Transdentinal protective role of sodium ascorbate against thecytopathic effects of H2O2 released from bleaching agentsAdriano Fonseca Lima, DDS, MS,a Fernanda Campos Rosetti Lessa, DDS, MS, PhD,b

Maria Nadir Gasparoto Mancini, DDS, MS, PhD,c Josimeri Hebling, DDS, MS, PhD,d

Carlos Alberto de Souza Costa, DDS, MS, PhD,e and Giselle Maria Marchi, DDS, MS, PhD,f

Piracicaba, Araraquara, and São José dos Campos, SP, BrazilSTATE UNIVERSITY OF CAMPINAS-UNICAMP AND UNIVERSITY OF SÃO PAULO STATE-UNESP

Objectives. The objectives of this study were to evaluate the transdentinal cytotoxicity of 10% and 16% carbamideperoxide gel (CP), as well as the ability of the antioxidant, 10% sodium ascorbate (SA), to protect the odontoblasts inculture.Study design. Human dentin discs of 0.5-mm thickness were obtained and were placed into artificial pulp chambers.MDPC-23 odontoblastlike cells were seeded on pulp surface of the discs and the following groups were established:G1-No Treatment (control), G2-10% SA/6hs, G3-10%/CP6hs, G4-10%SA/6hs�10%CP/6hs, G5-16%CP/6hs, andG6-10%SA/6hs�16%CP/6hs. The cell viability was measured by the MTT assay.Results. In groups where 16% CP was used, decreased cell viability was observed. Conversely, the application of 10%SA on the dentin discs, before the use of the CP, reduced the cytotoxic effects of these products on cells.Conclusions. The 16% CP cause a significant decrease in MDPC-23 cell viability and 10% SA was able to partially

prevent the toxic effects of CP. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:e70-e76)

Dental bleaching is one of the most requested cosmeticprocedures in aesthetic dentistry. Nevertheless, thistreatment may cause some side effects, such as (1)decreased bond strength of composite resin to thebleached substrate,1,2 (2) morphological changes in

This study was supported by the Fundação de Amparo à Pesquisa doEstado de São Paulo – FAPESP (Grants: 2006-58780-8 and 2008/05890-6) and Conselho Nacional de Desenvolvimento Científico eTecnológico - CNPq (Grants: 476137/2006 and 301029/2007-5).aPhD Student, Department of Restorative Dentistry, PiracicabaSchool of Dentistry, State University of Campinas-UNICAMP, Pi-racicaba, SP, Brazil.bPhD student, Department of Orthodontics and Pediatric Dentistry,Araraquara School of Dentistry, University of São Paulo State-UNESP, Araraquara, SP, Brazil.cAssociate Professor, Department of Physiological Sciences, São Josédos Campos School of Dentistry, University of São Paulo State-UNESP, São José dos Campos, SP, Brazil.dAssociate Professor, Department of Orthodontics and Pediatric Den-tistry, Araraquara School of Dentistry, University of São Paulo State-UNESP, Araraquara, SP, Brazil.eAssociate Professor, Department of Physiology and Pathology,Araraquara School of Dentistry, University of São Paulo State-UNESP, Araraquara, SP, Brazil.fAssociate Professor, Department of Restorative Dentistry, PiracicabaSchool of Dentistry, State University of Campinas-UNICAMP, Pi-racicaba, SP, Brazil.Received for publication Apr 22, 2009; returned for revision Dec 2,2009; accepted for publication Dec 9, 2009.1079-2104/$ - see front matter© 2010 Mosby, Inc. All rights reserved.

doi:10.1016/j.tripleo.2009.12.020

e70

dental structure,3,4 (3) dentin hypersensitivity,5,6 and(4) modifications in pulp enzyme activities.7

During tooth bleaching, the carbamide peroxide gelemployed breaks down to hydrogen peroxide, urea,ammonia, and carbon dioxide. The degradation ofH2O2 by the reactions of Fenton and Harber-Weiss,which occur in the tissues and cells containing metalions and copper, can produce the hydroxyl radical. Thisoxygen-derived free radical, in addition to being able tobleach the tooth structures, has important biologicalsignificance owing to its high reactivity and toxicity,8,9

causing deleterious effects to the pulp cells.10,11

Because the carbamide peroxide remains in contactwith teeth during the treatment period, H2O2 and somereactive oxygen species (ROS) are released, which dif-fuse across hard tissues to reach more vulnerable toothstructures, such as the pulp tissue.12-14 In this special-ized connective tissue, ROS can cause inflammation,5

consequently increasing intrapulp pressure, and theseeffects may result in dentin hypersensitivity.

