Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, CEP: 88040-900, Florianópolis, Santa Catarina, Brazil; Département Biologie et Sciences de La Terre, Université de Caen Normandie, Caen, Normandie, France.
Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, CEP: 88040-900, Florianópolis, Santa Catarina, Brazil.
Ecotoxicol Environ Saf. 2020 Oct 1;202:110876. doi: 10.1016/j.ecoenv.2020.110876. Epub 2020 Jun 18.
This study investigated the acute in vitro effect of low-concentration bisphenol A (BPA) on calcium (Ca) influx in zebrafish (Danio rerio) testis and examined whether intracellular Ca was involved in the effects of BPA on testicular toxicity. In vitro studies on Ca influx were performed in the testes after incubation with BPA for 30 min. Inhibitors were added 15 min before the addition of Ca 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 influx. In addition, BPA increased cytosolic Ca by activating inositol triphosphate receptor (IPR) and inhibiting sarco/endoplasmic reticulum calcium ATPase (SERCA) at the endoplasmic reticulum, contributing to intracellular Ca 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/IPR 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 and mediated by IPR. 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 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-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（Ca）内流的急性体外效应，并研究了细胞内 Ca 是否参与 BPA 对睾丸毒性的影响。在 BPA 孵育 30 分钟后，对睾丸中的 Ca 内流进行体外研究。在向睾丸中添加 Ca 和 BPA 之前 15 分钟加入抑制剂，以研究 BPA 的作用机制。在 BPA 体外孵育 1 小时后，通过向睾丸中体外孵育来研究细胞内钙库对乳酸脱氢酶（LDH）释放和三酰基甘油（TAG）含量的影响。此外，在体外孵育 D. rerio 1 小时后，分析肝脏中的γ-谷氨酰转肽酶（GGT）和天冬氨酸氨基转移酶（AST）活性。我们的数据表明，极低浓度的 BPA 急性体外处理斑马鱼睾丸会激活质膜离子通道，如电压依赖性钙通道和钙依赖性氯通道，以及蛋白激酶 C（PKC），从而刺激 Ca 内流。此外，BPA 通过激活肌醇三磷酸受体（IPR）和抑制内质网肌醇 1,4,5-三磷酸受体（SERCA）来增加细胞浆 Ca，导致细胞内 Ca 超载。蛋白激酶 PKC、MEK1/2 和 PI3K 参与了 BPA 的作用机制，这可能表明由 PLC/PKC/IPR 信号介导的非基因组起始效应与由雌激素受体（ESR）介导的 BPA 基因组反应之间存在串扰。体外暴露于较高浓度的 BPA 会导致细胞损伤和质膜损伤，导致 LDH 释放和 TAG 含量增加；这两种作用均依赖于细胞内 Ca，并由 IPR 介导。此外，BPA 可能会导致肝损伤，这可通过 GGT 活性增加来证明。总之，低浓度 BPA 的体外作用会引发细胞浆 Ca 超载，并激活下游蛋白激酶，表明其通过 ESR 介导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 会导致细胞内 Ca 依赖性睾丸细胞损伤和质膜损伤。这种急性毒性通过增加睾丸 LDH 释放和肝脏中的 GGT 活性得到加强。
