anisomycin 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

Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a critical upstream mediator of the ribotoxic stress response (RSR) to the trichothecene deoxynivalenol (DON) and other translational inhibitors. Here, we employed HeLa cell lysates to: (1) characterize PKR's interactions with the ribosome and ribosomal RNA (rRNA); (2) demonstrate cell-free activation of ribosomal-associated PKR and (3) integrate these findings in a unified model for RSR. Robust PKR-dependent RSR was initially confirmed in intact cells. PKR basally associated with 40S, 60S, 80S and polysome fractions at molar ratios of 7, 2, 23 and 3, respectively. Treatment of ATP-containing HeLa lysates with DON or the ribotoxins anisomycin and ricin concentration-dependently elicited phosphorylation of PKR and its substrate eIF2α. These phosphorylations could be blocked by PKR inhibitors. rRNA immunoprecipitation (RNA-IP) of HeLa lysates with PKR-specific antibody and sequencing revealed that in the presence of DON or not, the kinase associated with numerous discrete sites on both the 18S and 28S rRNA molecules, a number of which contained double-stranded hairpins. These findings are consistent with a sentinel model whereby multiple PKR molecules basally associate with the ribosome positioning them to respond to ribotoxin-induced alterations in rRNA structure by dimerizing, autoactivating and, ultimately, evoking RSR.

Topics: Anisomycin; Cloning, Molecular; eIF-2 Kinase; HeLa Cells; Humans; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Synthesis Inhibitors; Ribosomes; Ricin; RNA, Ribosomal, 18S; RNA, Ribosomal, 28S; Sequence Analysis, RNA; Trichothecenes

The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥25ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥10ng/ml) and ribosome-inactivating protein ricin (≥300ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism.

Topics: Animals; Anisomycin; Apoptosis; Blotting, Western; Caspase 8; Cathepsin L; Cells, Cultured; Enzyme Activation; Mice; Mitogen-Activated Protein Kinases; Nucleic Acid Synthesis Inhibitors; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-hck; Ricin; RNA Cleavage; RNA, Ribosomal; Trichothecenes; Tumor Suppressor Protein p53

CCAAT/enhancer-binding protein homologous protein (CHOP) is a crucial stress-responsive factor in various mucosal injuries, including cellular translational stress conditions. In this study, chemical ribosome-inactivating stresses were assessed for their effects on stress-inducible CHOP expression and its association with epithelial inflammatory cytokine production. Several representative ribotoxic agents (deoxynivalenol, anisomycin, and 15-acetyldeoxynivalenol) enhanced CHOP expression and its nuclear translocation in human intestinal epithelial cells. Moreover, CHOP was a strong positive regulator of IL-8 production, but CHOP-mediated IL-8 production was inversely associated with expression of the mucosal regulatory factor peroxisome proliferator-activated receptor γ (PPARγ). Based on our recent report that PPARγ is a negative regulator of mRNA stability of IL-8, PPARγ was linked to a notable mRNA stabilizing protein, HuR, since ribotoxin-induced IL-8 mRNA is stabilized by HuR protein. Expression of exogenous PPARγ suppressed ribotoxin-triggered cytoplasmic translocation of HuR. In contrast, PPARγ-regulating CHOP was a positive modulator of HuR protein export from nuclei. Taken together, the results indicate that ribotoxin-induced CHOP protein is positively associated with production of proinflammatory cytokine IL-8, but it downregulates PPARγ action, subsequently allowing the cytosolic translocation of HuR protein and stabilization of IL-8 mRNA in gut epithelial cells. CHOP and PPARγ may represent critical mechanistic links between ribotoxic stress and proinflammatory cytokine production, and they may have a broader functional significance with regard to gastrointestinal stresses by toxic mucosal insults.

