Effect of fluoride on the proteomic profile of the hippocampus in rats

2.1 Animals and fluoride treatment

The protocol for the study was reviewed and approved by the Institutional Animal Use and Care Committees of Jiangsu University and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. The male Sprague-Dawley rats (n=30) were received three weeks after weaning, an age at which all regions of the central nervous system are rapidly developing. All animals were housed in stainless-steel cages suspended in stainless-steel racks with relative humidity ranging from 30% to 55% and a temperature of 22 °C to 25 °C. They were fed a standard pellet diet and given distilled water ad libitum. The animals were allowed to acclimatize to the laboratory conditions for four days before experiments began.

The rats were weighed and randomly divided into two groups (15 in each group). The animals of the first group were injected i.p. with aqueous NaF (20 mg/kg/body weight/day), a dose selected on the basis of the LD₅₀ value of fluoride in mice (51.6 mg/kg body weight/day) [13] and maintained for 30 days. The second group served as control and was injected with physiological saline. The NaF solution (30 mL) was prepared fresh each day in double distilled water [10]. At the end of the 30-day treatment, the animals were killed by decapitation, the brains were rapidly dissected and the hippocampus removed. The tissue was immediately frozen in liquid nitrogen and stored at –80 °C until use. The experiments were performed in accordance with the regional legal regulations.

2.2 Determination of body weight and serum F content

Each animal was weighed before treatment and then weighed every 10 days. F levels in serum were determined according to Hall et al. [14] using a CSB-F-I fluoride ion electrode (ChangSa Analysis Instrumentation, ChangSa, China). Data were presented as means ± SD, and statistical significance was determined by Student’s t-test using the SPSS program.

2.3 Protein sample preparation

Approximately 100 mg of hippocampus tissue from five rats were minced on ice and subsequently homogenized with a hand-held tissue homogenizer in 500 uL of lysis buffer (20 mM Tris-HCl, pH 8.5, 7 M urea, 2 M thiourea, 4%(w/v) CHAPS, 10 mM DTT, 1 mM EDTA and 1 mM PMSF). The suspension was sonicated for approximately 30 s and centrifuged at 150,000×g for 45 min. The protein content in the supernatant was determined using the Coomassie blue stain.

2.4 Two-dimensional gel electrophoresis

IPG strips, 17-cm nonlinear pH 3-10 (Bio-Rad, Hercules, CA, USA) containing 3000 μg of protein were rehydrated in a buffer consisting of 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, 0.2% Bio-Lyte (pH 3-10) and 0.001% bromophenol blue (BPB), then subjected to isoelectric focusing (IEF) using a Protean IEF Cell (Bio-Rad). Focusing was at 50 V for 12 h, 250 V for 30 min, 1000 V for 1 h, 10000 V for 5 h, and then up to 10,000 V for approximately 5 h. The electric current was below 50 mA per strip. After IEF, the IPG strips were equilibrated for 15 min in equilibration buffer I containing 6 M urea, 2% SDS, 0.375 M Tris-HCl (pH 8.8), 20% glycerol and 2% (w/v) DTT followed by 15 min in buffer II (same as buffer I but containing 2.5% iodoacetamide instead of DTT). For electrophoresis in the second dimension, the strips were transferred onto a 12% polyacrylamide gel (SDS-PAGE) and overlaid with 0.5% agarose. Electrophoresis was performed at 4 °C with 70 V for 30 min followed by 120 V for 1.5 h in Tris-glycine buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3).

2.5 Protein visualization and image analysis

Gels were fixed overnight in a solution of 45.4% (v/v) methanol and 9.2% acetic acid and stained with Coomassie blue G-250 (0.01% w/v) or by silver staining. The resulting 2-DE protein patterns were scanned with a ScanMaker 9700XL (600 ppi, Microtek, Hsinchu, Taiwan) and analysed with the PDQuest software 16.0 (Bio-Rad).

2.6 In-gel digestion and mass spectrometry analysis

Proteins of spots the intensity of which detectably differed between the fluoride-treated and control group, respectively, were identified by peptide mass fingerprinting (PMF). The spots were excised with circular plugs 2–3 mm in diameter, and transferred to 1.5 mL EP tubes. Coomassie blue-stained gel pieces were first destained with 50 μL of a 1:1 (v/v) mixture of 50 mM NH₄HCO₃ and acetonitrile for 10 min, followed by three washes with 50 μL of MilliQ water. The gel pieces were then dehydrated with 100% acetonitrile for 5 min and dried in a SpeedVac (Thermo Savant, Holbrook, NY, USA) for 30 min. The dried gel particles were rehydrated at 4 °C for 30 min with trypsin (sequencing grade; Promega, Madison, WI, USA) in 50 mM NH₄HCO₃ (20 g/mL), and then incubated at 37 °C overnight. The peptide mixture (1 μL) was mixed with 1 μL 10 mg/mL α-cyano-4-hydroxycinnamic acid (Sigma) and spotted onto the MTP Anchor Chip (Bruker Daltonics, Bremen, Germany) and analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) (ultraflex tof/tof, Bruker, Bremen, Germany).

