Ultrastructural Pathology
ISSN: 0191-3123 (Print) 1521-0758 (Online) Journal homepage: http://www.tandfonline.com/loi/iusp20
ESEM Detection of Foreign Metallic Particles inside Ameloblastomatous Cells Luca Roncati MD, PhD, Antonietta Morena Gatti PhD, Teresa Pusiol MD, PhD, Giuseppe Barbolini MD, PhD, Antonio Maiorana MD, PhD & Stefano Montanari PhD To cite this article: Luca Roncati MD, PhD, Antonietta Morena Gatti PhD, Teresa Pusiol MD, PhD, Giuseppe Barbolini MD, PhD, Antonio Maiorana MD, PhD & Stefano Montanari PhD (2015) ESEM Detection of Foreign Metallic Particles inside Ameloblastomatous Cells, Ultrastructural Pathology, 39:5, 329-335, DOI: 10.3109/01913123.2015.1042608 To link to this article: http://dx.doi.org/10.3109/01913123.2015.1042608
Published online: 25 Jun 2015.
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Ultrastructural Pathology, 2015; 39(5): 329–335 ! Taylor & Francis Group, LLC ISSN: 0191-3123 print / 1521-0758 online DOI: 10.3109/01913123.2015.1042608
B ASIC RESEARCH
ESEM Detection of Foreign Metallic Particles inside Ameloblastomatous Cells Luca Roncati, MD, PhD1, Antonietta Morena Gatti, PhD2,3, Teresa Pusiol, MD, PhD4, Giuseppe Barbolini, MD, PhD1, Antonio Maiorana, MD, PhD1, and Stefano Montanari, PhD3
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1
Department of Diagnostic and Clinical Medicine and of Public Health, Section of Pathology, University of Modena and Reggio Emilia, Modena (MO), Italy, 2National Research Council, Institute of Science and Technology for Ceramics, Faenza (RA), Italy, 3Nanodiagnostics Laboratory, San Vito di Spilamberto, Modena (MO), Italy, and 4Provincial Health Care Services, Institute of Pathology, Santa Maria del Carmine Hospital, Rovereto (TN), Italy
ABSTRACT Ameloblastoma is a borderline tumor of odontogenic origin, with a high recurrence rate and possible local aggressiveness. The etiopathogenetic factors involved in its occurrence are not still defined and our study has been precisely aimed to search for novel factors associated with its development. Sections cut from paraffin blocks, containing the representative specimens of 18 different ameloblastomas, collected in a 15-year period (1999–2014), have been observed by an environmental scanning electron microscope, in order to search microand nano-sized particles and to identify their composition. In all the neoplastic cases, micro- and nano-sized metallic debris, differing in size and composition, have been detected inside the ameloblastomatous cells. On the contrary, the total absence of metallic particles in the healthy control cases has been emerged. Our results reveal a relationship between ameloblastoma and metallic particulate. The cigarette smoke and the routine dental practice appear the most probable source for the presence of these biopersistant inorganic particles inside the neoplastic cells. Keywords: Ameloblastoma, cigarette smoke, dental practice, environmental scanning electron microscope (ESEM), heavy metals, metallic particles
INTRODUCTION
which can nevertheless display malignant features, such as local recurrence and invasive behavior, with tendency to infiltrate beyond the observed radiographic margins [4]. It is the most common neoplasia of the jaws. It shows an even distribution between the sexes and accounts for 1% of all the oral tumors. It can occur at all the ages with a peak incidence in the third and fourth decade of life. The mandible is by far more frequently affected than the maxilla [5]. Classic ameloblastoma typically grows in intraosseous toothbearing areas of the jaws, especially in the molar region, and appears as a solid or cystic, (unilocular or
Described for the first time in 1827 by Cusack [1], ameloblastoma is one of the most controversial tumors of the maxillofacial region. Initially designated as adamantinoma [2], the term ‘‘ameloblastoma’’ was subsequently coined in 1934 by Ivey and Churchill [3], merging the early English word ‘‘amel’’ (enamel) with the Greek ‘‘blastos’’ (germ). In effect, ameloblasts are epithelial cells deputed to the enamel deposit during tooth development. Ameloblastoma is a borderline odontogenic tumor characterized by a slow growth,
Received 28 February 2015; Revised 4 April 2015; Accepted 9 April 2015; Published online 25 June 2015 Correspondence: Dr. Luca Roncati, MD, PhD, Department of Diagnostic and Clinical Medicine and of Public Health, Section of Pathology, University of Modena and Reggio Emilia, Policlinico Hospital, I-41124 Modena (MO), Italy. Tel: +390594224812. Fax: +390594224998. ORCID: 0000-0001-6949-2216. E-mail: [email protected]
