sq-23377 and deoxynivalenol

Previously, we studied the effects of deoxynivalenol (DON) and tributyltin oxide (TBTO) on whole genome mRNA expression profiles of human T lymphocyte Jurkat cells. These studies indicated that DON induces ribotoxic stress and both DON and TBTO induced ER stress which resulted into T-cell activation and apoptosis. The first goal of the present study was to provide final proof for these mode of actions by comparing the effects of 6 h exposure to DON and TBTO on mRNA expression to those of positive controls of ribotoxic stress (anisomycin), ER stress (thapsigargin) and T cell activation (ionomycin). Genes affected by anisomycin and the majority of genes affected by thapsigargin were affected in the same direction by DON and TBTO, respectively, confirming the expected modes of action. Pathway analysis further sustained that DON induces ribotoxic stress and both DON and TBTO induce unfolded protein response (UPR), ER stress, T cell activation and apoptosis. The second goal was to assess whether DON and/or TBTO affect other pathways above those detected before. TBTO induced groups of genes that are involved in DNA packaging and heat shock response that were not affected by thapsigargin. DON did not affect other genes than anisomycin indicating the effect of DON to be restricted to ribotoxic stress. This study also demonstrates that comparative gene expression analysis is a very promising tool for the identification of modes of action of immunotoxic compounds.

Topics: Anisomycin; Apoptosis; Carcinogens; Cell Survival; Chromosome Mapping; Data Interpretation, Statistical; Endoplasmic Reticulum Stress; Gene Expression Profiling; Heat-Shock Proteins; Humans; Ionomycin; Jurkat Cells; Microarray Analysis; Mitochondrial Proteins; NF-E2-Related Factor 2; Nucleic Acid Synthesis Inhibitors; RNA, Neoplasm; T-Lymphocytes; Thapsigargin; Trialkyltin Compounds; Trichothecenes

The mechanisms by which trichothecene mycotoxins cause immunological effects in leukocytes such as cytokine up-regulation, aberrant IgA production, or apoptotic cell death are not fully understood. In the present study, mRNA differential display analysis was used to evaluate changes in gene expression induced by the trichothecene vomitoxin (VT or deoxynivalenol) in a T-cell model, the murine EL-4 thymoma, that was stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION). Ten differentially expressed fragments of cDNA were isolated and sequenced and three of these were identified as the known genes GRP78/BiP, P58(IPK), and RAD17. Most notably, expression of GRP78/BiP (a 78-kDa glucose-regulated protein), a stress-response gene induced by agents or conditions that adversely affect endoplasmic reticulum (ER) function, was found to decrease in VT-exposed cells. Competitive RT-PCR analysis revealed that 250 ng/ml VT decreased GRP78/BiP mRNA expression in both unstimulated and PMA/ION-stimulated EL-4 cells at 6 and 24 h after VT treatment. Western blotting confirmed that VT (50 to 1000 ng/ml) also significantly diminished GRP/BiP protein levels in a dose-response manner in PMA/ION-stimulated cells. GRP78/BiP has been shown to play a role in regulation of protein folding and secretion, and to protect cells from apoptosis. When PMA/ION-stimulated cells were incubated with 50 to 1000 ng/ml VT for 24 h, 200-bp DNA laddering, a hallmark of apoptosis, increased in a dose-dependent manner. In addition to GRP78, mRNA expression of the cochaperone P58(IPK), which is the 58-kDa cellular inhibitor of the double-stranded RNA-regulated protein kinase (PKR), was also shown to be suppressed by VT-treatment. GRP78 and P58(IPK) are critical for maintenance of cell homeostasis and prevention of apoptosis. The down-regulation of these molecular chaperones by VT represent a novel observation and has the potential to impact immune function at multiple levels.

