Available online at Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone-marrow Marina Goumenou , Demetrios P. Matthopoulos a Department of Environmental and Natural Resources Management, University of Ioannina, Agrinio Campus, Greece b General Chemical State Laboratory, D Division of Athens, B Department, Athens, Greece Received 2 January 2007; received in revised form 10 April 2007; accepted 20 May 2007 Abstract
Imidacloprid and metalaxyl are two pesticides that are widely used in agriculture, either separately, or in combination. These agents were studied for their possible genotoxic effects with respect to the following cytogenetic end-points: (1) in vitro micronucleus(MN) formation and sister-chromatid exchange (SCE) induction in human lymphocytes and (2) in vivo micronucleus induction inpolychromatic erythrocytes (PCEs) of the rat bone-marrow. The results of the MN analysis indicate that MN frequencies aftertreatment with both pesticides, separately or as a mixture, do not significantly differ from those in the controls except after treatmentwith metalaxyl alone at 50 ␮g/ml (p < 0.05). The results of the SCE analysis show that SCE frequencies after treatment withimidacloprid do not differ significantly from those in the controls. A statistically significant increase (p < 0.05) in SCE frequencyresulted from treatments with metalaxyl at 5, 10 and 100 ␮g/ml and with the combination of imidacloprid and metalaxyl at 100and 200 ␮g/ml. Finally, the in vivo micronucleus assay with rat bone-marrow polychromatic erythrocytes showed a statisticallysignificant effect upon separate treatments with imidacloprid and metalaxyl at doses of 300 mg/kg body weight (b.w.) (p < 0.01) orupon combined treatment with 200 mg/Kg b.w. (p < 0.001) and 400 mg/kg b.w. (p < 0.05).
2007 Elsevier B.V. All rights reserved.
Keywords: Sister-chromatid exchange (SCE); Micronucleus (MN) test; Human lymphocytes; Rat bone-marrow; Genotoxicity; Pesticides 1. Introduction
Occupationally or incidentally, humans are exposed not only to single pesticides, but also to pesticide mix- The introduction of new chemicals in nature may be tures. Limited in vitro studies have been conducted on responsible for numerous negative effects in humans, possible genotoxic side effects of pesticide mixtures such as biochemical malfunctions or genetic instability Pesticides are widely used noxious chemicals in Imidacloprid and metalaxyl are two commonly used agriculture, either separately or in mixtures, and they pesticides applied either separately or as a mixture. In invade the environment in large quantities.
vivo results indicated that further studies are required toelucidate their genotoxic effects Imidacloprid, a systemic chloro-nicotinyl insecticide with soil, seed and foliar uses, blocks the nicotinergic Corresponding author. Tel.: +30 26410 74148; neuronal pathway, which is more abundant in insects E-mail address: (D. Vlastos).
than in warm-blooded animals. It is a General Use 1383-5718/$ – see front matter 2007 Elsevier B.V. All rights reserved.
G. Demsia et al. / Mutation Research 634 (2007) 32–39 Pesticide (GUP) classified by the US-EPA as a toxic- 2. Materials and methods
ity class II and class III agent Imidacloprid wasfound to induce DNA damage in a dose-related man- ner in earthworms as well as to increase the frequency ofadducts in pesticide-treated calf thymus DNA, indicating Blood samples were obtained from two healthy non- smokers without previous known contact with pesticides. The donors were between 20- and 25-year-old.
