TY - JOUR
AU1 - Fortoul, Teresa, Imelda
AU2 - Vélez-Cruz,, Madeleine
AU3 - Antuna-Bizarro,, Silvia
AU4 - Montaño, Luis, F.
AU5 - Rodriguez-Lara,, Vianey
AU6 - Saldivar-Osorio,, Liliana
AB - Abstract Air pollution by suspended particles has become a worldwide health problem. The main sources of these particles are fossils and additives combustion. Mn enters the body through inhalation, but part of the particles accesses contact with tongue's posterior surface where lingual tonsils and lingual papillae are placed. We decided to explore in a mouse model, the impact that the deposit of inhaled Mn has on the tongue's surface. Atrophy of the lingual tonsil, filiform papillae, as well as the swelling of taste buds in fungiform papillae, were the predominant changes. Ferropenic anemia is associated with the changes described and could be related to the interference of Mn in iron metabolism and riboflavin absorption. More research should be done to explore the participation of suspended particles trapped in the oral cavity in toxicology of Mn or other inhaled pollutants. manganese, oral cavity, tongue, inhalation exposure, scanning electron microscopy Introduction Air pollution, as a consequence of total suspended particles (TSP) has originated a worldwide health problem. In these particles, metals are adsorbed; they enter into the lungs and into the systemic circulation. The main source of suspended particles is combustion of fuel sources and additives [1,2]. In some countries, lead additives were substituted by manganese (Mn) in the form of methylcyclopentadienyl manganese tricarbonyl (MMT), and a sequel was the increase in manganese in the air [3]. Other sources of airborne Mn include wind erosion of dusts and soils, anthropogenic fugitive dusts, and emissions from power plants, coke ovens, municipal waste incinerators and metal smelting operations [3]. The nervous system is the main target of Mn, which induces an alteration similar to Parkinson's disease called manganism [1,2,4,5]; other systems affected by this element are reproductive, respiratory, hematologic and endocrine organs [6,7]. Inhalation is the main route for TPS lung and system access [2,8,9]; however, part of the particles are retained at the upper part of oropharyngeal mucosa, swallowed and part of these particles enter in contact with the tongue's dorsal surface [1,2] where the lingual tonsils and the lingual papillae are located [10]. There are four types of lingual papillae: filiform, fungiform, foliate, and circumvallated. The taste buds are present in all of them except in the filiform papillae [10]. No information is available in the literature about the effects that the deposition of swallowed air pollutants might have on the tongue epithelium, which is the largest organ in the oral cavity. With the previous information, we decided to implement a manganese inhalation model in mice to evaluate the morphological changes which might occur on tongue's dorsal surface. Methods Manganese exposure Forty-eight CD-1 male mice weighing 35 ± 2 g were placed in an acrylic box inhaling 0.02 M MnCl2 (Merk, Darmstadt, Germany) 1 h twice a week during 12 weeks under controlled light conditions (12-h light/dark regime), and fed with Purina rat chow and water ad libitum. Sixteen control mice inhaled only the vehicle saline for the same period of time. The experimental protocol was in accordance with the Animal Act of 1986 for Scientific Procedures. Inhalation exposures were performed as described by Fortoul et al. [11]. Light and scanning sample process Four exposed mice and two controls were sacrificed each week during the whole exposure protocol (12 weeks). Under i.p. sodium pentobarbital anesthesia, and via the aorta, all the animals were perfused with the saline (0.9%), followed by a fixative solution containing 2% glutaraldehyde and 2% paraformaldehyde in a 0.1 M phosphate buffer, pH 7.2, and for 2 h postfixed in 2% osmium tetroxide. From each animal, the whole tongue was extracted including the base, and sectioned in three longitudinal segments. Right half of the tongue from each animal was processed for standard light microscopy evaluation and the left half was processed for scanning electron microscopy analysis as follows [9]. Samples were dried in an E3000 Polaron Point Drying Apparatus [14]; then selected sections were mounted in stubs with silver double tape and first coated with a carbon layer, followed by a 20-nm layer of gold in a Polaron Ion Supputer-4D1. Samples were analyzed in a JEOL JSM 35 CF (Tokyo, Japan). From each animal, at least six fields were evaluated [12]. Taste bud counts From each sample, other papillae, besides filiform, were observed with Mallory trichrome stain in order to identify taste buds. From each sample taste buds from five papillae were evaluated, and the longitudinal axis was measured by means of an image processor software Image-Pro Plus 6 software (Silver Spring, MD, USA). ANOVA with Tukey's post hoc test was performed (SigmaStat 3.0, Systat Software Inc., San Jose, CA, USA) considering differences statistically significant between controls and exposed mice at P < 0.05. Manganese chamber concentration The concentrations of MnCl2 in the chamber were quantified as follows: a filter was positioned at the outlet of the ultranebulizer during the whole inhalation time at a flow rate or 10 L min−1. After each exposure, the filters were removed and weighed; the element was quantified following the same protocol as with tissue samples. Six filters for each inhalation were evaluated [13]. Samples were analyzed using a graphite furnace, atomic absorption spectrometer (Perkin Elmer model 2380). The light source came from a hollow cathode lamp. Formaldehyde and the blanks were