Venn diagram showing distribution of total kidney proteins identified with differences in expression from the 2D-PAGE and LC-MS/MS-based proteome.

<p>The numbers indicate the total protein identified from each comparison (control, 10 and 50 ppmF A/J and 129P3/J) and the number of proteins commonly identified between them.</p

Expression of unique kidney proteins between A/J and 129P3/J mice.

a<p>Experimental molecular weight (kDa)/p<i>I</i> of protein spot in the gel (Mean of min. and max.) based on the coordinates of landmark proteins. <i>^b</i>Theoretical molecular weight (kDa)/p<i>I</i> of theoretical protein. <i>^c</i>Number of peptides identified and score. <i>^d</i>Identification is based on protein ID from IPI (international protein index) protein database (<a href="http://www.uniprot.org/" target="_blank">http://www.uniprot.org/</a>). <i>^e</i>Category of protein based on its primary biological function according to Rison (2000) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053261#pone.0053261-Rison1" target="_blank">[18]</a>.</p

Renal Proteome in Mice with Different Susceptibilities to Fluorosis

<div><p>A/J and 129P3/J mouse strains have different susceptibilities to dental fluorosis due to their genetic backgrounds. They also differ with respect to several features of fluoride (F) metabolism and metabolic handling of water. This study was done to determine whether differences in F metabolism could be explained by diversities in the profile of protein expression in kidneys. Weanling, male A/J mice (susceptible to dental fluorosis, n = 18) and 129P3/J mice (resistant, n = 18) were housed in pairs and assigned to three groups given low-F food and drinking water containing 0, 10 or 50 ppm [F] for 7 weeks. Renal proteome profiles were examined using 2D-PAGE and LC-MS/MS. Quantitative intensity analysis detected between A/J and 129P3/J strains 122, 126 and 134 spots differentially expressed in the groups receiving 0, 10 and 50 ppmF, respectively. From these, 25, 30 and 32, respectively, were successfully identified. Most of the proteins were related to metabolic and cellular processes, followed by response to stimuli, development and regulation of cellular processes. In F-treated groups, PDZK-1, a protein involved in the regulation of renal tubular reabsorption capacity was down-modulated in the kidney of 129P3/J mice. A/J and 129P3/J mice exhibited 11 and 3 exclusive proteins, respectively, regardless of F exposure. In conclusion, proteomic analysis was able to identify proteins potentially involved in metabolic handling of F and water that are differentially expressed or even not expressed in the strains evaluated. This can contribute to understanding the molecular mechanisms underlying genetic susceptibility to dental fluorosis, by indicating key-proteins that should be better addressed in future studies.</p> </div

Expression of differentially significant kidney proteins between 10 ppmF A/J <i>vs</i> 10 ppmF 129P3/J mice.

a<p>Experimental molecular weight (kDa)/p<i>I</i> of protein spot in the gel (Mean of min. and max.) based on the coordinates of landmark proteins. <i>^b</i>Theoretical molecular weight (kDa)/p<i>I</i> of theoretical protein. <i>^c</i>Number of peptides identified and score. <i>^d</i>Differences in expression in relation to 129P3/J mice (↓ down-modulation; ↑ up-modulation); individual <i>P</i> value after ANOVA. <i>^e</i>Identification is based on protein ID from IPI (international protein index) protein database (<a href="http://www.uniprot.org/" target="_blank">http://www.uniprot.org/</a>). <i>^f</i>Category of protein based on its primary biological function according to Rison (2000) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053261#pone.0053261-Rison1" target="_blank">[18]</a>.</p

Expression of differentially significant kidney proteins between control A/J <i>vs</i> control 129P3/J mice.

a<p>Experimental molecular weight (kDa)/p<i>I</i> of protein spot in the gel (Mean of min. and max.) based on the coordinates of landmark proteins. <i>^b</i>Theoretical molecular weight (kDa)/p<i>I</i> of theoretical protein. <i>^c</i>Number of peptides identified and score. <i>^d</i>Differences in expression in relation to 129P3/J mice (↓ down-modulation; ↑ up-modulation); Individual <i>P</i> value after ANOVA. <i>^e</i>Identification is based on protein ID from IPI (international protein index) protein database (<a href="http://www.uniprot.org/" target="_blank">http://www.uniprot.org/</a>). <i>^f</i>Category of protein based on its primary biological function according to Rison (2000) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053261#pone.0053261-Rison1" target="_blank">[18]</a>.</p

Simplified scheme of F-induced alterations in the expression of proteins related to energetic metabolism pathways in liver.

