99m and the shape of the red blood cells.
S.Paoli MD1, T.S.Giani MD2, G.A.Presta MD3, C.G.Correa4, A.I.Maiworm5, S.D.Santos-Filho MD6 and M.Bernardo-Filho PhD7.
1. Dentistry; Professor, Faculdade de Odontologia (FONF) de Nova Friburgo, Nova Friburgo, RJ, and Centro Universitário Serra dos Órgãos (UNIFESO), Teresópolis, RJ, Brazil; Universidade Federal do Rio Grande do Norte (UFRN), Programa de Pós-Graduação em Ciências da Saúde, Natal, RN, Brazil;
2. Physiotherapist, Professor, Universidade Estácio de Sá, UFRN, Programa de Pós-Graduação em Ciências da Saúde, Natal, RN, Brazil.
3. Medical Doctor; Professor and researcher, UFRN, Programa de Pós-Graduação em Ciências da Saúde, Natal, RN, Brazil.
4. Biologist; Professor and researcher in FONF, Nova Friburgo, RJ, and UNIFESO, Teresópolis, RJ, Brazil. 5. Physiotherapist; Head of the Physiotherapy Department, UFRN, Programa de Pós-Graduação em Ciências da Saúde, Natal, RN, Brazil.
6. Biologist and Physiotherapist Professor in UNIFOA, Volta Redonda, RJ, Brazil; UFRN, Programa de Pós- Graduação em Ciências da Saúde, Natal, RN, Brazil;
7. Biomedical an Physiotherapist, PhD, Instituto Nacional do Câncer, Rio de Janeiro, RJ, Brazil and Universidade do Estado do Rio de Janeiro, Departamento de Biofísica e Biometria. Rio de Janeiro, RJ, Brazil.
Correspondence to author: Severo de Paoli
Universidade do Estado do Rio de Janeiro Instituto de Biologia Roberto Alcantara Gomes Departamento de Biofísica e Biometria Laboratório de Radiofarmácia Experimental Av. 28 de Setembro, 87, Vila Isabel 20551-030, Rio de Janeiro, Brasil
ABSTRACT. (170 words)
Zinc oxide and eugenol (OZE) mixture produces a cement utilized in almost all the specialities in dentistry all over the world. Blood constituents are labeled with technetium- 99m (99mTc) and used in nuclear medicine. Rationale and Objectives: The aim of this work was to study the effect of an OZE solution on the labeling of blood constituents with 99mTc and on the qualitative and quantitative evaluation of the shape of the red blood cells (RBC). Materials and Methods: Blood (Wistar rats) was incubated with OZE, stannous chloride and
99mTc. Samples of blood were isolated and fractions were separated. The percentage of
radioactivity (%ATI) was calculated and the morphology and morphological parameter were evaluated. Results: The %ATI on the blood constituents decreased significantly (p<0.05) due to the treatment with OZE. Qualitative and quantitative alterations were found. Conclusion: Although the experiments had been performed with rats, it is suggested precaution in the interpretation of the examinations of the nuclear medicine in patients that have been treated with OZE in the dentistry procedures.
Keywords: zinc oxide eugenol, dentistry, technetium-99m, blood constituents, red blood cell.
INTRODUCTION
In dentistry, the combination of the zinc oxide and eugenol substances produces a cement (OZE) with several characteristics as well as anesthesia, analgesic, anti-inflammatory, sedative and tissue cicatrisation.[1,2]. The cement OZE was proposed since 1855 and has been continuously utilized in the various dentistry specialties, such as (i) in pediatric dentistry, (ii) as managing removable prosthesis material, (iii) as temporary obturation material in dentistry, (iv) as surgery cement in periodontics and (V) as sealers in endodontics. [3-7] In
general, this cement is used in procedures of the dentistry in the proportion of 1 g of zinc oxide and up to 0.25 ml of eugenol [5].
