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Oxidative Biochemistry Disbalance and Changes on Proteomic Profile in Salivary Glands of Rats Induced by Chronic Exposure to Methylmercury.

1. Introduction

Mercury is a high toxic metal with a wide distribution on nature and has been considered as an important health concern [1, 2]. Mercury occurs naturally in the environment on organic and inorganic forms, in which methylmercury (MeHg) and mercury chloride (Hg[Cl.sub.2]) are the most toxic forms, respectively. MeHg is an alkylmercury compound that is derived from the methylation of [Hg.sup.+2] by methanogenic and sulfate-reducing bacteria on aquatic ecosystems and some soils [3-5].

The anthropogenic action leads to higher concentrations on biosphere and may expose animals of aquatic food chain to damage [5]. Many researches have reported that fishes and other seafood are the main sources of human contamination by low daily doses, in which about 90% of Hg is present on its organic form, leading to higher bioaccumulation and biomagnification [6, 7].

Hg also has been described as a huge pollutant able to accumulate and induce cellular impairments in organs related to metabolism and excretion, as the liver and kidneys [8-11]. Few studies [12-14] have demonstrated the effects of Hg[Cl.sub.2] in salivary glands, and there is no evidence of MeHg effects. In this way, it is important to describe the MeHg-induced alteration in salivary glands to elucidate the consequences of this metal exposure.

The parotid, submandibular, and sublingual are the main salivary glands, representing about 90% of the total salivary human production. Salivary glands are important organs on the maintenance of oral cavity homeostasis; their major function is salivary secretion that is responsible for balancing the microbiota, pH, and carbohydrates catalysis in the mouth [15, 16]. Moreover, studies of metal toxicological effects on salivary glands are still rare, especially about MeHg. The aim of this study was to evaluate whether the chronic exposure to low doses of MeHg is able to impair biochemical parameters and the proteomic profile, which could be related to dysfunctions in the organs.

2. Materials and Methods

2.1. Animals and Experimental Groups. A total of 40 male Wistar rats were obtained from the Federal University of Para (UFPA, Belem, Brazil), under the BIO 225-14 CEPAE --UFPA, following the NIH Guide for the Care and Use of Laboratory Animals. The animals were maintained in collective cages with 5 animals each and were kept in a climate-controlled room on a 12 h light/dark cycle with water and food ad libitum. Corn oil or MeHg with a daily dose of 0.04 mg/kg was orally administered by gavage over a period of 60 days of exposure according to an adapted protocol from Kong et al. [17]. To regulate the administered dose in animals, they were weekly weighted for adjustment of MeHg concentration and determination of the body mass curve.

2.2. Samples Collection. After 24 hours of the last administration, the animals were euthanized by cervical dislocation and had their three pairs of salivary glands (parotid, submandibular, and sublingual) collected and frozen in liquid nitrogen and posteriorly stored in ultrafreezer -80[degrees]C until further analysis. One gland from each animal was saved to the assays of oxidative biochemistry, proteomic analysis, and measurement of total mercury deposits.

2.3. Mercury Deposits. Total mercury content in the samples was quantified by wet digestion, reduction, and cold vapor atomic absorption spectrometry with gold amalgamation system Mercury Analyzer: SP3D (Nippon Corporation). The estimations were conducted following the protocol previously mentioned by Suzuki et al. [18], in which consists by converting the Hg into elementary Hg vapor in order to quantify in parts per million (ppm) by an absorption cell. The analyses were performed preparing duplicates of each sample of groups, and the values were tabulated to further inferential statistical analysis.

2.4. Oxidative Biochemistry Analysis. The samples were first thawed and homogenized in 700 [micro]L Tris-HCl buffer (20 mM, pH 7.4). A volume of 200 [micro]L from the total homogenate was used to perform the measurement of antioxidant capacity against peroxyl radicals (ACAP), whereas 300 [micro]L was used to measure nitrite levels and 100 [micro]L to lipid peroxidation assay (LPO).

First, ACAP was measured as previously described by Amado et al. [19]. The supernatant of each sample after centrifugation was, in triplicate, exposed to a peroxyl radical generator, the 2,2-azobis 2 methylpropionamidine dihydrochloride (ABAP; 4 mM; Aldrich), and, in another triplicate, they received only ultrapure water in a 96-well microplate. After reaction, the fluorescence generated was measured in a fluorimeter (Victor X3, Perkin Elmer) at 35[degrees]C. After the first reading for determination of background fluorescence values, a total of 10 [micro]l of 2;,7; (H2DCF-DA) 40 nM was added to the microplate, with a reading in every 5 minutes during 60 minutes [19, 20]. The results were expressed according to the difference between areas below the curves created by total fluorescence production of samples with and without ABAP after application to a second order polynomial function, being named as a relative area. High-relative areas mean low antioxidant activity, once the sample had low capacity to neutralize the peroxyl radicals. Results were expressed as percentages of control group.

The remaining homogenate was centrifuged at 21.000p for 20 minutes, and the supernatant was used to perform the assays. The concentration of nitrite was determined following the protocol previously adapted by Fagundes et al. [21], which is based on a reaction with Griess reagent (0.1% N-(1-naphthyl) ethylenediaminedihydrochloride; 1% sulfanilamide in 5% phosphoric acid; 1 : 1) proceeding the absorbance measurement on a spectrophotometer at 550 nm, and compared to standard solutions of sodium nitrite.