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330 L. Roncati et al. multilocular) lesion. A rare variant, known by the term ‘‘peripheral ameloblastoma’’, directly develops from the overlying gingival soft tissues [6]. The tumor is often asymptomatic, presenting as an incidental finding on X-ray. Its size can considerably vary: if clinical symptoms, such as facial swelling, pain, malocclusion, and loosening of teeth, occur, they are usually due to the physical presence of the tumor [7]. In 1933, Cahn first proposed that ameloblastoma could arise from a dentigerous cyst [8], and, over the years, several authors have tried to find the factors that could be at the basis of the cyst’s lining transformation [9–11]. Unspecified pressure and nutritional disturbances, rickets, extraction trauma, caries infection, severe oral sepsis and viruses have all been proposed [10]. In 1955, Small and Waldron collected and discussed the different hypotheses about the origin of ameloblastoma: odontogenic rests, surface epithelium, epithelium of dentigerous cysts and epithelial rests in follicles of unerupted teeth [12]. In 1960s, several authors investigated the possibility of a viral origin (polyoma virus) of ameloblastoma [13–15]. Later, there was a surge of interest in human papilloma viruses, which have been extensively studied using the recent advances in immunohistochemistry and molecular biology [16–19]. Taking into account the frequency of this neoplasm and the scanty knowledge regarding its etiologic factors, we have analyzed the tumor samples using an innovative technique of investigation, the environmental scanning electron microscopy (ESEM), in order to search micro- and nano-sized particles and to identify their composition.
MATERIALS AND METHODS This study was based on 18 cases of ameloblastoma (9 classic cases and 9 peripheral variants) diagnosed and treated between 1999 and 2014. In 14 patients (77.7%), the ameloblastoma was located in the mandibular area and in 4 cases (22.3%), the tumor was at the level of the maxillary region. All the patients were active smoker and all they had previously undergone dental practices. Ten of the patients were women, while eight were men, their age ranging between 16 and 88 years old, with a mean age of 42 years. The tumor size varied between 0.7 and 6 cm. Seven patients (38.9%) had one recurrence and only a patient had two recurrences, the second 41 months after the primary surgery. In 11 patients (61.1%), primary surgery was radical resection of the lesion, whereas 7 patients (38.9%) underwent non-radical surgery. Biopsies were performed during primary surgery and histological examinations were carried out. The negative controls used in this study were the specimens of both the maxillary and mandibular molar regions of six patients, who gave their consent to have a biopsy
during the extraction of their upper or lower third molar. Twenty micrometer thick sections were cut from representative formalin-fixed and paraffinembedded blocks. The slices were suspended in warm water and deposited on a clean rectangular acetate sheet (4 mm 2 mm). The samples were covered with some drops of xylene and 98% alcohol. After a few seconds, the excess of liquid was slid along the sheet edge and absorbed in blotting paper. A double-sided adhesive carbon disc was stuck between an acetate sheet and a 12-mm diameter aluminum stub and observed under an ESEM (Quanta 200, FEI-Company, The Netherlands). ESEM can work at room conditions and can be used to examine uncoated, non-conductive, and wet specimens, also without any dehydration of the biological sample. The ESEM used in this study was equipped with a tungsten filament, an ionization secondary electron sensor and an energy dispersive system (EDS by EDAX, Mahwah, NJ) for the chemical analysis, able to detect all the elements beyond beryllium (Be). Different kinds of observation were carried out: high vacuum (10–6 Torr), low vacuum (0.2–1.2 Torr), and air; secondary and backscattered electron mode; accelerating voltages from 12 to 25 kV; point to point resolution from 6.0 to 4.0 nm. This new specific equipment was deemed to be necessary and, enabling to observe unprocessed samples, it ensured that no foreign particulates or air-borne dusts were introduced after performing the biopsy.
