| Antibodies for macrocyclic trichothecene, satratoxin G (SG), were produced upon immunization of rabbits with SG bis-hemisuccinate conjugated to BSA. The antibodies could detect free SG using a competitive direct-ELISA with a range of detection from 0.1 to 100 ng/ml. The observed antibody cross-reactivity may facilitate simultaneous detection of other satratoxins and to a lesser extent, other macrocyclic trichothecenes. Methanol content up to 20% in samples did not affect the immunoassay and this stability expands applicability of the antibodies. The capacity of representative macrocyclic trichothecenes to alter TNF-α and IL-6 production and viability was assessed in a murine macrophage model. Macrocyclic trichothecenes can superinduce the proinflammatory cytokine, TNF-α, at low cytotoxic concentrations, whereas these compounds are cytotoxic and reduce cytokine production at higher concentrations. These immunomodulatory effects were observed at relatively low (ng/ml) concentrations, suggesting that macrocyclic mycotoxins may pose a hazard to humans exposed to Stachybotrys. In order to expand understanding of how trichothecenes affect gene regulation, we have isolated vomitoxin (VT)- and SG-responsive genes, macrophage inflammatory protein-2 (MIP-2) and complement 3a receptor (C3aR), from a murine macrophage cell line using differential display-PCR. Both VT and SG up-regulated expression of MIP-2, which is a chemokine responsible for chemotaxis of neutrophils to inflammation site, whereas VT selectively up-regulated C3aR, which is a receptor involved in activation by complement. VT up-regulated splenic MIP-2 mRNA and its protein in serum in a mouse model. To investigate roles of mitogen-activated protein kinase (MAPK) in cytokine superinduction by VT, the effects of VT on MAPK activation, as well as the relationship of MAPKs to VT-induced mRNA expression, TNF-α promoter activity, TNF-α mRNA stability, and TNF-α protein production were assessed in RAW 264.7 murine macrophage cells. The results indicate that VT may superinduce TNF-α expression by increasing its transcripts and mRNA stability through activation of MAPK. Trichothecene-induced activation of MAPK may play a critical role in superinduction of proinflammatory cytokine, TNF-α, and succeeding pathologic events. |