Dental materials or components that are able to dif-fuse through the dentinal tubules can cause irreversibledamage to the pulp or even induce the processes of celldeath and tissue necrosis.15 In these cases, the first cellsto be damaged are the odontoblast cells. As such, forthe development of in vitro studies to investigate theinfluence of dental materials on pulp tissue, cells of anodontoblast phenotype are the most appropriate foruse,16 allowing the laboratory stimulation of certain

clinical conditions.

OOOOEVolume 109, Number 4 Lima et al. e71

It has been demonstrated that 10% sodium ascorbatemay be used as an antioxidizing agent to restore thebond strength of composite resin to the bleached sur-face.2,17 This chemical agent, which is a powerful hy-dro-soluble antioxidant found in biological fluids, has aremarkable ability to promote reduction of reactivespecies derived from oxygen and nitrogen and, thus, isable to prevent oxidative damage to important biolog-ical macromolecules, such as DNA, proteins, and lip-ids.18,19 Therefore, the use of antioxidant substances,such as sodium ascorbate, during the clinical procedureof tooth bleaching, may be important not only to restorebond strength after treatment, but also for the possibleprotection of pulp cells against aggression imposed bytoxic components in bleaching materials with the abil-ity of transdentinal diffusion. Therefore, the objectivesof this in vitro study were to (1) evaluate the possibletransdentinal cytotoxic effects of bleaching agentsbased on carbamide peroxide (CP; 10% and 16% solu-tions), (2) examine whether the antioxidizing propertiesof 10% sodium ascorbate (SA) protect odontoblastic-like cells against the possible cytotoxic effects ofbleaching agents, and (3) evaluate the amount of hy-drogen peroxide that is able to diffuse through humandentin.

MATERIALS AND METHODSAfter approval of this research study by the Ethics

Committee of the Dentistry University of Piracicaba/UNICAMP, Brazil (protocol 01/2007), 70 dentin discsof approximately 0.6 mm in thickness were obtainedfrom intact human third molars. The discs were furthermanually ground with 400- and 600-grit abrasive pa-pers (T469-SF-Noton, Saint-Gobam Abrasivos Ltda.,Jundiai, Brazil) to a 0.5-mm final thickness. The smearlayer produced on both sides of the discs was removedby the active application of 0.5 M EDTA (pH 7.4) for30 seconds, followed by abundant rinsing with distilledwater.20 Thus, dentin discs with a standardized thick-ness (0.5 mm) were mounted on a filtering cameraconnected to a water column of 180 cm21 and thehydraulic conductance was calculated and determinedfor each disc. Finally, the dentin discs were dividedbetween the groups, such that the permeability averagevalues were not statistically different between them(Kruskal-Wallis, P � .05).

Transdentinal cytotoxic effectFor the cell viability analysis, a device simulating an

artificial pulp chamber (APC) was used (Fig. 1). In thisAPC, a dentin disc was adapted to evaluate the trans-dentinal diffusion and cytotoxicity of the bleachingagents. The dentin discs were reduced to a diameter of

8 mm, and then positioned in the APC in such way that

the pulpal surface remained facing the APC internalregion (pulp space), between 2 sterilized o-rings ofsilicone (Orion, São Paulo, SP, Brazil).

The APCs were individually placed in sterile acrylicplates of 24 wells (Coaster Corp., Cambridge, MA)with the pulp surface of the dentin discs facing upward.Immortalized cells of the MDPC-23 cell line22,23 wereseeded (50,000 cells/cm2) on the dentin surface. TheAPCs with discs and cells were maintained for 48 hoursin a humidified incubator at 37°C with 5% CO2 and95% air. The culture medium used in this experimentwas Dulbecco modified Eagle medium (DMEM, SigmaChemical Co., St. Louis, MO) complemented with 10%fetal bovine serum (FBS, Cultilab, Campinas, SP, Brazil),supplemented with 100 IU/mL of penicillin, 100 mg/mLstreptomycin, and 2 mmol/L glutamine (GIBCO, GrandIsland, NY). After this period, the APCs were invertedand individually placed into a new 24-acrylic-wellplate, with the cells immersed in the culture medium.The occlusal surfaces of the dentin discs were main-tained in an upward fashion, allowing the application ofthe bleaching agents on this tubular substrate. Thegroups were formed according to the following differ-ent treatments (n � 10):