RESULTS By histological examination, four different histological sub-types of ameloblastoma have been detected: follicular (eight cases), plexiform (six cases), basal cells (two cases), and spindle cells (two cases) (Figure 1). In all the ameloblastomatous samples, exogenous particles of different sizes and composition have been found. Finely entrapped inside the ameloblastomatous cells, there were micro- and nano-sized foreign particles, which EDS has revealed to be mostly metallic in origin. These particles contained wolfram (W) and titanium (Ti) (Figure 2), silver (Ag) (Figure 3), gold (Au) (Figure 4), or were lead (Pb) -based (Figure 5) alloys. The debris sizes ranged from 40 mm down to 100 nm. There was no apparent relationship between the histological type of ameloblastoma and the different particulate found. On the contrary, in all the control cases, there was no presence of foreign metallic debris. The EDS spectrum in fact revealed only the presence of carbon (C), oxygen (O), sodium (Na), phosphorous (P), sulfur (S), calcium (Ca), and iron (Fe) (Figure 6), that is the normal elemental composition of a fixed biological sample from a healthy tissue. Ultrastructural Pathology
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Heavy Metal Storage in Ameloblastomatous Cells 331
FIGURE 1. A surgical specimen of ameloblastoma from our series is seen on histological examination: the oral mucosa is appreciable just above the bleeding neoplasia (A, H&E, 4). Four different histological patterns have been encountered: follicular (B, H&E, 10), plexiform (C, H&E, 10), basal cells (D, H&E, 4), and spindle cells (E, H&E, 10).
FIGURE 2. ESEM images (A and B) with related EDS spectrum (C) of an intracellular tumor (histological insert) particulate characterized by the presence of two heavy metals, wolfram (W), and titanium (Ti). Both wolfram and titanium spectral peaks are noticeable, well beyond the trace level (X axis = keV; Y axis = counts).
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FIGURE 3. ESEM image (A) with related EDS spectrum (B) of an intracellular tumor (histological insert) storage of silver (Ag), whose spectral peak is well observable. On the contrary, iron (Fe) and chromium (Cr) are present at trace level (X axis = keV; Y axis = counts).
FIGURE 4. ESEM image (A) with related EDS spectrum (B) of an intracellular tumor (histological insert) particle of gold (Au) in a pure form, as confirmed by the presence of three exclusive spectral peaks (X axis = keV; Y axis = counts).
DISCUSSION Our results claim attention to the presence of microand nano-sized metallic particulate inside the ameloblastomatous cells in the totality of the examined cases. Metals are chemical elements with a shiny appearance and they are good conductors of electricity and heat. From a practical point of view, they are sub-divided into light and heavy metals. Different definitions of heavy metals have been proposed, based on the elemental density (45 g/cm3) or the atomic weight (420) [20]. From a biochemical point of view, it is inclined to think a heavy metal as a cationic element with different oxidation states and with high ability to form DNA adducts and molecular complexes in the cell cytoplasm [20]. Commonly encountered heavy metals are as follows: aluminum (Al), arsenic (As), bismuth (Bi), cadmium (Cd), chromium
(Cr), cobalt (Co), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), mercury (Hg), nickel (Ni), platinum (Pt), silver (Ag), titanium (Ti), and wolfram (W). In our series, we have observed the presence of four heavy metals (lead, wolfram, silver, titanium) beyond the trace levels inside the ameloblastomatous cells. As it is well known, heavy metals are able to interfere with the function of vital cellular components, such as structural proteins, enzymes, and nucleic acids [21], and a long-term exposure to their high concentrations can be related to tumorigenesis [22,23]. Moreover, it has been already demonstrated that the presence of metallic particles in a human tissue can depress the immunological response, favoring tumorigenesis, and infections [24,25]. During dental practice, the drilling of hard metallic alloys produce and release debris inside the mouth. For instance, the debris of wolfram detected in our patients could belong to dental burrs Ultrastructural Pathology
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FIGURE 5. ESEM image (A) with related EDS spectrum (B) of an intracellular tumor (histological insert) lead (Pb) -based alloy, as testified by the co-presence of silicon (Si) and aluminum (Al) (X axis = keV; Y axis = counts).
FIGURE 6. ESEM images (A and B) with related EDS spectrum (C) of a healthy gingival tissue, showing only the presence of carbon (C), oxygen (O), sodium (Na), phosphorous (P), sulfur (S), calcium (Ca), and iron (Fe), that is the normal elemental composition of a fixed biological sample. Oncogenic heavy metals are completely missing (X axis = keV; Y axis = counts).
used to drill dental prostheses, when they are removed. The co-presence of titanium is also explainable through the use of dental bars or orthodontic wires. It does not have origin from dental reamers, since they are alloy of nickel–titanium. Moreover, silver is present in dental materials bound to noble !