The level of LPO was established by measurement of malonaldehyde (MDA) levels based on Esterbauer and Cheeseman [22] method adapted by Fagundes et al. [21]. An aliquot of the supernatant was processed as described by the Bioxytech LPO-568 kit (Cayman Chemical). This kit is a chromogenic reagent that reacts with MDA at 45[degrees]C. The absorbance measurement on spectrophotometers was performed at 586 m wavelength.

The total protein content in the supernatants (20 [micro]L) was analyzed by Bradford [23] method. After the correction for protein concentration, results of lipid peroxidation and nitrite levels were expressed in percentages of control groups.

2.5. Proteomic. The frozen salivary glands were homogenized in a cryogenic mill (model 6770, Spex, Metuchen, NJ, EUA). For protein extraction, gland homogenate was incubated in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% IPG buffer pH 3-10, and 40 mM DTT) for 1 hour at 4[degrees]C with continuous shaking. In order to recovery the soluble proteins and the supernatant, the homogenate was centrifuged at 15.000 rpm for 30 minutes at 4[degrees]C. The proteins were precipitated using the kit PlusOne 2D Cleanup (GE Healthcare, Uppsala, Sweden), as recommended by the manufacturer. Pellets were resuspended in rehydration buffer (7 M urea, 2M thiourea, 0.5% CHAPS, 0.5% IPG buffer pH 3-10, 18 mM DTT, and 0.002% bromophenol blue). Twenty-five [micro]L of salivary gland proteins from each animal of the same group was combined to constitute a pool that was centrifuged for clarification. To each pool, 50 mM AMBIC containing 3 M urea was added. Each sample was filtered twice in 3 kDa AMICON (Millipore, St. Charles, MO, USA). The protein content was measured in the pooled samples by Bradford protein assay [23]. To each sample (50 fig of total protein for each pool in a volume of 50 [micro]L), 10 f L of 50 mM AMBIC was added. In sequence, 25 [micro]L of 0.2% RapiGEST[TM] (Waters Co., Manchester, UK) was added and incubated at 80[degrees]C for 15 min. Following, 2.5 [micro]L of 100 mM DTT was added and incubated at 60[degrees]C for 30 min. Also, 2.5 p L of 300 mM IAA was added and incubated for 30 min at room temperature (under dark). Then, 10 [micro]L of trypsin (100 ng; Trypsin Gold Mass Spectrometry, Promega, Madison, USA) was added and digestion occurred for 14 h at 37[degrees]C. After digestion, 10 pl of 5% TFA was added, incubated for 90 min at 37[degrees]C, and the sample was centrifuged (14,000 rpm for 30 min). The supernatant was collected, and 5 p L of ADH (1 pmol/[micro]L) plus 85 [micro]L 3% ACN was added.

After protein extraction is completed, separation and identification of peptides were performed on a nanoAcquity UPLC-Xevo QTof MS system (Waters, Manchester, UK). The difference in expression among the groups was obtained using PLGS software, consideringp < 0.05 for downregulated proteins andp > 0.95 for upregulated proteins. The bioinformatics analysis was performed using Uniprot protein ID accession numbers to map their associated encoding Uniprot gene entries for the comparison of control x MeHg. Gene ontology (GO) annotation of biological process was performed using Cytoscape v3.0 software, with the Cluego v2.0.7 plugin. Uniprot IDs were uploaded to the software and analyzed with default parameters, which specify an enrichment (right-sided hypergeometric test) correction method using Bonferroni step down, analysis mode "Function" and load gene cluster list for Rattus novergicus, evidence codes "All," set networking specificity "medium" (GO levels 3 to 8), and KappaScoreThreshold 0.4. The protein-protein interaction (ppi) network was built by ClusterMarker, another Cytoscape plugin. After the network creation, we edited nodes and edge colors according to each group.

2.6. Statistical Analysis. Statistical comparison of body weight gains between control and MeHg groups was performed using one-way analysis of variance (ANOVA).

All values obtained from the biochemical and total mercury quantification analysis were plotted on GraphPad Prism 5.0 software (San Diego, CA, USA) and were expressed, respectively, as percentage of control and mean [+ or -] SEM. All data were compared using Student's t-test with a level of significance of p < 0.05.

3. Results

3.1. Body Weight Measurement and Deposits of Total Hg in Salivary Glands. Although a normal increase on body mass over the 60 days of MeHg exposure was observed, no difference was observed at the final weight (Figure 1(a)). The MeHg chronic exposure was able to promote Hg deposits (ppm) on salivary glands (Figure 1(b)). Our results showed, after the 60th day of MeHg exposure, a significantly Hg deposit in all three types of rat glands compared to the control groups. We also showed a higher deposit on parotid, suggesting a major tropism than submandibular and sublingual glands.

3.2. Chronic Exposure of MeHg Impairs the Oxidative Biochemistry. In order to analyze the possible involvement of cellular stress, we performed oxidative/nitrosative biochemistry assay. Our data suggest that MeHg is able to induce impairment at the antioxidant defense system by reducing the total antioxidant capacity. Results showed lower ACAP levels in submandibular (Figure 2(c)) showing 36.92% [+ or -] 14.51 more RNS than in control group, whereas sublingual (Figure 2(b)) showed 58.24% [+ or -] 12.26 and parotid (Figure 2(a)) 59.67% [+ or -] 7.32 more than control, respectively.

