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Induction of micronuclei in tadpoles of Odontophrynus americanus (Amphibia:Leptodactylidae) by the pyrethroidinsecticide cypermethrinMariana C. Cabagna a , Rafael C. Lajmanovich b , Paola M. Peltzerb , Andrés M. Attademo b & Ezequiel Ale ca Faculty of Biochemistry and Biological Sciences – FBCB-UNL,Cathedra of Normal Morphology, Pje. El Pozo s/n (3000), Santa Fe,Argentinab Faculty of Biochemistry and Biological Sciences – ESS-FBCB-UNL,National Council for Scientific and Technical Research (CONICET),Pje. El Pozo s/n (3000), Santa Fe, Argentinac Laboratory of General Citogenetic, (UNaM), Félix de Azara 1552(3300), Posadas (Misiones), ArgentinaVersion of record first published: 01 Feb 2007.

To cite this article: Mariana C. Cabagna , Rafael C. Lajmanovich , Paola M. Peltzer , AndrésM. Attademo & Ezequiel Ale (2006): Induction of micronuclei in tadpoles of Odontophrynusamericanus (Amphibia: Leptodactylidae) by the pyrethroid insecticide cypermethrin, Toxicological& Environmental Chemistry, 88:4, 729-737

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Toxicological & Environmental Chemistry, Oct.–Dec. 2006; 88(4): 729–737

Induction of micronuclei in tadpoles ofOdontophrynus

americanus (Amphibia: Leptodactylidae) by thepyrethroid insecticide cypermethrin

MARIANA C. CABAGNA1, RAFAEL C. LAJMANOVICH2,PAOLA M. PELTZER2, ANDRES M. ATTADEMO2, & EZEQUIEL ALE3

1Faculty of Biochemistry and Biological Sciences – FBCB-UNL, Cathedra of Normal

Morphology, Pje. El Pozo s/n (3000), Santa Fe, Argentina, 2Faculty of Biochemistry and

Biological Sciences – ESS-FBCB-UNL, National Council for Scientific and Technical Research

(CONICET), Pje. El Pozo s/n (3000), Santa Fe, Argentina, and 3Laboratory of General

Citogenetic, (UNaM), Felix de Azara 1552 (3300), Posadas (Misiones), Argentina

AbstractCypermethrin (CY) is an active cyano pyrethroid effective against a wide range of pests encounteredin agriculture and forestry. Although CY is not mutagenic in in vitro assays for gene mutation, in vivoassays showed conflicting results. In vivo genotoxicity of the synthetic pyrethroid CY in erythrocytes ofOdontophrynus americanus tadpoles was examined. The frequency of micronuclei (MN) was recordedin blood smears obtained from tadpoles exposed in vivo to four different nominal concentrations5, 10, 20 or 40 mgL�1 of the compound and fixed at two sampling times 48 and 96h. As a positivecontrol larvae were exposed to 40mgL�1 of cyclophosphamide (CP). Tadpoles exposed to all CYtreatments showed a significant increase in single small MN compared to the negative control groupafter 48 h and at 5 and 10 mgL�1 of CY at 96 h. Results obtained here demonstrated the genotoxiceffects of the commercial formulation CY in the anuran larvae analyzed. Thus, data suggest thatmeasurements of MN and other erythrocytes morphological aberrations performed in circulatingblood samples of O. americanus tadpoles is a method for detecting cytogenetic damage in other nativespecies.

Keywords: Odontophrynus americanus, cypermethrin, tadpoles

Correspondence: Rafael C. Lajmanovich, Faculty of Biochemistry and Biological Sciences – FBCB-UNL, Laboratory ofEcotoxicology, Pje. El Pozo s/n (3000), Santa Fe, Argentina. Tel.: þ54 342 4740152. E-mail: rafalajmanovich@yahoo.com.ar

ISSN 0277-2248 print/ISSN 1029-0486 online � 2006 Taylor & FrancisDOI: 10.1080/02772240600903805

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Introduction

Pesticide contamination is considered an important factor in the decline of amphibianpopulations in agricultural regions [1]. Pyrethroid insecticides are synthetic moleculesstructurally related to natural pyrethrins, sharing many characteristics of their actions withDDT [2]. Pyrethroids have become increasingly popular for insect control in land andaquatic agricultural systems. Although pyrethroids are considered relatively non-toxic tobirds and mammals, they are extremely toxic to aquatic organisms, including invertebrates,fish, and amphibians [3,4].