OZE has several pharmacological and/or biological properties including: (i) non toxic in vivo and toxic in vitro, (ii) adherent to tissues, (iii) mucostatic or mucocodisplacive (depending on brand used), (iv) good surface detail in thin section, (v) good dimensional stability (little or no dimensional change on setting, 0.1% dimensional change during setting), (vi) can be added to with fresh OZE and (vii) stable on storage and good shelf life. [8]
Radiobiocomplexes, known as radiopharmaceuticals, are radioactive tracers employed in nuclear medicine to help in the diagnosis and/or treatment of diseases. [9, 10]
Red blood cells labeled with 99mTc (99mTc RBC) are radiobiocomplexes widely used in clinical nuclear medicine for several important applications. [11, 12] The labeling of blood constituents with 99mTc depends on the presence of a reducing agent and stannous chloride is widely utilized. The in vitro technique is easily carried out and produces a better and well controlled product. [9]
Some authors have reported that the presence of natural or synthetic drugs might alter the labeling of blood constituents with 99mTc. [13-17] The morphological analysis of RBC has been of importance on the clinical and on the laboratory investigations and has contributed to evaluate possible alterations in the area, shape, volume and perimeter/area ratio of this kind of cellular structure. [18]
As a patient that has been submitted to a treatment in dentistry with OZE solution can need to be submitted to an examination in nuclear medicine with some blood constituent labeled with Tc-99m. The purpose of this work was to study the effect of an OZE solution on the labeling of blood constituents with 99mTc and on morphology (shape) of red blood cells
MATERIALS AND METHODS Animals
In our laboratory, the experiments have been carried out with rats that are maintained in a controlled environment. The animals had free access to water and food and ambient temperature was kept at 25 ± 2ºC. Heparinized whole blood was withdrawn by cardiac puncture from adult male Wistar rats (3-4 months of age, 250±15g of weight) and a pool of blood from 6 animals was obtained. The animals are under anesthesia by sodic thiopental 40mg/kg. The protocols of the experiments were performed without sacrificing the animals and was approved (CEA/115/2006) by the Ethical Committee of the Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro.
Zinc oxide eugenol cement preparation
The zinc oxide (500 mg) (Super Dentaria Leão Ltda, Rio de Janeiro, Brazil, lot number 3/058, May 2002, validity May 2008) was mixed with eugenol 100 mg (Biodinâmica Química e Farmacêutica Ltda, Rio de Janeiro, Brazil, lot number 765/00, November/ 2002, validity November/2008) in a glass plaque to obtain a similar cement glass mass [19,20]. This cement was separated in 4 parts of 150 mg. One part was put in 0.9% NaCl (15 ml). After that, it was triturated and mixed in a vortex for 2 minutes. After filtration with a qualitative filter paper (Aldrich Chemical Co, 11cm, Lot number k932), the filtered solution was considered to be 10 mg/ml.
Labeling of blood constituents with 99mTc
The tubes used in these experiments were previously closed with a rubber cap and a syringe was used to reduce the air atmosphere (vacuum) inside the vials. Samples of 0.5 ml of the blood were incubated in vitro with OZE solution at different concentrations (0.625, 1.25, 2.5, 5.0 and 10 mg/ml). After this period of time, stannous chloride (1.2 μg/ml) was added
sequence, 99mTc (3.7 MBq) was added, as sodium pertechnetate (99Molibdenium/99mTechnetium generator, Instituto de Pesquisas Energéticas e Nucleares, Comissão Nacional de Energia Nuclear, Brazil) and the incubation continued for another 10 min. These samples were centrifuged (clinical centrifuge, 1500 rpm, 5 min and aliquots (20 µl) of plasma (P) and blood cells (BC) were separated. Aliquots (20 μl) of P and BC were also precipitated in trichloroacetic acid (5%) and soluble (SF) and insoluble fractions (IF) of P and BC were separated. The radioactivity in P, BC, IF-P, SF-P, IF-BC and SF-BC were determined in a well counter (Packard Instrument Company, mod C5002, USA). After that, the percentage of radioactivity (%ATI) was calculated as described previously [21]. All experiments were repeated at least three times.
After the incubation with 99mTc, one drop of each sample was smeared in glass slides (5 slides for each sample) and the May-Grünwald-Giemsa (MGG) method was performed. The smear blood was fixed with methanol (Vetec, Brazil) for 5 min, then stained with Giemsa (azure eosin methylene blue solution, Isofar, Brazil) for 10min and washed in methanol to remove excess of stain. The glass slides were stayed at room temperature to dry. The stained glass slides with MGG were analyzed by optical microscopy and morphometric parameter (perimeter/area ratio) of a total of five fields per each glass slide were evaluated (Software image pro-plus, media Cybernetics, USA).
