Transcriptomics analysis of primary mouse thymocytes exposed to bis(tri-n-butyltin)dioxide (TBTO).

The biocide bis(tri-n-butyltin)oxide (TBTO) causes thymus atrophy in rodents and is toxic to many cell types of which thymocytes are the most sensitive. To obtain insight in the mechanisms of action of TBTO, we exposed primary mouse thymocytes in vitro for 3, 6 and 11 h to 0.1, 0.5, 1 and 2 µM TBTO. Subsequently, the cells were subjected to whole-genome gene expression profiling. Biological interpretation of the gene expression data revealed that TBTO affects a wide range of processes. Cell proliferation related genes were downregulated by all treatments except for 3 and 6 h 0.5 µM TBTO which upregulated these genes. Treatment with TBTO resulted in upregulation of genes involved in endoplasmatic reticulum (ER) stress, NFkB and TNFα pathways, and genes involved in DNA damage, p53 signaling and apoptosis. Remarkably, TBTO also increased the expression of genes that are known to be upregulated during T cell activation or during negative selection of thymocytes. The effect of TBTO on expression of genes involved in ER stress and apoptosis was confirmed by qPCR. Induction of the T cell activation response was corroborated by demonstrating that TBTO exposure resulted in translocation of NFAT to the nucleus, which is an essential event for T cell activation.

The effects of deoxynivalenol on gene expression in the murine thymus.

Deoxynivalenol (DON) is a mycotoxin produced by several Fusarium species and is often detected in grains. Because of its high abundance, there has been a large interest in the effects of DON in animals and humans. DON is known to be immunosuppressive at high concentrations and immunostimulatory at low concentrations. The present study aimed to acquire insight into the modes of action of DON. For this, C57Bl6 mice were orally exposed to 5, 10, or 25mg/kg bw DON for 3, 6, or 24h and thymuses were subjected to genome-wide expression microarray analysis. Gene set enrichment analysis (GSEA) demonstrated that DON downregulated genes involved in proliferation, mitochondria, protein synthesis, and ribosomal proteins. Furthermore, GSEA showed a selective downregulation of genes highly expressed at the early precursor thymocytes stage. This indicates that early precursor thymocytes, particularly at the double-positive CD4+CD8+ stage, are more vulnerable to DON than very early or late precursor thymocytes. There was a large overlap of genes upregulated by DON with genes previously reported to be either upregulated during T cell activation or upregulated during negative selection of thymocytes that recognize "self-antigens". This indicates that DON induces cellular events that also occur after activation of the T cell receptor, for example, release of calcium from the endoplasmatic reticulum. This T cell activation in the thymus then evokes negative selection and depletion of thymocytes, which provides a plausible explanation for the high sensitivity of the thymus for DON exposure. The expression patterns of four genes indicative for some of the processes that were affected after DON treatment were confirmed using real-time PCR. Immunocytological experiments with primary mouse thymocytes demonstrated the translocation of NFAT from the cytoplasm into the nucleus upon exposure top DON, thus providing further evidence for the involvement of T cell activation.

Proteomic analysis of mouse thymoma EL4 cells treated with bis(tri-n-butyltin)oxide (TBTO).

Here, we report the results of proteomic analysis of the mouse thymoma EL4 cell line exposed to bis(tri-n-butylin)oxide (TBTO), an immunotoxic organotin compound. The objective of the work was to examine whether TBTO affects the expression of proteins in this cell line and to compare the differentially expressed proteins with the corresponding mRNA expression data. The identified proteins were quantified using a label-free quantitative method based on counting the observed peptides as an index of protein abundance. The calculation of the ratio of peptides obtained from exposed and control samples allowed us to evaluate the effect of TBTO on protein expression and to compare these results to those obtained in gene expression profiling studies. Correlation of some of the differentially expressed proteins and their corresponding mRNAs was observed. The analysis of the protein ratios revealed that 12 proteins were significantly affected. These proteins included cytoskeleton proteins myosin-9, spectrin beta 2 and plectin 8. The first two proteins were down-regulated 3-fold, whereas the third was up-regulated 2-fold. Ras-related Rab1, a GTP binding protein and T-complex protein-1 subunit alpha, a chaperonin, were decreased 2- and 3.6-fold, respectively. The ribosomal S10 and eukaryotic translation factor (eIf4G1), which are involved in protein synthesis, were down-regulated 2.6- and 3.7-fold, respectively. Also, proteins involved in splicing of pre-mRNA and in transcription, splicing factor arginine/serine-rich 2 and chromodomain-helicase-DNA binding protein 4 (Chd4), were decreased 2.6- and 4.5 times, respectively. Nuclear RNA helicase II was reduced 2.8-fold. Finally, prothymosin-alpha (ProTalpha), an essential protein for cell proliferation, and a protein similar to ProTalpha, (with a molecular weight and a pI (3.54) comparable to that of ProTalpha) were also down-regulated 6-and 8-fold, respectively. We propose that the observed down-regulation of the expression level of ProTalpha in the TBTO-exposed cells could account for the previously reported anti-proliferative effect of TBTO.