2.7 Protein identification

MS data were analysed using MASCOT (Matrix Science, London, UK) and the NCBInr eukaryotic protein sequence database. The parameters were set as follows: missed cleavages was 1, fixed modification was acetylation of carbamidomethyl (C), variable modification was Glu→pyro-Glu (N-term Q) or oxidation of methionine (M), mass tolerance was 0.3 Da, mass value was MiH⁺. A protein with a minimum ion score of 79 (p<0.05) was considered reliably identified.

2.8 Gene ontology (GO) analysis

Corresponding Gene Ontology IDs of proteins were obtained by InterProScan Sequence Search (http://www.ebi.ac.uk/Tools/pfa/ iprscan/). GO classification of these proteins was conducted with WEGO (http://wego.genomics.org.cn/) [15].

2.9 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis

The sequences of identified proteins were queried against KEGG GENES (Kyoto Encyclopedia of Genes and Genomes) (http://blast. genome.jp/) using BLASTP program with BLOUSM62 scoring matrix, and the resultant genes were used to search the KEGG reference pathway database (http://www.genome.jp/kegg/tool/search_pathway.html) to obtain reference pathways for these proteins [16].

3.1 Body weight and fluoride content

After 30 days, the body weight of rats treated with NaF was 305.61±21.48 g, those of rats treated with physiological saline was 361.26±26.80 g (Figure 1). The values were significantly different (p<0.05) already after 10 days of treatment, consistent with a previous study [10].

Figure 1:

Body weight of F-treated (20 mg/kg/body weight/day) and control rats as a function of time. The values are means ± SD for 15 animals. * Student’s t-test, p<0.05. ** Student’s t-test, p<0.01.

The F level in the serum of the F-treated group after 30 days was 106.70±15.58 μg/L, which was markedly higher (p<0.01) than in the control group with 80.71±10.06 μg/L. These data established the usefulness of the animal model in the present study.

3.2 Global identification of differentially expressed proteins

To understand the changes in the hippocampus of rats exposed to excessive fluoride at the proteomic level, we employed proteomic tools (2-DE and MS) to globally identify differentially expressed proteins. The gels were analyed by the Bio-Rad PDQuest software which is commonly used in proteomic research. Eventually, 27 proteins were excised from the 2-DE gels, subjected to in-gel trypsin digestion and subsequent MALDI-TOF/TOF identification. Identified proteins are shown in Table 1 and Figure 2. Spot volume comparisons of the spots’ intensity (Figure 3) indicated pronounced differences in the abundance of certain hippocampus proteins between the F-treated group and the control group. A total of 26 spots of different intensity between the F-treated group and the control represent 27 proteins, in which 19 were up-regulated and eight were down-regulated. Spot 16 contains 2 proteins, i.e. alcohol dehydrogenase and fructose-bisphosphate aldolase C.

Figure 2:

2-DE pattern of rat hippocampus proteins. A and B represent hippocampus of the control group and the F-treated group, respectively. Spots of different intensity in the two gels are indicated by Arabic numerals.

Figure 3:

Selected spots of 2-DE gels and corresponding spot volumes identified in the hippocampus of F-treated group (F) and control group (C). Image pairs and detection of protein spots with relative spot volumes were performed using PDQuest software (Bio-Rad). The gel is shown in the left panel, and the spot volume is shown in the right panel.

3.3 Gene ontology analysis

Gene ontology (GO) analysis is widely used in proteomic research to annotate the biochemical functions of proteins identified by MALDI-TOF. All 27 proteins were subjected to GO analysis. The results (Figure 4) showed that the up-regulated proteins are mainly involved in catalytic and metabolic processes, and the down-regulated proteins are involved in binding, catalytic, cellular process and metabolic process.

Figure 4:

Gene ontology analysis of the 27 differentially expressed proteins. The proteins were classified into the three main categories and 19 subcategories indicated below the graph.

3.4 KEGG pathway analysis

KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis is often used to characterize gene functions, and was used here to further functionally characterise the proteins used in the GO analysis. The proteins (Figure 5) fell into the phagosome, oxytocin signaling pathway, hypertrophic cardiomyopathy, biosynthesis of amino acids, insulin signaling pathway and carbon metabolism. Importantly, they are preferentially involved in amino acid biosynthesis, especially that of glutamine.

Figure 5:

KEGG pathway analysis. The differentially expressed proteins were analyzed for their respective KEGG pathways. The resultant pathways belong to eight categories.