ORIGINAL PAPERs
Yevhen Viktorovich Kuzenko
1, A, Antonina Politun
2, F, Larysa shaposhnyk
3, C,
Helena Vladimirovna Logvinova
4, E, Olena Hudymenko
5, BAccumulation of Heavy Metals in Epulis
Akumulacja metali ciężkich w nadziąślakach
1 Department of Pathology, Medical Institute of sumy state University, sumy, Ukraine 2 Head of the Department of Therapeutic Dentistry, Kiev University UANM, Ukraine 3 Medical Institute of sumy state University, sumy, Ukraine
4 Medical Institute of sumy state University, sumy, 6th course student, sumy, Ukraine
5 Masters Degree, Dnipropetrovsk Medical Academy of the Health Ministry of Ukraine, Dnipropetrovsk, Ukraine
A – research concept and design; B – collection and/or assembly of data; C – data analysis and interpretation; D – writing the article; E – critical revision of the article; F – final approval of article
Abstract
Background. Benign growths on the gums, called epulis, is often found in dental practice. There are five types:
fibrous epulis, ossifying fibroid epulis, angiomatosis (pyogenic granuloma), giant cell epulis and combined epu-lis. Epulis is more common in the front teeth, usually in patients between the ages of 20–40 years, 2 times more common in women than men, and during pregnancy occur more frequently and grow faster. There are different views regarding the origin of epulis. some authors consider epulis to have an inflammatory origin. Other authors attribute it to benign tumors. Precious metals are not available to many dental clinics. However cobalt-chromium alloys are used for structures in the oral cavity (hence, possible chronic cobalt-chromium intoxication). Cobalt-chromium (Cr-Co) alloys are commonly used in the Third World.
Objectives. The object of this study was to study heavy metals accumulation in different types of epulis tissues. Material and Methods. For recognition of various types of epulis tissues and the rate of accumulation of heavy
metals in it, a method of scanning was used with an electron microscope equipped with Energy Dispersive X-ray spectroscope (EDs) capability.
Results. The giant cell epulis corresponding to Cr and Ni were higher. This can clearly be related to the origin
of giant cells. Cr and Ni levels increased to a statistically significant extent. As for Cl and K levels, there was no remarkable difference between the normal and epulis tissue. The lowest levels of P and Ca were observed in giant cell epulis. Hydroxyapatite is a mineral component of ossifying fibroid epulis. Accordingly, we observed an increase in calcium and phosphorus (compared to the control).
Conclusions. This study shows exogenous accumulation of heavy metals in giant cell epulis. This is due to the fact
that they are macrophages by descent and, as a result, significantly accumulate the elements (Dent. Med. Probl.
2014, 51, 3, 375–381).
Key words: heavy metals, oral epulis, toxic levels.
Streszczenie
Wprowadzenie. W praktyce stomatologicznej często są spotykane łagodne rozrosty dziąsła zwane nadziąślakami.
Wyróżnia się pięć typów nadziąślaków: włókniste, kostniejące, naczyniakowate (ziarniniaki ropotwórcze), olbrzy-miokomórkowe i mieszane. Zmiany te są szczególnie częste w zębach przednich u osób między 20. a 40. rokiem życia i odnotowuje się je dwa razy częściej u kobiet, u których występują w ciąży i wówczas rosną szybciej. Zauważalne są zróżnicowane poglądy na temat ich etiologii – jedni autorzy uważają je za zmiany zapalne, inni za łagodne nowo-tworowe. Metale szlachetne nie są dostępne dla większości pacjentów stomatologicznych. stopy chromo-kobaltowe są natomiast często stosowane w jamie ustnej, szczególnie w krajach mniej zamożnych.
Cel pracy. Ocena akumulacji metali ciężkich w różnych typach nadziąślaków.
Materiał i metody. Do oceny typów nadziąślaków oraz zawartości w ich tkance metali ciężkich zastosowano
elek-tronowy mikroskop skaningowy wyposażony w mikrodetektor rentgenowski EDs.
Dent. Med. Probl. 2014, 51, 3, 375–381
The tissues of the oral cavity may be affect-ed by toxic agents both by direct action and sys-temic exposure such as poisoning with heavy met-als, e.g. lead and mercury [1]. Metal ions are im-portant components of the human body and have the following functions: metal ions act as catalytic centers of enzymes; metal ions, not primarily in-volved in catalysis, act as binding groups to bring the enzyme and substrate together; metal ions help maintain physiological control (antagonism with other metals).
“Dental amalgam is widely used in restorative care and is a compound of mercury and silver-based alloys; however, some concerns have been expressed about the possible health effects of mer-cury in amalgam and about contamination of the environment by mercury.” [2].
