This study investigated the acute in vitro effect of low-concentration bisphenol A (BPA) on calcium (⁴⁵Ca²⁺) 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 ⁴⁵Ca²⁺ influx were performed in the testes after incubation with BPA for 30 min. Inhibitors were added 15 min before the addition of ⁴⁵Ca²⁺ 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.

这项研究调查了低浓度双酚A（BPA）对斑马鱼（Danio rerio）睾丸中钙（⁴⁵ Ca ²⁺）流入的急性体外作用，并研究了细胞内Ca ²⁺是否参与了BPA对睾丸毒性的影响。与BPA孵育30分钟后，在睾丸中进行了⁴⁵ Ca ^2+内流的体外研究。在添加⁴⁵ Ca ²⁺之前15分钟添加抑制剂和双酚A到睾丸研究双酚A的作用机理。睾丸与BPA体外孵育1小时后，进行了细胞内钙离子与乳酸脱氢酶（LDH）释放和三酰甘油（TAG）含量的关系。此外，在体外BPA温育里氏螺旋体后1小时，在肝脏中分析了γ-谷氨酰转肽酶（GGT）和天冬氨酸转氨酶（AST）的活性。我们的数据表明，以非常低的浓度用BPA进行的体外对D. rerio睾丸的急性治疗会激活质膜离子通道，例如电压依赖性钙通道和钙依赖性氯离子通道以及蛋白激酶C（PKC），从而刺激钙²⁺涌入。此外，BPA通过激活肌醇三磷酸受体（IP ₃ R）并抑制内质网的肌浆/内质网钙ATPase（SERCA）来增加胞质Ca ²⁺，从而导致细胞内Ca ²⁺超载。蛋白激酶PKC，MEK 1/2和PI3K参与了BPA的作用机制，这可能表明PLC / PKC / IP ₃ R信号介导的非基因组起始效应与BPA的基因组反应之间存在串扰由雌激素受体（ESR）介导。体外暴露于较高浓度的BPA中会导致细胞损伤和质膜损伤，同时LDH释放和TAG含量增加；两种作用均依赖于细胞内Ca ²⁺，并由IP ₃ R介导。此外，如GGT活性增加所证实，BPA可能引起肝损伤。总之，低浓度BPA的体外作用会触发胞质Ca ²⁺超载并激活下游蛋白激酶，从而表明其ESR介导的BPA的非基因组和基因组效应之间存在串扰。此外，体外暴露于较高浓度的BPA会引起细胞内Ca ²⁺依赖性睾丸细胞损伤和质膜损伤。肝脏中睾丸LDH释放的增加和GGT的活性增强了这种急性毒性。