Nivalenol and deoxynivalenol affect rat intestinal epithelial cells: a concentration related study - PubMed (original) (raw)

Giuseppe Bianco et al. PLoS One. 2012.

Abstract

The integrity of the gastrointestinal tract represents a crucial first level defence against ingested toxins. Among them, Nivalenol is a trichotecenes mycotoxin frequently found on cereals and processed grains; when it contaminates human food and animal feed it is often associated with another widespread contaminant, Deoxynivalenol. Following their ingestion, intestinal epithelial cells are exposed to concentrations of these trichothecenes high enough to cause mycotoxicosis. In this study we have investigated the effects of Nivalenol and Deoxynivalenol on intestinal cells in an in vitro model system utilizing the non-tumorigenic rat intestinal epithelial cell line IEC-6. Both Nivalenol and Deoxynivalenol (5-80 µM) significantly affected IEC-6 viability through a pro-apoptotic process which mainly involved the following steps: (i) Bax induction; (ii) Bcl-2 inhibition, and (iii) caspase-3 activation. Moreover, treatment with Nivalenol produced a significant cell cycle arrest of IEC-6 cells, primarily at the G(0)/G(1) interphase and in the S phase, with a concomitant reduction in the fraction of cells in G(2). Interestingly, when administered at lower concentrations (0.1-2.5 µM), both Nivalenol and Deoxynivalenol affected epithelial cell migration (restitution), representing the initial step in gastrointestinal wound healing in the gut. This reduced motility was associated with significant remodelling of the actin cytoskeleton, and changes in expression of connexin-43 and focal adhesion kinase. The concentration range of Nivalenol or Deoxynivalenol we have tested is comparable with the mean estimated daily intake of consumers eating contaminated food. Thus, our results further highlight the risks associated with intake of even low levels of these toxins.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Apoptosis detection by propidium iodide (PI) staining of hypodiploid nuclei after IEC-6 incubation with NIV or DON (0.5–80 µM) for 24 h.

Both NIV and DON exibited a significant and concentration-related pro-apoptotic effect on IEC-6 at concentrations higher than 5 µM and 10 µM respectively (***P<0.001 vs control). NIV at the concentrations between 5 and 80 µM exibits a stronger pro-apoptotic effect compared to the same concentrations of DON (°°°P<0.001, °°P<0.01 vs DON). Data are expressed mean ± s.e.m. from at least three-independent experiments.

Figure 2

Figure 2. Apoptotic nuclear morphological changes highlighted by DAPI staining in cells treated with graded concentration of NIV and DON (0.5–20 µM) for 24 h.

Apoptotic cells showed pyknotic nuclei and apoptotic bodies were clearly identified after incubation with higher concentration of NIV while DON induces a less marked effect. Cultures were examined and photographed using a confocal microscope as described in method section.

Figure 3

Figure 3. Flow cytometric analysis of IEC-6 cycle phase distribution.

Cells were treated with either NIV or DON (10 µM) for 24 h, incubated with PI and analysed for cell cycle analysis using a Becton Dickinson FACScan flow cytometer and ModFit software (***P<0.001,**P<0.01 vs control). Data are expressed mean ± s.e.m. from at least three-independent experiments.

Figure 4

Figure 4. Concentration dependent effect in NIV and DON treated cells on apoptosis-related proteins Bax (panel A) and Bcl-2 (panel B) expression.

Cells were treated with increasing concentration of NIV or DON (5–20 µM) for 24 h and protein expression was detected by Western blot. Tubulin protein expression was used as a loading control. (***P<0.001,**P<0.01,*P<0.05 vs control; °°P<0.01,°P<0.05 vs DON). Western blot is representative of at least three-independent experiments.

Figure 5

Figure 5. Concentration dependent effect in NIV and DON treated cells on caspase-3 (full and cleaved form) expression.

Cells were treated with increasing concentration of NIV or DON (5–20 µM) for 24 h and protein expression was detected by Western blot. Tubulin protein expression was used as a loading control. The Western blot is representative of independent experiments (panel A). Apoptosis detection by propidium iodide (PI) staining of hypodiploid nuclei with a broad spectrum caspase inhibitor (zVAD-fmk) (panel B). IEC-6 were treated with NIV or DON (20 and 40 µM) for 24 h in presence of zVAD-fmk (50 µM). The pro-apoptotic effect of NIV or DON was significantly reduced by the caspase inhibitor (***P<0.001 NIV+zVAD vs NIV alone; ***P<0.001 DON+zVAD vs DON alone). Data are expressed mean ± s.e.m. from at least three-independent experiments.

Figure 6

Figure 6. Representative pictures of wound repair from mechanical scratch 24 h after NIV and DON cells treatment (0.5–2.5 µM).

Black dotted-line indicates the edge of the wounded area at the starting time (panel A). Quantitative analysis of the wound repair 24 h after making the scratch. For each condition ten different cells were randomly selected to measure the migration distances covered every 10 min from the initial time up to the end of incubation time (***P<0.001 vs control; °°°P<0.001 vs DON; panel B). Data are expressed mean ± s.e.m. from at least three-independent experiments. Bar = 200 µM.

Figure 7

Figure 7. Effect of mycotoxins NIV and DON on IEC-6 cytoskeleton highlighted by FITC-coniugated phalloidin staining.

The microtubule cytoskeleton is displayed in control IEC-6. The microtubules are evident as actin filaments that originate near the nucleus and radiate through the cytosol. Incubation for 24 h of cultured cells with NIV and DON (0.5–2.5 µM) caused a reorganisation of actin filaments characterised by redistribution to the cell subcortical compartment and subsequent cell rounding in concentration dependent manner. Cultures were examined and photographed using a confocal microscope as described in method section.

Figure 8

Figure 8. Concentration dependent effect in NIV (Panel A) and DON (Panel B) treated cells on Cx43and its phosphorilated form (pCx43) expression.

Cells were treated with increasing concentration of NIV or DON (0.5–10 µM) for 24 h and protein expression was detected by Western blot (***P<0.001,*P<0.05 vs control; °°°P<0.001,°°P<0.05 vs DON). Tubulin protein expression was used as a loading control. The Western blot is representative of at least three-independent experiments.

Figure 9

Figure 9. Concentration dependent effect in NIV (Panel A) and DON (Panel B) treated cells on FAK and its phosphorilated form expression (pFAK).

Cells were treated with increasing concentration of NIV or DON (0.5–10 µM) for 24 h and protein expression was detected by Western blot. (***P<0.001, **P<0.01, *P<0.05 vs control; °°°P<0.001, °P<0.05 vs DON). Tubulin protein expression was used as a loading control. The Western blot is representative of at least three-independent experiments.

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