Our result showed that nitrite concentrations are higher at all three types of rat salivary gland than at the control group. The results showed higher levels in parotid (Figure 3(a)) showing 86.89% [+ or -] 15.71 more RNS than in control group, whereas submandibular (Figure 3(b)) showed 56.42% [+ or -] 11.65 and sublingual (Figure 3(c)) 50.45% [+ or -] 8.09 more than the control, respectively.

We further showed that MeHg is able to induce lipid peroxidation, by the yields of MDA (Figure 4), on salivary glands of MeHg group. The results showed higher levels of MDA in all three major glands. All glands of MeHg group showed 100% more damaged than those of the control animals. The parotid (Figure 4(a)) showed about 121.16% [+ or -] 18.2 higher, submandibular (Figure 4(b)) about 141.94% [+ or -] 27.18 higher, and sublingual (Figure 4(c)) about 106.59% [+ or -] 29.38 more when compared to the control.

3.3. MeHg Changes the Proteomic Profile of Rat Salivary Glands. This exposure model revealed a total of 15 proteins downregulated and 22 proteins upregulated on parotid glands (Table 1); 6 proteins downregulated and 7 upregulated on submandibular glands (Table 2); and 7 proteins downregulated and 2 upregulated on sublingual glands (Table 3). The proteomic analysis of the three salivary glands also revealed several proteins that were only found in one of the groups, being absent in the other (Supplementary Tables available online at https://doi.org/10.1155/2017/5653291).

In parotid gland, MeHg was able to change 20 categories of proteins classified by Cytoscape according to GO (Figure 5(a)). Our data showed that they are related to the following biological processes: nucleosome (20.31%), aerobic respiration process (7.81%), NAD binding (7.81%), mesenchyme migration (6.65%), hydrolyase activity (6.25%), oxygen binding (4.69%), and response to mercury ion (4.69%). Regulation of cellular respiration, protein transmembrane transport, melanosome, nucleosomal DNA binding, retina homeostasis, benzaldehyde dehydrogenase (NAD+) activity, myelin sheath, intermediate filament, pigment granule, mRNA 5-UTR biding, cellular response to epidermal growth factor stimulus, and The ppi network of parotid gland (Figure 5(b)) showed a central protein (mitogen-activated protein kinase 3; P21708), which interacts with proteins related to stress response (heat shock protein HSP 90-alpha, P82995; heat shock protein HSP 90-beta, P34058), molecular function proteins (synaptic vesicle membrane protein VAT-1 homolog, Q3MIE4; protein ERGIC-53, Q62902; 40S ribosomal protein S14, P13471; 40S ribosomal protein AS, P38983; Sec61 beta subunit, B2RZD1; elongation factor 1-alpha 2, P62632), oxidative stress enzymes (superoxide dismutase (Cu-Zn), P07632), microtubule component (tubulin alpha-4A chain, Q5XIF6), and energetic metabolism process (trifunctional enzyme subunit alpha, mitochondrial, Q64428; ATP synthase subunit beta, mitochondrial, P10719).

As for the submandibular gland, our results showed that 10 categories of proteins classified by Cytoscape, according to GO, were significantly altered upon chronic exposure to MeHg (Figure 6(a)). The myelin sheath proteins correspond to 31.43%; the structural constituent of cytoskeleton and the substance nigra development represent 11.43%, each. Moreover, the nuclear nucleosome represents 8.57% and regulation of sodium ion transmembrane transporter activity symbolizes 8.57% of the proteins with alterations. Adenylate cyclase binding, mitochondrial proton-transporting ATP synthase complex, catalytic core F (1), phosphoserine binding, bHLH transcription factor binding, and eukaryotic translation elongation factor 1 complex corresponded to 3.13% each.

Similarly to what was found for the parotid gland, most of the proteins with altered expression in the submandibular gland interacted with mitogen-activated protein kinase 3 (P21708), which was found in the center of the ppi network. The interacting proteins were those related to stress response (P14659), energetic metabolic process (ATP synthase subunit alpha, mitochondrial, P15999; ADP/ATP translocase 2, Q09073; ATP synthase subunit delta, mitochondrial, P35434 and citrate synthase, mitochondrial, Q8VHF5), microtubule constituent (tubulin alpha-1B chain, Q6P9V9), molecular functions (elongation factor 1-alpha 2, P62632; uncharacterized protein, F1M446; 14-3-3 protein theta, P68255; elongation factor 1-alpha 2, P62632), and cellular component (endoplasmic reticulum resident protein 29, P52555; 78kDa glucose-regulated protein, P06761) (Figure 6(b)).

Sublingual glands presented the lower number of protein groups identified on Cytoscape, following ClueGO app GO classification (Figure 7(a)). Each of the 6 groups of proteins is represented by myelin sheath (33.33%), unfolded protein binding (19.05%), retina homeostasis (14.29%), structural constituent of cytoskeleton (14.29%), detection of chemical stimulus involved in sensory perception of bitter taste (9.52%), and alpha-amylase activity (9.52%).