Vernon et al. [3] reviewed 41 published cas-es of allergy to dental amalgam, which includ-ed 30 female and 11 male patients. Twenty of the 41 patients recovered after removal of their amal-gam restorations. The most frequent symptoms were of the remote cutaneous type (38 of 41 cases), while local symptoms, particularly gingivitis and stomatitis, occurred in 17 cases. The authors sug-gested that the figures probably underestimate the true prevalence of the condition because of under-reporting of cases. Mercury was found to be the most common sensitizing agent, but other metals, particularly copper, zinc, and silver, could also be implicated [4].
Cobalt-chromium alloys are the most com-mon material for metal parts of dental prosthesis, and usually such metal alloys contain about 60% to 65% cobalt. They are regarded as biocompatible because of the absence of nickel and beryllium in their composition. Cobalt is nevertheless listed as a sensitizing metal.
Chromium compounds, on the other hand, can induce contact dermatitis and even cause se-vere corrosive irritation of the epithelium. Howev-er, it has been found that neither chromium con-taining alloys nor chromium-plated objects, such as dental alloys, produce allergic contact dermati-tis in cases of application to chromium-sensitive individuals.
The interactions between metals and cells are diverse, but can be divided into 3 major categories:
metals are essential for metabolism and toxic met-als can stop metabolic reactions; metmet-als can accu-mulate in cells via intracellular uptake and bind-ing; metals can undergo biochemical transforma-tion (inclusive of leaching).
Iron is a component of the peroxidase and ni-trous oxide-generating enzymes that participate in the immune response to infections and is probably involved in the regulation processes of production and activation of cytokines [5, 6].
The investigators did not evaluate several met-als, such as Сr, which are important components of dental alloys. In this paper, we study contam-ination induced by metallic elements released by dental prostheses.
Heavy metals are inducers of neoplastic pro-cesses. Epulis is overgrowth induced by chronic inflammatory processes. Kramer’s investigation convincingly proved that periapical irritation in-duces periodontal disease [7]. In severe cases, it is associated with loss of alveolar crest bone. How-ever such cases are uncommon and generally confined only to the soft tissues. More common-ly among females, probabcommon-ly due to hormonal dif-ferences, the affected areas are mostly the anteri-or of molar teeth [8]. Epulides are classified (ac-cording to clinical appearance, histopathological appearance and sometimes according to the or-igin) as: fibrous epulis, ossifying fibroid epulis, pyogenic granuloma (also known as hemangio-ma), giant cell epulis (also known as osteoclas-toma, giant cell reparative granuloma, peripher-al giant cell granuloma or giant cell hyperplasia) and combined epulis.
We analyzed the epulis and soft periodon-tal tissue location for meperiodon-tal presence by scanning with an electron microscope (sEM – РЕМ-100Е) equipped with Energy Dispersive X-ray spectro-scope (EDs). Lastly, we have underlined the im-portance of scanning electron microscope diagno-sis to prevent possible damage.
Material and Methods
The study samples consisted of the periodon-tal and epulis tissues of patients. The subjects were divided into two equal groups:
Wyniki. Nadziąślaki olbrzymiokomórkowe zawierały duże stężenia chromu i niklu. Nie wykazano istotnych różnic
zawartości chloru i potasu między tkanką pochodzącą z nadziąślaków a tkanką kontrolną. Mniejsze stężenia fosforu i wapnia stwierdzono w nadziąślakach olbrzymiokomórkowych. Hydroksyapatyt jest częścią składową nadziąśla-ków kostniejących i dlatego obserwowano większą zawartość wapnia i fosforu w tej tkance w odniesieniu do kon-trolnej.
Wnioski. W nadziąślakach olbrzymiokomórkowych wykazano akumulację egzogennych metali ciężkich, co wynika
z ich nagromadzenia się w makrofagach (Dent. Med. Probl. 2014, 51, 3, 375–381).
Patient Group (Group I) consisted of 51 peo-ple who had a clinical diagnosis of epulis. Only pa-tients that had epulis formations were included in the study cohorts.
Control group (Group II) consisted of 9 pa-tients who had died in sumy Regional Hospital. The patients had various diagnoses (not athero-sclerotic ones).
Briefly, 4 µm thick tissue sections were placed on graphite plates (Fig. 1).
Paraffin sections were immersed into three sets of xylene for 10 min each followed by three sets of absolute ethanol for 10 min and
final-ly rinsed with distilled water. slides were placed into hematoxylin for 5 min and 96% alcohol for 10 min and rinsed thoroughly under distilled wa-ter for approximately 4–5 min.
