Dooley Gregory P, Tjalkens Ronald B, Hanneman William H
Center for Environmental Medicine, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.
Toxicol Res (Camb). 2013 May 1;2(3):180-186. doi: 10.1039/C3TX20088D.
The primary metabolite of the herbicide atrazine (ATRA), diaminochlorotriazine (DACT), has been suggested to cause disruption in the hypothalamic-pituitary-gonadal axis leading to inhibition of luteinizing hormone (LH) release. DACT is a reactive electrophile known to form covalent protein adducts both and following ATRA exposure and maybe targeting proteins involved in GnRH-induced calcium signaling and subsequent LH release. To test this hypothesis, LβT2 pituitary cells were exposed to 300 μM DACT for 24 hrs and examined by fluorescence microscopy for GnRH-induced changes in intracellular calcium and LH release. LβT2 cells exposed to DACT had markedly diminished GnRH-induced intracellular calcium transients and a significant decreased LH release in response to GnRH. DACT appeared to cause a selective decrease in caffeine-sensitive ryanodine receptor-operated calcium stores in LβT2 cells, rather than in thapsigargin-sensitive ER calcium stores. This sensitivity correlated with the formation of covalent protein adducts by DACT, as determined by mass spectrometry. ERp57 was identified by mass spectrometry as a target of DACT adduction in the ER that could potentially mediate the effects of DACT on inhibition of GnRH-induced calcium signaling and inhibition of LH release. Intracellular calcium responses to GnRH and release of LH were restored in DACT-treated cells with the addition of a calcium ionophore (A23187). These data suggest that DACT forms adducts on proteins involved in calcium handling within the ER and that dysfunction in this critical signaling system is associated with loss of normal sensitivity to GnRH and subsequent decreased release of LH.
除草剂莠去津（ATRA）的主要代谢产物二氨基氯三嗪（DACT），被认为会导致下丘脑 - 垂体 - 性腺轴功能紊乱，进而抑制促黄体生成素（LH）的释放。DACT是一种活性亲电试剂，已知在ATRA暴露后会在体内外形成共价蛋白加合物，并且可能靶向参与促性腺激素释放激素（GnRH）诱导的钙信号传导及随后LH释放的蛋白质。为了验证这一假设，将LβT2垂体细胞暴露于300μM DACT中24小时，并用荧光显微镜检查GnRH诱导的细胞内钙变化和LH释放情况。暴露于DACT的LβT2细胞中，GnRH诱导的细胞内钙瞬变明显减弱，并且对GnRH的反应中LH释放显著减少。DACT似乎导致LβT2细胞中对咖啡因敏感的兰尼碱受体操纵的钙储存选择性减少，而不是对毒胡萝卜素敏感的内质网（ER）钙储存减少。这种敏感性与DACT形成的共价蛋白加合物相关，这是通过质谱法测定的。通过质谱法鉴定出内质网蛋白57（ERp57）是内质网中DACT加合的靶点，它可能介导DACT对GnRH诱导的钙信号传导抑制和LH释放抑制的作用。添加钙离子载体（A23187）后，DACT处理的细胞中对GnRH的细胞内钙反应和LH释放得以恢复。这些数据表明，DACT在内质网中参与钙处理的蛋白质上形成加合物，并且这个关键信号系统的功能障碍与对GnRH的正常敏感性丧失以及随后LH释放减少有关。
