Reproductive Toxicology 21 (2006) 216–219

Short communication

Mancozeb exposure in vivo impairs mouse oocyte fertilizability

Gianna Rossia, Roberto Buccioneb, Massimiliano Baldassarreb, Guido Macchiarellic,Maria Grazia Palmerinic, Sandra Cecconia,∗

a Dipartimento di Scienze e Tecnologie Biomediche, Universita degli Studi di L’Aquila, 67100 L’Aquila, Italyb Dipartimento di Biologia Cellulare ed Oncologia, Consorzio Mario Negri Sud, Santa Maria Imbaro, 66030 Chieti, Italy

c Dipartimento di Medicina Sperimentale, Universita degli Studi di L’Aquila, 67100 L’Aquila, Italy

Received 22 March 2005; received in revised form 24 August 2005; accepted 26 August 2005Available online 4 October 2005

Abstract

Mancozeb is known to alter reproductive performance in exposed animals, but its specific mechanism of action is still unclear. We investigatedwhether in female mice of the F1 generation, mancozeb could affect oocyte ability to undergo complete meiotic maturation and fertilization. Femalemice were treated with 50 and 500 mg/kg of mancozeb (or vehicle in the controls) from gestational day 2 to postnatal day 20. Results demonstratedt zeb caused as©

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hat only at the highest dose, mancozeb induced a significant decrease in the number of ovulated eggs. Moreover, at this dose mancoignificant decrease of fertilizability related to a reduction of the formation of male and female pronuclei.2005 Elsevier Inc. All rights reserved.

eywords: Mouse; Meiotic maturation; In vitro fertilization; Mancozeb; PN

. Introduction

Among the 25,000 types of pesticides available for var-ous home and agricultural uses, more than 80% are eitherrganophosphate or carbamate compounds. DithiocarbamatesDTCs) and their derivatives like ethylenebisdithiocarbamatesEBDCs) are generally used to protect fruits, vegetables and fieldrops against a large spectrum of fungal diseases. Despite havingshort environmental resistance, one of their main degrada-

ion products, ethylenethiourea (ETU), shows a long persistence5–10 weeks) in soil[1].

Although the risk of acute intoxication by DTCs and EBDCsainly concerns agricultural and industrial workers[2], the pop-lation at large can be chronically exposed to residues present

n foods[3]. Moreover, as EBDC residues are present also inobacco, smoke can represent a chronic source of absorption bymokers and non-smokers[4]. EBDCs are metabolized to ETUhat in exposed animals exerts various toxic effects, includinghyroid and hepatic neoplasms[1].

To date few studies have investigated the effects of chronicxposure to these chemicals in humans, yet a potential role

as thyroid carcinogens, modulators of TSH homeostasi[5]and of the immune system[6] has been considered likely. Bcontrast, numerous studies have been performed to evDTC and EBDC toxicity in laboratory animals. Although toresponse varies depending on the species utilized[7,8], it isgenerally accepted that long-term exposure to these ccals induces either tumours[8–10], or mutagenic and teratgenic effects[10,11]. Concerning fertility, chronic exposure cinduce degenerative changes in testes[12–14], and impairmenof ovulatory LH surge[15–17].

Mancozeb is one of the most widely used EBDCs, becof its lower toxicity in mammals (LD50 = 8 g/kg in rats)[18].However, in vivo exposed female rodents show a signifidecrease in size and number of healthy follicles[19–22], andin embryo implantation rate[23], that are dose-dependent.our knowledge, the specific effects exerted by exposure todose mancozeb on the ability of mammalian oocytes to commeiotic maturation and to be fertilized, that are fundamentarequisites for a successful embryonic development[24], are stillunknown.

Thus, we have collected oocytes from female mexposed to mancozeb from foetal life to postnatal day

∗ Corresponding author. Tel.: +39 0862 433459; fax: +39 0862 433433.E-mail address: cecconi@univaq.it (S. Cecconi).

and have focused our attention on their ability to formboth male and female pronuclei after in vitro fertilization(IVF).

890-6238/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.reprotox.2005.08.004

G. Rossi et al. / Reproductive Toxicology 21 (2006) 216–219 217

2. Materials and methods

2.1. Chemicals

All chemicals were obtained from Sigma (St. Louis, MO, USA), unless statedotherwise. Mancozeb (78.3% wettable Powder, technical mixture, PESTANAL[manganese–zinc ethylenebis (dithio-carbamate)], #45553) was obtained fromRiedel-de Haen–Sigma–Aldrich. ECL from Amersham (Little Chalfont,UK).

2.2. Animals and treatments

Swiss CD1 mice (Harlan Italy, Udine, Italy) were housed in individual cageswith 12:12 h light:dark regimen, and at a room temperature of 21± 1◦C. All ani-mal experimentation described in this article was conducted in accordance withaccepted standards of animal care. All experimental protocols were approved bythe local committees on animal care and use and according to accepted veterinarymedical practice.

Adult female mice (n = 15; 8–10-weeks-old) were mated with adult malemice (10–12-weeks-old). After assessment of vaginal plugs (day 0), animalswere dosed orally every 2 days with 50 mg/kg (low exposure) and 500 (highexposure) mg/kg mancozeb in sesame oil, from gestational day 2 to postnatalday 20. This schedule was chosen because in mice about 90% of mancozebis excreted within 24–48 h[1,25]. Exposure of the offspring to the fungi-cide occurred through either placenta[26] or mother milk [27]. The dosesof fungicide administered were selected on the basis of: (1) LD50, that forrodents was reported to be greater than 8000 mg/kg/day[18], and (2) lackof evident metabolic and organic effects in treated animals[19,20]. Con-trol animals (n = 12) were treated with vehicle under the same experimentalconditions.