At the specified times, the specimens were ex-amined for topographical changes using a scan-ning Electron Microscope (sEM) equipped with Energy Dispersive X-ray spectroscope (EDs). The surface levels of copper elements were measured as weight percentage. Each sample was exposed to radiation at the center and in two additional equi-distant areas at a voltage of 15KV for 60 s and the average figure was calculated for each specimen.
The data was analysed using sTATIsTICA 8.0 software, user version sTA862D175437Q. The re-sults are presented as average values (± sD). The K-s test was used in order to evaluate the normal-ity of the data. Also, the student method was used to perform simple comparative analysis. A value of p < 0.05 was considered significant.
Results
A normal epithelial cell is shown in Fig. 2. Mi-croscopic examination revealed tissue with des-mosome (Fig. 2b) and nuclei contours (Fig. 2a).
These microscopic features are typical for a pyogenic epulis (Fig. 3). The connective tissue (Fig. 3a) was presented in the form of fibrocellu-lar structures with abundant vascufibrocellu-larity. Numer-ous endothelium were observed lining the vascu-lar spaces (Fig. 3b), with apparent proliferation of endothelial cells and fibroblasts. A moderate de-gree of chronic inflammatory cell infiltrate, com-posed chiefly of lymphocytes and plasma cells, was present.
Fig. 1. a – graphite plates with tissue sections,
b – graphite plates without tissue sections
Ryc. 1. a – płytki grafitowe z naniesioną tkanką,
b – płytki grafitowe bez tkanki
Fig. 2. Normal epithelium of
peri-odontal tissue: a – nuclei contours of epithelium cells, b – desmo-somes
Ryc. 2. Nabłonek fizjologicznego
dziąsła: a – kontury jąder keraty-nocytów, b – desmosomy
Fig. 5. Giant-cell epulis:
a – nuclei contours of giant cells, b – membrane pores of giant cells, c – areas of hem-orrhages
Ryc. 5. Nadziąślak
olbrzy-miokomórkowy: a – kontury jąder komórek olbrzymich, b – pory błonowe komórek olbrzymich, c – miejsca wynaczynienia krwi
Fig. 3. Pyogenic epulis:
a – connective tissue, b – vas-cular spaces
Ryc. 3. Ziarniniak ropotwórczy
(nadziąślak naczyniakowaty): a – tkanka łączna, b – naczynia krwionośne
Fig. 4. Fibrous epulis: a –
con-nective tissue
Ryc. 4. Nadziąślak włóknisty:
The microscopic examination of the sEM sec-tion showed a tissue with epithelium and underly-ing fibrous connective tissue structures that com-posed a stroma of the tumor (Fig. 4a). The sections revealed well-circumscribed fibroblasts, capillar-ies and edema areas and occasionally there are bone resorption areas.
The sEM sections revealed well-circum-scribed, unencapsulated cellular mass contain-ing oval to spindle-shaped fibroblasts, abundant multinucleated giant cells (Fig. 5a and b), numer-ous capillaries and areas of hemorrhage (Fig. 5c). These giant cells were localized in the deep corion in a vascular stroma of ovoid and spindle-shaped fibroblasts. The multinucleated giant cells were of various shapes and sizes containing open-faced nuclei ranging from 10 to 25 in number
conform-ing to the type I giant cells described in literature. There were also several areas with hemorrhage, with deposits of hemosiderin.
The diagnosis was ossifying fibroid epu-lis with spindle-shaped fibroblasts (Fig. 6). There was no pathologic mitosis that is usual for malig-nant tumors. The epulis contained calcifications with concentric and oscellular mineralization at the center. Other areas presented recently formed osteoid with peripheral osteoblasts (Fig. 6a) and signs of progressing calcification.
The average content of the micro- and macro-elements under study are shown in Table 1.
