Food and Chemical Toxicology 134 (2019) 110832 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox Acute uterine effects and long-term reproductive alterations in postnatally exposed female rats to a mixture of commercial formulations of endosulfan T and glyphosate ∗ Paola I. Ingaramo1, , Marlise Guerrero Schimpf1, María M. Milesi, Enrique H. Luque, Jorgelina Varayoud Instituto de Salud y Ambiente Del Litoral (ISAL), Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional Del Litoral – Consejo Nacional de Investigaciones Científicas y Técnicas, Santa Fe, Argentina ARTICLE INFO ABSTRACT Keywords: Endosulfan and glyphosate are widely used pesticides and have been associated to reproductive disorders. We Endosulfan examine the acute and long-term effects of postnatal exposure to commercial formulations of endosulfan (EF), Glyphosate glyphosate (glyphosate-based herbicide, GBH) and a mixture of both pesticides (MIX). After birth, female pups of Fertility Wistar rats received saline solution (CONTROL), EF (600 μg/kg of b.w/day), GBH (2 mg/kg of b.w/day) or a Uterus mixture (at the same doses) from postnatal day (PND) 1 to PND7. The uterine histology and expression of Pesticide Hoxa10, estrogen (ERα) and progesterone (PR) receptors were evaluated on PND8. Reproductive performance was evaluated on gestational day 19. GBH and MIX rats showed an increment of 1) the incidence of luminal epithelial hyperplasia, 2) PR and Hoxa10 expression. EF modified ERα and Hoxa10 expression. During adult- hood, MIX and GBH rats showed higher post-implantation losses while EF alone produced an increase of pre- implantation losses. We showed that the co-administration of both pesticides produced acute uterine effects and long–term deleterious reproductive effects that were similar to those induced by GBH alone. We consider im- portant to highlight the necessity to evaluate the commercial pesticide mixture as a more representative model of human exposure to a high number of pesticides. 1. Introduction Endosulfan is an organochlorine pesticide belonging to class cy- clodiene (Menezes et al., 2017). Despite its life-threatening toxic ef- Pesticides are a large and heterogeneous group of chemicals widely fects, endosulfan continues to be one of the most widely used agri- used in agriculture and in public health for prevention and control of cultural pesticides, largely in the developing countries, due to its high pests; however it is assumed they may have adverse health effects efficacy, low cost and environmental stability. However, is a con- (Hernandez et al., 2017; Mostafalou and Abdollahi, 2017). Even to very troversial agrochemical due to its potential for bioaccumulation, high low levels of exposure, a number of pesticides is suspected may act as persistence, low volatility, acute toxicity, and role as EDC (Ahmad endocrine disruptor compounds (EDCs) and may be harmful to exposed et al., 2018; Milesi et al., 2015; Ingaramo et al., 2016a). Other polemic people (Kim et al., 2017; Combarnous, 2017). The prenatal and post- pesticide is glyphosate and its commercial formulation, glyphosate- natal periods of development until puberty are particularly sensitive to based herbicides (GBHs). Glyphosate is the most widely used herbicide EDCs exposure, which might interfere with the physiology of normal in the world for non-selective control of annual and perennial weeds, endocrine-regulated events, leading to adverse effects later in life grasses and broadleaf plants (Siroski et al., 2016). Previous in vivo and (Crain et al., 2008; Varayoud et al., 2014). in vitro studies showed that glyphosate and endosulfan could act as Abbreviations: CL, corpora lutea; DAB, diaminobenzidine; DES, diethylstilbestrol; EDCs, endocrine disruptor compounds; EF, formulations of endosulfan; ERα, estrogen receptor alpha; GBH, glyphosate-based herbicide; GD, gestational day; GE, glandular epithelium; Hoxa10, Homeobox A10; IHC, immunohistochemistry; IOD, integral optical density; IS, implantation sites; LE, luminal epithelium; LEH, Luminal epithelial hyperplasia; PND, postnatal day; PR, progesterone receptor; RS, resorption sites; SEM, standard error of the mean; SS, subepithelial stroma ∗ Corresponding author. ISAL, Facultad de Bioquímica y Ciencias Biológicas, Casilla de Correo 242, 3000, Santa Fe, Argentina. E-mail address: [email protected] (P.I. Ingaramo). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.fct.2019.110832 Received 1 July 2019; Received in revised form 17 September 2019; Accepted 20 September 2019 Available online 21 September 2019 0278-6915/ © 2019 Elsevier Ltd. All rights reserved. P.I. Ingaramo, et al. Food and Chemical Toxicology 134 (2019) 110832 EDCs affecting endocrine events in different organs/cells (Li et al., bottles with rubber stoppers surrounded by a steel ring. 2013; Senthilkumaran, 2015; Myers et al., 2016; Varayoud et al., 2008a, 2017). Transgenic crops are associated with different pesticide formula- 2.2. Experimental design tions, the most used worldwide are GBHs, followed by different in- secticides including endosulfan, among others (Burella et al., 2018). Female rats (90 days old) were housed with males of proven ferti- Several studies have demonstrated presence of glyphosate and en- lity. The day on which sperm was found in the vaginal smear was de- dosulfan residues, in soil, water or sediments in Argentina (Regaldo signated as Gestational day 1 (GD1). Pregnant rats were housed singly, et al., 2018; Primost et al., 2017; Grondona et al., 2019; Aparicio et al., and, at delivery, pups were sexed according to the anogenital distance. 