Proteomic analysis of urine in fluoride-treated rats

Urine is an ideal source of materials to search for potential disease-related biomarkers as it is produced by the affected tissues and can be easily obtained by noninvasive methods. Objectives: Two-dimensional gel electrophoresis (2D-PAGE) based proteomics approach was used to better understand the molecular mechanisms of injury induced by fluoride (F) and define potentials biomarkers of fluorosis. Methods: Three groups of weanling male Wistar rats (21 days old) were treated with drinking water containing 0 (control), 5, or 50 ppm F for 60 days (n=15/group). During the experimental period, the animals were kept individually in metabolic cages, in order to analyze the water and food consumption, as well as fecal and urinary F excretion. For proteomic analysis, urine samples were collected in protease-inhibitors cocktail for 8 hours in ice box. After protein isolation, urinary proteome profiles were examined using 2D-PAGE and coomassie brilliant blue staining. Image Master 2D Platinum software was used for matching and analyzing protein spots. Spots presenting a 2-fold change in % volume were excised and identified by MALDI-TOF MS and MASCOT search engine (ion score=30 and protein score=60). Results: A dose-response regarding F intake and excretion was detected. Quantitative intensity analysis revealed 9, 10 and 13 proteins increased or decreased in control X 5 ppm F, 5 ppm F X 50 ppm F and control X 50 ppm F groups, respectively. Three proteins were identified: androgen-regulated 20 KDa protein and α-2µ-globulin (both regulated by androgens) and aflatoxin-B1-aldehyde-reductase (related to detoxification). Conclusions: These data indicate that proteomic analysis in urine of F-treated animals can identify differentially expressed proteins, even in cases of low F doses. Thus, this approach can contribute to clarify the mechanisms underlying fluorosis, by indicating key-proteins that should be better addressed, as well as to identify potential toxicity biomarkers. Support: (FAPESP 05/02744-0 and 05/03273-1).