<p>F^- (fluoride ion); red arrows indicate increased expression and green arrows indicate decreased expression; P (phosphate); Fructose-bisP-AldoB (Fructose-bisphosphate aldolase B); L-PK (R-type isoform of piruvate kinase isozymes R/L); PDHE1-B (Pyruvate dehydrogenase E1 component subunit beta, mitochondrial); LDH-A (L-lactate dehydrogenase A chain); Mdh2 (Malate dehydrogenase); OAA (oxaloacetate); VLCAD (Very long-chain specific acyl-CoA dehydrogenase, mitochondrial); 3-Ketoacyl-CoA thiolase – m.(3-Ketoacyl-CoA thiolase, mitochondrial). </p

Proteomic Analysis of Liver in Rats Chronically Exposed to Fluoride

<div><p>Fluoride (F) is a potent anti-cariogenic element, but when ingestion is excessive, systemic toxicity may be observed. This can occur as acute or chronic responses, depending on both the amount of F and the time of exposure. The present study identified the profile of protein expression possibly associated with F-induced chronic hepatotoxicity. Weanling male <i>Wistar</i> rats (three-weeks old) were divided into three groups and treated with drinking water containing 0, 5 or 50 mg/L F for 60 days (n=6/group). At this time point, serum and livers were collected for F analysis, which was done using the ion-sensitive electrode, after hexamethyldisiloxane-facilitated diffusion. Livers were also submitted to histological and proteomic analyses (2D-PAGE followed by LC-MS/MS). Western blotting was done for confirmation of the proteomic data A dose-response was observed in serum F levels. In the livers, F levels were significantly increased in the 50 mg/L F group compared to groups treated with 0 and 5 mg/L F. Liver morphometric analysis did not reveal alterations in the cellular structures and lipid droplets were present in all groups. Proteomic quantitative intensity analysis detected 33, 44, and 29 spots differentially expressed in the comparisons between control <i>vs.</i> 5 mg/L F, control <i>vs.</i> 50 mg/L F, and 5 mg/L <i>vs.</i> 50 mg/L F, respectively. From these, 92 proteins were successfully identified. In addition, 18, 1, and 5 protein spots were shown to be exclusive in control, 5, and 50 mg/L F, respectively. Most of proteins were related to metabolic process and pronounced alterations were seen for the high-F level group. In F-treated rats, changes in the apolipoprotein E (ApoE) and GRP-78 expression may account for the F-induced toxicity in the liver. This can contribute to understanding the molecular mechanisms underlying hepatoxicity induced by F, by indicating key-proteins that should be better addressed in future studies.</p> </div

Photomicrographs of liver of rats showing macrovesicular lipid droplets (LD) in all groups.

<p>(A) control, (B) 5 mg/L F and (C) 50 mg/L F. In the left images, the regions bounded by the square are shown in greater increase in right images.</p

Effect of chronic treatment with F on the expression of protein GRP78 in the liver of rats chronically treated with F or not.

<p>(A) Representative blotting of GRP78 (~78 kDa) and constitutive GAPDH (~36 kDa) in samples of individual animals from each group (n=3). Samples are identified as indicated. Gels were performed in duplicate, each containing 3 different samples per respective group. (B) Densitometric analysis was done using imageJ software. The average of arbitrary values obtained for control group (n=6) was considered 1 and the averages of the other groups (n=6) were calculated in respect to control. Distinct letters indicate statistical significance (p<0.05).</p

Functional distribution of proteins identified with differential expression in the liver of rats chronically treated with F or not.

<p>Category of protein based on its primary biological function according to Rison et al. (2000).</p