● Group 1 – No treatment (Control)● Group 2 – 10% sodium ascorbate (SA) (Sigma

Chemical Co.)● Group 3 – 10% carbamide peroxide (CP) applied for

6 hours● Group 4 – 10% SA/6 hours � washing of the discs �

10% CP/6 hours● Group 5 – 16% CP applied for 6 hours● Group 6 – 10% SA/6 hours � washing of the discs �

16% CP/6 hours

Of the total of 10 discs per group, 8 were used toevaluate cell viability, and 2 were used for the evalua-tion of cell morphology by scanning electron micros-copy (SEM). For groups 4 and 6, 6 hours after appli-cation of 10% sodium ascorbate (3 �L), the dentinalsubstrate was vigorously rinsed to completely removethe antioxidant substance from the dentin, enabling thetreatment of the occlusal surface of the discs with 18

Fig. 1. Artificial pulp chamber (A), and this apparatus withdentin disc adapted (B). OS, occlusal surface; PS, pulpalsurface.

mg of bleaching gels containing 10% or 16% carbam-

OOOOEe72 Lima et al. April 2010

ide peroxide (Whiteness, FGM, Joinvile, SC, Brazil).After an application period of 6 hours, the bleachinggels were removed from the occlusal surface of thediscs through vigorous rinsing with distilled water. Thedentin discs were then removed from the APCs andplaced individually in wells, such that their pulp sur-faces (where the cells were cultured) were maintainedfacing up. These cells were submitted to cell metabolicactivity analysis using a methyltetrazolium assay (MTTassay), as described in previous studies.10,24

Measurement of H2O2 transdentinal diffusionFor groups 3 and 5 only, 5 additional discs were used

to measure the concentration of hydrogen peroxide thatdiffused through the dentin to reach the pulp space ofthe APCs. The measurement of hydrogen peroxidewithin the APC was performed using the leukocrystalviolet/enzyme horseradish peroxidase (LCV/HRP) as-say.25 In each well of a microtiter plate, 900 �L of 2 Macetate buffer, pH 4.5, was added and the APCs wereimmersed with dentin, with the pulp surface facingdown and in contact with the buffer solution.

The bleaching gels were applied on the occlusalsurface of the discs and incubated for 6 hours at 37°C.After this incubation period, 100 �L of the acetatebuffer was removed from the wells and transferred toglass tubes. Then, 100 �L of LCV 0.5-mg/mL solutionwas added to each tube (Sigma Chemical Co.) togetherwith 50 �L HRP solution (1 mg/mL; Sigma ChemicalCo.).

After 5 minutes of reaction, the optical density of theresultant blue color in the tubes was measured using aUV spectrophotometer (UV-Vis SpectrophotometerUV-1203; Shimadzu, Kyoto, Japan) at a wavelength of596 nm. The optical density values obtained from thesamples were converted into micrograms equivalent tothe H2O2 concentration in each solution, using a cali-bration curve obtained with hydrogen peroxide in therange of 0.5 to 4.5 mg.

Cell morphology analysis by scanning electronicmicroscopy

For each group, 2 representative samples were pre-pared for analysis by SEM. For this, the dentin discswere carefully removed from the APCs, fixed in 1 mLof buffered 2.5% glutaraldehyde for 24 hours, submit-ted to three 5-minute rinses with 1 mL phosphate-buffered saline, and postfixed in 1% osmium tetroxidefor 60 minutes. Afterward, the discs with cells weredehydrated in increasing concentrations of ethanol so-lutions (30%, 50%, 70%, 90%, 100%). Finally, the cellson discs were subjected to drying by low surface ten-sion in 98% 1,1,1,3,3,3-hexamethyldisilazane (HMDS)

solvent (Acros Organics, Morris Plains, NJ) and kept in

desiccators for 12 hours. The dentin discs were fixed onmetal stubs and gold sputtered. These procedures al-lowed cell morphology analysis in SEM (JEOL-JMS-T33A Scanning Microscope, JEOL-USA Inc., Pea-body, MA).

Statistical analysisValues obtained for the MTT technique, referring to

the production of SDH, were subjected to statisticalanalysis of Kruskal-Wallis, complemented by Mann-Whitney test, to compare the groups in pairs. For valuesobtained for the transdentinal diffusion of carbamideperoxide, the Student t test was used. All statistical testswere considered with a preestablished level of signifi-cance of 5%. Statistical analysis was carried out usingSPSS statistical software (SPSS Inc., Chicago, IL) witha confidence interval of 95%.