Metalaxyl, a systemic benzenoid fungicide used in Imidacloprid (RDH 37894; purity, 99.9% by HPLC) and mixtures as foliar spray for tropical and subtropical metalaxyl (RDH 36153; purity, 99.9% by HPLC) obtained crops, is a General Use Pesticide (GUP) classified by from Pestanal Sigma–Aldrich, were dissolved in sterile dis- the US-EPA as a toxicity class III agent is tilled water at defined concentrations either as pure substances not considered genotoxic However, a significant or in 1:1 mixtures. The test concentrations are below the lim- dose-dependent induction of chromosomal aberrations its of solubility for imidacloprid metalaxyl in cultured human peripheral blood lymphocytes was derived from our preliminary studies, while they correspond to observed after high-dose treatments, while in vivo administration of 75–300 mg/kg b.w. to mice did notresult in any significant change in the frequency of 2.2. Micronucleus (MN) test in human lymphocytes in micronuclei (MN) in the polychromatic erythrocytes The genotoxic and mutagenic activities of cer- Whole blood (0.5 ml) was added to 6.5 ml Ham’s F-10 tain pesticides have been studied both in in vitro medium (Invitrogen), 1.5 ml foetal calf serum (Invitrogen)and 0.3 ml phytohaemagglutinin (Invitrogen) to stimulate cell and in vivo cytogenetic test systems such as the division. Cultures were incubated at 37 ◦C for 72 h. The appro- cytokinesis-block micronucleus (CBMN) test and the priate chemicals were added 41 h after the start of the culture sister-chromatid exchange (SCE) assay in human lym- at final concentrations of 0.1, 1, 5, 10, 50 and 100 ␮g/ml in phocytes in vitro and the micronucleus test on rat separate treatments and of 10, 25, 50, 100 and 200 ␮g/ml in bone-marrow cells after in vivo exposure of the animals mixed treatments. Mitomycin-C (Sigma) at a final concen- tration of 0.5 ␮g/ml served as positive control. Three hours The CBMN assay in human lymphocytes devel- after the addition of the pesticides, i.e. at 44 h post-culture oped by Fenech and Morley cytochalasin-B, initiation, 6 ␮g/ml cytochalasin-B (Sigma) was added. Cells an inhibitor of actin polymerisation, which pre- were collected by centrifugation at 72 h, fixed with freshly vents cytokinesis while permitting nuclear division made methanol/acetic acid (Riedel-de Haen/Merck) mixture As a result, binucleated (BN) cells are pro- (3:1 v/v) after mild hypotonic treatment, and stained withGiemsa (Fluka) At least 1000 binucleate (BN) cells duced, which are scored for the presence of MN with preserved cytoplasm were scored, for each donor and for each case, in order to calculate the frequency of MN. Standard The SCE analysis has often been applied as a cytogenetic assay for biomonitoring and genotoxic- The cytokinesis block proliferation index (CBPI) is given ity testing of potentially mutagenic and carcinogenic by equation: CBPI = M1 + 2M2 + 3 (M3 + M4)/N, where M1, M2, M3 and M4 correspond to the numbers of cells with one, two, nation as well as alternative RAD-51 independent three and four nuclei and N is the total number of cells The CPBI was calculated by counting at least 2000 cells, to determine possible cytotoxic effects.
The rodent bone-marrow MN test is the most widely used short-term in vivo assay for the identification of 2.3. Sister-chromatid exchange (SCE) assay in human genotoxic effects such as chromosome damage and ane- uploidy associated with mutagenesis and carcinogenesis Lymphocyte cultures from one healthy donor were set up as described for the MN assay. At the start of the culture, The present work focuses on the in vitro and in 5-bromodeoxyridine (5-BrdU) (Sigma) was added at a final vivo analysis of genotoxic effects of imidacloprid and concentration of 7.5 ␮g/ml. The appropriate chemicals were metalaxyl, as single agents and in combination, using added 48 h after the initiation of the cultures at final con- cytogenetic tests such as the cytokinesis-block micronu- centrations of 0.1, 1, 5, 10, 50 and 100 ␮g/ml in separate cleus assay and sister-chromatid exchange analysis in treatments and of 10, 25, 50, 100 and 200 ␮g/ml in mixture human lymphocytes in vitro and the micronucleus test treatments. Mitomycin-C (Sigma) at a final concentration of 0.1 ␮g/ml served as positive control. Cultures were incubated G. Demsia et al. / Mutation Research 634 (2007) 32–39 in the dark at 37 ◦C for 72 h and demecolcine (Gibco) at a final reason they can be distinguished from mature, norchromatic concentration of 0.3 ␮g/ml was added 2 h before harvesting erythrocytes (NCEs) by selective ribosome staining. In addi- slight modification of the original Moorhead et al. tion, 200 total (immature and mature) erythrocytes were protocol was used for cell harvesting and chromosomal prepa- scored for PCE frequency per animal and for each treatment ration. Chromosome staining was performed according to the fluorescence-plus procedure with minor modifications.
Briefly, air-dried slides were immersed for 15 min in a solu- tion of 0.5 ␮g/ml Hoechst 33258 (Sigma) in Sorensen’s buffer,exposed for 30 min to UV radiation, washed and finally stained The one-way ANOVA test and the Student’s t-test of the Ori- with Giemsa (Fluka) solution in Sorensen’s buffer, pH 6.8, for gin 7.0 software (OriginLab Corporation, Northampton, USA) were applied to statistically analyse the results obtained with The frequency of SCE was evaluated in 50 second-division metaphases for each treatment. Calculating the replicationindex (RI) from 200 metaphases was the criterion for thedetermination of possible cytotoxic effects. The RI was given 3. Results
by equation: RI = M1 + 2M2 + 3M3/N, where M1, M2 and M3denote those metaphases corresponding to first, second and Cytogenetic end-points such as CBMN and SCE third or subsequent divisions, and N is the total number of induction in human lymphocytes in vitro and the in vivo MN formation in rat bone-marrow were used to evaluatethe genotoxic effects of imidacloprid and metalaxyl.