also analyzed to identify metal contamination from this source. Accuracy was assured by three random determinations of seven different standard solutions, prepared with the same chemical reactives used during the metal analysis. For MnCl2, the wavelength was 279.5 nm, the detection limit was 0.05 ppm and the slit 0.2 nm. Each sample was analyzed in triplicate [14]. Results Histological findings Control mice tongue's surface was covered with slender structures which correspond to filiform papillae covered with the stratified squamous keratinized epithelium (Fig. 1a). In the exposed mice, filiform papillae were scarce and wider than those observed in controls (Fig. 1b). Fungiform papillae were scarce in control and exposed mice. A central taste bud was identified with cells arranged as cocoons with dark and clear cells in controls (Fig. 2a). In exposed mice, taste bud swelling was observed (Fig. 2b, c and d) and when its length was measured (Fig. 3), a statistically significant difference was observed at all exposures times compared with controls (P < 0.05), a change that supports the swelling previously mentioned. Fig. 1. Open in new tabDownload slide (a) The normal structure of the tongue's surface is observed, with several filiform papillae (arrows) covering it. The squamous epithelium is observed as part of the papillae structure. (b) When exposed animals were evaluated, a decrease in the presence of filiform papillae was observed (arrows). The remaining structures were wider than those observed in controls (hematoxylin & eosin stain. Bar = 315 μm). Fig. 1. Open in new tabDownload slide (a) The normal structure of the tongue's surface is observed, with several filiform papillae (arrows) covering it. The squamous epithelium is observed as part of the papillae structure. (b) When exposed animals were evaluated, a decrease in the presence of filiform papillae was observed (arrows). The remaining structures were wider than those observed in controls (hematoxylin & eosin stain. Bar = 315 μm). Fig. 2. Open in new tabDownload slide (a) Control fungiform papilla with its central taste bud (arrow). A single papilla is surrounded by several filiform papillae. (b) At fourth week the cells in the taste bud (arrow) are separated and a decrease in filiform papillae is evident. (c) An increase in size of the taste bud at the eighth week is observed (arrow). (d) The changes, swelling (arrow) and loss of papillae persisted after 12 weeks of exposure (hematoxylin & eosin stain. Bar = 117 μm). Fig. 2. Open in new tabDownload slide (a) Control fungiform papilla with its central taste bud (arrow). A single papilla is surrounded by several filiform papillae. (b) At fourth week the cells in the taste bud (arrow) are separated and a decrease in filiform papillae is evident. (c) An increase in size of the taste bud at the eighth week is observed (arrow). (d) The changes, swelling (arrow) and loss of papillae persisted after 12 weeks of exposure (hematoxylin & eosin stain. Bar = 117 μm). Fig. 3. Open in new tabDownload slide Differences in the longitudinal axis length (μm) of taste buds in fungiform papillae, comparing controls versus exposed mice. Fig. 3. Open in new tabDownload slide Differences in the longitudinal axis length (μm) of taste buds in fungiform papillae, comparing controls versus exposed mice. Ultrastructural findings Lingual tonsil In controls, crypts were surrounded by lymphoid tissue covered by non-keratinized stratified epithelium (Fig. 4a). Fig. 4. Open in new tabDownload slide (a) Control lingual tonsil is shown (arrow) with some epithelial cells sloughed, and folds of connective tissue surrounding it. (b) An exposed mouse (5 weeks) presented atrophy of two tonsil crypts (arrows), as well as similar changes in the surrounding tissue. In (c), a central bud is evident in control mice (*) as well as the tubercles forming demilunes with some sloughed cells. (d) Atrophy is evident (*) in the central bud, as well as in the tubercles (arrows); deep furrows and an increase in cellular sloughing were evident. Also, evagination of the circumvallated furrow is shown (white arrow) (scanning electron microscopy). Fig. 4. Open in new tabDownload slide (a) Control lingual tonsil is shown (arrow) with some epithelial cells sloughed, and folds of connective tissue surrounding it. (b) An exposed mouse (5 weeks) presented atrophy of two tonsil crypts (arrows), as well as similar changes in the surrounding tissue. In (c), a central bud is evident in control mice (*) as well as the tubercles forming demilunes with some sloughed cells. (d) Atrophy is evident (*) in the central bud, as well as in the tubercles (arrows); deep furrows and an increase in cellular sloughing were evident. Also, evagination of the circumvallated furrow is shown (white arrow) (scanning electron microscopy). A single circumvallated papilla in controls was located in the posterior third of the tongue. These calyx-like papillae were evident with a circumvallated furrow (deep cleft) notoriously large, as well as some tubercles which form two demillunes at each side (Fig. 4c). Fungiform papillae were identified by their fungi-like shape, distributed over the surface with their main location at the tip of the tongue. These structures in mice have a central taste bud (Fig. 5a). Fig. 5. Open in new tabDownload slide (a) Regular and compact papillae are observed with multiple layers of epithelial cells surrounding the taste bud (arrow). (b) An increase in sloughing was evident. (c) Several filiform papillae are evidenced with double or triple tip ends. (d) At 12 weeks of the exposure, three irregular filiform papillae are presented, with some foldings (arrows) and other three with fractures (*). A layer of