Cypermethrin (CY) [(RS)-alpha-cyano-3-phenoxybenzyl (1RS)-cis-,trans-3-(2,2,-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate] is a highly active synthetic pyrethroidinsecticide. Because of its effectiveness against a wide range of arthropods, CY is routinelyused not only in agriculture, but also in the control of human head lice and animal externalparasite infestations [5]. CY is one of the most light-stable pyrethroids [6] and itsdegradation rates was similar in both laboratory and field surveys [7]. The average rate ofCY application to crops is 10–200 g of active ingredient (CYha�1) [8]. Aside from directdeposition or drift, pyrethroid insecticides reach aquatic habitats via runoff, which dependon soil conditions, rainfall, and slope of the catchments area [9]. Four hours afteroverspraying ponds with CY (100 g ha�1), a concentration of 100 mgL�1 was measured inthe surface water (depth of 2.5–10 cm; [10]). The range of half-lives in the model ecosystemof CY was 4.7–30.8 days [7]. It was also noted that the degradation rates of the pyrethroidsfollowed first-order kinetics, and that only fenvalerate and CY residues remained atdetectable levels 56 days post-application [11].

Although CY is not mutagenic in in vitro assays for gene mutation [12], in contrast in vivo

assays showed conflicting results. Positive results were obtained in a mouse bone marrowmicronuclei test (MNT) following oral and dermal administration of CY and increasedfrequency of sister chromatid exchanges in vivo [13,14] and in Drosophila melanogaster usingthe alkaline Comet assay [15]. In addition, [16] reported a dose-dependent micronuclei(MN) induction by pyrethroid lambda-cyhalothrin in Rana catesbeiana. Currentgenotoxicity tests in amphibians are based on the observation of MN in vivo [17,18].The objective of this study was to evaluate the effects of commercial formulation CY inaquatic organisms using the MN test in erythrocytes of Odontophrynus americanus tadpoles.This study integrated a project to investigate lack of effects from exposure to pesticides onwild populations of amphibians in Parana River floodplain, Argentina [19–21].

Materials and methods

Chemical

Cypermethrin (CAS No. 52315-07-8) commercial grade, trade name ‘Cipermetrina’,was obtained from Argengric, Argentina. The commercial product consisted of 2.5% w/wCY formulated in aqueous xylene. As a positive control, tadpoles were exposed tocyclophosphamide (CP) (CAS No. 50-18-0, Filaxis) at a concentration of 40 ppm(mgL�1). All test solutions were freshly prepared before each experiment.

Anuran tadpoles

Tadpoles of O. americanus were selected as the test organisms. This anuran has an extensiveNeotropical distribution [22], and inhabits natural areas, agricultural land and urbanterritories [23]. Indeed, this species is easy to handle and acclimates to laboratory conditions.

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The tadpoles used in the bioassay were collected from a temporary pond of the ParanaRiver floodplain (31�430S; 60�340 W-Argentina). Prometamorphic larvae (from stages 26 upto 36) [24] were used for the bioassay. The average total size (snout-tail) was 15�0.5mmand weight was 0.07� 0.02 g. The tadpoles were acclimatized to a 12 : 12 h light : dark cyclesin glass tanks (12.5 cm diameter and 13.5 cm high) with artificial pond water (APW) [25]of pH 6.8, conductivity 175 mmhos cm�1, dissolved oxygen concentration 4.5� 1mgL�1,hardness 50.5mgL�1 of CO3Ca at 24� 2�C for 7 days.

