Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824-1224, USA.
J Toxicol Environ Health A. 2009;72(20):1242-51. doi: 10.1080/15287390903129234.
Satratoxin G (SG), a macrocyclic trichothecene produced by Stachybotrys chartarum, induces apoptosis in cultured neuronal cells as well as nasal olfactory sensory neurons (OSN) in the nose and brain of mice exposed intranasally to this toxin. The purposes of this study were to (1) develop a facile method for production and purification of both SG and its putative biosynthetic precursor, roridin L2 (RL2), from S. chartarum cultures and (2) compare their relative neurotoxicity in vitro and in vivo. Stachybotrys chartarum 29-58-17 was cultured in Fernbach flasks on rice (5 x 10(5) spores/250 g rice) for 4 to 6 wk. Following extraction with acetonitrile, the extract was dried, dissolved in dichloromethane, and subjected to Michel-Miller silica-gel chromatography using a stepwise acetonitrile-dichloromethane gradient with SG and RL2 eluting in the 30 and 40% acetonitrile fractions, respectively. Purification of the two compounds was completed by C18 semipreparative reverse-phase liquid chromatography using an acetonitrile-water gradient, and purity was confirmed by electrospray ionization/collision-induced dissociation (ESI-CID) tandem mass spectroscopy. Although viability significantly decreased in PC-12 neuronal cells treated with 10 to 25 ng/ml of SG, RL2 at concentrations up to 1000 ng/ml was not toxic. Flow cytometry and agarose DNA fragmentation assays revealed that SG at 10 to 25 ng/ml induced apoptotic death in the PC-12 cells, while RL2 at concentrations up to 1000 ng/ml was without effect. In a similar fashion, intranasal exposure of mice (female B6C3F1) to SG at 100 microg/kg body weight (bw) induced marked OSN apoptosis and atrophy of the olfactory epithelium, whereas RL2 at the equivalent dose did not exhibit toxicity. Taken together, an optimized protocol for production and isolation of trichothecenes from S. chartarum cultures is described and further demonstrates that while the macrocyclic SG was neurotoxic in vitro and in vivo, its biosynthetic precursor, RL2, was nontoxic.
曲古抑菌素 G（SG）是一种由链格孢产生的中环三萜烯，可诱导培养的神经元细胞以及经鼻腔暴露于该毒素的小鼠鼻腔和大脑中的嗅感觉神经元（OSN）凋亡。本研究的目的是：（1）从链格孢培养物中开发一种简便的方法来生产和纯化 SG 及其假定生物合成前体roridin L2（RL2）；（2）比较它们在体外和体内的相对神经毒性。链格孢 29-58-17 在米（5 x 10(5)个孢子/250 克米）的 Fernbach 瓶中培养 4 至 6 周。用乙腈提取后，提取物干燥，溶解在二氯甲烷中，用 Michel-Miller 硅胶柱层析，用逐步的乙腈-二氯甲烷梯度洗脱，SG 和 RL2 分别在 30%和 40%乙腈部分洗脱。用乙腈-水梯度通过 C18 半制备反相液相色谱法完成两种化合物的纯化，通过电喷雾电离/碰撞诱导解离（ESI-CID）串联质谱法确认纯度。虽然用 10 至 25 ng/ml 的 SG 处理 PC-12 神经元细胞后细胞活力明显下降，但 RL2 的浓度高达 1000 ng/ml 时没有毒性。流式细胞术和琼脂糖 DNA 片段化试验显示，10 至 25 ng/ml 的 SG 在 PC-12 细胞中诱导凋亡性死亡，而浓度高达 1000 ng/ml 的 RL2 则无影响。同样，用 100μg/kg 体重（bw）的 SG 对雌性 B6C3F1 小鼠进行鼻腔暴露，会导致明显的 OSN 凋亡和嗅上皮萎缩，而等效剂量的 RL2 则没有毒性。综上所述，描述了一种从链格孢培养物中生产和分离三萜烯的优化方案，进一步表明中环 SG 在体外和体内具有神经毒性，而其生物合成前体 RL2 则没有毒性。