2.4. Micronucleus (MN) test in rat bone-marrow in vivo 3.1. Micronucleus test in human lymphocytes in Healthy male Wistar rats, 6 weeks of age and weighing 138 ± 1.54 g were obtained from the Pasteur Institute, Athens,Greece and housed in groups of five in plastic chambers under shows the results obtained with peripheral controlled temperature and light conditions, having free access blood lymphocyte cultures treated with imidacloprid and to standard rodent chow and water. The rats were treated orally metalaxyl, separately or in combination.
either with separate or combined doses of imidacloprid and Neither separate nor mixed imidacloprid and met- metalaxyl. The Student’s t-test did not reveal statistically sig- alaxyl treatments were able to induce a statistically nificant differences in animal weights between study groups.
The pesticides were tested at three different doses, cor- significant increase in the frequency of MN and BNMN, responding to 22, 45 and 67% of the published LD except the concentration 50 ␮g/ml of metalaxyl. Anal- for imidacloprid (450 mg/kg) and 11, 22 and 45% of the pub- ysis of the CBPI indicated a minor decrease at all test concentrations (except the imidacloprid concentration of received either imidacloprid at 100, 200 and 300 mg/kg b.w. or 0.1 ␮g/ml) compared with the controls, but the level of metalaxyl at 75, 150 and 300 mg/kg b.w., or the 1:1 mixture of significance was not reached. In addition, in the case of these two agents at 150, 200 and 400 mg/kg b.w. Cyclophos- mixed pesticide treatments, a twofold increase in MN phamide (Sigma) at 20 mg/kg b.w. was used as the positive and BNMN frequencies was observed at concentrations control. At 24 h after the treatment, animals were anaesthetized of 100 and 200 ␮g/ml. However, these effects were also with CO2 and sacrificed by cervical dislocation in accordance with the approved procedure of the Ioannina University Insti-tutional Animal Care and Use Committee.
The micronucleus (MN) test was performed as described by 3.2. Sister-chromatid exchange analysis in human Schmid with minor modifications. Bone-marrow, from both epiphyseal cartilage-decapitated femurs, was collectedby mild centrifugation in 5 ml centrifuge tubes containing 2 ml of foetal calf serum (Invitrogen). Pelleted marrow cells blood lymphocyte cultures treated with imidacloprid and were washed by centrifugation at 1000 rpm for 3 min in 4 ml metalaxyl in separate or mixed treatments.
foetal calf serum, resuspended in a small amount of serum, The results of SCE analysis showed that imidacloprid spread on clean microscope slides, air-dried, methanol fixed, did not significantly alter SCE frequencies compared and finally stained with May-Grunwald (Fluka) followed by with the control. Statistically significant differences To determine MN frequencies, 2000 polychromatic ery- (p < 0.05) – without a dose–response pattern – in compar- throcytes (PCEs) were analysed per animal and for each ison with the control in the SCE frequencies were seen treatment. Polychromatic erythrocytes are developmentally at metalaxyl concentrations of 5, 10 and 100 ␮g/ml, as immature erythrocytes that still contain ribosomes. For this well as at the 100 and 200 ␮g/ml mixed treatments.
G. Demsia et al. / Mutation Research 634 (2007) 32–39 Table 1Frequencies of binucleated micronucleated cells (BNMN) and micronuclei (MN), as revealed after Giemsa staining, induced in vitro in humanlymphocyte cultures in separate or mixed treatments with imidacloprid and metalaxyl BN, binucleated cells; BNMN, micronucleated binucleated cells; MN, micronuclei; CBPI, cytokinesis block proliferation index; MF(‰) ± S.E.,mean frequencies (‰) ± standard error; ap < 0.05; bp < 0.05 1, 2, 3; cp < 0.0011, 2, 3 (one-way ANOVA test).
3.3. Micronucleus analysis in rat bone-marrow PCEs in total erythrocytes (PCEs and NCEs) in cyto- genetic assays, such as the in vitro cytokinesis-blockmicronuclei and SCE analysis in human lymphocytes The results obtained with PCEs from orally treated and the in vivo MN test on rat bone-marrow. Regard- rats are shown in with different separate or ing the cytotoxicity index in all cytogenetic end-points combined doses of imidacloprid and metalaxyl.
considered (no statistically significant dif- The results of the MNPCE analysis indicate statisti- ferences were observed between controls and metalaxyl- cally significant effects up to 300 mg/kg b.w. (p < 0.01) or imidacloprid-treated cultures or animals.
imidacloprid and metalaxyl when given separately, andup to 200 mg/kg b.w. (p < 0.001) and 400 mg/kg b.w.
