Basic ResearchBiology

Diabetes Induces Metabolic Alterations in Dental Pulp

Mariana Ferreira Leite, DDS, PhD,*, Emily Ganzerla, DDS, PhD,* Mrcia Martins Marques, DDS, PhD, and Jos Nicolau, DDS, PhD*
Abstract
Diabetes can interfere in tissue nutrition and can impair dental pulp metabolism. This disease causes oxidative stress in cells and tissues. However, little is known about the antioxidant system in the dental pulp of diabetics. Thus, it would be of importance to study this system in this tissue in order to verify possible alterations indicative of oxidative stress. The aim of this study was to evaluate some parameters of antioxidant system of the dental pulp of healthy (n 8) and diabetic rats (n 8). Diabetes was induced by streptozotocin in rats. Six weeks after diabetes induction, a pool of the dental pulp of the 4 incisors of each rat (healthy and diabetic) was used for the determination of total protein and sialic acid concentrations and catalase and peroxidase activities. Data were compared by a Student t test (p 0.05). Dental pulps from both groups presented similar total protein concentrations and peroxidase activity. Dental pulps of diabetic rats exhibited significantly lower free, conjugated, and total sialic acid concentrations than those of control tissues. Catalase activity in diabetic dental pulps was significantly enhanced in comparison with that of control pulps. The result of the present study is indicative of oxidative stress in the dental pulp caused by diabetes. The increase of catalase activity and the reduction of sialic acid could be resultant of reactive oxygen species production. (J Endod 2008;34:12111214)

Key Words
Antioxidants enzymes, dental pulp, diabetes, sialic acid

*Centro de Pesquisa em Biologia Oral Departamento de Biomateriais e Bioqumica Oral, School of Dentistry, Universidade de So Paulo, So Paulo, Brazil. Departamento de Dentstica, School of Dentistry, Universidade de So Paulo, So Paulo, Brazil.Centro de Cincias Biolgicas e da Sade, Universidade Cruzeiro do Sul, So Paulo, So Paulo, Brazil. Supported by Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP). Address requests for reprints to Dr Mariana Ferreira Leite, Centro de Pesquisa em Biologia Oral, Faculdade de Odontologia da Universidade de So Paulo, Av Lineu Prestes, 2227, So Paulo, Brazil. E-mail address: maricota@usp.br. 0099-2399/$0 - see front matter Copyright 2008 American Association of Endodontists. doi:10.1016/j.joen.2008.07.010