Statistical analysis
Results were reported as means ± standard deviation of %ATI and perimeter/area ratio. Statistical analysis was performed using ANOVA with Turkey post-test with the significance of p<0.05. Statistical analysis was used to Graph Pad InStat version 3.01 for Windows 95/NT (Graph Pad Software, USA).
RESULTS
The table 1 shows the effect of different concentrations of the OZE solution on the distribution of the radioactivity between cellular and plasma compartments of the Wistar rats’ blood. The studied OZE solution decreased significantly (p<0.05) the radioactivity on the cellular compartment from 96.00±3.31 to 85.11±1.74.
The table 2 shows the effect of different concentrations of the OZE solution on the fixation of the radioactivity in the soluble and insoluble fraction of the plasma of the Wistar rats’ blood. The studied OZE solution decreased significantly (p<0.05) the fixation of the 99mTc on IF-P from 73.94±1.04 to 62.54±3.34.
The table 3 shows the effect of different concentrations of the OZE solution on the fixation of the radioactivity in the insoluble and soluble fraction of the blood cells obtained from the blood treated with OZE solution. The studied OZE solution decreased significantly (p<0.05) the radioactivity fixation on IF-BC from 91.30±1.17 to 71.16±3.87.
The qualitative evaluation of the RBC shape (no treated and treated with different concentrations of the OZE solution), under optical microscopy, is shown in the figures 1, 2 and 3. Qualitative alterations were found when the blood samples were treated with OZE solutions (2.5 and 10 mg/ml). Similar results were obtained with other concentrations evaluated (data not shown). Figure 1 shows the histological preparation of a sample of blood not treated (control). Figure 2 shows the histological preparation treated with an OZE solution (2.5 mg/ml), where cells had the shape altered (crenation). Figure 3 shows the histological preparations of blood treated with an OZE solution (10 mg/ml) and an unexpected finding was observed due to the shape of RBC seems to be equal to the control preparation.
The evaluation of the perimeter/area ratio of the RBC (no treated and treated with different concentrations of the OZE solution) has revealed alterations due to the treatment of blood with an OZE solution (2.5 mg/ml). These results are shown in Figure 4.
DISCUSSION
The observation of the alterations on the fixation of the radionuclide 99mTc on blood constituents has been observed in studies in which whole blood has been treated with natural or synthetic products. Due to these findings has been possible to propose an experimental model to evaluate some biological effects of chemical compounds [22-25].
The OZE cement has been used in several treatments in dentistry and the total quantity of this product can be up, in general, up to 1250 mg by treatment [5]. It is expected, if this material is totally absorbed, a concentration up to 0.25 mg/ml in blood from a man with 5000 ml of blood. In our study 0.1 ml of a solution with 10 mg/ml was utilized. In consequence the highest concentration in presence of the blood was about 1 mg/ml (100%) and the lowest was about 0.062 mg/ml (6.25%).
The analysis of the results shown in tables I, II and II indicated that there was a significant (p<0.05) decrease on the labeling of the blood constituents when the incubation with OZE solution was made in vitro. These results could be explained by a possible oxidant and citotoxic in vitro property of the chemical compounds that are present in the OZE solution. [8] These compounds could oxidize the stannous ions that are necessary to the reduction of the pertechnetate ion, as suggested by other authors to several natural products, as Maytemus icilifolia [26], Ginkgo biloba [27] and “guaraná” [28] and Mentha crispa [29]. Other possibility, it would be due to the physiological/pharmacological properties of the OZE in the RBC membrane. This fact could be justified by an effect in another kind of cells, as reported by [29] that has observed that extractable components of some commonly used root
canal sealing materials may interfere with immunocompetent cells in vitro. This effect was considerably reduced (p< 0.01) when solutions of the solid material were employed.
Morphometric analysis has been used in research in several methodologies, as to verify (i) changes with time in optic disc structure and thickness of retinal nerve fiber layer in chronic ocular hypertensive monkeys [30], (ii) the relationship between infarct-related artery stenosis and capillary density [31] and (iii) the effects of two sex hormones on normal mamma gland of female rats [24, 32, 33] show possible alterations in the morphometric analysis (area, shape and volume) of the red blood cells. The qualitative analysis suggest OZE solution components alter the shape the RBC (figures 1, 2 and 3). The quantitative evaluation, however, shows a light tendency to an increase of perimeter/area ratio from RBC treated with OZE solutions (figure 4).
