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, 3 (12), 1484-501

Trichothecene Mycotoxins Inhibit Mitochondrial Translation--Implication for the Mechanism of Toxicity

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Trichothecene Mycotoxins Inhibit Mitochondrial Translation--Implication for the Mechanism of Toxicity

Mohamed Anwar Bin-Umer et al. Toxins (Basel).

Abstract

Fusarium head blight (FHB) reduces crop yield and results in contamination of grains with trichothecene mycotoxins. We previously showed that mitochondria play a critical role in the toxicity of a type B trichothecene. Here, we investigated the direct effects of type A and type B trichothecenes on mitochondrial translation and membrane integrity in Saccharomyces cerevisiae. Sensitivity to trichothecenes increased when functional mitochondria were required for growth, and trichothecenes inhibited mitochondrial translation at concentrations, which did not inhibit total translation. In organello translation in isolated mitochondria was inhibited by type A and B trichothecenes, demonstrating that these toxins have a direct effect on mitochondrial translation. In intact yeast cells trichothecenes showed dose-dependent inhibition of mitochondrial membrane potential and reactive oxygen species, but only at doses higher than those affecting mitochondrial translation. These results demonstrate that inhibition of mitochondrial translation is a primary target of trichothecenes and is not secondary to the disruption of mitochondrial membranes.

Keywords: trichothecenes; Fission; Fusarium; ROS; T-2 toxin; deoxynivalenol; diacetoxyscirpenol; fusion; mitochondria; ribosomes; translation.

Figures

Figure 1
Figure 1
Growth of wild type BY4743 cells (rho+ & rho0) in media containing dextrose vs. glycerol. Rho+ and rho0 cells were grown in liquid media supplemented with 2% dextrose (Dex) or with 3% glycerol (Glyc) in the presence or absence of varying concentrations of trichothecenes for 18 h. OD600 of cells treated with increasing concentrations of trichothecenes were compared to that of the untreated cells (control) to determine relative growth. The red lines indicate growth in dextrose-containing media and the blue lines indicate growth in glycerol-containing media. The green lines indicate growth of rho0 in dextrose-containing media. Error bars indicate S.E. where n = 3 independent replicates.
Figure 2
Figure 2
Effects of trichothecenes on total and mitochondrial protein synthesis. (A) Total and mitochondrial translation in wild type yeast cells treated with low doses of trichothecenes for 6 h prior to measuring [35S]-Met incorporation; (B) Total translation in wild type yeast cells treated with low doses of trichothecenes for 18 h prior to measuring [35S]-Met incorporation; (C) Mitochondrial translation in yeast cells treated with increasing concentrations of T-2 for 6 h prior to measuring [35S]-Met incorporation; (D) Mitochondrial translation in yeast cells treated with increasing concentrations of DAS for 6 h prior to measuring [35S]-Met incorporation; (E) In organello translation using equal amounts of mitochondria, isolated from wild type W303 yeast. 35[S]-methionine incorporation was measured after 10 min treatment with different concentrations of trichothecenes. Final counts (CPM) for all experiments were normalized to the OD600 of each sample. Translation levels of trichothecene-treated samples were expressed as a percentage of the control samples set to 100%. Error bars indicate S.E. where n = 3 independent replicates.
Figure 3
Figure 3
Effects of trichothecenes on mitochondrial morphology. Wild type yeast transformed with pVT100U-mtGFP, containing GFP targeted to the mitochondrial matrix was treated for 6 h with high (A) or low (B) doses of trichothecenes. Representative images are shown at 100X magnification using epifluorescence microscopy. Differential Interference Contrast (DIC) images of each cell are presented on the left (Scale bar, 10 µm).
Figure 4
Figure 4
Mitochondrial membrane potential and ROS production in cells treated with trichothecenes for 6 h. Petite (rho0) cells and wild type (rho+) cells treated with 3 mM H2O2, 50 µM CCCP and stained with MitoTracker Red for changes in mitochondrial membrane potential (A) and DCFH-DA for ROS generation (B) Wild type (rho+) cells treated with high doses of trichothecenes and stained with MitoTracker Red for changes in mitochondrial membrane potential (C) and DCFH-DA for ROS generation (D). Median fluorescence unit for each treatment was normalized to that of the untreated control and represented as relative fluorescent unit. 25-50,000 cells from each sample were analyzed using an Accuri C6 flow cytometer. Error bars indicate S.E. where n = 3 independent replicates.
Figure 5
Figure 5
Mitochondrial membrane potential and ROS production and in cells treated with trichothecenes for 6 h and 18 h at low doses, which inhibit mitochondrial, but not total translation. Wild type yeast, treated with trichothecenes for 6 h and stained with MitoTracker Red for changes in mitochondrial membrane potential (A) and DCFH-DA for ROS generation (B). At 18 h post treatment with trichothecenes, wild type yeast cells were stained with MitoTracker Red for changes in mitochondrial membrane potential (C) and DCFH-DA for ROS generation (D). Median fluorescence unit for each treatment was normalized to that of the untreated control and represented as relative fluorescent unit. 25-50,000 cells from each sample were analyzed using an Accuri C6 flow cytometer. Error bars indicate S.E. where n = 3 independent replicates.

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