College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan 410128, PR China.
College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan 410128, PR China.
Toxicol Lett. 2015 Feb 3;232(3):573-9. doi: 10.1016/j.toxlet.2014.12.016. Epub 2014 Dec 24.
T-2 toxin is a secondary metabolite produced by Fusarium genus and is a common contaminant in food and feedstuffs of cereal origin. In porcine granulosa cells(GC), T-2 toxin has been shown to inhibit the steroidogenesis; however, the mechanism has not been well understood. Gonadotropin-stimulated steroidogenesis is regulated by the cAMP-PKA pathway. In this study, we investigated potential mechanisms for T-2 toxin-induced reproductive toxicity focusing on the critical steps of the cAMP-PKA pathway affected by T-2 toxin. We first analyzed the effects of T-2 toxin on progesterone and estrogen production in rat granulosa cells. For this purpose the granulosa cells were cultured for 48 h in 10% fetal bovine serum-containing medium followed by 24h in serum-free medium containing FSH (10 ng/ml) and androstenedione (3 ng/ml), both are required for normal steroidogenesis. Treatment of these cells with T-2 toxin dose-dependently inhibited the growth of cells and the steroid hormone production. Cellular cyclic AMP levels were dose-dependently inhibited by T-2 toxin (0, 1, 10 and 100 nM, 24 h). Furthermore, we found that although the induction of progesterone by 8-Br-cAMP (a FSH mimetic) and 22R-HC (substrate for progesterone) could both be inhibited by T-2 toxin treatment, the T-2-imposed inhibitory effects could be reversed by increasing doses of 22R-HC, while increasing 8-Br-cAMP had no effects, suggesting that T2 toxin targeted at distinct mechanisms. cAMP-stimulated steroidogenic acute regulatory protein (StAR) is a rate limiting protein in progesterone synthesis. Exposure to T2 toxin caused significant suppression of StAR expression as determined by Western blotting and semi-quantitative RT-PCR suggesting StAR is a sensitive target for T-2 toxin. Taken together, our results strongly suggest that T2 toxin inhibits steroidogenesis by suppressing cAMP-PKA pathway and StAR is a target for T-2-toxin. The antisteroidogenesis effects were observable at low T-2 dose (1 ng/ml) suggesting T-2 toxin has an endocrine disruptive effect.
T-2 毒素是真菌属产生的一种次生代谢物,是谷物来源的食物和饲料的常见污染物。在猪的颗粒细胞(GC)中,T-2 毒素已被证明抑制类固醇生成;然而,其机制尚未得到很好的理解。促性腺激素刺激的类固醇生成受 cAMP-PKA 途径调节。在这项研究中,我们研究了 T-2 毒素诱导生殖毒性的潜在机制,重点关注 T-2 毒素影响的 cAMP-PKA 途径的关键步骤。我们首先分析了 T-2 毒素对大鼠颗粒细胞孕激素和雌激素产生的影响。为此,将颗粒细胞在含有 10%胎牛血清的培养基中培养 48 小时,然后在不含血清的培养基中培养 24 小时,其中含有 FSH(10ng/ml)和雄烯二酮(3ng/ml),这两者都是正常类固醇生成所必需的。用 T-2 毒素处理这些细胞可剂量依赖性地抑制细胞生长和类固醇激素产生。T-2 毒素(0、1、10 和 100 nM,24 小时)可剂量依赖性地抑制细胞内环腺苷酸水平。此外,我们发现,尽管 8-Br-cAMP(FSH 模拟物)和 22R-HC(孕激素的底物)诱导的孕激素产生均可被 T-2 毒素处理抑制,但 T-2 毒素施加的抑制作用可通过增加 22R-HC 的剂量来逆转,而增加 8-Br-cAMP 则没有效果,这表明 T2 毒素针对不同的机制。cAMP 刺激的类固醇急性调节蛋白(StAR)是孕激素合成中的限速蛋白。Western blot 和半定量 RT-PCR 分析表明,暴露于 T2 毒素会导致 StAR 表达显著抑制,这表明 StAR 是 T-2 毒素的敏感靶点。总之,我们的结果强烈表明,T2 毒素通过抑制 cAMP-PKA 途径抑制类固醇生成,StAR 是 T-2 毒素的靶点。低剂量(1ng/ml)的 T-2 毒素就具有抗类固醇生成作用,表明 T-2 毒素具有内分泌干扰作用。