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elements in prosthetic alloys or it can be found with mercury and copper in amalgams [26,27]. The particle of gold found in a case could be originated from dental prosthesis during a drilling. It is pure and for this reason it can be part of a gold inset, which had been notoriously installed in the patient. Moreover,
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334 L. Roncati et al. some detected debris are made up by an element mixture that induce to believe that it derives from an uncontrolled combustion (smoking or ingestion of polluted food). As a matter of fact, the tobacco of the cigarettes can contain the environmental pollution where it was grown [28], and the presence of heavy metals has been already described in cigarette smoke [29–31]. In conclusion, the ESEM investigation has allowed to successfully correlate the presence of inorganic metallic particles to ameloblastoma, for the first time in literature. However, the finding of metallic particles in ameloblastoma does not indicate a causal relationship. The source of this pollution can have different origins by dental practice, such as drilling and blasting. The possible side effects induced by the generation of ultrafine metallic particulate inside the mouth could lead to further recommendations for the dentists in their routinely activity, encouraging the use of rubber-dam. There is also the concrete possibility of pollution from the cigarette smoke, which is so confirmed as a broad-spectrum risk factor for tumors of the oral cavity [32]. In Taiwan, a statistical association between heavy metal exposure and oral cancer has been already described [33,34]; future researches, aimed to testify the presence of metal particles inside the neoplastic cells from other tumors of the oral cavity (e.g. squamous cell carcinoma), are advocated.
ACKNOWLEDGEMENTS The authors would express their thanks to Federico Capitani and Luca Fabbiani for their technical assistance.
DECLARATION OF INTEREST The authors report no conflicts of interest.
FUNDING The authors alone are responsible for the content and writing of this article. This study was supported by Nanodiagnostics Laboratory (Modena, IT).
REFERENCES 1. Cusack JW. Report of the amputations of the lower jaw. Dublin Hosp Rec 1827;4: 1–38. 2. Malassez L. Sur le role des debris epitheliaux papdentaires. Arch Physiol Norm Pathol 1885;5: 309–40. 3. Ivey RH, Churchill HR. The need of a standardized surgical and pathological classification of tumors and anomalies of dental origin. Am Assoc Dent Sch Trans 1930; 7: 240–5.
4. More C, Tailor M, Patel HJ, et al. Radiographic analysis of ameloblastoma: A retrospective study. Indian J Dent Res 2012;23: 698. 5. Mendenhall WM, Werning JW, Fernandes R, et al. Ameloblastoma. Am J Clin Oncol 2007;30: 645–8. 6. Bertossi D, Favero V, Albanese M, et al. Peripheral ameloblastoma of the upper gingiva: Report of a case and literature review. J Clin Exp Dent 2014;6: e180–4. 7. Dandriyal R, Gupta A, Pant S, Baweja HH. Surgical management of ameloblastoma: Conservative or radical approach. Natl J Maxillofac Surg 2011;2: 22–7. 8. Cahn LR. The dentigerous cyst is a potential adamantinoma. Dent Cosmos 1933;75: 889–93. 9. Reichart PA, Philipsen HP, Sonner S. Ameloblastoma: biological profile of 3677 cases. Eur J Cancer B Oral Oncol 1995;31B: 86–99. 10. Gomes CC, Duarte AP, Diniz MG, Gomez RS. Current concepts of ameloblastoma pathogenesis. J Oral Pathol Med 2010;39: 585–91. 11. Jhamb T, Kramer JM. Molecular concepts in the pathogenesis of ameloblastoma: implications for therapeutics. Exp Mol Pathol 2014;97: 345–53. 12. Small IA, Waldron CA. Ameloblastoma of the jaws. Oral Surg 1955;8: 281–97. 13. Fleming HS. Polyoma virus tumor and the teeth. J Dent Res 1963;42: 1405–15. 