muconomycin-a and deoxynivalenol

Macrocyclic trichothecenes have been frequently detected in fungi in water-damaged buildings and exhibited higher toxicity than the well-studied trichothecenes; however, the mechanism underlying their toxicity has been poorly understood. In this study, transcriptional regulation of the cytokine interleukin (IL)-8 by a macrocyclic trichothecene, verrucarin A (VA), in human monocytic THP-1 cells is reported. Consistent with previous findings, VA was 100-fold more cytotoxic than deoxynivalenol (DON), while ochratoxin A (OA) was not cytotoxic. In cells transduced with the wild-type IL-8 promoter luciferase construct, VA induced a biphasic dose response composed of an upregulation of luciferase expression at low concentrations of 0.01-1 ng/ml and a downregulation at high levels of 10 ng/ml and higher. In contrast, DON induced a sigmoid-shaped dose response with the EC50 of 11.6 ng/ml, while OA did not markedly affect the IL-8 expression. When cells were transduced with IL-8 promoter with a mutation of transcription factor nuclear factor-κB (NF-κB)-binding site, VA (1 ng/ml), DON (1000 ng/ml), and tumor necrosis factor (TNF) α (20 ng/ml)-induced luciferase expression were impaired. In addition, the NF-κB inhibitor caffeic acid phenethyl ester inhibited VA-, DON-, and TNFα-induced luciferase expression. Mutation of the CCAAT/enhancer-binding protein (CEBP) β binding site of the IL-8 promoter affected only DON-, but not VA- and TNFα-induced luciferase expression. Taken together, these results suggested that VA activated IL-8 promoter via an NF-κB-dependent, but not CEBPβ-dependent, pathway in human monocytes.

Topics: CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Interleukin-8; Lentivirus; Luciferases; Monocytes; NF-kappa B; Ochratoxins; Promoter Regions, Genetic; Signal Transduction; Trichothecenes; Tumor Necrosis Factor-alpha; Up-Regulation

The macrocyclic trichothecenes are a group of potent protein synthesis inhibitors that have been encountered in indoor air and food as a result of infestation by the fungus Stachybotrys. To evaluate the capacity of these mycotoxins to alter immune functions, the effects of satratoxin G, H, F, roridin A, and verrucarin A on interleukin 2 (IL-2) production and viability were evaluated in a murine T-cell model. EL-4 thymoma cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin and concurrently exposed to various concentrations of the trichothecenes. Enzyme-linked immunosorbent assay (ELISA) of supernatants revealed that IL-2 concentrations at 24 and 72 h were significantly increased in cultures that were incubated in the presence of 0.5 to 1 ng/ml of satratoxin H, 1 to 5 ng/ml of isosatratoxin F, 0.1 to 0.5 ng/ml of roridin A, and 0.25 to 0.5 ng/ml of verrucarin A. However, IL-2 levels at these time points were significantly depressed when incubated in the presence of higher concentrations of satratoxin G (> or =2.5 ng/ml), satratoxin H and isosatratoxin F (> or =5 ng/ml), and roridin A and verrucarin A (> or =1 ng/ml). Cell viability, as measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, was depressed by each of the trichothecenes in a concentration-dependent manner. MTT responses were significantly decreased by as little as 0.5 ng/ml satratoxin G, roridin A, and verrucarin A and by 2.5 ng/ml of isosatratoxin F and satratoxin H. When these data were compared to those found in EL-4 cells for the 8-ketotrichothecene vomitoxin (deoxynivalenol), a common food contaminant, the macrocyclic trichothecenes were at least 100 times more potent. The results indicate that, at low concentrations, macrocyclic trichothecenes as a group could superinduce IL-2 production even while partially decreasing cell viability, whereas higher concentrations suppressed cytokine production and were markedly cytotoxic. The capacity of these compounds to dysregulate cytokine production in a biphasic fashion may play an etiologic role in outbreaks of human illnesses associated with indoor Stachybotrys contamination.

Topics: Air Microbiology; Air Pollution, Indoor; Animals; Cell Line; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Food Microbiology; Humans; Immunosuppressive Agents; Interleukin-2; Mice; Protein Synthesis Inhibitors; Stachybotrys; T-Lymphocytes, Helper-Inducer; Trichothecenes

The effects of trichothecene structure on cytokine secretion and gene expression were assessed in primary CD4+ T-cells from murine spleen. CD4+ T-cells were stimulated with concanavalin A (Con A) for 2 or 7 days in the presence of various concentrations of the trichothecenes, vomitoxin (VT or deoxynivalenol), nivalenol (NIV), 15-acetyl deoxynivalenol (15-ADON), 3-acetyl deoxynivalenol (3-ADON), T-2 toxin (T-2) and verrucarin A (Ver A). Culture supernatants were subsequently analyzed for interleukin (IL)-2, IL-4 and IL-5 by ELISA. At day 2, all trichothecenes were found to have inhibited production of IL-2, IL-4, and IL-5. However, at day 7, supernatant IL-2 was significantly increased (2-5.5-fold) in cultures containing VT, NIV, 3-ADON, and 15-ADON at 250, 250, 2500, and 1000 ng/ml doses, respectively, when compared to control Con A-stimulated cultures; significant increases in IL-2 were not observed with T-2 and Ver-A. Similarly, at day 7, IL-4 and IL-5 were significantly increased in the presence of VT (100 ng/ml), NIV (100 ng/ml), 3-ADON (1000 ng/ml), 15-ADON (500 ng/ml), T-2 (1 ng/ml), and Ver A (50 pg/ml, only IL-5) when compared to control cultures. IL production was inhibited at trichothecene concentrations exceeding the aforementioned optima. When total RNA of 2-day cultures was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) in conjunction with Southern analysis, IL-2 mRNA was also found to be superinduced by VT (50 and 100 ng/ml), NIV (50, 100 and 250 ng/ml), 3-ADON (1500 ng/ml), 15-ADON (100 ng/ml), T-2 (0.5 ng/ml) and Ver A (25, 50 and 100 pg/ml); IL-4 mRNA by VT (50 ng/ml), NIV (50 ng/ml), and Ver A (25, 50 and 100 pg/ml); IL-5 mRNA by VT (50 ng/ml); and IL-6 mRNA by 15-ADON (100 ng/ml) and Ver A (50 pg/ml). As the trichothecene concentration increased from these levels, inhibition of mRNA transcript levels was also observed for many of the interleukins. Taken together, the results suggest that trichothecenes as a group can either inhibit or superinduce both IL secretion and mRNA levels in CD4+ T-cells. Superinduction exhibited a rank order of macrocyclic > type A > type B trichothecenes and was dependent on acylation of the trichothecene nucleus.

Topics: Acylation; Animals; Antibiotics, Antineoplastic; Blotting, Southern; CD4-Positive T-Lymphocytes; Cells, Cultured; Concanavalin A; Cytokines; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Interleukin-2; Interleukin-4; Interleukin-5; Mice; RNA, Messenger; Structure-Activity Relationship; T-2 Toxin; Transcription, Genetic; Trichothecenes