Stachybotrys chartarum (SC) is a mycotoxin-producing indoor mold, growing on humidified cellulose-based materials in water-damaged buildings. For the past two decades, SC has been linked to indoor mold-related health problems in residents and office personnel. SC produces highly toxic mycotoxins including several macrocyclic trichothecenes. Submicron sized trichothecene-containing air aerosols have been detected in moldy indoor environments. SC trichothecenes have been tested positive in the serum of workers exposed to moldy working environments. Experimentally, purified SC trichothecene or crude SC toxin extract and SC spores haven been shown to induce extensive cytotoxicity in various cultured cell lines or lung tissue toxicity in animal models (mice and rats).
The inhaled toxin-containing aerosols pass nasal cavity and deposit in the deeper alveolar where resident macrophages could interact via phagocytosis and induction of host response. To study the toxicity of SC trichothecenes to alveolar macrophages, we used the murine alveolar macrophage cell line MH-S and the methanol extracted toxin-mixture from the SC spores. SC toxin-treated MH-S cells showed apoptosis (including DNA ladder fragmentation, activation of Caspase 3/7, and sub-G1 peak in flow cytometry analysis), DNA damage (comet assay and micronuclei formation assay), and activation of stress-inducible MAPK and DNA-damage responsive p53 signaling pathways (Chapter II).
To study the underlying mechanism of interaction of alveolar macrophage cells with SC toxin, genome-wide transcriptional changes were evaluated in SC toxin-treated MH-S cell line using gene expression microarray and qRT-PCR (Chapter III). The results implied that SC toxin regulated various genes involved in multiple cellular processes (cell growth, proliferation and death, inflammatory/immune response, genotoxic stress and oxidative stress) and signal transduction pathways (MAPK-, NF-kB-, TNF-, and p53-mediated signaling), indicating that SC toxin activated cascades of cellular signaling events leading to apoptosis.
Several trcichothecene mycotoxins including both simple (T-2 toxin and DON) and macrocyclic (satratoxin G) types are known to bind to ribosome and inhibit protein synthesis. Since ribosomal proteins have also been reported to be involved in extra-ribosomal functions including cell cycle progression, cell proliferation, turmorigenesis, and apoptosis, we hypothesized that ribosomal component proteins might play a critical role in SC toxin (trichothecene)-induced toxicity in alveolar macrophage. In order to test this, we used yeast mat-a haploid yeast gene knockout library as a tool. The library was screened using both the SC toxin mixture and pure macrocyclic trichothecenes (Verrucarin A and Roridin A). The results revealed multiple ribosome-related genes and DNA damage/repair genes which could mediate SC toxin-induced toxicity, and could function as transducers in SC toxin-induced apoptosis signaling events (Chapter IV).