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Fig. 1. Effects of in vivo exposure to low (50 mg/kg) and high (500 mg/kg) dosesof mancozeb on the % of PB1 emission and 2PN formation. Different letters (a,b, c) indicate differences in statistical significance.

3. Results

3.1. Effects of mancozeb on meiotic maturation andfertilization

The average number of oocytes retrieved from the oviductsof mice of the F1 generation treated with either low (50 mg/kg)or high (500 mg/kg) doses of mancozeb was not significantlydifferent compared to controls (about 16 oocytes/mouse formancozeb-treated and control samples;P > 0.05). However, thepercentage of oocytes capable of completing meiotic maturationup to MII was lower for germ cells derived from the high dosetreatment group (control: 96%± 1; low: 94%± 2 versus high:84%± 3, P < 0.05;Fig. 1). Following in vitro fertilization, weobserved that about 82% of the oocytes derived from control orlow dose treatment groups formed male and female pronuclei(2PN;P > 0.05), in comparison with 71%± 2 of oocytes derivedfrom the high dose treatment (P < 0.05;Fig. 1).

4. Discussion

The data reported in this study demonstrate that in vivo expo-sure to the fungicide mancozeb dose-dependently impairs mousereproductive functions, by altering the maturation/fertilizationprocess, and by inducing DNA damage to ovaries and uteri.

oft ntly,r an-c inef-f ingso asingd endentdo ncen-t icea rfor-m -m o thef eral-i etion

During treatment, clinical symptoms, mortality or increased ratepontaneous abortion in parent generation were not observed. Twenay-old female mice of the F1 generation were induced to ovulat

ntra-peritoneal injection of 5 IU of Pregnant Mare Serum GonadotrPMSG; Folligon, Intervet Italia, Milan, Italy) and 5 IU of human Corioonadotropin (hCG; Serono Pharma, Rome, Italy), 44–48 h apart fromther.

.3. Preparation of spermatozoa

Spermatozoa, obtained from the cauda epididymis of adult male miceapacitated in T6 fertilization medium[28] for 2 h at 37◦C and 5% CO2 in air.nly suspensions showing excellent sperm motility, evaluated by using p

ontrast microscopy, were utilized for insemination at a final concentrati× 104 spermatozoa ml−1 [29].

.4. In vitro fertilization

Ovulated oocytes were collected from the oviducts at 16 h post hC2 medium[30], and briefly incubated in hyaluronidase (1 mg/ml) to disp

xpanded cumulus-corona cells. Only oocytes arrested at metaphase IIs evidenced by extrusion of the first polar body (PB1), were deprivedona pellucida by a brief treatment with Tyrode’s solution (pH 2.5)[31], andransferred for 1 h in M2 medium. Afterwards, oocytes were inseminateh, then were washed to remove excess adherent spermatozoa, andally cultured at 37◦C and 5% CO2 in air in T6 fertilization medium. Eggere examined 6 h later for the presence of male and female pronucle

32–35].

.5. Statistic analysis

All the experiments were performed at least in triplicates. Dere expressed as the mean± S.E.M., and multiple groups were coared by ANOVA followed by Tukey–Kramer test (GraphPad InStat 2raphPad Software for Science).P < 0.05 was considered statistica

ignificant.

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The production of MII-arrested oocytes by female micehe F1 generation appears to be slightly, although significaeduced in mice exposed to high doses (500 mg/kg) of mozeb, whereas exposure to low doses (50 mg/kg) wasective. This observation is consistent with previous findbtained in adult female rats exposed for 30 days to increoses of mancozeb, whose ovaries showed a dose-depecrease in the number of the healthy follicles[20,22]. More-ver, rats exposed for several months to increasing corations of mancozeb, ranging from 140 to 1400 mg/kg/tw

week, showed a significantly worse of reproductive peance for both sexes and at all doses[21,36]. These detriental effects in the reproductive process can be due t

act that mancozeb and related DTCs might induce a genzed hormonal imbalance, by affecting gonadotropin secr

218 G. Rossi et al. / Reproductive Toxicology 21 (2006) 216–219

[37]. In our study, we found that oocytes from high-dose-treated mice showed a reduced ability to sustain the early stepsof fertilization, i.e. 2PN formation. The formation of pronu-cleate oocytes results from a complex series of ordered pro-cesses, both metabolic and regulatory[29]. Therefore, it canbe proposed that mancozeb exposure may interfere directlywith one or more of the steps leading to successful reproduc-tion.

Both at low and high doses of mancozeb the morpholog-ical features of the ovaries of treated mice were not signifi-cantly different from that of control animals (data not shown).Indeed, also the ovaries of rats treated for several days with500 mg/kg/day of mancozeb did not show significant morpho-logical changes as compared with control[19]. In conclusion,the toxicity of DTCs and EBDCs towards several animal speciesmakes it likely that they are involved in the induction of infer-tility, due to their ability to interfere with normal cell cycleprogression.

Acknowledgements

This study was granted by the Ministero della Salute (Pro-getto di Ricerca Finalizzato 1998) and by MIUR 60% to SCand GM. Thanks are due to Prof. Mauro Maccarrone (Univer-sity of Teramo, Italy) and Dott. Alberto Mantovani (IstitutoS em

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