iabetes mellitus is defined as a metabolic disease characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both (1). The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, or failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels. Diabetes mellitus presents a wide spectrum of oral manifestation such as xerostomia, taste impairment, and sialosis and may affect the progression of some diseases, such as dental caries, periodontal disease, soft-tissue lesions, and fungal infections (1). Diabetes mellitus may be a modulating factor of endodontic infections and may compromise the healing of periapical lesions, especially under poorly controlled conditions (2). Diabetes mellitus causes a reduction in the leukocyte counts, migration of polymorphonuclear cells, and an increase in the detection rate of anaerobic bacteria in the dental pulp of diabetic rats (3). Impaired vasculature caused by diabetes could interfere in tissue nutrition and pulp repair and could create a microaerophilic state for the development of anaerobic microorganisms (4). Moreover, diabetic patients present circadian rhythms of pulp sensitivity that differ from those of healthy people (5). There are some evidences that show a reduction of antioxidant defenses in diabetes (6). Hyperglycemia induces overproduction of superoxide that contributes to pathogenesis of the micro- and macrovascular diabetic complications (7). These complications occur because of increased polyol pathway flux, increased advanced glycation end-products formation, activation of protein kinase C isoforms, and increased hexosamine pathway flux (8). The first defense against the superoxides is the enzymatic system, which is mainly composed of superoxide dismutase, catalase, glutathione peroxidase, and peroxidase. The second defense is the nonenzymatic system, most of them being dietary derivates, such as vitamins C and E and carotenes (9). Other compounds may act as hydrogen peroxide scavengers and antioxidant agents, such as sialic acid (10). Sialic acid is a compound derived from neuraminic acid and may exist in a free form or as terminal residues of oligosaccharide chains linked to peptides, gangliosides ,and glycoprotein in the body fluids, mucosa, epithelium, cell membranes, and nervous system (11). Sialic acid is oxidized by hydrogen peroxide and converted into a stable and nontoxic decarboxylated compound called ADOA (4-acetylamino-2,4-dydeoxy-D-glycero-D-galacto-octonic acid), preventing tissue damage against reactive oxygen species (ROS), especially under physiological conditions of pH and temperature (12). Diabetes causes alterations of sialic acid metabolism correlated with retinopathy, nephropathy, and cardiovascular risk (13, 14). Epidemiological studies have shown that diabetic patients experienced decreased retention of endodontically treated teeth and increased incidence of retreatment (15). People with diabetes have increased periodontal disease in endodontically involved teeth and have a reduced likelihood of successful endodontic treatment in cases with preoperative periradicular lesions (16). On the other hand, other authors have shown that diabetes is not a significant factor affecting outcomes for endodontic restorations (17). However, there are few studies that are able to explain the damage caused by diabetes in the dental pulp and periapical tissues. Thus, the study of the biochemical parameters of the antioxidant system would be of importance. Knowledge of the biochemical status of diabetic dental pulp could offer preventive alternatives for maintaining the integrity of this tissue. Considering the function of sialic acid as a hydrogen peroxide scavenger, the aim of this study was to evaluate its concentration in the dental pulp of streptozotocin (SZT)-induced diabetic rats. Moreover, other parameters of the antioxidant system, such as peroxidase and catalase activities, were also evaluated.

JOE Volume 34, Number 10, October 2008

Diabetes Induces Metabolic Alterations in Dental Pulp

1211

Basic ResearchBiology
Material and Methods
Animals and Diabetes Induction Experiments were conducted with 10 female Wistar rats weighing 180 to 220 g. The protocol of this study was approved by the Ethics Committee of the Dentistry Faculty, Universidade de So Paulo. The animals were fasted overnight, and the diabetes was induced by a single intraperitoneal injection of SZT at a dose of 60 mg/kg body weight dissolved in 0.1 mol/L sodium citrate (pH 4.5). The control group received the vehicle only (citrate buffer). Each animal was individually caged with free access to water and food, which were measured throughout the experiment. The fasting blood glucose was assessed 72 hours after diabetes induction (initial glycemia) by Accucheck Advantage Blood Glucose Meter (Roche, Germany). Only those presenting fasting blood glucose levels higher than 300 mg/dL were considered as diabetics for the purpose of this study. The final glycemia was determined at the time of sacrifice. Sample Preparation After 6 weeks of SZT-induced diabetes, all animals were killed, always in the morning (9 11 AM). The heads were immediately removed, precooled in dry ice, and stored at 80C until the moment of the analysis. Maxillary and mandibular incisor dental pulps were removed by using Hedstrom files. Each sample represented a pool of four incisor pulps from one animal to obtain an adequate amount of tissue. The samples were homogenized at 10% in 10 mmol/L sodium phosphate buffer (pH 7.4) and centrifuged for 10 minutes at 3,020 g, resulting in the precipitate and supernatant parts. This supernatant was used for the biochemical analysis. Biochemical Assays The free and total sialic acid concentrations were measured by using the thiobarbituric acid assay using N-acetylneuraminic acid solution as the standard (18). Free, total, and bound (conjugated to glycoprotein and gangliosides) sialic acid concentrations were measured. Briefly, the total sialic acid concentration was determined after the supernatant was hydrolyzed in 0.1 N sulfuric acid at 80C for 1 hour. The samples were then treated with periodate reagent (25 mmol/L periodic acid in 0.125 N sulfuric acid) and incubated at 37C for 30 minutes. The reaction was terminated by adding sodium arsenite (2% sodium arsenite in 0.22 mmol/L hydrochloric acid). After the disappearance of the iodine color, a solution of thiobarbituric acid (0.1 mol/L, pH 9.0) was added. The solution was heated in boiling water for 7.5 minutes and then cooled. Dimethyl sulphoxide was added, and the intensity of the developed color was measured at 549 nm in a DU-68 Spectrophotometer (Beckman, Fullerton, CA). The levels of free sialic acid in samples were determined following the same steps, except for the hydrolysis in sulfuric acid. Bound sialic acid represented the difference between total and free sialic acid (18).
The protein concentration was quantified with the Folin phenol reagent using bovine serum albumin as the standard following the method of Lowry et al. (19). The catalase activity was determined in a reaction mixture containing the supernatant, 10 mol/L sodium phosphate (pH 7.4), and 100 mmol/L H2O2 according to Aebi (20). The decomposition of H2O2 was monitored for 5 minutes at 240 nm in a Beckman DU-68 Spectrophotometer (20). Peroxidase activity was assayed by the method described by Chandra et al. (21) method and modified by Andersons method (22) using lactoperoxidase as the standard. The interference of pseudoperoxidase activity was eliminated by performing duplicate assays in the presence of 10 mmol/L 3-amino-1,2 triazole, an inhibitor of peroxidase activity. Peroxidase activity was measured in a medium containing 8 mmol/L phosphate buffer (pH 6.0), 1 mmol/L O-dianisidine, and supernantant. The reaction was started by adding H2O2 to a final concentration of 0.02 mmol/L. The absorbance of the developed color was determined at 460 nm in a Beckman DU-68 Spectrophotometer (21, 22).