EDs analyses revealed that inorganic phases of epulis were mainly composed of calcium and phosphorus as the major constituents with some minor components such as Cr, Zn, Mg, Fe Ni
Table 1. Average concentrations of microelement in samples Tabela 1. Średnie stężenie mikroelementów w badanych tkankach
Micro-elements % Control Group n = 9 mean ± sD Fibrous epulis n = 20 mean ± sD Ossifying fibro-id epulis n = 4 mean ± sD Pyogenic granu-loma n = 9 mean ± sD
Giant cell epulis n = 14 mean ± sD Combined epulis n = 4 mean ± sD P 9.77 ± 0.56 10.94 ± 0.71 20.38 ± 4.87*** 6.21 ± 0.78 5.75 ± 0.51** 18.29 ± 5.12* Cl 3.71 ± 0.35 3.51 ± 0.4 3.98 ± 0.96 4.39 ± 0.67 4.39 ± 0.7 5.94 ± 1.3 K 3.68 ± 0.41 3.74 ± 0.21 4.06 ± 0.51 4.25 ± 0.21 4.03 ± 0.19 5.80 ± 1.08 Ca 15.40 ± 1.5 15.68 ± 0.91 35.68 ± 2.8*** 10.89 ± 1.7 11.45 ± 0.5** 14.89 ± 3.88 Cr – – – – 0.70 ± 1.98 ± 0.71 Zn 0.45 ± 0.03 0.51 ± 0.04 1.65 ± 0.46 3.56 ± 0.81*** 3.79 ± 0.7* 3.48 ± 0.93 Mg 0.64 ± 0.02 0.56 ± 0.03 – 0.69 ± 0.12 1.10 ± 0.09** – Fe 1.89 ± 0.08 2.88 ± 0.16 4.33 ± 1.56 25.60 ± 0.9*** 6.69 ± 0.05** 12.34 ± 2.78 Ni – – – – 11.70 ± 0.14 9.12 ± 1.56 Cu – – – – 2.61 ± 1.3 5.36 ± 1.9 * P < 0.05 ** P < 0.01 ***P < 0.001.
Fig. 6. Ossifying fibroid epulis:
a – osteoblasts with degenera-tive changes, b – vessels
Ryc. 6. Nadziąślak kostniejący:
a – osteoblasty ze zmianami zwyrodnieniowymi, b – naczy-nia
and K. The giant cell epulis corresponding to Ni was higher. It can clearly be seen from Table 1 that Ni levels increased to a statistically significant ex-tent. As for Cl and K levels, there was no remark-able difference between the normal and epulis tis-sue. The lowest levels of P and Ca were observed in giant cell epulis.
Discussion
There are physiological effects of different heavy metals. It was suggested that their indis-criminate and injudicious use produces anemia, leucopenia, hypoglycemia, hypoproteinemia, in-creased enzymatic activity and toxic effects.
Oral tattoos are usually caused by heavy met-als (amalgam) particles or graphite in lead pen-cils. A tattoo is localized, dark gray to black, non- -tender, and either macular or slightly thickened. A tattoo sometimes increases in size due to in-gestion of the foreign material by phagocytes and then migration of these cells [9].
Giant cell epulis had the most diverse elemen-tal composition. In our opinion, this is related to the origin of giant cells. Giant cell epulis is strongly positive to antibody CD34 and reaction to it is pos-itive in the areas of aggregation of multinucleate giant cells. This data may suggest that multinucle-ate giant cells are macrophages or osteoclasts [10].
The “direct” damage may involve conforma-tional changes of bio-molecules or alter specific binding sites, as in the case of heavy metals. On the other hand, “indirect” damage is the
conse-quence of metal driven formations in reactive ox-ygen/nitrogen species involving superoxide, hy-droxyl radicals or nitric oxide, hydrogen peroxide and/or endogenous oxidants. Apart from ROs- -induced oxidative stress, binding of these heavy metals to proteins rich in -sH groups aggravates cellular toxicity [11].
The chromium absorption of giant cell epu-lis is different in concentration and could be due to the fact that the hexavalent form given orally was reduced to Cr3+ in the acidic environment of
the stomach [12]. The removal of hexavalent and trivalent chromium from synthetic solutions has been extensively studied by a number of research-ers. According to some investigators, the remov-al of Cr(VI) occurs through severremov-al steps of inter-facial reactions. Our findings are consistent with studies using animal cells, that supraphysiological concentrations of chromium ions induce apopto-sis in osteoblast cells in a dose dependent man-ner [13], and suppress osteoblast synthetic func-tion [14]. In our opinion, it is ferric in pyogenic epulis, coming from erythrocytes.
However, not all people accumulate toxic lev-els of metals or exhibit symptoms of metal toxicity, suggesting that genetics play a role in their poten-tial to damage health. Metal toxicity creates pro-tein rich -sH group dysfunction, which appears to be mediated primarily through mitochondrial damage from glutathione depletion [15].
This study shows exogenous accumulation of heavy metals in giant cell epulis. This is due to the fact that they are macrophages by descent and, as a result, significantly accumulate the elements.
Acknowledgments. On behalf of the group of authors, we thank Zolotarova Vira, who provided the laboratory for
tis-sue sections.
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Address for correspondence:
Yevhen Viktorovich Kuzenko
Medical Institute of sumy state University Department of Pathology
Illinska 12/2 st. f. 44 40009 sumy Ukraine
E-mail: kuzenko_yevhen@rambler.ru Conflict of interest: None declared Received: 14.05.2014
Revised: 18.05.2014 Accepted: 3.06.2014
Praca wpłynęła do Redakcji: 14.05.2014 r. Po recenzji: 18.05.2014 r.