The ppi network of the sublingual gland (Figure 7(b)) showed the same protein in the center of the network (mitogen-activated protein kinase 3; P21708), which the proteins with altered expression interacted. The interaction proteins were related mainly to biological processes, such as mitosis and cytoskeletal rearrangements. In addition, proteins associated to stress response (heat shock cognate 71 kDa protein, P63018 and heat shock-related 70kDa protein 2, P14659) and microtubule constituent (tubulin alpha-1B chain, Q6P9V9) were clustered by ClusterMarker app.

4. Discussion

In this study, we showed for the first time that MeHg, in a chronic period, causes damage to the salivary gland proteomic profile and oxidative stress. A significant total Hg deposit in the gland tissue structure was verified, as well as a membrane cell damage by higher LPO levels, nitrogen species formation due to oxidative stress, and low antioxidant capacity against peroxyl radicals. In addition, the novelty of the toxicological evaluation on the proteomic profile of rat glands after MeHg exposure brings the association with differences in expression at important proteins of metabolic pathways, oxidative biochemistry, and mainly in the composition of cytoarchitecture.

Most of the studies about MeHg toxicity are under acute exposures and with high MeHg concentrations, even though the reality faced by the population of endemic regions of this metal intoxication is different. Recent studies showed that long-term intoxication includes other effects such as cardiovascular diseases and genotoxicity [24-27]. This latter one includes oxidative stress and alterations of cytoskeleton proteins as main causes for these effects. The known effects of MeHg are mainly given by chronic exposures, thus, the model proposed by Kong et al. [17], in which we based our study, mimics a daily and continuous exposure in low doses. The exposure time of our study brings the possibility to find interesting results on an oxidative biochemistry and proteomic profiles at rat glands, showing that even at low doses, a chronic MeHg exposure is able to induce molecular and cellular damages in living organisms.

For understanding, the nomination of low dose used in this researcher is important to consider other anatomic regions and the toxic effects. The lowest observed adverse effect level (LOAEL) for neurotoxic effects such as paresthesia is 50 ppm of mercury in hair [28]. This level in hair corresponds approximately to 1 ppm of mercury in brain, according to the proportion of 250: 50: 1 for hair: brain: whole blood contents (reviewed by Branco et al. [29]).

The mercury content in brains of rats exposed to the dose used in our work (0.04 mg/kg per day for 8 weeks) is about a half of that level, that is, about 0.5 ppm [17, 30], characterizing a low exposure. Moreover, animals exposed to this low level of methylmercury did not develop neurotoxic effects such as alterations of forelimb grip strength, running wheel performance, or hind limb cross [30], supporting that this mercury level in the brain is not sufficient to cause deleterious neurobehavioral consequences.

Looking at Figure 1, an interesting fact is that salivary glands accumulated about 10 times less mercury than 0.5 ppm. However, our results show for the first time that this low dose is sufficient to cause significant oxidative stress and alterations of protein regulation in salivary glands, revealing the high susceptibility of this tissue to the toxic effects of methylmercury.

The main form of MeHg intoxication is the oral through food [6], so the preferential site of absorption is in the intestinal lumen by enterocytes. The experimental model of intragastric gavage, used in this work, illustrates the MeHg intake by food consumption. MeHg is formed by a methyl group attached to the Hg atom (C[H.sub.3]Hg), and this allows a greater affinity with thiol groups, mainly of protein components [31].

The MeHg gains systemic circulation from the efflux through the basolateral membrane of enterocytes and on this part of membrane surface. There are multidrug resistance-associated proteins (MRPs), neutral amino acid transporters, as LAT2, and organic anions transporter (OAT) [32]; then, it is suggested that these proteins' action is related to MeHg efflux. Moreover, some MRPs need GSH as cosubstrate for their effectiveness, so MeHg may also undergo efflux to systemic blood circulation. According to Martinez-Madrigal and Micheau [33], blood vessels as external carotid, sublingual, submental, and facial supply all glands, suggesting that vascularization is the main path by which Hg forms deposits by intragastric gavage intoxication.

In this work, we found significant Hg concentration (ppm) in the three main salivary glands in comparison to the control group. Studies about the proteomic profile of salivary glands have suggested that parotid is responsible to produce the major part of the protein components of saliva [34-36], and this could be the reason to a higher absorption to this gland showed by our group, once this metal has an affinity to structures present in many proteins.

Studies appoint that MeHg induces multiple effects within cell homeostasis. MeHg is able to induce cell death [37], impairment on cell proliferation and differentiation [38], disorganization of cytoskeleton [39, 40], DNA mutation [41], impairments on transcriptome [42], and proteome [43]. In addition, according to the literature, these damages on biological processes and structures could be associated with oxidative/nitrosative biochemistry misbalance due to oxidative stress.

Cellular membranes are crucial targets ofMeHg-induced toxicity, and due to its constitution of unsaturated lipids, the products MDA and 4-HNE are the most mutagenic and toxic products of LPO (for review see Ayala et al. [44]); in addition, organelle membranes also may suffer due to LPO [45]. In this study, we showed an increase on MDA levels in rat salivary glands after MeHg exposure, suggesting a high susceptibility of glandular tissue to oxidative/nitrosative stress.

The protein peroxidation products, that is, peroxyl radical, are highly harmful to cell membranes. Beyond the importance of stress in the glandular homeostasis after MeHg exposure, the production of peroxyl radical seems to be an important linkage between lipid peroxidation and protein oxidation, once peroxyl could be an initiator factor of LPO [46-48]. This way, our results suggest that MeHg chronic exposure can decrease the ACAP in salivary glands and lead the glandular stress, seen by nitrite and MDA levels, which may lead to damage at membrane lipid and to cellular proteins.

