J Vet Sci.  2010 Jun;11(2):107-113. 10.4142/jvs.2010.11.2.107.

Induction of apoptotic lesions in liver and lymphoid tissues and modulation of cytokine mRNA expression by acute exposure to deoxynivalenol in piglets

Affiliations
  • 1Safety Research Team, National Institute of Animal Health, Tsukuba, 305-0856, Japan. mikami@affrc.go.jp
  • 2Research Team for Environmental/Enzootic Diseases, Tohoku Research Station, National Institute of Animal Health, Shichinohe, 039-2586, Japan.
  • 3Saga Central Livestock Hygiene Service Center, Saga, 849-0928, Japan.
  • 4Department of Animal Medical Science, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, 603-8555, Japan.
  • 5Hokkaido Research Station, National Institute of Animal Health, Sapporo, 062-0045, Japan.

Abstract

Six 1-month-old piglets were intravenously injected with deoxynivalenol (DON) at the concentration of 1 mg/kg body weight, with three pigs each necropsied at 6 and 24 h post-injection (PI) for investigation of hepatotoxicity and immunotoxicity with special attention to apoptotic changes and cytokine mRNA expression. Histopathological examination of the DON-injected pigs revealed systemic apoptosis of lymphocytes in lymphoid tissues and hepatocytes. Apoptosis of lymphocytes and hepatocytes was confirmed by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemical staining against single-stranded DNA and cleaved caspase-3. The number of TUNEL-positive cells in the thymus and Peyer's patches of the ileum was increased at 24 h PI compared to 6 h PI, but the peak was at 6 h PI in the liver. The mRNA expression of interleukin (IL)-1beta, IL-6, IL-18, and tumor necrosis factor (TNF)-alpha in the spleen, thymus and mesenteric lymph nodes were determined by semi-quantitative RT-PCR, and elevated expression of IL-1beta mRNA at 6 h PI and a decrease of IL-18 mRNA at 24 h PI were observed in the spleen. IL-1beta and IL-6 mRNA expressions increased significantly at 6 h PI in the thymus, but TNF-alpha decreased at 6 h PI in the mesenteric lymph nodes. These results show the apoptosis of hepatocytes suggesting the hepatotoxic potential of DON, in addition to an immunotoxic effect on the modulation of proinflammatory cytokine genes in lymphoid organs with extensive apoptosis of lymphocytes induced by acute exposure to DON in pigs.

Keyword

apoptosis; cytokine; deoxynivalenol; hepatocytes; pigs

MeSH Terms

Animals
Apoptosis/*drug effects/immunology
Cytokines/*biosynthesis/genetics
Gene Expression Regulation/drug effects
Histocytochemistry/veterinary
In Situ Nick-End Labeling/veterinary
Liver/*drug effects/immunology
Lymphoid Tissue/*drug effects/immunology
RNA, Messenger/*biosynthesis/genetics/immunology
Reverse Transcriptase Polymerase Chain Reaction/veterinary
Specific Pathogen-Free Organisms
Swine/*immunology
Trichothecenes/*toxicity

Figure

  • Fig. 1 Microscopic lesions of thymus, Peyer's patches of ileum and liver. Increased numbers of apoptotic lymphocytes (arrows) are observed in the thymus and Peyer's patches of deoxynivalenol (DON) -injected pigs at 24 h PI. Apoptotic changes of hepatocytes (arrow heads) and activation of Kupffer cells are found in the liver of DON-injected pigs, especially at 6 h PI. (Inset: high-power view of apoptotic hepatocyte, Scale bar = 10 µm.) Thy: thymus, PP: Peyer's patches of ileum, Li: liver. H&E stain, Scale bars = 20 µm.

  • Fig. 2 TdT-mediated dUTP-biotin nick end-labeling (TUNEL) (A) and immunohistochemical staining against single stranded DNA (ssDNA) (B) and cleaved caspase-3 (C) for thymus and liver of DON-injected (6 h PI) and control pigs. Increased number of positive reactions are observed in DON-injected pigs. Positive reactions are in the nuclei of apoptotic cells in TUNEL and anti-ssDNA staining, but in the cytoplasm in anti-cleaved caspase-3 staining. C: control, D: DON-injected pig. Scale bars = 20 µm.

  • Fig. 3 Number of TUNEL-positive cells in thymus (A), Peyer's patches (B), and liver (C) of DON-injected and control pigs. TUNEL-positive cells in the thymus and Peyer's patches reach a maximum at 24 h PI (A, B), while the peak in the liver is at 6 h PI (C). Data are shown as mean ± SD from three pigs. *Significant difference compared with controls (p ≤ 0.05).

  • Fig. 4 Expression of proinflammatory cytokine mRNA in the lymphoid organs from DON-injected and control pigs. (A) spleen; (B) thymus; (C) mesenteric lymph nodes. Bars show the mean expression of mRNA of three pigs from each group as the ratio of the band intensity of each PCR product to the corresponding β-actin PCR product. Error bars show standard deviation. *Significant difference compared with control (p ≤ 0.05).


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