Role of bisphenol A on calcium influx and its potential toxicity on the testis of Danio rerio

This study investigated the acute in vitro effect of low-concentration bisphenol A (BPA) on calcium (45Ca(2+)) influx in zebrafish (Danio rerio) testis and examined whether intracellular Ca2+ was involved in the effects of BPA on testicular toxicity. In vitro studies on 45Ca(2+) influx were performed in the testes after incubation with BPA for 30 min. Inhibitors were added 15 min before the addition of 45Ca(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 Ca2+ influx. In addition, BPA increased cytosolic Ca2+ by activating inositol triphosphate receptor (IP3R) and inhibiting sarco/endoplasmic reticulum calcium ATPase (SERCA) at the endoplasmic reticulum, contributing to intracellular Ca2+ 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/IP3R 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 Ca2+ and mediated by IP3R. 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 Ca2+ 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 Ca2+-dependent testicular cell damage and plasma membrane injury. This acute toxicity was reinforced by increased testicular LDH release and GGT activity in the liver.