RESULTSDentin permeability

The permeability of the discs was measured througha filter chamber; data obtained were tabulated and sub-jected to statistical analysis (Kruskal-Wallis P � .05),as presented in Table I.

Cell viabilityMedian (P25/P75) values for the production of SDH,

as detected by the MTT assay, are shown in Table II,according to the experimental groups. Application of16% CP for 6 hours resulted in lower values of SDHproduction (P � .05), whereas the use of 10% SA for6 hours resulted in an increased production of thisenzyme, although this production was not statisticallyhigher than that of the control group (P � .05). Groups3, 4, and 6 did not differ statistically between them, orwhen compared with controls (P � .05). Consideringthe control group as presenting 100% cell viability, thepercentage of MDPC-23 cell viability was calculatedfor the different experimental groups, as presented in

Table I. Median of hydraulic conductance accordingto the experimental groupsGroups Hydraulic Conductance* (gL/cm2/min/cm H2O)

1 0.014162 0.014463 0.014204 0.014045 0.014166 0.01422

*No statistical difference between the groups (Kruskal-Wallis, P �.05).

Fig. 2.

OOOOEVolume 109, Number 4 Lima et al. e73

H2O2 transdentinal diffusionGroup 5 (carbamide peroxide 16%) presented a sta-

tistically higher transdentinal diffusion of hydrogenperoxide (4.23 �g/mL) than group 3 (carbamide per-oxide 10% - 1.699 ìg/mL) (Student t test, P � .01).

Cell morphology (SEM)Groups 1 and 2 presented a large number of

MPDC-23 cells that remained attached to the dentinalsubstrate; the quantity of cells was close to confluence.In both groups, the MDPC-23 cells were observed aslarge cells with a number of thin cytoplasmic prolon-gations originating from the cell membrane that seemedto adhere to the dentin (Fig. 3, A and B). For all theother groups, a decrease in the number of cells thatremained attached to the dentin substrate was observed;although it was still possible to observe morphologicchanges presented by the cells, as characterized by thedecrease in their size and reduction in the amount andextension of cytoplasm (Fig. 3, C-F). These importantmorphological changes observed in the MDPC-23 cellsthat remained attached to the dentin, led them to adopta round-shaped morphology. Nevertheless, these mor-phological cell changes (reduction in size and decreasein number) were more evident in group 5, where largeareas of the dentin with dentinal tubules exposed re-mained open, demonstrating the occurrence of signifi-cant cell death following their detachment from thesubstrate (Fig. 3, F).

DISCUSSIONDentin permeability was measured before the initia-

tion of experiments to make certain that all groups werestatistically similar to each other and to ensure thatdentin permeability would not affect H2O2 diffusion, aswell as cell viability. Transdentinal diffusion of H2O2

occurred in all the groups in which the bleaching gels

Table II. Succinic dehydrogenase enzyme (SDH) ac-tivity according to the experimental groups representedby absorbent value in the control and experimentalgroupsGroups SDH activity Statistical comparison*

1 0.1989 (0.0975-0.2398)† AB2 0.2002 (0.1807-0.2540) A3 0.1733 (0.0629-0.2064) B4 0.1897 (0.0714-0.2298) B5 0.0697 (0.0595-0.1485) C6 0.1605 (0.0796-0.1914) B

*For SDH activity, different letters indicate statistically significantdifference (Mann-Whitney, P � .05).†Values represent the median (P25-P75).

were applied and had a direct toxic effect on the cells

cultured on the pulp surface of the dentin discs. A greatdecrease in cell viability was observed in group 5 (16%CP), and this viability was statistically lower than thatof group 1 (control) and all the other experimentalgroups. These negative results observed for group 5,were in part because a larger quantity of H2O2 diffusedthrough the dentinal tubules to reach the pulpal surfaceof the dentin discs, where the cells MDPC-23 werecultured. In group 5, the high concentration of H2O2

that reached the pulpal space of the APC (4.2 g/mL)was clearly observed using the LCV/HRP technique.Conversely, approximately 1.7 g/mL of H2O2 was de-tected for group 3 in the pulpal space of the APC; thisconcentration was significantly lower than that of group5. Therefore, when a higher concentration of carbamideperoxide gel is applied to the dentin substrate, a signif-icantly larger amount of H2O2 is able to diffuse throughthe dentin and cause cellular damage. Thus, althoughdentin acts as an organic barrier to protect the pulptissue against external toxic agents,11 this tubular tis-sue, with a standardized width of 0.5 mm, does notprevent components released from the bleaching agent,such as H2O2, from diffusing across the dentin to de-crease the metabolic activity of the MDPC-23 cells.