Topics: Animals; Blotting, Western; Calcimycin; Carrier Proteins; DNA Fragmentation; DNA, Complementary; Down-Regulation; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Heat-Shock Proteins; HSP40 Heat-Shock Proteins; Ionomycin; Ionophores; Mice; Molecular Chaperones; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thapsigargin; Thymoma; Trichothecenes

Trichothecene mycotoxins have been reported to suppress or superinduce cytokine mRNA expression by leukocytes both in vitro and in vivo. Modulation of transcription factor activities may be critical for these observations. Here, the effect of trichothecene vomitoxin (VT, deoxynivalenol) on activator protein-1 (AP-1) activity was determined in the murine EL-4 thymoma. Electrophoretic mobility shift assay (EMSA) revealed that VT modulated AP-1 binding activity in a concentration- and time-dependent manner when using a synchronous model in which VT was added concurrently with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION) to EL-4 cells. Induction of AP-1 binding activity by PMA/ION was suppressed in the presence of VT for a short period (1 to 12 h), but was enhanced upon prolonged VT exposure (48 to 72 h). VT also enhanced AP-1 binding activity when added to the cell culture 12 h after PMA/ION activation (delayed synchronous model). Using specific antibodies against AP-1 complex proteins, it was demonstrated by gel supershift assay that VT preferentially affected phosphorylated c-Jun, Jun B, c-Fos, and Fra-2 binding activities, whereas it did not alter Jun D and Fra-1 binding. A transient transfection assay demonstrated that these increased binding activities are associated with enhanced AP-1 transactivation potential. Elevation of AP-1 activity may contribute to cytokine dysregulation and immunotoxic effects associated with exposure to trichothecene mycotoxins such as VT.

Topics: Animals; DNA-Binding Proteins; Down-Regulation; Fos-Related Antigen-2; Ionomycin; Kinetics; Mice; Phosphorylation; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Tetradecanoylphorbol Acetate; Thymoma; Transcription Factor AP-1; Transcription Factors; Transcriptional Activation; Trichothecenes; Tumor Cells, Cultured

The effects of the trichothecene vomitoxin (VT) on the kinetics of cell proliferation and cytokine production were evaluated in murine CD4+ T cells. The CD4+ cultures were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin to activate protein kinase C and increase cytoplasmic free calcium, respectively, in a range of VT concentrations. Total and viable cell counts at 3, 5, 7, 9, and 11 days revealed delayed or impaired cell proliferation in cultures containing between 50 and 100 ng/ml VT, with complete inhibition being observed at 250 and 500 ng/ml of VT. The VT concentration required to inhibit protein synthesis in a 3-day culture by 50% in this model was 40 ng/ml. When enzyme-linked immunosorbent assay (ELISA) was used to quantitate cytokines, IL-2 levels in control cultures were highest at Day 1 and declined rapidly thereafter, whereas, in VT groups, IL-2 levels were highest at Day 3 and remained elevated up to 11 days. IL-2 levels were elevated by continuous exposure to 100-500 ng/ml of VT with more than 100-fold differences being observed between control and 250 ng/ml VT from Days 5 to 11. When IL-2 levels were expressed on a per viable cell basis, increases were even more marked with as much as 6 log differences being observed between the treatments at 250-500 ng/ml VT and control cultures at Day 7. Supernatant IL-4 and IL-5 levels were also elevated by 100 and 250 ng/ml VT in a dose- and time-dependent fashion compared to control cultures, whereas 500 ng/ml VT was inhibitory. When relative IL mRNA abundance was analyzed during the first 3 days of culture by reverse transcriptase-polymerase chain reaction (RT-PCR) in conjunction with Southern hybridization analysis, IL-2 mRNA levels in Days 1, 2 and 3 in cultures containing 100 and 250 ng/ml VT were greater than corresponding controls. IL-2 mRNA abundance in both control and VT-treated cultures was maximal at Day 1 and decreased rapidly thereafter in controls, whereas much slower rates of IL-2 disappearance were noted in 100 and 250 ng/ml of VT. IL-4 and IL-5 mRNA levels at VT doses of 50 and 100 ng/ml were also elevated compared to controls. Pulsed VT (8 to 48 hr) or cycloheximide (4 to 48 hr) exposure of CD4+ cells enhanced supernatant levels of IL-2 but not IL-4 upon incubation for 24 hr in fresh medium. This effect was not persistent. Taken together, VT enhanced and/or delayed peak IL-2, IL-4, and IL-5 gene expression and secretion in CD4+ T cells stimulated with PMA and ionomyc

Topics: Animals; CD4-Positive T-Lymphocytes; Cell Cycle; Drug Synergism; Female; Interleukin-2; Interleukin-4; Interleukin-5; Interleukins; Ionomycin; Lymphocyte Activation; Mice; Mice, Inbred C57BL; RNA, Messenger; Tetradecanoylphorbol Acetate; Trichothecenes