14. Stanley HR, Dawe CJ, Law LW. Oral tumors induced by polyoma virus in mice. Oral Surg Oral Med Oral Pathol 1964;17: 547–58. 15. Main JHP, Dawe CJ. Tumor induction in transplanted tooth buds infected with polyoma virus. J Nat Cancer Ins 1966;36: 1121–8. 16. Kahn MA. Demonstration of human papillomavirus DNA in a peripheral ameloblastoma by in situ hybridization. Hum Pathol 1992;23: 188–91. 17. Sand L, Jalouli J, Larsson PA, et al. Presence of human papilloma viruses in intraosseous ameloblastoma. J Oral Maxillofac Surg 2000;58: 1129–34. 18. Namin AK, Azad TM, Eslami B, et al. A study of the relationship between ameloblastoma and human papilloma virus. J Oral Maxillofac Surg 2003;61: 467–70. 19. Migaldi M, Pecorari M, Rossi G, et al. Does HPV play a role in the etiopathogenesis of ameloblastoma? An immunohistochemical, in situ hybridization and polymerase chain reaction study of 18 cases using laser capture microdissection. Mod Pathol 2005;18: 283–9. 20. Duffus JH. ‘‘Heavy metals’’ a meaningless term? In: Burrows H, Weir R, Stohner J, eds. Pure and applied chemistry (IUPAC Technical Report). Research Triangle Park: De Gruyter, 2009:793–807. 21. Hamilton JW, Kaltreider RC, Bajenova OV, et al. Molecular basis for effects of carcinogenic heavy metals on inducible gene expression. Environ Health Perspect 1998;4: 1005–15. 22. Zhao Q, Wang Y, Cao Y, et al. Potential health risks of heavy metals in cultivated topsoil and grain, including correlations with human primary liver, lung and gastric cancer, in Anhui province, Eastern China. Sci Total Environ 2014;470–471: 340–7. 23. Khlifi R, Olmedo P, Gil F, et al. Arsenic, cadmium, chromium and nickel in cancerous and healthy tissues from patients with head and neck cancer. Sci Total Environ 2013;452–453: 58–67. 24. Sighinolfi GL, Artoni E, Gatti AM, Corsi L. Carcinogenic potential of metal nanoparticles in BALB/3T3 cell transformation assay. Environ Toxicol 2014. doi: 10.1002/ tox.22063. 25. Roncati L, Gatti AM, Pusiol T, et al. Acquired immunodeficiency similar to Gulf War illness in a dead Ultrastructural Pathology
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30. Pappas RS, Fresquez MR, Martone N, Watson CH. Toxic metal concentrations in mainstream smoke from cigarettes available in the USA. J Anal Toxicol 2014;38: 204–11. 31. Stephens WE, Calder A, Newton J. Source and health implications of high toxic metal concentrations in illicit tobacco products. Environ Sci Technol 2005;39: 479–88. 32. Motgi AA, Chavan MS, Diwan NN, et al. Assessment of cytogenic damage in the form of micronuclei in oral epithelial cells in patients using smokeless and smoked form of tobacco and non-tobacco users and its relevance for oral cancer. J Cancer Res Ther 2014;10: 165–70. 33. Lin WC, Lin YP, Wang YC, et al. Assessing and mapping spatial associations among oral cancer mortality rates, concentrations of heavy metals in soil, and land use types based on multiple scale data. Int J Environ Res Public Health 2014;21: 2148–68. 34. Su CC, Tsai KY, Hsu YY, et al. Chronic exposure to heavy metals and risk of oral cancer in Taiwanese males. Oral Oncol 2010;46: 586–90.
ISSN: 0191-3123 (Print) 1521-0758 (Online) Journal homepage: http://www.tandfonline.com/loi/iusp20
ESEM Detection of Foreign Metallic Particles inside Ameloblastomatous Cells Luca Roncati MD, PhD, Antonietta Morena Gatti PhD, Teresa Pusiol MD, PhD, Giuseppe Barbolini MD, PhD, Antonio Maiorana MD, PhD & Stefano Montanari PhD To cite this article: Luca Roncati MD, PhD, Antonietta Morena Gatti PhD, Teresa Pusiol MD, PhD, Giuseppe Barbolini MD, PhD, Antonio Maiorana MD, PhD & Stefano Montanari PhD (2015) ESEM Detection of Foreign Metallic Particles inside Ameloblastomatous Cells, Ultrastructural Pathology, 39:5, 329-335, DOI: 10.3109/01913123.2015.1042608 To link to this article: http://dx.doi.org/10.3109/01913123.2015.1042608
Published online: 25 Jun 2015.