It is well known that oxidative/nitrosative stress is able to impair gene expression [49] and protein translation [50] driving to oxidized and nonfunctional proteins. In this way, we further characterized for the first time the proteomic changes under oxidative stress after MeHg exposure on salivary rat glands. The global proteomic results of proteins up- and downregulated in parotid, submandibular, and sublingual glands are described in Tables 1, 2, and 3, respectively.

The literature has pointed that the imbalance in oxidative stress may lead to disorders on cellular pathways of energetic metabolism, such as the heat shock class of proteins [51-53]. We showed that in parotid glands, MeHg was able to induce downexpression of heat shock cognate 71 proteins (D4A4S3 and M0R8M9) and induce exclusively the expression of heat shock proteins (HSP) 90 (P82995 and P34058) in MeHg group. In submandibular, we also found heat shock proteins (M0R660 and P14659) that were unique in MeHg group, while in the sublingual gland, we found the exclusive expression of heat shock cognate 71 (P63018) and heat shock-related 70 (P14659) and expression of heat shock protein beta-6 (P97541) only in the control group. HSPs have been already proposed as new therapeutic tools for some disorders as cancer and cardiovascular diseases, with a protective role especially for HSP90 [54]. The results of the present study support a main role for this protein also in methylmercury intoxication.

In addition, glutathione S-transferase P (P04906) and components of cytochrome (Q62737 and Q68FY0) were exclusively expressed in parotid control group, suggesting an important association on protein changes and MeHg-induced oxidative stress.

The metabolic pathway class of proteins was also changed after MeHg exposure. In parotid, the proteins ATP synthase subunit alpha (F1LP05) and ATP synthase subunit beta, mitocondrial (P10719), were downregulated and malate dehydrogenase, cytoplasmic (O88989), and aconitate hydratase (G3V6S2) were unique in the control group. In submandibular, ATP synthase subunit alpha, mitochondrial (P15999), was downregulated, while ADP/ATP translocase 2 (Q09073), ATP synthase subunit delta, mitochondrial (P35434), citrate synthase (G3V936), citrate synthase, and mitochondrial (Q8VHF5) were present only in the control glands. Our results corroborate with metabolic pathway disorders established in others experimental models [17, 55].

MeHg treatment also impaired negatively the proteins of cellular components, such as cytoskeleton, from the main constituents of salivary glands. We have showed that the proteins related to muscle cell contraction (actin, alpha skeletal muscle, P68136; actin, aortic smooth muscle, P62738; and actin, gamma-enteric smooth muscle, P63269), in parotid glands were downregulated after MeHg exposure, which may suggest possible damages in myoepithelial cells present in the glands. We also showed downregulation of keratin, type II cytoskeletal 8 (Q10758) that is an important component of conjunctive tissue.

Concerning to microtubule constitution, we showed here that tubulin beta chain (B4F7C2) and tubulin beta-4B chain (Q6P9T8) were downregulated, corroborating with the literature, which suggests that microtubule depolymerization is one of the mechanisms of Hg damage on living cells [25, 56, 57].

A collagen chain protein (collagen alpha-1(I) chain, P02454), cytokeratins (keratin, type I cytoskeletal 10, Q6IFW6; keratin, type I cytoskeletal 14, Q6IFV1; keratin, type I cytoskeletal 15, Q6IFV3; keratin, type I cytoskeletal 17, Q6IFU8; keratin, type I cytoskeletal 24, Q6IFX1; keratin, type I cytoskeletal 42, Q6IFU7; and keratin, type II cytoskeletal 7, Q6IG12), and tubulin chains (tubulin beta chain, Q4QQV0 and tubulin alpha-4A chain, Q5XIF6) were found exclusively in control parotid glands. In sublingual glands, we also found conjunctive constituent (collagen alpha-2(I) chain, P02466), cytoskeleton (Q5BJY9) and microtubule (tubulin alpha-1A chain, P68370; tubulin alpha-1B chain, Q6P9V9; and tubulin alpha-1C chain, Q6AYZ1) components uniquely in the nonexposed group.

As previously mentioned, salivary glands are responsible for salivary formation and flow; this way, the acinar and tubular constituents have great importance in physiology. In the proteomic analysis, downregulation of alpha-amylase (E9PSQ1) was observed, as well as prolactin-induced protein, isoform CRA_d (G3V812), and prolactin-inducible protein homolog (O70417) in the parotid. An interesting protein related to biomineralization (submandibular gland secretory Glx-rich protein CB, P08462) was upregulated in the submandibular of the exposed group. Salivary flow is first derived by ionic exchanges by sodium/potassium bombs in acini and intercalated portions of secretory duct system [58], and in this study, we noticed that sodium/potassium-transporting ATPase subunit alpha-1 (P06685) was exclusive of control group in sublingual gland, similarly to sodium channel subunit beta-3 (Q9JK00) and sodium/potassium-transporting ATPase subunit alpha-2 (P06686) that were unique of submandibular gland of animals not submitted to MeHg exposure.