cellular debris covers the surface. Fig. 5. Open in new tabDownload slide (a) Regular and compact papillae are observed with multiple layers of epithelial cells surrounding the taste bud (arrow). (b) An increase in sloughing was evident. (c) Several filiform papillae are evidenced with double or triple tip ends. (d) At 12 weeks of the exposure, three irregular filiform papillae are presented, with some foldings (arrows) and other three with fractures (*). A layer of cellular debris covers the surface. Filiform papillae were exuberant and their shape was like thin hair prolongation, some of them ending in double tips. They were covered by keratinized stratified epithelium (Fig. 5c). Exposed animals Changes in the tonsil were evident at the fourth week of inhalation, and were more evident at the sixth week of inhalation. Atrophy was identified because of its flattened surface (Fig. 4b). The same changes described for the tonsils were observed in the circumvallated papillae. Also, the loss of some of the tubercles which formed the demillunes was evidenced. In addition, desquamated cells were noticed (Fig. 4D). Fungiform papillae presented an increase epithelial cell shedding, losing their usual morphology at the sixth week (Fig. 5b). In exposed animals since the fourth week of exposure, the partial loss and rupture of the filiform papillae were notorious, changes which persisted until the end of the study (Fig. 5d). Manganese average concentration in the inhalation chamber, during the whole experiment was 14.1 μg m−3. Discussion The normal breathing pattern changes during life; observing that more children are mouth breathers compared with adults, this may alter the deposition of inhaled pollutants and predisposes children to decrease the conditioning and cleansing of the inhaled air. The nose partially filters toxic particles and gases, making the mode of breathing, oral versus nasal, an important determinant of toxicant dose to the lungs or to other surfaces, such as oral cavity and specially the larger organ in the mouth: the tongue [15]. The oral cavity might be the reflection of individual's health. Changes indicating disease are seen as alterations in the oral mucosa lining the mouth, which might reveal systemic conditions, such as diabetes, vitamin deficiency, altered breathing patterns [15] or the local effects of chronic tobacco or alcohol use [16]. Nothing has been mentioned in the literature about air pollution, and if air-suspended particles (ASP) with metals could be deposited on the mucosa and the tongue's surface, the metals adsorbed on the ASP might be ionized and liberated in the cavity, generating local injuries or being absorbed and entering into the systemic blood stream [17]. Manganese is found in the air as the result of industrial emissions and combustion of fossil fuels [1]. A previous report from our group referred its presence in the lung tissue from autopsy cases with a moderate, but not statistically significant increase throughout three decades [14]. One of the clinical findings in manganese poisoning is ferropenic anemia associated with glossitis, characterized by the loss of filiform papillae, as we identified in this murine model [18]. Also, changes like glossitis and selective filiform papillae atrophy have been mentioned in riboflavin deficiency [19]. In this regard, an explanation for this association might be because of the interference of manganese in iron absorption, and this situation might result in secondary iron deficiency, which at the same time could limit riboflavin absorption, giving the changes that we are reporting here [20]. As another source of oral toxic reactions, manganese is referred only as capable of inducing oral mucosa pigmentation, but other oral structures being affected has not been referred [21]. The defoliation and swelling of taste buds in fungiform papillae observed in this model would decrease the contact surface of taste buds, as well as their function, decreasing taste sense, and probably inducing changes in food intake behavior as a consequence of the exposure to suspended toxic air particles [1]. A common mechanism of tissue damage is oxidative stress [22,23] and we suggest that manganese might be inducing the changes observed by this mechanism, because of its chemical reactivity [24]. Because of the scarceness of information about the impact of air pollution on tongue's surface in general and inhaled metals in particular, we were not able to compare our results with others. For this reason, we might only report that the changes we are showing here were associated with the inhalation of Mn. More research is needed in order to dilucidate the pathophysiology of inhaled metals and oral cavity changes, especially on tongue's papillae, structures in which taste takes place and could modify nutritional behavior in mammals. Also, the impact that mouth breathing might have on oral cavity health should be evaluated, because this breathing pattern will decrease air conditioning, and will increase the concentration of a variety of xenobiotics in the saliva, the teeth and the tongue, with insufficiently evaluated effects. Funding Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, Universidad Nacional Autónoma de Mexico, PAPIIT-UNAM IN200606. 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TI - Morphological changes in the tongue as a consequence of manganese inhalation in a murine experimental model: Light and scanning electron microscopic analysis
JF - Journal of Electron Microscopy
DO - 10.1093/jmicro/dfp041
DA - 2010-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/morphological-changes-in-the-tongue-as-a-consequence-of-manganese-eQgCZxX7ln
SP - 71
EP - 77
VL - 59
IS - 1
DP - DeepDyve
ER -