Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv Role of bisphenol A on calcium influx and its potential toxicity on the testis of Danio rerio Hemily Batista-Silva a,d , Keyla Rodrigues a , Kieiv Resende Sousa de Moura b , Glen Van Der Kraak c , Christelle Delalande-Lecapitaine d , Fátima Regina Mena Barreto Silva a,∗ a Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, CEP: 88040-900, Florianópolis, Santa Catarina, Brazil b Departamento de Ciências Morfológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil c Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada d Département Biologie et Sciences de La Terre, Université de Caen Normandie, Caen, Normandie, France ARTICLE INFO Keywords: Calcium BPA Inositol trisphosphate receptor Zebrafish Testis ABSTRACT This study investigated the acute in vitro effect of low-concentration bisphenol A (BPA) on calcium ( 45 Ca 2+ ) influx in zebrafish (Danio rerio) testis and examined whether intracellular Ca 2+ was involved in the effects of BPA on testicular toxicity. In vitro studies on 45 Ca 2+ influx were performed in the testes after incubation with BPA for 30 min. Inhibitors were added 15 min before the addition of 45 Ca 2+ and BPA to testes to study the mechanism of action of BPA. The involvement of intracellular calcium from stores on lactate dehydrogenase (LDH) release and on triacylglycerol (TAG) content were carried out after in vitro incubation of testes with BPA for 1 h. Furthermore, gamma-glutamyl transpeptidase (GGT) and aspartate aminotransferase (AST) activities were analyzed in the liver at 1 h after in vitro BPA incubation of D. rerio. Our data show that the acute in vitro treatment of D. rerio testes with BPA at very low concentration activates plasma membrane ionic channels, such as voltage-dependent calcium channels and calcium-dependent chloride channels, and protein kinase C (PKC), which stimulates Ca 2+ influx. In addition, BPA increased cytosolic Ca 2+ by activating inositol triphosphate receptor (IP 3 R) and inhibiting sarco/endoplasmic reticulum calcium ATPase (SERCA) at the endoplasmic re- ticulum, contributing to intracellular Ca 2+ overload. The protein kinases, PKC, MEK 1/2 and PI3K, are involved in the mechanism of action of BPA, which may indicate a crosstalk between the non-genomic initiation effects mediated by PLC/PKC/IP 3 R signaling and genomic responses of BPA mediated by the estrogen receptor (ESR). In vitro exposure to a higher concentration of BPA caused cell damage and plasma membrane injury with increased LDH release and TAG content; both effects were dependent on intracellular Ca 2+ and mediated by IP 3 R. Furthermore, BPA potentially induced liver damage, as demonstrated by increased GGT activity. In conclusion, in vitro effect of BPA in a low concentration triggers cytosolic Ca 2+ overload and activates downstream protein kinases pointing to a crosstalk between its non-genomic and genomic effects of BPA mediated by ESR. Moreover, in vitro exposure to a higher concentration of BPA caused intracellular Ca 2+ -dependent testicular cell damage and plasma membrane injury. This acute toxicity was reinforced by increased testicular LDH release and GGT activity in the liver. 1. Introduction A wide variety of exogenous compounds function as Endocrine Disrupting Chemicals (EDC), and cause deleterious effects on the male reproductive system including effects on fertility (Hill and Janz, 2003; Brouard et al., 2016). Bisphenol A [2,2-bis(4-hydroxyphenyl)propane] (BPA) is an EDC that is considered to be a xenoestrogen. Xenoestrogens are compounds that produce estrogenic or anti-androgenic responses in several animal species by mimicking the action of 17β-estradiol (E2) and, therefore, interfering with endogenous endocrine regulation (Molina-Molina et al., 2013; Urriola-Munoz et al., 2014). BPA is used for manufacturing epoxy resins to coat metal cans and for polymerizing polycarbonate plastic for the manufacture of food utensils, plastic containers, packaging, dental sealants, bottles and water supply tubes (Kang et al., 2006; Koch and Calafat, 2009). As such, human exposure to BPA is a frequent occurrence, since BPA is released from https://doi.org/10.1016/j.ecoenv.2020.110876 Received 9 March 2020; Received in revised form 7 June 2020; Accepted 8 June 2020 ∗ Corresponding author. Departamento de Bioquímica, Centro de Ciências Biológicas, UFSC. Campus Universitário, Bairro Trindade, CEP: 88040-900, Florianópolis, Santa Catarina, Brazil. E-mail address: mena.barreto@ufsc.br (F.R. Mena Barreto Silva). Ecotoxicology and Environmental Safety 202 (2020) 110876 0147-6513/ © 2020 Elsevier Inc. All rights reserved. T