Statistical Analysis The data are presented as a mean standard deviation. Each biochemical parameter of diabetic and healthy dental pulps was compared by a Student t test. The level of significance was 5% (p 0.05).

Results
Table 1 shows the data with regard to the general conditions of the animals, showing the severity of the induced diabetic disease. Diabetic rats maintained blood glucose higher than 300 mg/100 mL blood throughout the experimental time. These rats presented a weight loss, and after 6 weeks the body weight was significantly lower than that of control rats. The diabetic rats also presented a significant increase in liquid consumption and food ingestion in comparison with the control group. Table 2 shows the total protein concentration (mg/mL) and the total and free sialic acid concentrations ( g/mg protein). Both groups presented similar total protein concentration. The free, conjugated, and total sialic acid concentrations were significantly lower in dental pulps from the diabetic group (26%, 25%, and 30%, respectively) when compared with the control group values. Table 3 shows the activity of catalase and peroxidase enzymes, which are antioxidant system parameters. In the dental pulps of SZTinduced diabetic rats, a significant increase in the catalase activity (25%) was observed in comparison with the healthy control group. The peroxidase activity was similar in both groups.

Discussion
Diabetes mellitus is a metabolic disease that presents oral manifestations. The effects of diabetes on the metabolism of dental pulp tissue have already been studied through the analysis of systems other

TABLE 1. Body Weight (g), Blood Glucose Level (mg/100 mL), Food Intake (g/d/g body weight), and Fluid Intake (mL/d/g body weight) from the Control Group and the SZT-induced Diabetic Group Body Weight (g)
Control Initial Final Diabetic Initial Final 213.80 230.80 205.42 157.08 21.90 17.30 16.44 36.4*

Blood Glucose (mg/100 mL)

83.14 80.50 334.8 336.8 10.96 13.92 46.0* 115.5*

Food Intake (g/d/g body weight)

0.072 0.206 0.007 0.03*

Fluid Intake (mL/d/g body weight)

0.109 0.989 0.017 0.193*

SZT, streptozotocin. Data presented as mean SD (n 10). *Indicate statically significant differences compared with control group (p

0.05).

1212

Leite et al.