H2O2 and its subproducts, such as the hydroxyl rad-ical (OH–), are considered to be ROS that are able tocause damage to several cellular components, includingthe cell membrane and DNA, by direct action or byreacting with other cellular components to generatenew ROS.26 This damage begins in the membrane,triggering an autocatalytic reaction, denominated aslipid peroxidation.27 Damage to the cell membraneresults in the decreased fluidity of this structure andincreased permeability, as well as the inactivation ofreceptors, enzymes, and ion channels.27 These cellmembrane changes allow a marked and sustained in-crease in the cytosolic free Ca2� concentration, whichcan cause calcium-dependent catabolic processes, suchas activation of phospholipases, endonucleases, andproteases.28 These changes may cause extensive dam-age to the cell membrane or even cell death.27

In group 5, most cells that were attached to the pulpsurface of the dentin discs were lethally damaged afterapplication of bleaching gel with 16% CP. This wasconfirmed by SEM analysis. For this group, after treat-ment, several dead cells detached from the dentinalsubstrate. Thus, only a few MDPC-23 cells with around-shaped morphology and reduced size remainedon the broad areas of tubular dentin (Fig. 3, E). Thesemorphological changes in cells were less significant ingroup 3 (10% CP), which clearly shows that the dam-age to the MDPC-23 cells is directly related to theconcentration of H2O2 released by the CP gel applied to

the dentin substrate.

bility

n subsof thettache

OOOOEe74 Lima et al. April 2010

Oxidative stress, which occurs because of severeunbalance between the production of ROS and the

Fig. 2. Graphical representation in percentage of the cell via

Fig. 3. Representative SEM photomicrographs (increased frostudy. A, Group 1 (control): A large number of odontoblastlikobserved. Note that the cells exhibited a large volume of cytfrom the cell membrane. B, Group 2: The morphological chacontrol group (G1). C, Group 3, and D, Group 4: A numbesubstrate exhibit round-shaped morphology with a few cytoalthough some cells still have a large cytoplasmic volume, mGroup 5: Only a few cells remain attached to the tubular dentihave detached from the dentin (arrows). F, Group 6: In spitewith a few cytoplasmic processes on the membrane remain a

presence of endogenous antioxidants,29 was not very

expressive in group 3. This indicates that perhaps theendogenous enzymatic antioxidants such as catalase,

in function of different experimental groups.

inal �500) of experimental and control groups used in thisat near confluence on the pulp surface of the dentin disc arewith numerous short cytoplasmic prolongations originating

istics of MDPC-23 cells are similar to those observed in theattered MDPC-23 cells that remained attached to the dentinic prolongations originating from the membrane. Note thatve a reduced size when compared with the control group. E,trate. Note a number of residual membranes of dead cells thathigh cell loss, note that the higher number of MDPC-23 cellsd to the substrate when compared with Group 5.

m orige cells

oplasmracter

r of scplasmost ha

superoxide dismutase, and glutathiose peroxidase, may

OOOOEVolume 109, Number 4 Lima et al. e75

have been sufficient to prevent serious cellular damage.Moreover, it can be speculated that the concentration ofapproximately 1.7 g/mL of H2O2 in the APC pulpspace, where the cells were cultured, was not sufficientto cause cell death, as observed in group 5. However,despite the lower H2O2 dentin diffusion, SEM cellanalysis of group 3 demonstrated significant morpho-logical changes, without a remarkable decrease in thenumber of cells that remained attached to the dentinsubstrate.

With the objective of simulating the daily period ofapplication of carbamide peroxide employed for night-guard vital bleaching; in this study, cell viability wasassessed after the bleaching gels were applied for 6hours on the tooth structure. The nightguard vitalbleaching technique, however, usually has an averageperiod of application of 2 weeks. As such, it may bespeculated that with consecutive applications, the lowercytotoxicity of 10% CP gel for MDPC-23 cells, aspresented in this in vitro study, may be aggravatedbecause of the increase in the transdentinal diffusion ofoxidizing agents, which may cause intense damage orcell death. A previous study reported that the applica-tion of 10% CP to first premolars leads to slight pulpinflammation30; however, these teeth present greaterdentin thickness than that of the upper and lower inci-sors, which are the teeth that are most affected by thebleaching treatment. Future investigations are, there-fore, needed to clarify the effects of this long-termbleaching therapy on the pulp cells.