Exposure design

The 96h sub-lethal tests were conducted according to USEPA Standard Methods [26],with 10 prometamorphic larvae (from stages 26 up to 36) [24] per treatment group. In thesub-lethal test, the nominal concentrations used were: 5, 10, 20 or 40mg of CYL�1.Negative controls were conducted in APW during the same period. CP was used as apositive control, at a concentration of 40 ppm (mgL�1). All test solutions were prepared intriplicate immediately before each experiment. The water, containing the compound andthe food (boiled lettuce) changed for every 24 h. The MN frequency in each group wasmeasured after 48 and 96h.

Micronuclei test

Red blood cells (RBCs) in amphibians are nucleated and undergo cell division in thecirculation, particularly during the developmental stages [27]. The blood was obtaineddirectly from the heart of anesthetized tadpoles (30% ethyl alcohol). Peripheral blood smears,two for each tadpole, were prepared on clean slides, fixed and stained by theMay–Grunwald–Giemsa method [28]. It is important to note that, [29] found that no significant differencebetween data from preparations stained by Giemsa and acridine orange. The MN frequencywas determined in 1000 erythrocytes from each tadpole using 1000�magnification [16,30].Coded and randomized slides were scored blind by a single observer. The criteria for MNdeterminations are (1) the intensity of stained MN and the diameter of the MN shouldbe less than one-third of themain nuclei, (2) the intensity of stainedMN is similar to themainnuclei, (3) is not connected to the main nuclei, (4) is round with a nuclear membrane,(5) there is no overlap with the main nuclei, and (6) is within the cytoplasm [31,32].In addition, other alterations of the erythrocytes were recorded.

Data analysis

The non-parametric Kruskal–Wallis test [33] was used to analyze data from controls andexperimental groups. The criterion for significance was p5 0.05.

Results

The MN frequencies and time-responses at different concentrations of test compounds areshown in Figure 1. Tadpoles exposed to all CY treatments showed a significant increase insingle small MN compared to the negative control group after 48 h and at 5 and 10mgL�1

of CY at 96 h. Tadpoles exposed to CP showed a significant increase in micronucleatederythrocytes at 48 and 96h exposure.

Control tadpole’s mature erythrocytes are oval cells with centrally placed and similarlyshaped nuclei (Figure 2a). The nuclei are clearly structured and possess a well-definedboundary, which facilitates the identification of fragments in cytoplasm. For all treatments

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Figure 1. Induction of micronuclei in red blood cells (per 1000 cells) of O. americanus larvaetreated with different concentrations of test compounds (Cypermethrin) (*p5 0.05 in relation to thenegative control).

Figure 2. May–Grunwald–Giemsa stained blood smear of O. americanus tadpoles (1000�).(a) Control, normal erythrocytes and heterophils (HT). (b) Erythrocytes exposed to the sub-lethaldoses of CY (5mg CYL�1 – 48 h), arrow indicating one small MN. (c) Binucleated cells (BN) in shortexposed (5mg CYL�1 – 48 h) erythrocytes. (d) Long exposed (10 mg CYL�1 – 96 h) erythrocytesshowing multiple MN.

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single MN were predominant in the erythrocytes analyzed (Figure 2b). Indeed, binucleatedcells and erythrocytes showing multiple MN in the way of apoptotic-like cells [34] werefound in tadpoles exposed to all CY concentrations in low frequencies (520%) at differenttimes (Figure 2c,d), except at 20 and 40mgL�1 of CY at 96 h treatment because thetadpoles did not survive.

Discussion

Pesticides have become indispensable in modern agriculture and are also harmfulpollutants, especially in aquatic environments [9,35,36]. Results obtained here showedgenotoxic effects of the CY on erythrocytes of O. americanus tadpoles.

Most toxicological studies on pyrethroids have been performed in the laboratory withrodents and limited data are available for other vertebrates [37]. Pyrethroids are morehydrophobic than other classes of insecticides and this feature indicates that the site ofaction is biological membranes [38]. It is possible that pyrethroids are transportedthrough blood to the liver for metabolism and may produce cellular damage toerythrocytes [39].