JOE Volume 34, Number 10, October 2008

Basic ResearchBiology
TABLE 2. Total Protein Concentration (mg/mg of tissue) and Sialic Acid Content (Free, Total, and Conjugated) ( g/mg prot) of Dental Pulp from the Control Group and the SZT-induced Diabetic Group Protein (mg/mg of tissue)
Control Diabetic 0.104 0.105 0.0084 0.0078

Sialic Acid ( g/mg prot) Free

18.5 13.6 4.8 3.6*

Total
53.7 37.2 8.3 10.1*

Conjugated
36.0 26.1 7.4 5.3*

SZT, streptozotocin. Data presented as mean SD (n 10). *Indicates statically significant differences compared to control group (p

0.05).

than the antioxidant system (23). The role of antioxidant enzymes in the wound healing process and its therapeutic potential has been the subject of interest in medical sciences. Diabetes induces the expression of inflammatory mediators and can cause modifications in the structural components of dental pulp. Kallikrein-kinin system and myeloperoxidase (MPO) activity are altered in the dental pulp of STZ-induced diabetic rats (23). Moreover, a longer period of diabetes causes a reduction in the collagen concentration and an increase in alkaline phosphatase activity, which could be related to irreversible damages in the dental pulp leading to necrosis (23). In this study, the biochemical parameters studied were the sialic acid concentration and catalase and peroxidase activities. These parameters are involved in the antioxidant system, preventing damage caused by the ROS and advanced glycation end-products, which are increased in the hyperglycemic state. Overproduction of ROS causes stimulation of matrix metalloproteinase (MMP) members by regulating both MMP gene expression and MMP activation (24). MMPs are zinc-dependent endopeptidases capable of cleaving most of the basement membrane and extracellular matrix components, including collagen, fibrin, laminin, and proteoglycans in connective tissue (25), such as dental pulp. The MMP stimulation could explain the degradation of collagen in the dental pulp of diabetic rats (23). The activation of MMPs may be prevented by antioxidant agents, such as catalase and glutathione peroxidase (26). Sialic acid was present in the dental pulp of diabetic and healthy rats. Indeed, dentin sialoprotein (27), bone sialoprotein (28), and rich sialic acid proteins were already found in the mesenchymal cells of dental pulp. An interesting result was the significant reduction in sialic acid in the diabetic rats. This reduction was observed in all the forms of acid sialic (eg, free, total, and conjugated). This alteration in sialic acid concentration could be related to the physiopathology of diabetic complications, as revised by Sillanaukee et al. (14). Other studies have shown alterations in sialic acid metabolism related to diabetes. Rellier et al. (13) reported changes in the enzymes involved in sialic acid metabolism. They showed that the retinas of diabetic rats presented a change in glycosilation by an increase of neuraminidase and a reduction of sialyltransferase. Additionally, diabetic rats also presented a reduction of mucin concentration, a rich sialic acid glycoprotein that protects the oral mucosa (29).
TABLE 3. Catalase (mU/mg prot) and Peroxidase ( g/mg prot) Activities of Dental Pulp from the Control Group and the SZT-induced Diabetic Group Catalase (mU/mg prot)
Control Diabetic 2.74 3.44 0.35 0.67*

Peroxidase ( g/mg prot)

18.65 15.8 2.88 4.07

SZT, streptozotocin. Data presented as mean SD (n 10). *Indicates statically significant differences compared to control group (p

0.05).