The proteomic profile changes shown by this work suggest that MeHg exposure leads to salivary gland damage, since several proteins were shown to be affected, compromising essential biological functions and components that under normal circumstances are fundamental do cell and tissue viability.

5. Conclusion

The proteomic profile verified after MeHg exposure in this study allowed us to see what is underlying the oxidative stress effects in salivary glands. Our findings have showed that salivary glands are also targets for MeHg in systemic intoxication, at low doses and chronic exposure, being able of triggering imbalance in oxidative biochemistry and, consequently, the modulation of proteomic profile of the glands.

https://doi.org/10.1155/2017/5653291

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Acknowledgments

This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq), Fundacao de Amparo a Pesquisa do Estado do Para (FAPESPA), and Pro-Reitoria de Pesquisa e Pos-Graduacao da UFPA (PROPESP, UFPA, Brazil). Rafael R. Lima is an investigator from CNPq (Edital MCTI/CNPQ/Universal 14/2014). Leonardo O. Bittencourt's scholarship is supported by the Brazilian National Council for Scientific and Technological Development (CNPq).

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Leonardo Oliveira Bittencourt, (1) Bruna Puty, (1) Senda Charone, (2) Walessa Alana Braganca Aragao, (1) Paulo Mecenas Farias-Junior, (1) Marcia Cristina Freitas Silva, (1) Maria Elena Crespo-Lopez, (3) Aline de Lima Leite, (2) Marilia Afonso Rabelo Buzalaf, (2) and Rafael Rodrigues Lima (1)

(1) laboratory of Functional and Structural Biology, Institute of Biological Sciences, Federal University of Para, Belem, PA, Brazil

(2) Department of Biological Sciences, Bauru Dental School, University of Sao Paulo, Bauru, Sao Paulo, SP, Brazil

(3) Laboratory of Molecular Pharmacology, Institute of Biological Sciences, Federal University of Para, Belem, PA, Brazil

Correspondence should be addressed to Rafael Rodrigues Lima; rafalima@ufpa.br

Received 14 April 2017; Revised 7 June 2017; Accepted 13 June 2017; Published 24 July 2017

Academic Editor: Laura Giusti

Caption: Figure 1: (a) Effects of MeHg chronic exposure at a dose of 0.04 mg/kg/day during 60 days on body mass gain of rats. The results are expressed as mean [+ or -] SEM (n = 20 animals per group) with one-way ANOVA with repeated measures followed by Tukey test. (b) Total mercury deposits on salivary glands of rats exposed to MeHg for 60 days. The values are expressed as mean [+ or -] SEM of Hg concentrations. * p < 0.05 compared to control group and # compared to others glands (one-way ANOVA and Tukey posttest).

Caption: Figure 2: Levels of antioxidant capacity against peroxyl radical parotid (a), submandibular (b), and sublingual salivary glands (c) of animals exposed chronically to methylmercury. The values are expressed as percentage of control [+ or -] SEM. * p < 0.05 (Student's t-test).

Caption: Figure 3: Levels of nitrite concentration in parotid (a), submandibular (b), and sublingual salivary glands (c) of animals exposed chronically to methylmercury. The values are expressed as percentage of control [+ or -] SEM. * p < 0.05 (Student's t-test). cellular response to gamma radiation corresponded to 3.13% each.

Caption: Figure 4: Levels of lipid peroxidation in parotid (a), submandibular (b), and sublingual (c) glands of animals exposed chronically to methylmercury. The values are expressed as percentage of control [+ or -] SEM. * p < 0.05 (Student's t-test).
Table 1: Identified proteins with expression significantly altered in
rat parotid glands of group exposed to Mehg (Hg).

(a) Access              Protein name description
number

P68136                Actin. alpha skeletal muscle
P62738                 Actin. aortic smooth muscle
P63269             Actin. gamma enteric smooth muscle
ni.P05                 ATP synthase subunit alpha
P10719          ATP synthase subunit beta, mitochondrial
P21701                     Deoxyribonuclease-1
D3ZXS6                  Elongation factor 1-alpha
M0R757                  Elongation factor 1-alpha
P62630                 Elongation factor 1-alpha 1
P62632                 Elongation factor 1-alpha 2
D1A1S3              Heat shock cognate 71 kDa protein
M0R8M9              Heat shock cognate 71 kDa protein
P01916                Hemoglobin subunit alpha-1/2
P02091                  Hemoglobin subunit beta-1
D3ZWE0                         Histone H2A
P02262                     Histone H2A type 1
P0C169                    Histone H2A type 1-C
P0C170                    Histone H2A type 1-E
Q61598                    Histone H2A type 1-F
P0CX89                    Histone H2A type 2-A
Q1FCT6                     Histone H2A type 3
Q0O728                     Histone H2A type 4
A9UMV8                        Histone H2A.J
P0C8S7                        Histone H2A.Z
D3ZK97                         Histone H3
P62901                         Histone H4
Q10758               Keratin, type II cytoskeletal 8
D3ZUQ1                           Lipase
G3V812          Prolactin induced protein, isoform CRA_d
070117             Prolactin-inducible protein homolog
FILRA1                      Protein ERGIC-53
F1M6C2                    Protein LOC103691939
F1LZI1                      Protein LOC680121
P02770                        Serum albumin
B1F7C2                       Tubulin beta-4
Q6P9T8                    Tubulin beta-4B chain
M0RCB1                   Uncharacterized protein