Submit your article to this journal
Article views: 58
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View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iusp20 Download by: [University of Florida]
Date: 07 November 2015, At: 00:46
Ultrastructural Pathology, 2015; 39(5): 329–335 ! Taylor & Francis Group, LLC ISSN: 0191-3123 print / 1521-0758 online DOI: 10.3109/01913123.2015.1042608
B ASIC RESEARCH
ESEM Detection of Foreign Metallic Particles inside Ameloblastomatous Cells Luca Roncati, MD, PhD1, Antonietta Morena Gatti, PhD2,3, Teresa Pusiol, MD, PhD4, Giuseppe Barbolini, MD, PhD1, Antonio Maiorana, MD, PhD1, and Stefano Montanari, PhD3
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1
Department of Diagnostic and Clinical Medicine and of Public Health, Section of Pathology, University of Modena and Reggio Emilia, Modena (MO), Italy, 2National Research Council, Institute of Science and Technology for Ceramics, Faenza (RA), Italy, 3Nanodiagnostics Laboratory, San Vito di Spilamberto, Modena (MO), Italy, and 4Provincial Health Care Services, Institute of Pathology, Santa Maria del Carmine Hospital, Rovereto (TN), Italy
ABSTRACT Ameloblastoma is a borderline tumor of odontogenic origin, with a high recurrence rate and possible local aggressiveness. The etiopathogenetic factors involved in its occurrence are not still defined and our study has been precisely aimed to search for novel factors associated with its development. Sections cut from paraffin blocks, containing the representative specimens of 18 different ameloblastomas, collected in a 15-year period (1999–2014), have been observed by an environmental scanning electron microscope, in order to search microand nano-sized particles and to identify their composition. In all the neoplastic cases, micro- and nano-sized metallic debris, differing in size and composition, have been detected inside the ameloblastomatous cells. On the contrary, the total absence of metallic particles in the healthy control cases has been emerged. Our results reveal a relationship between ameloblastoma and metallic particulate. The cigarette smoke and the routine dental practice appear the most probable source for the presence of these biopersistant inorganic particles inside the neoplastic cells. Keywords: Ameloblastoma, cigarette smoke, dental practice, environmental scanning electron microscope (ESEM), heavy metals, metallic particles
INTRODUCTION
which can nevertheless display malignant features, such as local recurrence and invasive behavior, with tendency to infiltrate beyond the observed radiographic margins [4]. It is the most common neoplasia of the jaws. It shows an even distribution between the sexes and accounts for 1% of all the oral tumors. It can occur at all the ages with a peak incidence in the third and fourth decade of life. The mandible is by far more frequently affected than the maxilla [5]. Classic ameloblastoma typically grows in intraosseous toothbearing areas of the jaws, especially in the molar region, and appears as a solid or cystic, (unilocular or
Described for the first time in 1827 by Cusack [1], ameloblastoma is one of the most controversial tumors of the maxillofacial region. Initially designated as adamantinoma [2], the term ‘‘ameloblastoma’’ was subsequently coined in 1934 by Ivey and Churchill [3], merging the early English word ‘‘amel’’ (enamel) with the Greek ‘‘blastos’’ (germ). In effect, ameloblasts are epithelial cells deputed to the enamel deposit during tooth development. Ameloblastoma is a borderline odontogenic tumor characterized by a slow growth,
Received 28 February 2015; Revised 4 April 2015; Accepted 9 April 2015; Published online 25 June 2015 Correspondence: Dr. Luca Roncati, MD, PhD, Department of Diagnostic and Clinical Medicine and of Public Health, Section of Pathology, University of Modena and Reggio Emilia, Policlinico Hospital, I-41124 Modena (MO), Italy. Tel: +390594224812. Fax: +390594224998. ORCID: 0000-0001-6949-2216. E-mail: [email protected]
329
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330 L. Roncati et al. multilocular) lesion. A rare variant, known by the term ‘‘peripheral ameloblastoma’’, directly develops from the overlying gingival soft tissues [6]. The tumor is often asymptomatic, presenting as an incidental finding on X-ray. Its size can considerably vary: if clinical symptoms, such as facial swelling, pain, malocclusion, and loosening of teeth, occur, they are usually due to the physical presence of the tumor [7]. In 1933, Cahn first proposed that ameloblastoma could arise from a dentigerous cyst [8], and, over the years, several authors have tried to find the factors that could be at the basis of the cyst’s lining transformation [9–11]. Unspecified pressure and nutritional disturbances, rickets, extraction trauma, caries infection, severe oral sepsis and viruses have all been proposed [10]. In 1955, Small and Waldron collected and discussed the different hypotheses about the origin of ameloblastoma: odontogenic rests, surface epithelium, epithelium of dentigerous cysts and epithelial rests in follicles of unerupted teeth [12]. In 1960s, several authors investigated the possibility of a viral origin (polyoma virus) of ameloblastoma [13–15]. Later, there was a surge of interest in human papilloma viruses, which have been extensively studied using the recent advances in immunohistochemistry and molecular biology [16–19]. Taking into account the frequency of this neoplasm and the scanty knowledge regarding its etiologic factors, we have analyzed the tumor samples using an innovative technique of investigation, the environmental scanning electron microscopy (ESEM), in order to search micro- and nano-sized particles and to identify their composition.