In addition to sialic acid, the present study has also studied other parameters of the antioxidant system in the dental pulp of diabetic rats. These parameters were the catalase and peroxidase activities. These enzymes are responsible for hydrogen peroxide degradation in the water and oxygen, reducing toxicity in the tissues. Although a reduced catalase and no peroxidase activities in the dental pulp were reported (30), the activity of both enzymes was detected in this tissue in the present study. The diabetic rats presented an increase in catalase activity, which is indicative of oxidative stress. This could be because of the presence of superoxides produced by the diabetes in the dental pulp. Indeed, catalase has been indicated for controlling dental pulp inflammation, which is also in oxidative stress. The topical application of catalase in dental pulps may act as a direct capping agent, inducing a slight inflammatory cell infiltrate after exposure of the dental pulp, with a normal soft tissue response after 90 days (31). It was difficult to discuss the results of the present study further because there are no other reports on the enzymatic parameters of the antioxidant system in diabetic dental pulp. Some of the enzymatic parameters of the antioxidant system were studied in pulpitis (32) and direct pulp capping (31). With regard to the nonenzymatic parameters of the antioxidant system in diabetic dental pulp, there is a report showing that a long-term supplementation of vitamin C prevents the reduction of pulpal blood flow caused by diabetes (33). Catalase and peroxidase are enzymes responsible for the degradation of hydrogen peroxide, a known dental bleaching agent. Hydrogen peroxide has been used for tooth bleaching, and some studies have shown that this agent can penetrate into the pulp chamber through the dentin (34) causing moderate vasodilatation and an inflammatory pulp response (35). The alteration of catalase activity in the dental pulp of a diabetic rat is indicative of oxidative stress in this tissue. This finding suggests that diabetes may cause a different dental pulp response to the bleaching procedure with hydrogen peroxide. It could be of importance when planning dental bleaching in diabetic patients. The present study showed some of the dental pulp indications of oxidative stress resulting from diabetes. However, clinicians must be aware of other indications of oxidative stress in diabetic dental pulps, which are also of importance when planning dental treatment. One of these indications are the advanced glycation end-products, which are free radicals involved in diabetic microangiopathy, a common complication caused by endothelial dysfunction (36). This would lead to an impaired oxygenation, which, in turn, could explain another pathological finding in diabetic patients, which is the difficulty of periapical lesion repair (2, 3). Thus, the association of microvasculature malfunction with the natural anatomic condition of the dental pulp located inside hard walls could worsen the response of this tissue in diabetic patients to oral stimulus, such as dental caries, trauma, and periodontal diseases and their management. Based on the limitations of this study, it was concluded that diabetes can lead to important changes in the parameters of the antioxidant system of dental pulp tissue. These changes, mainly represented by the 1213