In group 2 (10% SA), cell viability was found to beslightly, but not significantly, higher than that of thecontrol group. For this experimental group, only aslight change was observed in cell morphology, indi-cating that the concentration of 10% SA applied on theocclusal surface of the dentin discs is not able to causetoxic effects on MDPC-23 cells. In groups 4 and 6, inwhich the 10% SA solution was applied to the dentinbefore the use of 10% or 16% CP gel respectively, lessdamage to the MDPC-23 cells was found, suggestingthat the SA solution may protect against the deleteriouseffects of H2O2.

The dentinal diffusion of SA was not directly eval-uated in this study. However, the capacity of resinmonomers, such as triethyleneglycol-dimethacrylate(TEGDMA) (286.3 g/mol molecular weight) and hy-droxyethyl methacrylate (HEMA) (130 g/mol molecu-lar weight), to diffuse through the dentin and reach thepulp tissue should be considered in different clinicalsituations.31-34 SA, the antioxidant agent used in thisstudy, has a molecular weight of 198.11 g/mol, which isan intermediate molecular weight when compared withthe TEGDMA and HEMA resin components. There-

fore, it may be speculated that SA may have diffused

through the dentin disc, reaching the pulp space of theAPC, possibly explaining the lower cell damage ob-served in the groups in which the dentin tissue wastreated with 10% SA before application of the bleach-ing agents. SA may interact with ROS, making themmore stable (Fig. 4), decreasing their reactivity,35 and,consequently, preventing and/or decreasing cell dam-age caused by free radicals. The protective ability ofSA has also been observed in a previous study, wherethis antioxidant agent was applied with eluates obtainedfrom experimental composite resins, on cultured fibro-blast. In this in vitro study, the authors demonstratedthat the cytotoxicity of the resin products released fromthe resin-based materials in an aqueous environmentwere clearly reduced.18

The dentin thickness of 0.5 mm used in this studycan be found mainly in regions that present cervicalabrasion or erosion injuries; this thickness has beenused in previous studies.11 The dentinal diffusion ofH2O2 observed in group 5 (16% CP) was statisticallyhigher than that observed in group 3 (10% CP). It hasbeen demonstrated that the release of H2O2 frombleaching gels is approximately 4.7% for 16% CP and3.5% for 10% CP,36 which may explain the notablediffusion of this oxidative agent across dentinal tubulesand the, consequently, intense cytotoxicity of the dentalproducts that are widely used to bleach teeth.

In the present study, transdentinal diffusion of H2O2

released from both concentrations of CP tested (10%and 16%) was observed. However, 16% CP causedgreater cytotoxic effects on cultured odontoblastlikeMDPC-23 cells than 10% CP. Data also indicate that10% SA, when used before application of the bleachinggel (16% CP) on dentin substrate, can prevent theharmful effects of this kind of dental product to the pulpcells in culture. Despite the interesting data presented inthis investigation, results from in vitro studies cannot

Fig. 4. Hydrogen peroxide (H2O2) is neutralized by the trans-fer of a single electron from ascorbate (Asc), producing waterand ascorbyl free radicals (AFR). The pairs of AFR form 1molecule of dehydroascorbic acid (DHAA) and 1 Asc. Thelactone ring of DHAA breaks down forming the inert prod-ucts, diketogulonic acid, threonic acid, and oxalic acid; alter-natively, DHAA can be reduced to the useful Asc.

necessarily be directly extrapolated to clinical situa-

OOOOEe76 Lima et al. April 2010

tions.37 This is because in the in vivo situations, severalfactors such as presence of immune system, lymphaticdrainage, variable thickness, and morphological char-acteristics of the dentin substrate may modulate thepossible toxic effects of the bleaching agent compo-nents to the pulp cells.

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Reprint requests:

Carlos Alberto de Souza Costa, DDS, MS, PhDDepartamento de Fisiologia e PatologiaFaculdade de Odontologia de Araraquara - UNESPRua Humaitá, 1680 - Centro. CEP: 14801-903, Araraquara, SP,Brasil

casouzac@foar.unesp.br