Evaluation of enteric innervation and proteomic analysis of the small intestine of rats exposed to acute or chronic fluoride dose

Author(s)

Carina Guimarães de Souza Melo

• Document type: Doctoral Thesis
• Press: Bauru.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia de Bauru (FOB/SDB)
• Defense date: 2015-12-03

Examining board members

Marilia Afonso Rabelo Buzalaf; Eduardo José de Almeida Araújo; Ricardo Brandt de Oliveira; Juliana Vanessa Colombo Martins Perles; Antonio de Castro Rodrigues

• Advisor: Marilia Afonso Rabelo Buzalaf; Jacqueline Nelisis Zanoni

Abstract

The gastrointestinal tract (GIT) is the main route of fluoride (F) exposure, and the most important site of its absorption. It is believed that F toxicity compromises the intestine physiology, due to the relevant gastrointestinal symptomatology reported in consequence to excessive exposure. The intestinal function is controlled by a complex neuronal net, which is interconnected and embedded in the wall of this organ, named Enteric Nervous System (ENS). Although the toxic effects of F on the Central Nervous system are described in the literature, there are no studies related to its toxicity on the ENS. Therefore, in this study performed in rats, the effects of chronic and acute F exposure were evaluated, on the general population of enteric neurons and on the subpopulations that express the main enteric neurotransmitters: Acetylcholine (Ach), Nitric Oxide (NO), Vasoactive Intestinal Peptide (VIP), Calcitonin gene related peptide (CGRP), and Substance P (SP). The animals were divided into 5 groups: 3 designed to the chronic exposure (0 ppm, 10 ppm ou 50 ppm de F in the drinking water) and 2 to the acute exposure (0 ou 25 mgF/Kg - gastric gavage). Three intestinal segments were collected (duodenum, jejunum, and ileum) and processed for the immunofluorescence techniques to detect HuC/D, ChAT, nNOS, VIP, CGRP and SP, on the myenteric plexus. Images were obtained for the quantitative analysis of the general population of neurons (HuC/D immunoreactive) and the nitrergic neurons (nNOS immunoreactive), for the morphometric analysis of the general population and nitrergic neurons and also for the immunoreactive varicosities to ChAT, VIP, CGRP or SP. Samples of the 3 intestinal segments were prepared and stained with hematoxylin and eosin for histological analysis of the basic morphology. Duodenum, the intestinal segment considered the most affected in the morphological analysis of the general population of neurons, was selected for the proteomic analysis, with the objective to offer the entire protein profile and to determine differences in the protein expression due to chronic or acute F exposure. Plasma F concentration was analyzed to confirm the exposure. In the quantitative analysis, the 50 ppm F group presented a significant decrease in the density of the general population of neurons in the jejunum and ileum, and in the density of the nitrergic neurons in the duodenum and jejunum. Regarding the morphometric analysis, the general population of neurons and all the neuronal subpopulations evaluated presented significant morphological alterations, for both exposures, chronic and acute. In the proteomic analysis of the duodenum, it was verified that both exposures caused alterations in the expression of intestinal proteins. These proteins identified with differential expression were distributed according the association of their genes to a specific term, and by this term classified in different biological process. In the group that received the acute dose the biological process with the highest percentage of associated genes was the generation of precursor metabolites and energy (27% of proteins), and for the 10 and 50 ppm F groups, they were pyridine nucleotide metabolic process (41%) and protein polymerization (33%), respectively. (AU)

• FAPESP's process: 11/10233-7 - Evaluation of enteric innervation and proteomic analysis of small intestin of rats exposed to chronic or acute fluoride dose
• Grantee: Carina Guimarães de Souza Melo
• Support Opportunities: Scholarships in Brazil - Doctorate