JOE Volume 34, Number 10, October 2008

Diabetes Induces Metabolic Alterations in Dental Pulp

Basic ResearchBiology
increase of catalase activity and the reduction of sialic acid concentration, indicate that diabetic dental pulp could have impaired responses. This would be of importance when planning any dental treatment that would depend on the dental pulp response, such as direct pulp capping, restoration of deep cavities, and dental bleaching, among others. Thus, further studies of other parameters of antioxidant systems are required, especially using human tissue, to reach conclusions about evidences of oxidative injury in the dental pulp under diabetic conditions and their clinical implications.
15. Mindiola MJ, et al. Endodontic treatment in an American Indian population: a 10-year retrospective study. J Endod 2006;32:828 32. 16. Fouad AF, Burleson J. The effect of diabetes mellitus on endodontic treatment outcome: data from an electronic patient record. J Am Dent Assoc 2003;134:4351. 17. Doyle SL, et al. Factors affecting outcomes for single-tooth implants and endodontic restorations. J Endod 2007;33:399 402. 18. Siqueira WL, Nicolau J. Stimulated whole saliva components in children with Down syndrome. Spec Care Dentist 2002;22:226 30. 19. Lowry OH, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:26575. 20. Aebi H. Catalase in vitro. Methods Enzymol 1984;105:121 6. 21. Chandra T, Das R, Datta AG. Role of thyroid gland on the peroxidase and iodinating enzymes of submaxillary gland. Eur J Biochem 1977;72:259 63. 22. Anderson LC. Peroxidase release from rat submandibular salivary acinar cells in vitro. Arch Oral Biol 1986;31:5013. 23. Catanzaro O, et al. Diabetes and its effects on dental pulp. J Oral Sci 2006;48:1959. 24. Nelson KK, Melendez JA. Mitochondrial redox control of matrix metalloproteinases. Free Radic Biol Med 2004;37:768 84. 25. Nelson AR, et al. Matrix metalloproteinases: biologic activity and clinical implications. J Clin Oncol 2000;18:1135 49. 26. Kheradmand F, et al. Role of Rac1 and oxygen radicals in collagenase-1 expression induced by cell shape change. Science 1998;280:898 902. 27. DSouza RN, et al. Developmental expression of a 53 KD dentin sialoprotein in rat tooth organs. J Histochem Cytochem 1992;40:359 66. 28. Garcia JM, et al. Immunolocalization of bone extracellular matrix proteins (type I collagen, osteonectin and bone sialoprotein) in human dental pulp and cultured pulp cells. Int Endod J 2003;36:404 10. 29. Belce A, et al. Evaluation of salivary sialic acid level and Cu-Zn superoxide dismutase activity in type 1 diabetes mellitus. Tohoku J Exp Med 2000;192:219 25. 30. Bowles WH, Burns H Jr. Catalase/peroxidase activity in dental pulp. J Endod 1992;18:52734. 31. Alacam A, et al. Effects of topical catalase application on dental pulp tissue: a histopathological evaluation. J Dent 2000;28:3339. 32. Tulunoglu O, et al. Superoxide dismutase activity in healthy and inflamed pulp tissues of permanent teeth in children. J Clin Pediatr Dent 1998;22:3415. 33. Amatyakul S, et al. The effect of long-term supplementation of vitamin C on pulpal blood flow in streptozotocin-induced diabetic rats. Clin Hemorheol Microcirc 2003;29:3139. 34. Camargo SE, et al. Penetration of 38% hydrogen peroxide into the pulp chamber in bovine and human teeth submitted to office bleach technique. J Endod 2007;33:1074 7. 35. Robertson WD, Melfi RC. Pulpal response to vital bleaching procedures. J Endod 1980;6:6459. 36. Vlassara H. Advanced glycation end-products and atherosclerosis. Ann Med 1996; 28:419 26.

References
1. Manfredi M, et al. Update on diabetes mellitus and related oral diseases. Oral Dis 2004;10:187200. 2. Fouad AF. Diabetes mellitus as a modulating factor of endodontic infections. J Dent Educ 2003;67:459 67. 3. Iwama A, et al. Increased number of anaerobic bacteria in the infected root canal in type 2 diabetic rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006; 101:681 6. 4. Bender IB, Bender AB. Diabetes mellitus and the dental pulp. J Endod 2003; 29:3839. 5. Guo B, et al. Altered circadian rhythm of pulp sensibility in elderly diabetic and hypertensive patients. Chin Med J (Engl) 2007;120:1024 6. 6. West IC. Radicals and oxidative stress in diabetes. Diabet Med 2000;17:171 80. 7. Rolo AP, Palmeira CM. Diabetes and mitochondrial function: role of hyperglycemia and oxidative stress. Toxicol Appl Pharmacol 2006;212:16778. 8. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001;414:81320. 9. Gate L, et al. Oxidative stress induced in pathologies: the role of antioxidants. Biomed Pharmacother 1999;53:169 80. 10. Iijima R, et al. Novel biological function of sialic acid (N-acetylneuraminic acid) as a hydrogen peroxide scavenger. FEBS Lett 2004;561:163 6. 11. Ashwell G, Berlin WK, Gabriel O. Alpha 2-8 sialic acid polymers: size, structure, and compositional analysis. Anal Biochem 1994;222:495502. 12. Iijima R, et al. Characterization of the reaction between sialic acid (N-acetylneuraminic acid) and hydrogen peroxide. Biol Pharm Bull 2007;30:580 2. 13. Rellier N, et al. In vitro and in vivo alterations of enzymatic glycosylation in diabetes. Life Sci 1999;64:1571 83. 14. Sillanaukee P, Ponnio M, Jaaskelainen IP. Occurrence of sialic acids in healthy humans and different disorders. Eur J Clin Invest 1999;29:41325.

1214

Leite et al.

JOE Volume 34, Number 10, October 2008