Figure 2. Continued.

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In fact, it is well known that CY is toxic at lower concentrations, particularly for tadpolesof different amphibian species [34]. In the present study CY, at 96 h exposure producedlarvae mortality. This elevated mortality at longer periods and in higher concentrationssimulated biological animal responses to CY and confirmed mutagenic effects induced byMN test [32]. Although the occurrence of MN was relatively lower (1–3MN/1000 cells),this result is to find in tadpoles exposed to pyrethroids, copper, acetylaminofluorene, andorganochlorines [16,32,40,41].

CP produced a significant increase in the frequency of micronucleated erythrocytes atalmost all times observed. This substance, a bifunctional alkylating agent, is extensivelyused for genetic effects in a wide variety of animal species [42]. This drug produced amarked increase in the number of MN and is recommended for use as a positivecontrol [43,44] in amphibian tadpoles genotoxicological test at concentrations of5–40mgL�1 [16,41].

Different studies on the genotoxicity of technical grade CY are available in theliterature [13,45] but few reports used aquatic animals for evaluation. To ourknowledge the results presented here appear to be the first to demonstrate thegenotoxic effect of CY on erythrocytes of anurans tadpoles. On the other hand,Vanderkerken et al. [46] classified MN into two major types of morphology, classifyingsmall and large MN relative to cell size. They postulated that aneugens induced MNof the large type while clastogens induced small MN. Further, Ramadan et al. [47]postulated that, generally, the clastogens induced many more MN than aneugens,mostly of the small type, and more frequently induced multiple MN. Data suggest aclastogenic effect of CY [48] on erythrocytes of O. americanus.

Moreover, the finding of apoptotic-like cells at 96 h treatment was similar to the resultsof [49]. The authors demonstrated the CY induction (12.9–100 mgL�1) of apoptotic cellin prometamorphic larvae developing brain of another sympatric leptodactylid,Physalaemus biligonigerus. During the process of apoptosis and at the stage of chromatincondensation the original nuclei splits into a number of dense MN, scattered throughoutthe cytoplasm [50]. These MN generally appear surrounded by a double membranesystem, externally outlined by ribosomes. The functional role of these MN is stillunknown, but it is generally accepted that they contain sequestered inactive geneticmaterial [51]. Consequently, in the MNT a possible explanation is that the very earlysteps of chromatin condensation due to apoptosis are not easily distinguishable from MNinduced by chemicals using Giemsa staining [31]. Moreover, Simko et al. [52] showedthat the increase in apoptotic cells is positively correlated with the appearance of MN.It is important to note that the occurrence of nuclear morphological aberrations inO. americanus, as well as the general degenerative changes in the erythrocytes during thetadpole stages studied, corresponded to a period of intense hematopoiesis with active celldivision in the circulating blood [18,53].

Finally, measurements of MN and other erythrocytes morphological aberrationsperformed in circulating blood samples of O. americanus tadpoles is a method fordetecting cytogenetic damage. The general measurement of tadpole’s blood character-istics, including erythrocyte morphology, may provide a sensitive means of early warningfor some water quality changes adverse to aquatic life [40,54,55]. Extrapolation from thepresent study is easy, by the use of a representative species of native anuran commonlyfound in agroecosystems [56]. Before definitive conclusions are made about the genotoxiceffect of CY on anuran larvae, more assessments are urgently need to be conducted underlaboratory conditions and on agricultural systems with other native species. Finally,further work needs to be undertaken on the factors affecting rates of increased MN in

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tadpoles subject to the range of chemical and physical conditions found in theenvironment.

Acknowledgment

We thank Dr Sergio Guerrero, Chief of CIEN-FBCB, for providing laboratoryfacilities and Lic. Hugo G. Pitoco for helping with the translation. We also thank SamKacew PhD.

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