Toxicology 320 (2014) 34–45 Contents lists available at ScienceDirect Toxicology j ournal homepage: www.elsevier.com/locate/toxicol Mechanisms underlying the neurotoxicity induced by glyphosate-based herbicide in immature rat hippocampus: Involvement of glutamate excitotoxicity Daiane Cattani, Vera Lúcia de Liz Oliveira Cavalli, Carla Elise Heinz Rieg, Juliana Tonietto Domingues, Tharine Dal-Cim, Carla Inês Tasca, ∗ Fátima Regina Mena Barreto Silva, Ariane Zamoner Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil a r a t i b s c l t r e a i n f o c t Article history: Previous studies demonstrate that glyphosate exposure is associated with oxidative damage and neu- Received 23 August 2013 rotoxicity. Therefore, the mechanism of glyphosate-induced neurotoxic effects needs to be determined. Received in revised form 12 February 2014 ® The aim of this study was to investigate whether Roundup (a glyphosate-based herbicide) leads to Accepted 6 March 2014 neurotoxicity in hippocampus of immature rats following acute (30 min) and chronic (pregnancy and Available online 15 March 2014 lactation) pesticide exposure. Maternal exposure to pesticide was undertaken by treating dams orally ® with 1% Roundup (0.38% glyphosate) during pregnancy and lactation (till 15-day-old). Hippocampal Keywords: ® slices from 15 day old rats were acutely exposed to Roundup (0.00005–0.1%) during 30 min and experi- Glyphosate 45 2+ Calcium ments were carried out to determine whether glyphosate affects Ca influx and cell viability. Moreover, 14 we investigated the pesticide effects on oxidative stress parameters, C-␣-methyl-amino-isobutyric acid Glutamatergic excitotoxicity 14 Oxidative stress ( C-MeAIB) accumulation, as well as glutamate uptake, release and metabolism. Results showed that ® 45 2+ Kinase pathways acute exposure to Roundup (30 min) increases Ca influx by activating NMDA receptors and voltage- 2+ dependent Ca channels, leading to oxidative stress and neural cell death. The mechanisms underlying ® Roundup -induced neurotoxicity also involve the activation of CaMKII and ERK. Moreover, acute expo- ® 3 sure to Roundup increased H-glutamate released into the synaptic cleft, decreased GSH content and increased the lipoperoxidation, characterizing excitotoxicity and oxidative damage. We also observed ® 3 that both acute and chronic exposure to Roundup decreased H-glutamate uptake and metabolism, 45 2+ 14 while induced Ca uptake and C-MeAIB accumulation in immature rat hippocampus. Taken together, ® these results demonstrated that Roundup might lead to excessive extracellular glutamate levels and consequently to glutamate excitotoxicity and oxidative stress in rat hippocampus. © 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Agricultural Defense Products (Sindicato Nacional da Indústria de Produtos para Defesa Agrícola – SINDAG), in 2008 Brazil became the The annual consumption of pesticides in Brazil has increased largest consumer of pesticides in the world. The increase in pesti- alarmingly in recent years. The justification for the use of these sub- cide consumption leads to high rates of intoxication among farmers. stances is based on the improvement of agricultural productivity, Moreover, it has been suggested important associations between which places the country as one of the world’s largest produc- the bulk sale of pesticides and the increased rates of several types of ers of food. However, the risks to the environment and health are cancer, endocrine disorders and a high prevalence of neurodegen- neglected. According to the Brazilian National Trade Union for the erative diseases in agricultural workers (Londres, 2011), reinforcing the need for mechanistic investigations. Glyphosate-based herbicides lead the pesticide world market. Moreover, glyphosate is the primary active ingredient present ∗ Corresponding author at: Departamento de Bioquímica, Centro de Ciências ® in Roundup (Monsanto Company, St. Louis, MO). It has been Biológicas, UFSC, Campus Universitário, Bairro Trindade, CEP: 88040-970, Flori- ® suggested that the toxicity of Roundup is probably due to syner- anópolis, Santa Catarina, Brazil. Tel.: +55 48 3721 4747; fax: +55 48 3721 9672. gistic effects between glyphosate and other formulation products E-mail addresses: [email protected], [email protected], [email protected] (A. Zamoner). (Marc et al., 2002; El-Shenawy, 2009), such as the surfactant http://dx.doi.org/10.1016/j.tox.2014.03.001 0300-483X/© 2014 Elsevier Ireland Ltd. All rights reserved. D. Cattani et al. / Toxicology 320 (2014) 34–45 35 polyethoxylated tallowamine (POEA) which might facilitate neurotransmitters metabolism, and poor radical scavenging mech- glyphosate penetration through plasma