Topics: Anisomycin; Antigens, Surface; Blotting, Western; Cell Line; ELAV Proteins; ELAV-Like Protein 1; Enzyme-Linked Immunosorbent Assay; Gene Expression; Gene Expression Regulation; Humans; Immunity, Mucosal; Interleukin-8; Intestinal Mucosa; Microscopy, Confocal; PPAR gamma; Protein Synthesis Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; Transcription Factor CHOP; Transfection; Trichothecenes

The effects of the ribotoxic trichothecene deoxynivalenol (DON) on mitogen-activated protein kinase (MAPK)-mediated IL-8 expression were investigated in cloned human monocytes and peripheral blood mononuclear cells (PBMC). DON (250 to 1000 ng/ml) induced both IL-8 mRNA and IL-8 heteronuclear RNA (hnRNA), an indicator of IL-8 transcription, in the human U937 monocytic cell line in a concentration-dependent manner. Expression of IL-8 hnRNA, mRNA and protein correlated with p38 phosphorylation and was completely abrogated by the p38 MAPK inhibitor SB203580. DON at 500 ng/ml similarly induced p38-dependent IL-8 protein and mRNA expression in PBMC cultures from healthy volunteers. Significantly increased IL-6 and IL-1beta intracellular protein and mRNA expression was also observed in PBMC treated with DON (500 ng/ml) which were also partially p38-dependent. Flow cytometry of PBMC revealed that DON-induced p38 phosphorylation varied among individuals relative to both threshold toxin concentrations (25-100 ng/ml) and relative increases in percentages of phospho-p38(+) cells. DON-induced p38 activation occurred exclusively in the CD14(+) monocyte population. DON was devoid of agonist activity for human Toll-like receptors 2, 3, 4, 5, 7, 8 and 9. However, two other ribotoxins, emetine and anisomycin, induced p38 phosphorylation in PBMC similarly to DON. Taken together, these data suggest that (1) p38 activation was required for induction of IL-8 and proinflammatory gene expression in the monocyte and (2) DON induced p38 activation in human monocytes via the ribotoxic stress response.

Topics: Anisomycin; Emetine; Gene Expression Regulation; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Leukocytes, Mononuclear; Monocytes; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Synthesis Inhibitors; RNA, Messenger; Trichothecenes; U937 Cells

Trichothecene mycotoxins and other protein synthesis inhibitors activate mitogen-activated protein kinase (MAPKs) via a mechanism that has been termed the "ribotoxic stress response." MAPKs are believed to mediate the leukocyte apoptosis that is observed following experimental exposure to these chemical agents in vitro and in vivo. The purpose of this research was to test the hypothesis that double-stranded, RNA-activated protein kinase R (PKR) is a critical upstream mediator of the ribotoxic stress response induced by the trichothecene deoxynivalenol (DON) and other translational inhibitors. DON was found to readily induce phosphorylation of JNK 1/2, ERK 1/2, and p38 in the murine macrophage RAW 264.7 cell line, within 5 min of culture addition, in a concentration-dependent fashion. Effects were maximal from 15 to 30 min and lasted up to 6 h. The translational inhibitors anisomycin and emetine also had similar effects when added to cultures at equipotent concentrations to DON. DON rapidly activated PKR within 1 to 5 min, as evidenced by autophosphorylation and by phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha). Interestingly, the latter effect was associated with rapid degradation of eIF2alpha. Pretreatment of RAW 264.7 cells with two inhibitors of PKR, 2-aminopurine (2-AP) or adenine (Ad), markedly impaired MAPK phosphorylation in RAW 264.7 cells according to the following rank order JNK>p38>ERK. The capacity of DON to induce MAPK phosphorylation was also markedly suppressed in a stable transformant of the human promonocytic U-937 cell line containing an antisense PKR expression vector. This suppression followed a rank order of JNK>p38>ERK in this PKR-deficient cell line when compared to control cells transfected with vector only. Apoptosis induction by DON and two other translational inhibitors, anisomycin and emetine, was almost completely abrogated in PKR-deficient cells. Together, the results indicate that PKR plays a critical upstream role in the ribotoxic stress response inducible by translational inhibitors.

Topics: Animals; Anisomycin; Apoptosis; Dose-Response Relationship, Drug; eIF-2 Kinase; Emetine; Enzyme Activation; Humans; Macrophages; Mice; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Monocytes; Protein Biosynthesis; Protein Synthesis Inhibitors; Ribosomes; Trichothecenes; U937 Cells