2013). In agricultural topsoil samples from across the European Union, To minimize the use of siblings and avoid potential litter effects, off- glyphosate was one of the most frequently compound found in soil spring of the same litter were distributed between different mothers. samples and at the highest concentration (Silva et al., 2019). Cross-fostered litters were adjusted to eight pups, prioritizing a max- In the agricultural practices the application of more than one pes- imum of the female pups per litter when possible. When fewer than ticide at the same time is common so the human exposure to environ- eight females were available, an appropriate number of males were mental contaminants is not limited to an individual compounds retained. Female pups were assigned to one of four neonatal treatment (Perobelli et al., 2010; dos Santos and Martinez, 2014). Therefore, se- groups: 1) control group treated with saline solution (Control, n = 34); parate toxicity assessment of single chemicals may not estimate ade- 2) GBH group treated with a commercial formulation of glyphosate quately combined adverse effects of pesticides (Hernandez et al., 2017). diluted with saline solution at a dose of 2 mg of glyphosate/Kg of body A more realistic study of pesticide effects may be through evaluation of weight/day (n = 38); 3) EF group treated with a commercial formula- exposure effects to mixture of pesticides simultaneously. However, the tion of endosulfan diluted with saline solution at a dose of 600 μgof large number of different combinations of chemicals, exposure patterns, endosulfan/kg of body weight/day (n = 38); 4) MIX group treated with and complex interactions makes it impossible to test all possible com- a mixture of both pesticides at the same doses of GBH and EF groups binations, and experimental data sets for toxicity of mixtures are (n = 38). The GBH was a liquid water-soluble formulation containing lacking (Hernandez et al., 2017). It has been demonstrated that the 66.2% of glyphosate potassium salt, as its active ingredient, coadju- exposure to mixtures of pesticides increase the risk of spontaneous vants and inert ingredients. EF and GBH doses were selected based on abortion and birth defects (Bhatt, 2000). In addition, adult rats exposed previous evidence of adverse effect on uterine development and func- to a mixture of EDCs during perinatal period shows signs of early re- tionality using pure endosulfan and GBH (Ingaramo et al., 2016a, 2017; productive senescence (Johansson et al., 2016). The impact of exposure Milesi et al., 2012, 2015). The selected dose of GBH is in the order of of pesticides on health in addition is conditionate by chemical for- magnitude of 1) the environmental levels detected in our country mulation of these pesticides. Some commercial formulations of different (Aparicio et al., 2013; Arregui et al., 2004; Peruzzo et al., 2008) and 2) pesticides contain surfactants and other compounds to increase the oral reference dose defined by EPA (EPA, 1993). The EF used was spreading and penetrating power but the identity of adjuvants is gen- Thionex EC (Makhteshim Chemical Works Ltd.) a formulation con- erally not disclosed (Huston and Pignatello, 1997). A previous work taining 400 g of ensosulfan/L. The dose of EF is according to the no determined the effects of glyphosate, endosulfan and mixtures of them observed effect level (NOEL) defined by EPA, 600 μg/kg/d (EPA, 2007) on a South American caiman, Caiman latirostris, after in ovo exposure for endosulfan. As we mentioned before, the MIX group received the (Poletta et al., 2011). All parameters analyzed indicated a higher mixture of pesticides administrated simultaneously (at above-described toxicity for the mixture of pesticides than for glyphosate formulation doses). The different treatments were given on postnatal days (PND) 1, alone (Poletta et al., 2011; Hayes et al., 2006). 3, 5 and 7 by sc injections in the nape of the neck. The MIX group In the present study we evaluated the acute and long-term effects of received two injections daily with individual doses of formulation of a brief postnatal exposure to EF, GBH, and a mixture of them. We as- each pesticide. No signs of acute toxicity were observed by exposure to sessed: 1) the acute effects evaluating uterine differentiation on PND8, the pesticides, and no significant differences in weight between exposed and 2) the long-term effects determining the reproductive performance and control pups were recorded during the experiment (data no of female adult rats. shown).