MATERIALS AND METHODS This study was based on 18 cases of ameloblastoma (9 classic cases and 9 peripheral variants) diagnosed and treated between 1999 and 2014. In 14 patients (77.7%), the ameloblastoma was located in the mandibular area and in 4 cases (22.3%), the tumor was at the level of the maxillary region. All the patients were active smoker and all they had previously undergone dental practices. Ten of the patients were women, while eight were men, their age ranging between 16 and 88 years old, with a mean age of 42 years. The tumor size varied between 0.7 and 6 cm. Seven patients (38.9%) had one recurrence and only a patient had two recurrences, the second 41 months after the primary surgery. In 11 patients (61.1%), primary surgery was radical resection of the lesion, whereas 7 patients (38.9%) underwent non-radical surgery. Biopsies were performed during primary surgery and histological examinations were carried out. The negative controls used in this study were the specimens of both the maxillary and mandibular molar regions of six patients, who gave their consent to have a biopsy
during the extraction of their upper or lower third molar. Twenty micrometer thick sections were cut from representative formalin-fixed and paraffinembedded blocks. The slices were suspended in warm water and deposited on a clean rectangular acetate sheet (4 mm 2 mm). The samples were covered with some drops of xylene and 98% alcohol. After a few seconds, the excess of liquid was slid along the sheet edge and absorbed in blotting paper. A double-sided adhesive carbon disc was stuck between an acetate sheet and a 12-mm diameter aluminum stub and observed under an ESEM (Quanta 200, FEI-Company, The Netherlands). ESEM can work at room conditions and can be used to examine uncoated, non-conductive, and wet specimens, also without any dehydration of the biological sample. The ESEM used in this study was equipped with a tungsten filament, an ionization secondary electron sensor and an energy dispersive system (EDS by EDAX, Mahwah, NJ) for the chemical analysis, able to detect all the elements beyond beryllium (Be). Different kinds of observation were carried out: high vacuum (10–6 Torr), low vacuum (0.2–1.2 Torr), and air; secondary and backscattered electron mode; accelerating voltages from 12 to 25 kV; point to point resolution from 6.0 to 4.0 nm. This new specific equipment was deemed to be necessary and, enabling to observe unprocessed samples, it ensured that no foreign particulates or air-borne dusts were introduced after performing the biopsy.
RESULTS By histological examination, four different histological sub-types of ameloblastoma have been detected: follicular (eight cases), plexiform (six cases), basal cells (two cases), and spindle cells (two cases) (Figure 1). In all the ameloblastomatous samples, exogenous particles of different sizes and composition have been found. Finely entrapped inside the ameloblastomatous cells, there were micro- and nano-sized foreign particles, which EDS has revealed to be mostly metallic in origin. These particles contained wolfram (W) and titanium (Ti) (Figure 2), silver (Ag) (Figure 3), gold (Au) (Figure 4), or were lead (Pb) -based (Figure 5) alloys. The debris sizes ranged from 40 mm down to 100 nm. There was no apparent relationship between the histological type of ameloblastoma and the different particulate found. On the contrary, in all the control cases, there was no presence of foreign metallic debris. The EDS spectrum in fact revealed only the presence of carbon (C), oxygen (O), sodium (Na), phosphorous (P), sulfur (S), calcium (Ca), and iron (Fe) (Figure 6), that is the normal elemental composition of a fixed biological sample from a healthy tissue. Ultrastructural Pathology
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Heavy Metal Storage in Ameloblastomatous Cells 331
FIGURE 1. A surgical specimen of ameloblastoma from our series is seen on histological examination: the oral mucosa is appreciable just above the bleeding neoplasia (A, H&E, 4). Four different histological patterns have been encountered: follicular (B, H&E, 10), plexiform (C, H&E, 10), basal cells (D, H&E, 4), and spindle cells (E, H&E, 10).
FIGURE 2. ESEM images (A and B) with related EDS spectrum (C) of an intracellular tumor (histological insert) particulate characterized by the presence of two heavy metals, wolfram (W), and titanium (Ti). Both wolfram and titanium spectral peaks are noticeable, well beyond the trace level (X axis = keV; Y axis = counts).
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FIGURE 3. ESEM image (A) with related EDS spectrum (B) of an intracellular tumor (histological insert) storage of silver (Ag), whose spectral peak is well observable. On the contrary, iron (Fe) and chromium (Cr) are present at trace level (X axis = keV; Y axis = counts).
