Purification and Comparative Neurotoxicity of the Trichothecenes Satratoxin G and Roridin L2 from Stachybotrys Chartarum

Abstract

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 × 10⁵ 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 µg/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.

This research was funded by the Michigan State University Respiratory Research Initiative, Michigan State University Foundation Strategic Partnership Grant and Public Health Service Grant ES03358 (JJP) from the National Institute for Environmental Health Sciences. We thank Evelyne Mbandi, Lijun Chen, Lori Bramble, Sarah Godbehere, and Mary Rosner. Animal studies were conducted in accordance with National Institutes of Health guidelines as overseen by the All University Committee on Animal Use and Care at Michigan State University.