(a) Access      PLGS        Fold
number         scare     change Hg

P68136         1873.1       -0.9
P62738         1713.4       -0.9
P63269         1713.4       -0.9
ni.P05         548.2        -0.8
P10719         353.9        -0.8
P21701         268.7        -0.6
D3ZXS6          327         2.4
M0R757         343.66       2.5
P62630         343.66       2.5
P62632         327.0        2.5
D1A1S3         151.3        -0.7
M0R8M9         739.22       -0.8
P01916         1975.8       1.2
P02091         1841.8       1.8
D3ZWE0         764.11       2.0
P02262         764.11       1.9
P0C169         764.11       1.8
P0C170         764.11       2.0
Q61598         764.11       1.9
P0CX89         764.11       1.8
Q1FCT6         764.11       1.8
Q0O728         764.11       1.7
A9UMV8         764.11       1.9
P0C8S7         764.11       1.9
D3ZK97         258.66       1.2
P62901        2231.90       1.3
Q10758         863.19       -0.8
D3ZUQ1         396.87       1.3
G3V812        7686.80       -0.9
070117        7084.70       -0.9
FILRA1         400.1        1.2
F1M6C2         343.66       2.5
F1LZI1         538.95       -0.7
P02770         489.45       1.3
B1F7C2         455.5        -0.7
Q6P9T8         455.5        -0.7
M0RCB1         769.68       -0.8

The identified proteins are organized according to the alphabetical
order.Relative differential expression is indicated by positive value,
when the protein is upregulated, and by negative values (-), when the
protein is downregulated in the comparison between groups.(a)
Identification is based on protein ID from UniProt protein database
(http://www.uniprot.org/).

Table 2: Identified proteins with expression significantly altered in
rats' submandibular glands of group exposed to Mehg (Hg).

(a) Access                  Protein name description
number

Q08163               Adenylyl cyclase-associated protein 1
P15999             ATP synthase subunit alpha, mitochondrial
M0R757                     Elongation factor 1-alpha
P62630                    Elongation factor 1-alpha 1
P62632                    Elongation factor 1-alpha 2
O88752                          Epsilon 1 globin
P11517                     Hemoglobin subunit beta-2
D3ZNH4                            Histone H2B
F1LQ08                            Protein Car6
F1M6C2                        Protein LOC103691939
E9PTY1                           Protein Prol1
P08462         Submandibular gland secretory Glx-rich protein CB
D4A5P1                      Uncharacterized protein

(a) Access        PLGS           Fold
number           score        change Hg

Q08163           148.55          -0.2
P15999           560.23          -0.6
M0R757           225.49          1.6
P62630           225.49          1.6
P62632           188.54          1.8
O88752          2060.91          -0.3
P11517          2099.64          -0.3
D3ZNH4          3909.79          -0.7
F1LQ08           439.43          1.4
F1M6C2           193.93          1.7
E9PTY1           325.88          2.0
P08462           944.25          1.3
D4A5P1           364.33          -0.6

The identified proteins are organized according to the alphabetical
order. Relative differential expression is indicated by positive
value, when the protein is upregulated, and by negative values (-),
when the protein is downregulated in the comparison between groups.
(a) Identification is based on protein ID from UniProt protein
database (http://www.uniprot.org/).

Table 3: Identified proteins with expression significantly altered in
the rats' sublingual glands of group exposed to MeHg (Hg).

(a) Access              Protein name description
number

E9PSQ1                        Alpha-amylase
F1LPK5                 Acidic mammalian chitinase
G3V812          Prolactin induced protein, isoform CRA_d
G3V844                        Alpha-amylase
M0R7K9                        Protein Csap1
O70417             Prolactin-inducible protein homolog
P00689                  Pancreatic alpha-amylase
P02770                        Serum albumin
P12020              Cysteine-rich secretory protein 1

(a) Access     PLGS score    Fold change
number                            Hg

E9PSQ1          2696.09          -0.3
F1LPK5          1384.72          -0.2
G3V812          6144.39          -0.2
G3V844           344.21          -0.3
M0R7K9          1852.75          -0.3
O70417          5900.39          -0.2
P00689           344.21          1.7
P02770           600.4           -0.6
P12020           221.34          2.5

The identified proteins are organized according to the alphabetical
order.Relative differential expression is indicated by positive value,
when the protein is upregulated, and by negative values (-), when the
protein is downregulated in the comparison between groups.
(a) Identification is based on protein ID from UniProt protein
database (http://www.uniprot.org/).

Figure 5: Functional distribution of proteins identified with
differential expression (a) in rat parotid glands exposed to MeHg
versus control group. Categories of proteins based on GO annotation
biological process, molecular function, and cellular component.
Terms significant (Kappa = 0.4) and distribution according to
percentage of number of genes association. And subnetwork (b)
created by ClusterMarker app to establish the interaction among
identified proteins of parotid glands with differentiated
expression on exposed group when compared to the control group. The
node colors indicate the differential expression of respective
protein named with its accession ID. Red and dark green indicate
proteins found in the control and exposed groups, respectively.
Light green and pink indicate upregulated and downregulated
proteins, respectively. Gray nodes represent those proteins that
were not identified on this study, but they interacted on the
network. The access identification number in nodes corresponds to
P17046: lysosome-associated membrane glycoprotein 2, P82995: heat
shock protein HSP 90-alpha, P34058: heat shock protein HSP 90-beta,
P21708: mitogen-activated protein kinase 3, P10719: synaptic
vesicle membrane protein VAT-1 homolog, Q3MIE4: protein ERGIC-53,
Q62902: protein ERGIC-53, P13471: 40S ribosomal protein S14,
P38983: 40S ribosomal protein SA, B2RZD1: protein Sec61b, P07632:
superoxide dismutase (cu-Zn), Q5XIF6: tubulin alpha4A chain,
Q64428: trifunctional enzyme subunit alpha, mitochondrial, and
P62632: elongation factor 1-alpha 2.