The analysis of the results indicates that the studied solution has substances that could have redox potential, altering the distribution of the radioactivity between cellular and plasma compartments and the fixation of 99mTc on the plasma and cellular proteins. These data could be also justified by alterations on the cell membrane. However, the morphometric parameters obtained showed a light alteration only. Other experiments have been performed in our laboratory to evaluate the possible mechanisms involved in the effects of OZE cement on labeling of blood constituents with 99mTc.
In conclusion, although the experiments had been performed with rats, it is suggested precaution in the interpretation of the examinations of the nuclear medicine in patients that have been treated with OZE in the dentistry procedures.
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Table 1
Percentage of radioactivity incorporated (%ATI) between cellular and plasma compartments of blood treated with OZE solutions
OZE (mg/ml) Blood Cells Plasma
0.00 96.00±3.31 4.00±3.31 0.63 92.48±1.38 7.52±1.38 1.25 90.02±1.78 8.27±1.78 2.50 89.46±3.31 10.54±3.31 5.00 86.74±3.50 * 13.26±3.50 10.00 85.11±1.74 * 14.89±1.74
Heparinized blood was withdrawn from Wistar rats under anesthesia by sodic thiopental (40 mg/kg). Samples of 0.5 ml of the blood were incubated in vitro with 100μl of OZE solutions (0.625, 1.25, 2.5, 5.0 and 10 mg/ml), 0.5 ml of stannous chloride (1.2 μg/mL) and 0.1µl of 99mTc (3.7MBq). These samples were centrifuged (1500 rpm, 5min) and aliquots (20 µl) of plasma and blood cells were separated. The radioactivity in all fractions was determined and the %ATI calculated. Significance was accepted at the p<0.05 level (*).
Table 2
Percentage of radioactivity incorporated (%ATI) between different concentrations of the OZE solution on the labeling of plasma proteins.
OZE (mg/ml) Fraction insoluble Plasma Fraction soluble Plasma
0.00 73.94±1.04 26.06±1.04 0.63 71.85±3.17 28.15±3.19 1.25 66.91±2.82 31.09±2.82 2.50 66.96±3.97 * 33.04±3.97 5.00 64.88±1.96 * 35.12±1.96 10.00 62.54±3.34 * 37.46±3.
Heparinized blood was withdrawn from Wistar rats under anesthesia by sodic thiopental (40 mg/kg). Samples of 0.5 ml of the blood were incubated in vitro with 100 μl of OZE solutions (0.625, 1.25, 2.5, 5.0 and 10 mg/ml), 0.5 ml of stannous chloride (1.2 μg/mL) and 0.1μl of 99mTc (3.7MBq). These samples were centrifuged (1500 rpm, 5min) and aliquots (20 µl) of fraction insoluble plasma and fraction soluble plasma were separated. The radioactivity in all fractions was determined and the %ATI calculated. Significance was accepted at the p<0.05 level (*).
Table 3
Percentage of radioactivity incorporated (%ATI) between different concentrations of the OZE solution on the labeling of of cells proteins
OZE (mg/ml) Fraction insoluble cells Fraction soluble cells
0.00 91.39±1.17 8.61±1.17 0.63 82.03±3.38 * 7.97±3.38 1.25 79.65±2.27 * 20.35±2.27 2.50 76.01±3.05 * 23.99±3.05 5.00 73.53±3.47 * 26.47±3.47 10.00 71.16±3.87 * 28.84±3.87
Heparinized blood was withdrawn from Wistar rats under anesthesia by sodic thiopental (40 mg/kg). Samples of 0.5 ml of the blood were incubated in vitro with 100 μl of OZE solutions (0.625, 1.25, 2.5, 5.0 and 10 mg/ml), 0.5 ml of stannous chloride (1.2 μg/mL) and 0.1μl of 99mTc (3.7 MBq). These samples were centrifuged (1500 rpm, 5min) and aliquots (20 µl) of fraction insoluble cells and fraction soluble cells were separated. The radioactivity in all fractions was determined and the %ATI calculated. Significance was accepted at the p<0.05 level (*).
Figure 1
Figure 1: Samples of whole blood were incubated with NaCl 0.9% solution for 60 min. After that, stannous chloride solution was added and the