FIGURE 4. ESEM image (A) with related EDS spectrum (B) of an intracellular tumor (histological insert) particle of gold (Au) in a pure form, as confirmed by the presence of three exclusive spectral peaks (X axis = keV; Y axis = counts).
DISCUSSION Our results claim attention to the presence of microand nano-sized metallic particulate inside the ameloblastomatous cells in the totality of the examined cases. Metals are chemical elements with a shiny appearance and they are good conductors of electricity and heat. From a practical point of view, they are sub-divided into light and heavy metals. Different definitions of heavy metals have been proposed, based on the elemental density (45 g/cm3) or the atomic weight (420) [20]. From a biochemical point of view, it is inclined to think a heavy metal as a cationic element with different oxidation states and with high ability to form DNA adducts and molecular complexes in the cell cytoplasm [20]. Commonly encountered heavy metals are as follows: aluminum (Al), arsenic (As), bismuth (Bi), cadmium (Cd), chromium
(Cr), cobalt (Co), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), mercury (Hg), nickel (Ni), platinum (Pt), silver (Ag), titanium (Ti), and wolfram (W). In our series, we have observed the presence of four heavy metals (lead, wolfram, silver, titanium) beyond the trace levels inside the ameloblastomatous cells. As it is well known, heavy metals are able to interfere with the function of vital cellular components, such as structural proteins, enzymes, and nucleic acids [21], and a long-term exposure to their high concentrations can be related to tumorigenesis [22,23]. Moreover, it has been already demonstrated that the presence of metallic particles in a human tissue can depress the immunological response, favoring tumorigenesis, and infections [24,25]. During dental practice, the drilling of hard metallic alloys produce and release debris inside the mouth. For instance, the debris of wolfram detected in our patients could belong to dental burrs Ultrastructural Pathology
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FIGURE 5. ESEM image (A) with related EDS spectrum (B) of an intracellular tumor (histological insert) lead (Pb) -based alloy, as testified by the co-presence of silicon (Si) and aluminum (Al) (X axis = keV; Y axis = counts).
FIGURE 6. ESEM images (A and B) with related EDS spectrum (C) of a healthy gingival tissue, showing only the presence of carbon (C), oxygen (O), sodium (Na), phosphorous (P), sulfur (S), calcium (Ca), and iron (Fe), that is the normal elemental composition of a fixed biological sample. Oncogenic heavy metals are completely missing (X axis = keV; Y axis = counts).
used to drill dental prostheses, when they are removed. The co-presence of titanium is also explainable through the use of dental bars or orthodontic wires. It does not have origin from dental reamers, since they are alloy of nickel–titanium. Moreover, silver is present in dental materials bound to noble !
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elements in prosthetic alloys or it can be found with mercury and copper in amalgams [26,27]. The particle of gold found in a case could be originated from dental prosthesis during a drilling. It is pure and for this reason it can be part of a gold inset, which had been notoriously installed in the patient. Moreover,
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334 L. Roncati et al. some detected debris are made up by an element mixture that induce to believe that it derives from an uncontrolled combustion (smoking or ingestion of polluted food). As a matter of fact, the tobacco of the cigarettes can contain the environmental pollution where it was grown [28], and the presence of heavy metals has been already described in cigarette smoke [29–31]. In conclusion, the ESEM investigation has allowed to successfully correlate the presence of inorganic metallic particles to ameloblastoma, for the first time in literature. However, the finding of metallic particles in ameloblastoma does not indicate a causal relationship. The source of this pollution can have different origins by dental practice, such as drilling and blasting. The possible side effects induced by the generation of ultrafine metallic particulate inside the mouth could lead to further recommendations for the dentists in their routinely activity, encouraging the use of rubber-dam. There is also the concrete possibility of pollution from the cigarette smoke, which is so confirmed as a broad-spectrum risk factor for tumors of the oral cavity [32]. In Taiwan, a statistical association between heavy metal exposure and oral cancer has been already described [33,34]; future researches, aimed to testify the presence of metal particles inside the neoplastic cells from other tumors of the oral cavity (e.g. squamous cell carcinoma), are advocated.
ACKNOWLEDGEMENTS The authors would express their thanks to Federico Capitani and Luca Fabbiani for their technical assistance.
DECLARATION OF INTEREST The authors report no conflicts of interest.
FUNDING The authors alone are responsible for the content and writing of this article. This study was supported by Nanodiagnostics Laboratory (Modena, IT).
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