Nucleosome                                                20.31%
Hydrolyase activity                                        6.25%
Aerobic respiration                                        7.81%
Regulation of cellular respiration                         3.13%
Oxygen binding                                             4.69%
Protein transmembrane transport                            3.13%
Melanosome                                                 3.13%
Nucleosomal DNA binding                                    3.13%
Retina homeostasis                                         3.13%
Benzaldehyde dehydrogenase (NAD+) activity                 3.13%
Myelin sheath                                              3.13%
Intermediate filament                                      3.13%
Pigment granule                                            3.13%
mRNA 5'-UTR binding                                        3.13%
Response to mercury ion                                    4.69%
Cellular response to epidermal growth factor stimulus      3.13%
Cellular response to gamma radiation                       3.13%
Mesenchyme migration                                       6.65%
Eukaryotic translation elongation factor 1 complex         4.69%
NAD binding                                                7.81%

(a)
Note: Table made from pie chart.

Figure 6: Functional distribution of proteins (a) identified with
differential expression in rat submandibular glands exposed to MeHg
versus control group. Categories of proteins based on GO annotation
biological process, molecular function, and cellular component.
Terms significant (Kappa = 0.4) and distribution according to
percentage of number of gene association. Subnetwork (b) created by
ClusterMarker app to establish the interaction among identified
proteins of submandibular glands with differentiated expression on
exposed group when compared to control group. The node colors
indicate the differential expression of respective protein named
with its accession ID. Red and dark green indicate proteins found
in control and exposed groups, respectively. Light green and pink
indicate upregulated and downregulated proteins, respectively. Gray
nodes represent those proteins that were not identified on this
study, but they interacted on the network. The access
identification number in nodes corresponds to P52555: endoplasmic
reticulum resident protein 29, P06761: 78kDa glucose-regulated
protein, P14659: heat shock-related 70kDa protein 2, P15999: ATP
synthase subunit alpha, mitochondrial, Q09073: ADP/ATP translocase
2, Q6P9V9: tubulin alpha-1B chain, P62632: elongation factor
1-alpha 2, P35434: ATP synthase subunit delta, mitochondrial,
F1M446 protein RGD1306148, P68255: 14-3-3 protein theta, Q8VHF5:
citrate synthase, mitochondrial, and P62632: elongation factor
1-alpha 2.

Adenylate cyclase binding                                       5.71%
Nuclear nucleosome                                              8.57%
Myelin sheath                                                  31.43%
Mitochondrial proton-transporting ATP synthase complex,         5.71%
  catalytic core (f1)
Substantia nigra development                                   11.43%
Phosphoserine binding                                           5.71%
bHLH transcription factor binding                               5.71%
Eukaryotic translation elongation factor 1 complex              5.71%
Regulation of sodium ion transmembrane transporter activity     8.57%
Structural constituent of cytoskeleton                         11.43%

(a)

Note: Table made from pie chart.

Figure 7: Functional distribution of proteins (a) identified with
differential expression in rat submandibular glands exposed to MeHg
versus control group. Categories of proteins based on GO annotation
biological process, molecular function, and cellular component.
Terms significant (Kappa = 0.4) and distribution according to
percentage of number of gene association. Subnetwork (b) created by
ClusterMarker app to establish the interaction among identified
proteins of sublingual glands with differentiated expression on
exposed group when compared to control group. The node colors
indicate the differential expression of respective protein named
with its accession ID. Red and dark green indicate proteins found
in control and exposed groups, respectively. Light green and pink
indicate upregulated and downregulated proteins, respectively. Gray
nodes represent those proteins which were not identified on this
study, but they interacted on the network. The access
identification number in nodes corresponds to P80067: dipeptidyl
peptidase 1, P63018: heat shock cognate 71 kDa protein, P21708:
mitogen-activated protein kinase 3, Q6P9V9: tubulin alpha-1B chain,
P14659: heat shock-related 70kDa protein 2, P06761: 78 kDa
glucose-regulated protein, P52555: endoplasmic reticulum resident
protein 29.

Alpha-amylase activity                                          9.25%
Myelin sheath                                                  33.33%
Unfolded protein binding                                       19.05%
Detection of chemical stimulus involved in sensory              9.52%
  perception of bitter taste
Retina homeostasis                                             14.29%
Structural constituent of cytoskeleton                         14.20%

(a)

Note: Table made from pie chart.
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Title Annotation:Research Article
Author:Puty, Leonardo Oliveira BittencourBruna; Charone, Senda; Aragao, Walessa Alana Braganca; Farias-Juni
Publication:Oxidative Medicine and Cellular Longevity
Article Type:Report
Date:Jan 1, 2017
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