Effects of Mycotoxins on Mononuclear Cells (MNCs) in Normal Blood, T-Cell Leukemia and Lymphoma Cell Lines.

Abstract Background: T cell lymphoma, comprising 15% of diagnosed US lymphomas, currently has no standard of care in the relapsed setting. Exposure to molds diminishes the numbers of T-helpers cells in peripheral blood. Aspergillus and Penicillium are known to produce small molecules called mycotoxins. Mycotoxins have previously been associated with immunosuppressive effects (Sutton et al., 1994, 1995), and a reduction of cytokine secretion (Rossano et al., 1999.) This study investigates the effects of two of these mycotoxins, Gliotoxin and Patulin, on the cellular components of mononuclear cells in peripheral blood of normal donors and T-cell lymphoma cells, examining viability and apoptotic damage in CD3+ (T cells), CD4+ (cytotoxic t cells), CD8+ (t helper cells), and CD16+ (NK cells), BDCA1+ and BDCA2+ (dendritic cells), CD14+ (monocytes), and CD19+ (B cells). Methods: Two mycotoxins, Gliotoxin and Patulin were solubilized into methanol or ethanol and cultured with either normal ficolled blood samples, T-cell lymphoma or T-cell leukemic cells. We plated cells at a concentration of 1x 106 cells per well, treated them with concentrations of 0, 1, 10, 100, 1,000, or 10,000 ng/mL, and incubated for 24 hours. After incubation the cells were removed and washed, and stained with fluorescent antibodies. Cellular content was determined by flow cytometry (FACS). Prior to acquisition by FACS, counting beads were added to each sample to determine absolute counts of each population of cells. Populations of interest were CD3+, CD4+, CD8+, and CD16+, BDCA1+ and BDCA2+, CD14+, and CD19+. Annexin and Propidium Iodide (PI) were added to evaluate apoptotic and non-viable cells. FloJo software was used to analyze the FACS data. Absolute count of viable cells was then converted to percentages using solvent only control as 100% viable. Results: Cell Viability after 24 hr Incubation with Mycotoxins Concentration Normal Peripheral Blood (n=9) T Cell Lymphoma (n=2) Gliotoxin (ng/mL) CD3+/CD4+ CD3+/CD8+ DC1 DC2 NK B CD3+ 0 100 100 100 100 100 100 100 1 127 128 219 143 104 127 96 10 126 129 36 69 109 117 82 100 15 15 3 25 2 18 110 1,000 23 33 3 11 13 11 37 10,000 47 46 63 13 26 27 4 Patulin (ng/mL) 0 100 100 100 100 100 100 100 1 66 68 74 86 60 81 93 10 94 100 101 114 84 88 333 100 84 101 79 141 110 138 152 1,000 39 38 2 26 35 27 69 10,000 53 45 0 34 39 78 1 Median Percent Viable when 0 ng/mL = 100% Viable The dose of Gliotoxin, most effective for inducing apoptosis and cell death in normal donor T cells is 100 ng/mL, whereas the most effective dose of Patulin was 1,000 ng/mL. In the cell line, the lowest dose of both mycotoxins that produced significant apoptosis and cell death was 1,000 ng/mL. Conclusion: Our results demonstrate that the mycotoxins, Patulin and Gliotoxin kill PBMC, and confirm the results found by Stanzani et al that Gliotoxin is most cytotoxic against the antigen presenting cells (APC), which impairs T cell response. Our experiment showed that Patulin had the same effect against APC. T cell viability itself was greatly influenced by the addition of Gliotoxin or Patulin, which supports previous evidence that mycotoxins suppress some populations of T cells. Certain issues, such as a less toxic solvent must be addressed; the data, however, shows that mycotoxins do have the ability to kill both normal and T cell leukemic/lymphoma cells.