The fungicide mancozeb induces toxic effects on mammalian granulosa cells

Abstract

The ethylene-bis-dithiocarbamate mancozeb is a widely used fungicide with low reported toxicity in mammals. In mice, mancozeb induces embryo apoptosis, affects oocyte meiotic spindle morphology and impairs fertilization rate even when used at very low concentrations. We evaluated the toxic effects of mancozeb on the mouse and human ovarian somatic granulosa cells. We examined parameters such as cell morphology, induction of apoptosis, and p53 expression levels. Mouse granulosa cells exposed to mancozeb underwent a time- and dose-dependent modification of their morphology, and acquired the ability to migrate but not to proliferate. The expression level of p53, in terms of mRNA and protein content, decreased significantly in comparison with unexposed cells, but no change in apoptosis was recorded. Toxic effects could be attributed, at least in part, to the presence of ethylenthiourea (ETU), the main mancozeb catabolite, which was found in culture medium. Human granulosa cells also showed dose-dependent morphological changes and reduced p53 expression levels after exposure to mancozeb. Altogether, these results indicate that mancozeb affects the somatic cells of the mammalian ovarian follicles by inducing a premalignant-like status, and that such damage occurs to the same extent in both mouse and human GC. These results further substantiate the concept that mancozeb should be regarded as a reproductive toxicant.

Introduction

Mancozeb is a metal ethylene-bis-dithiocarbamate fungicide used to protect many fruits (e.g. apples, citrus), vegetables, (including tomatoes, beans, celery, carrots, onions, ginseng) and field crops against a large spectrum of fungal diseases (EPA 2/92). Occupational exposure to mancozeb occurs in manufacturing or formulating facilities, during field application or by contact with treated foliage or crops. Food intake represents the main source of contamination for the general population. Mancozeb has low acute toxicity in mammals and is rapidly excreted from the body (48–96 h) via urine. Its main catabolite is ethylenthiourea (ETU). In 1988, The World Health Organization (WHO, 1988) estimated that exposure to mancozeb and ETU was 0.01–1 μg/kg b.w./day for the general population. Small quantities of ETU have been detected in tomatoes, potatoes, wine and tobacco, thus suggesting that the population can be chronically exposed to mancozeb starting from childhood. Despite its capacity to induce thyroid and hepatic cancers in rodents (Chhabra et al., 1992), ETU has been degraded from being a group 2B to a group 3 chemical by IARC (2001).

Mancozeb is considered a multipotent carcinogen in laboratory animals (Belpoggi et al., 2002, Cecconi et al., 2007) with evidence for some genotoxic effects (Cecconi et al., 2007). Moreover, mancozeb exposure determines a reduction in T(4) levels in female rats, and may therefore adversely affect the developing brain (Axelstad et al., 2011). In humans, a moderate association between mancozeb exposure and neural tube defects but not thyroid cancer has been reported (Nordby et al., 2005). A recent study reported mancozeb-induced neoplastic potential in human skin in vitro and in mouse skin in vivo (Tyagi et al., 2011). In vitro experiments show that the fungicide mainly targets mitochondrial enzymes (Domico et al., 2006, Leiphon and Picklo, 2007), and that in rat fibroblasts it stimulates the formation of reactive oxygen species (Calviello et al., 2006).

As for reproductive toxicity in general, it is difficult to ascertain whether the adverse effects observed in laboratory animals exposed to pesticides are due to alterations of the gonad-hypothalamic axis, to damages occurring in specific cells of the reproductive organ, or both. In this respect, the use of isolated mouse oocytes provides information about the subtle effects of many pesticides on the acquisition of germ cell developmental capacity (Can and Albertini, 1997, Pocar et al., 2003). Moreover, mancozeb impairs female reproductive performance, with toxic effects becoming already evident at very low concentrations (≤ 1 μg/ml) (Cecconi et al., 2007). Indeed, mouse oocytes exposed to such concentrations (0.01–1 μg/ml) during in vitro maturation showed dose-dependent alterations of meiotic spindle structure and reduction of the fertilization rate in vivo and in vitro (Rossi et al., 2006a, Rossi et al., 2006b). These results are in agreement with a previous report by Greenlee et al. (2004) demonstrating mouse blastomere apoptosis in embryos exposed to 0.003 μg/ml mancozeb. Very little data, however, are available concerning fungicide toxicity on somatic ovarian cells, and in particular on granulosa cells (GC) (Cecconi et al., 2007). These are somatic cells of the mammalian ovary that interact with germ cells from development throughout adult life by forming, together with the oocyte, a functional and metabolic system that ensures the correct development of the whole follicle (Zuccotti et al., 2011). GC can undergo neoplastic transformation thus giving rise to about 5% of all ovarian cancers (Schumer and Cannistra, 2003). GC can thus be used as a model system to test for toxic effects, especially in terms of transformation potential by exogenous substances.

Here we show the effects of mancozeb exposure on GC obtained from laboratory mice and from women undergoing assisted reproduction procedures by evaluating morphology, acquisition of migratory and proliferative features, induction of apoptosis, as well as expression levels of p53.