Deoxynivalenol Impairs Weight Gain and Affects Markers of Gut Health after Low-Dose, Short-Term Exposure of Growing Pigs

Abstract

Deoxynivalenol (DON) is one of the major mycotoxins produced by Fusarium fungi, and exposure to this mycotoxin requires an assessment of the potential adverse effects, even at low toxin levels. The aim of this study was to investigate the effects of a short-term, low-dose DON exposure on various gut health parameters in pigs. Piglets received a commercial feed or the same feed contaminated with DON (0.9 mg/kg feed) for 10 days, and two hours after a DON bolus (0.28 mg/kg BW), weight gain was determined and samples of different segments of the intestine were collected. Even the selected low dose of DON in the diet negatively affected weight gain and induced histomorphological alterations in the duodenum and jejunum. The mRNA expression of different tight junction (TJ) proteins, especially occludin, of inflammatory markers, like interleukin-1 beta and interleukin-10 and the oxidative stress marker heme-oxigenase1, were affected along the intestine by low levels of DON in the diet. Taken together, our results indicate that even after low-level exposure to DON, which has been generally considered as acceptable in animal feeds, clinically-relevant changes are measurable in markers of gut health and integrity.

Keywords: cytokines; deoxynivalenol; histomorphology; intestine; tight junctions; weight gain.

Figure 1

DON induces histomorphological changes in the piglet duodenum even at low exposure levels. Piglets were fed a control or DON diet (0.9 mg/kg feed) for 10 days; two hours after the DON bolus (0.28 mg/kg BW), segments obtained from duodenum were fixed in 10% formalin, and paraffin sections were H&E-stained for histomorphometric analysis of villus height (A), crypt depth (B), epithelial cell area (C) and area without epithelial layer (D); representative photomicrographs of villi in duodenum (Figure 1E,F) from control (E) and DON-fed piglets (F). Magnification: 100×. Results are expressed as means ± SEM; n = 9–10 animals/group. * p ≤ 0.05; significantly different from the control group.

Figure 2

DON induces histomorphological changes in the piglet jejunum even at low exposure levels. Piglets were fed a control or DON diet (0.9 mg/kg feed) for 10 days; two hours after the DON bolus (0.28 mg/kg BW), segments obtained from jejunum were fixed in 10% formalin, and paraffin sections were H&E-stained for histomorphometric analysis of villus height (A), crypt depth (B), epithelial cell area (C) and area without epithelial layer (D); representative photomicrographs of villi in jejunum from control (E) and DON-fed piglets (F). Magnification: 100×. Results are expressed as means ± SEM; n = 9–10 animals/group. * p < 0.05, ** p < 0.01, *** p < 0.001; significantly different from the control group.

Figure 3

The mRNA expression levels of markers of barrier integrity are affected by short-term, low-dose exposure to DON. Piglets received a control (blue line) or DON diet (0.9 mg/kg feed, red line) for 10 days; two hours after the DON bolus, samples from different parts of the intestine (duodenum, jejunum, Ileum, cecum and colon) were collected, and mRNA levels of various tight junction (TJ) proteins (CLDN1, 2, 3, 4, 5, ZO-1, ZO-1 and OCLN) were measured by RT-PCR. Results are expressed as the relative mRNA expression as means ± SEM; n = 9–10 animals/group.

Figure 4

The protein expression of occludin in different parts of the intestine are affected by short-term, low-dose exposure to DON. Piglets received a control diet or DON diet (0.9 mg/kg feed) for 10 days; two hours after the DON bolus, samples from different parts of the intestine (duodenum, jejunum, Ileum, cecum and colon) were collected, and protein levels were measured by Western blot analysis. Results are expressed as the relative protein expression (normalized to β-actin) as means ± SEM; n = 8 animals/group. * p ≤ 0.05, ** p < 0.01; significantly different from the control group.

Figure 5

The mRNA expression levels of markers of inflammation are affected by short-term, low-dose exposure to DON. Piglets received a control (blue line) or DON diet (0.9 mg/kg feed, red line) for 10 days; two hours after the DON bolus, samples from different parts of the intestine (duodenum, jejunum, Ileum, cecum and colon) were collected, and mRNA levels of inflammatory markers (COX-1, COX-2, IL-10, IL-1β and TLR4) were measured by RT-PCR. Results are expressed as the relative mRNA expression as means ± SEM; n = 9–10 animals/group.

Figure 6

The mRNA expression levels of markers of oxidative stress, efflux transporter, proliferation and apoptosis are affected by short-term, low-dose exposure to DON. Piglets received a control (blue line) or DON diet (0.9 mg/kg feed, red line) for 10 days; two hours after the DON bolus, samples from different parts of the intestine (duodenum, jejunum, Ileum, cecum and colon) were collected, and mRNA levels of stress markers (HIF-1α, HMOX1, HMOX2), an apoptotic and a proliferative marker (caspase-3 and Ki67) and an efflux transporter (ABCB1) were measured by RT-PCR. Results are expressed as the relative mRNA expression as means ± SEM; n = 9–10 animals/group.

Figure 7

No obvious changes in the expression of proliferation marker Ki67 and apoptotic marker caspase-3 between jejunum of control and DON-treated piglets. For immunohistochemical staining, Swiss-rolled paraffin sections obtained from the jejunum of control (A,C) and DON-treated (B,D) piglets were detected by antibodies for Ki67 or caspase-3, as described in the Materials and Methods. Representative images are shown (n = 5 piglets/group). Magnification: 200×.

Figure 8

chematic overview of DON-related effects in different segments of the intestine. Red arrows indicate absorption of DON. Green arrows indicate secretion of DON. The rectangles below each segment of the intestine summarize the impact of DON on villus architecture (duodenum and jejunum) and on mRNA expression levels of different genes related to intestinal barrier function (and OCLN protein expression), oxidative stress and inflammation. Negative effects of DON () are mainly observed in the jejunum (downregulation of different genes). Up arrows indicate increase and down arrows indicate decrease.