Detection of Ochratoxin A on intestinal mucosa in intoxicated BALB/c mice

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Abstract

Ochratoxin A (OTA) is a mycotoxin produced by filamentous fungi, which
contaminates plant-based products and is known to cause harmful effects in animal and human health. This study aimed to evaluate, by immunohistochemistry, the presence of OTA on the surface of duodenal mucosa after oral exposure in mice. Two groups of one-day-old mice (BALB/c) were separated in: control group (n=6), which was treated with a single dose of PBS, and experimental group (n=6), which was intoxicated with a single oral dose of OTA (1.4 mg/kg/body weight). The experiment was approved by the Ethics Committee on the Use of Animals at the State University of Londrina (CEUA No. 1995.2020.02). The OTA and PBS inoculation was made on the 1st day of the experiment, and after the 14th day, mice were euthanized and their duodenum fragments
were collected, fixed in 10% buffered formalin solution, and stored in 70% ethanol. The tissues were dehydrated in ethanol, diaphonized in xylol, embedded in paraffin and sectioned in a 5 µm thickness microtome. Sections were blocked for endogenous peroxidase using a 1% H2O2 solution for 1 hour, and for nonspecific binding sites using a 1% BSA solution for 3 hours, both at room temperature. Then were incubated with monoclonal antibody to OTA (mAbOTA) (1/20 dilution) overnight, washed with PBS, and incubated with biotinylated anti-mouse IgG labeled antibody (1/1000 dilution) for 1 and ½ hour, washed with PBS 1x, and incubated with streptavidin-peroxidase conjugate for another 1 and ½ hour. Then, slides were washed, followed by revelation with diaminobenzidine (DAB) 0.05% + 0.3% H2O2 + TBS solution. Finally, they were counterstained with Harris hematoxylin and analyzed under an optical microscope.Images of each section were analyzed using ImageJ 1.53k software (400xmagnification) to determine the relative area of the tissue occupied with OTA.
Statistical analysis was performed using SigmaPlot 12.0 by comparing the two groups with a T-test, and the data were considered significantly different when p-value <0.05. Results showed significant difference when comparing the two groups (p=0.012). Control mice and experimental mice showed, respectively, 0.319% and 1.624% of area occupied by OTA on the surface of duodenal mucosa. In conclusion,
immunohistochemistry using streptavidin-biotin system allows OTA detection on
duodenal mucosa surfaces in BALB/c mice. Also, it was shown that even 14 days after a single oral OTA exposure, the OTA remained on the duodenum mucosa in BALB/c mice.

Institutions
  • 1 universidade estadual de londrina
  • 2 Centro de Ciências Biológicas / Universidade Estadual de Londrina
  • 3 State University of Londrina
  • 4 Departamento de Ciência e Tecnologia de Alimentos / Centro de Ciências Agrárias / Universidade Estadual de Londrina
  • 5 Departamento de Microbiologia / Centro de Ciências Biológicas / Universidade Estadual de Londrina
Track
  • Veterinary Microbiology
Keywords
Duodenum
DAB
Immunohistochemistry
Mycotoxin