membranes potentiating anisms (Chong et al., 2005). its toxicity (Williams et al., 2000; Tsui and Chu, 2003; Richard et al., Taking into account the previous studies demonstrating that 2005; Benachour and Séralini, 2009). exposure to glyphosate might be associated with neurotoxicity and The planting of transgenic soybeans resistant to glyphosate- oxidative damage, the aim of this study was to determine whether ® ® Roundup (Glyphosate resistant soybeans, GRS) has greatly Roundup leads to neurotoxicity in hippocampus of immature enhanced the consumption of this herbicide in Brazilian crops. rats following acute (30 min) and chronic (pregnancy and lacta- In this context, it was recently reported a teratogenic action of tion) pesticide exposure. Then, we investigated the involvement 2+ herbicides based on glyphosate to vertebrates (Paganelli et al., of Ca , intracellular signaling pathways and oxidative damage on ® 2010), warning of the need to evaluate the use of the product the mechanisms underlying Roundup -induced neurotoxicity in in agriculture. Moreover, Benachour and Séralini (2009) recently rat hippocampus. Moreover, this study investigated whether acute demonstrated that glyphosate formulations induce apoptosis and or maternal exposure to a glyphosate-based herbicide alters glu- necrosis in human umbilical, embryonic, and placental cells. This tamatergic system by interfering in the neurotransmitter uptake, herbicide was also associated with induction of oxidative stress release and/or metabolism within the hippocampal cells. and neuroinflamation (El-Shenawy, 2009; Astiz et al., 2012). More- over, glyphosate was able to provoke oxidative stress in specific 2. Material and methods brain regions: substantia nigra, cerebral cortex and hippocampus. However, most of these studies were carried out by using the com- 2.1. Radiochemical and compounds mercial formulations, and not pure glyphosate, suggesting that 3 3 further studies are necessary to examine whether the pesticide L-[2,3- H] glutamic acid ([ H] glutamate) (specific activity formulation may lead to neurotoxicity. 49 Ci/mmol) was purchased from Amersham (Oakville, Ontario, 45 2+ It has been suggested that pesticide exposure could be a risk Canada). CaCl2 (specific activity of 321 kBq/mg of Ca ) and factor for neurodegenerative disorders (Le Couteur et al., 1999; Optiphase Hisafe III biodegradable liquid scintillation were 14 Kirby et al., 2001; Barlow et al., 2005; Patel et al., 2006). In this purchased from PerkinElmer (Waltham, MA). ␣-[ C] methy- 14 context, occupational pesticide exposure leads to oxidative dam- laminoisobutyric acid ([ C] MeAIB) (sp.act. 1.85 GBq/mmol) was age, increases the risk of incidence of Parkinson’s and Alzheimer’s purchased from Du Pont, NEN Products, MA, USA. The herbicide ® disease, and also might accelerate age-related neurodegeneration Roundup Original (Homologation number 00898793) containing (Wang et al., 2006; Peng et al., 2007; Hayden et al., 2010); how- glyphosate 360 g/L is a commercial formulation registered in the ever, it is unclear as to which pesticides and what mechanisms Brazilian Ministry of Agriculture, Livestock and Supply (Ministério of action may contribute to the neurodegenerative condition. In da Agricultura, Pecuária e Abastecimento – MAPA). Nifedipine, this context, it has been reported that acute and chronic expo- N-[2-(p-Bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide sure to glyphosate might cause parkinsonism, a condition similar (H89), (Bisindoylmaleimidine IX, 2-{1-[3-(Amidinothio)propyl]- } to Parkinson’s disease (Barbosa et al., 2001; Wang et al., 2011). 1H-indol-3-yl -3-(1-methylindol-3-yl)maleimide ethanesulfonate Barbosa and colleagues (2001) reported a case of a 54-year-old salt) Ro 31-8220, d(–)-2-amino-5-phosphonopentanoic acid (AP5), man who accidentally sprayed himself with the chemical agent KN-93, (+)-␣-methyl-4-carboxyphenylglycine (MCPG) and flunar- glyphosate and one month later he developed parkinsonism. One izine were purchased from Sigma Chemical Company (St. Louis, year later, the patient presented a slow resting tremor in the left MO, USA). The G6PD assay kit was kindly provided by INTERCIEN- hand and arm, accompanied by impairment of short-term mem- TÍFICA (São José dos Campos, SP, Brazil). All other chemicals were ory. Further, Wang et al. (2011) reported a case of parkinsonism of analytical grade. following chronic exposure to glyphosate in a previously healthy 44-year-old woman who worked for 3 years in a chemical factory, 2.2. Animals exclusively in the glyphosate production division. The glyphosate neurotoxicity was associated with rigidity, slowness and resting Wistar rats were bred in animal house and maintained