis) was used as a positive control, at a concentra-

It is important to note that red blood cells (RBCs) in amphibians are nucleated and undergo cell division in the circulation, particularly during the developmental stages [29]. In tadpoles, blood was obtained directly from the heart of anesthetized animals (30% ethyl alcohol). Two peripheral blood smears for each tadpole were prepared on clean slides, fixed and stained by the May–Grunwald–Giemsa method [30]. In this context, Majone et al. [31] found that no significant difference

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between data from preparations stained by Giemsa and acridine orange. The MN frequency was determined in 1000 erythrocytes from each tadpole using 1000× magnification [15,32]. Coded and randomized slides were scored blind by a single observer. The criteria for MN determinations are that the intensity of stained MN is similar to the principal nucleus, its diameter is inferior to the principal nucleus, it is round with a nuclear membrane, it is not connected to the principal nucleus, there is no overlap with the principal nucleus, and it is within the cytoplasm [33,34]. 2.5. Statistical analysis Data from controls and experimental groups were analyzed using the non-parametric Kruskal–Wallis test [35]. A value of p < 0.05 was considered to indicate significance.

3. Results The mature erythrocytes of H. pulchella tadpoles are oblong-oval shape with a centric nucleus. The nucleus is clearly structured and has a well-defined boundary, which facilitates the identification of fragments in their cytoplasm. MN observed are spherical nuclear fragments separated from the parent nucleus (Fig. 1). Single MN is predominant in the erythrocytes cells analyzed. However, some few erythrocytes presented

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Fig. 2. Induction of micronucleated erythrocyte (per 1000 cells) in H. pulchella larvae treated with different concentrations of test compounds. * p < 0.05, compared to the controls (Kruskal–Wallis test). E: endosulfan.

nuclear morphological aberrations, binucleated cells and cells, showing multiple MN. The data obtained with the MN frequency measured in erythrocytes of H. pulchella tadpoles not showed a dose-dependence for the different endosulfan concentrations. These data are shown in Fig. 2. Tadpoles exposed to CP showed a significant increase in micronucleated erythrocytes at 48 and 96 h exposure (p < 0.05). The relative MN increases after endosulfan treatment (5 and 10 ␮g/l) is quite small, although statistically significant compared to the negative control at 48 and 96 h (p < 0.05). The MN frequency of tadpoles exposed to 2.5 ␮g/l of insecticide was similar to the negative control.

4. Discussion

Fig. 1. Micronucleated erythrocyte (arrow) in H. pulchella exposed to the organochlorine insecticide endosulfan (10 ␮g/l, 96 h). Giemsastained blood smear, 1000×.

Studies with pesticides show differences between active ingredients and their formulations with respect of mutagenicity [36]. The first step taken in this assessment was an evaluation of clastogenic properties of commercial formulations, which really are going to the field. However, several independent studies have shown that endosulfan is genotoxic, but few reports used aquatic animals for evaluation. Data from in vitro and in vivo mutagenicity studies generally provide evidence that endosulfan is mutagenic, clastogenic, induces effects on cell cycle kinetics and DNA strand breaks [1,11,37]. Endosulfan was also found to cause chromosomal aberrations in hamster and mouse, sexlinked recessive mutations in Drosophila, and dominant lethal mutations in mice [37–39]. It is also known to cause mutations in mammals [40]. It may induce

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mutations in human, if exposure is great. It is also a potential tumor promoter [41]. To our knowledge the results presented here appear to be the first to demonstrate the genotoxic effect of endosulfan on erythrocytes of amphibian tadpoles. It is important to note that the occurrence of nuclear morphological aberrations in H. puchella, as well as the general degenerative changes in the erythrocytes during the tadpole stages studied, corresponded to a period of intense hematopoiesis with active cell division in the circulating blood [16]. Cyclophosphamide, a well-known genotoxic substance, was used as a positive control for the MN test in amphibian tadpoles [42]. It is an indirect alkylating agent, and in our experiments it was used as a positive control. This agent caused a significant increase in the frequency of micronucleated erythrocytes at almost all times observed. This drug, and vinblastine sulfate, an aneugenic agent, are mutagenic drugs usually used as positive controls in vivo MN tests of short duration [43]. However, the CP is most mutagenic of the two drugs and is recommended for use as a positive control [44]. The length of the cell cycle critical to MN formation depends upon the time needed to replicate DNA and perform nuclear division [21]. In man and mice the duration of the cell cycle has been well documented. There is, however, little information on the duration of the cell cycle in the tissues of amphibians species since the cell cycle varies with temperature in poikilotherms [45]. Moreover, understandably the importance of sampling time for micronucleus induction has been highly emphasized [46,47]. Results obtained here showed a genotoxic effect of the endosulfan on erythrocytes of H. puchella. It important noticed that commercial formulations of endosulfan utilized, contains many ‘inert’ ingredients that can increase the toxicity of the product when compared to the technical-grade material. Furthermore, ‘inert’ ingredients used in formulated pesticide products are usually not disclosed and not included in most of the testing required in order to register these pesticides. Moreover, although ‘inert’ ingredients have no pesticide activity, they may be biologically active and sometimes the most toxic component of a pesticide formulation [48]. Endosulfan, in the laboratory in the amphibian tadpoles test showed 96-h LC 50 within the range of 11.75–123.0 ␮g/l [49,50]. Thus, we demonstrated genotoxic effects on erythrocytes at lower concentra-

tions that those calculated to cause mortality. In addition, Vanderkerken et al. [51] classified MN into two major types of morphology, classifying small and large MN relative to cell size. They postulated that aneugens induced MN of the large type while clastogens induced small MN. Although our study is preliminary, we can suggest a clastogenic effect of endosulfan on erythrocytes of H. pulchella. Finally, the results indicate that the frequency of MN performed in circulating blood samples of H. pulchella tadpoles is a rapid method for detecting cytogenetic damage, and could be used to detect their response to environmental toxicants.

Acknowledgements The authors thank two anonymous reviewers for their valued comments and suggestions.

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