4. Discussion
(p < 0.05) when given in combination.
The reported control frequencies of MN, SCEs and Pesticides form an important group of man-made MNPCEs are in accordance with the published values for noxious chemicals. Their potential synergistic or antag- the cytogenetic end-points used here In addi- onistic side effects in humans have not yet been tion, the MN and SCE frequencies after treatment with extensively investigated. Bolognesi viewed cyto- mitomycin C as positive control as well as the genetic biomarkers in pesticide-exposed workers and MNPCEs frequencies after treatment with cyclophos- reported indications of dose-dependent effects with phamide as positive control consistent with the increasing duration or intensity of exposure. Further- literature and authenticate our experimental procedure, test animal response, and treatment observations.
chemicals might be dependent on the dose and dose The cytotoxic effects of imidacloprid and metalaxyl rate at which the chemicals reach the target tissues.
were evaluated by the determination of CBPI, RI and (%) This possible variable response to the level, length and G. Demsia et al. / Mutation Research 634 (2007) 32–39 Table 2Frequencies of sister-chromatid exchange (SCE) induced in human lymphocyte cultures in vitro in separate or mixed treatments with imidaclopridand metalaxyl MF ± S.E., mean frequencies ± standard error; RI, replication index; ap < 0.05; bp < 0.00011, 2, 3 (t-test).
Table 3Frequencies of micronuclei in polychromatic erythrocytes (PCEs), induced in rat bone-marrow cells treated in vivo with imidacloprid and metalaxyl MF(‰) ± S.E., mean frequencies(‰) ± standard error; MNPCEs, micronucleated polychromatic erythrocytes; CP, Cyclophosphamide; ap < 0.01;bp < 0.001; cp < 0.05 (t-test).
G. Demsia et al. / Mutation Research 634 (2007) 32–39 frequency of exposure to various pesticides could be Taking into consideration the suggestions of Bolog- correlated to the variable response observed in other mammalian tissues to other environmental stressors observed genotoxic effects may be the result of either such as heat treatment or viral infection In the dose applied to the test system and/or the rate by an attempt to further address these issues we focused which the test pesticides reach the target tissue.
on the possible synergistic effect of imidacloprid and Our results do not indicate a clear-cut genotoxic effect of imidacloprid and metalaxyl when tested separately In a study of the potential in vivo genotoxic effects of on human lymphocytes in vitro or on rat bone-marrow imidacloprid and metalaxyl on occupationally exposed in vivo. In treatments with a mixture of these pesticides farmers, smokers and non-smokers, statistically sig- we observed statistically significant differences at the nificant differences were observed in MN frequencies highest test concentrations for both SCE and MNPCEs.
between controls and exposed smokers, as well as This effect was not dose-dependent. However, Dolara between controls and all the exposed farmers et al. an in vitro dose-dependent induction observations provided the basis for the idea that the of SCE in human lymphocytes exposed to a mixture of observed effects may have resulted from an interaction four different pesticides, while no significant increase in SCEs was seen when the various pesticides were tested Significant differences in MN and SCE frequen- separately. This latter result is in agreement with our cies were observed in in vitro studies when 0.1 and observations when we tested imidacloprid and metalaxyl 0.5 ␮g/ml of imidacloprid were added to human lym- alone. In addition, Bianchi-Santamaria et al. phocytes at 24 h after the start of the culture Our a dose-independent induction of MN in human lympho- results show that MN and SCE frequencies after treat- cyte cultures with mixtures of various phytochemicals ment with imidacloprid are not different from the control at environmental concentrations. Taking into considera- values. However, the differences between our MN and tion that to some extent controversial results have been SCE control values and those in the above-mentioned reported on the genotoxicity of the various agrochemi- study may explain the controversial results in MN and cals, tested alone or in combination, it appears important to conduct studies using different genotoxicity tests in order to reach to a comprehensive understanding of their 1000 ␮g/ml) resulted in a significant dose-dependent induction of CAs in human lymphocytes Treat- In conclusion, our study revealed an increase in MN ments with metalaxyl doses lower than those mentioned frequency in human lymphocytes as well as in rat bone- above did not result in a statistically significant increase marrow cells and an induction of SCEs in cultured human in MN frequencies. On the other hand, a significant lymphocytes treated with a mixture of imidacloprid and increase in SCE frequency – without a dose–response metalaxyl. These observations are indicative of a possi- pattern – was observed after treatment with metalaxyl.
ble synergistic effect of these two pesticides.
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