Determination of para-Chloroaniline and Reactive Oxygen Species in Chlorhexidine and Chlorhexidine Associated with Calcium Hydroxide

Basic Research—Technology

Determination of para-Chloroaniline and Reactive Oxygen Species in Chlorhexidine and Chlorhexidine Associated with Calcium Hydroxide Luiz Eduardo Barbin, DDS, MSc, PhD,* Paulo César Saquy, DDS, MSc, PhD,* Débora Fernandes Costa Guedes, BCH, MSc, PhD,† Manoel Damião Sousa-Neto, DDS, MSc, PhD,* Carlos Estrela, DDS, MSc, PhD,‡ and Jesus Djalma Pécora, DDS, MSc, PhD* Abstract The aim of this study was to determine whether parachloroaniline (PCA) and/or reactive oxygen species (ROS) are generated by chlorhexidine (CHX) alone or after CHX is mixed with calcium hydroxide at different time points. Mass spectrometry was performed to detect PCA in samples of 0.2% CHX and Ca(OH)2 mixed with 0.2% CHX. High-performance liquid chromatography was used to confirm the presence of CHX in the mixture with Ca(OH)2. The samples were analyzed immediately after mixing and after 7 and 14 days. During the intervals of the experiment, the samples were maintained at 36.5°C and 95% relative humidity. PCA was detected in the 0.2% CHX solution after 14 days. The mixture of CHX with Ca(OH)2 liberated ROS at all time points, but no traces of CHX were present in the mixture as a result of immediate degradation of the CHX. (J Endod 2008;34:1508 –1514)

Key Words Calcium hydroxide, Ca(OH)2, chlorhexidine, intracanal medicaments, p-chloroaniline, reactive oxygen species

From the *Department of Restorative Dentistry and †Laboratory of Dental Waste Management, Department of Restorative Dentistry, Dental School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP; and ‡Department of Endodontics, Federal University of Goiás, Goiânia, GO, Brazil. Address requests for reprints to Prof Dr Carlos Estrela, Centro de Ensino e Pesquisa Odontológica do Brazil (CEPOBRAS), Rua C-245, Quadra 546, Lote 9, Jardim América, CEP 74.290-200, Goiânia, GO, Brazil. E-mail address: [email protected]. 0099-2399/$0 - see front matter © 2008 Published by Elsevier Inc. on behalf of the American Association of Endodontists. doi:10.1016/j.joen.2008.08.032

1508

Barbin et al.

E

ndodontic therapy aims to eliminate microorganisms from the root canal system by an effective debridement and disinfection process. Therefore, biomechanical preparation, irrigation with different chemical solutions, and use of intracanal dressings are considered essential components for microbial control in endodontic infections (1– 4). Calcium hydroxide, sodium hypochlorite, and chlorhexidine gluconate (CHX) are widely used medicaments in the treatment of infected root canal systems. These materials have distinct characteristics, and their antimicrobial activity has been demonstrated (5– 8). CHX, chemically 2,4,11,13-tetraazatetradecanediimidamide, N=N-bis(4-chlorophenyl)-3,12-diimino-, di-D-gluconate, has a molecular mass of 505.4 g/mol (9,10) and has been widely used as an antiseptic agent for routine dental plaque control and irrigation of root canals for reduction of endodontic microbiota (11). However, this substance has been shown to have no capacity to detoxify endotoxin (12). The effectiveness of Ca(OH)2 on the inactivation of microorganisms and tissue healing is directly related to its ionic dissociation into calcium and hydroxyl ions. The antimicrobial mechanism of action of Ca(OH)2 is directly influenced by its high pH, which causes inactivation of enzymes in bacteria. It has been shown that the direct contact of Ca(OH)2 with bacteria is more effective than its diffusion through dentinal tubules (5). The neutralization of bacterial toxins is another essential issue to be considered in the selection of an antimicrobial agent. Ca(OH)2 hydrolyzes the lipids of bacterial lipopolysaccharide (LPS), leading to the release of free hydroxyl fatty acids, which suggests that Ca(OH)2-mediated degradation of LPS might have an important contribution to the beneficial effects of Ca(OH)2 use in clinical endodontics (13). Several of these agents have been evaluated in combination. For example, CHX mixed with Ca(OH)2 has been shown not to influence the time required and efficacy for microbial inactivation, including Enterococcus faecalis (14). However, other studies have demonstrated that this association presents a greater antimicrobial action (6, 8). The generation of para-chloroaniline (PCA) is a potential consequence of the use of CHX. Recent investigations (9, 15–18) have demonstrated that CHX liberates PCA and, when associated with Ca(OH)2, might produce reactive oxygen species (ROS). PCA is generated by the hydrolysis of CHX as a function of time, alkaline environment (high pH), and heat (17, 18). The presence of PCA has been detected in CHX solutions (9, 15). The International Agency for Research on Cancer (IARC, 2006) categorizes PCA in their 2B Group, which means that this agent is possibly carcinogenic to humans (16). The interaction of CHX with Ca(OH)2 might generate ROS, which play a critical role on the cellular wall and membrane structure of microorganisms. Accordingly, tissue damage should be considered during endodontic practice when higher concentrations of CHX are extruded into the periapical tissue (17). Waris and Ahsan (19) related that various carcinogens might also partly exert their effect by generating ROS during metabolism. Oxidative damage to cellular DNA can lead to mutations and play an important role in the initiation and progression of multistage carcinogenesis. The aim of this study was to determine the time course for the generation of both PCA and ROS in CHX solutions and when CHX was mixed with Ca(OH)2.

JOE — Volume 34, Number 12, December 2008

Basic Research—Technology

Figure 1. (A) Mass spectrometry analysis of a stock solution of 0.2% CHX immediately after its preparation demonstrated peaks at 505 and 701 m/z, which confirms the high degree of purity of this sample. The presence of PCA was not detected at this time point. (B) Mass spectrometry analysis of a solution of 0.2% CHX after 7 days of incubation. The chromatographic analysis showed decomposition into several sub-products with different values (101, 125, 152, 170, 177, 201, 319, 336, 353, and 505 m/z). (C) Mass spectrometry analysis of a solution of 0.2% CHX after 14 days of incubation. There was a total degradation of CHX, as indicated by the loss of the initial peaks (505 and 701 m/z). In addition, PCA appears to be produced, as indicated by peaks at 129 m/z (references 9, 18) and 149 and 167 m/z (observed in our own control studies by using a standard solution of PCA, data not shown). (D) Mass spectrometry analysis of a solution of 0.2% CHX and Ca(OH)2 immediately after mixture, with peaks observed at 169, 195, 256, and 351 m/z. The peak of 169 m/z corresponds to the breakdown product of chlorophenyl guanide. These results are consistent with an ability of Ca(OH)2 to degrade the CHX molecule in positions containing groups NHn. The peak of 195 m/z probably originated by production of reactive compounds, as a result of the high concentration of hydroxyl ions (alkaline environment) in the presence of CHX. There is no evidence of PCA formation. (E) Mass spectrometry analysis of a solution of 0.2% CHX and Ca(OH)2 after 7 days of incubation. No peaks were observed at either 505 or 701 m/z, which suggest a total degradation of CHX (compare with A). ROS were observed, but indicators for PCA were not detected. (F) Mass spectrometry analysis of a solution of 0.2% CHX and Ca(OH)2 after 14 days of incubation. No peaks were observed at either 505 or 701 m/z, which suggest a total degradation of CHX (compare with A). ROS were observed, but indicators for PCA were not detected.

JOE — Volume 34, Number 12, December 2008

Detection of P-Chloroaniline in Chlorhexidine

1509

Basic Research—Technology

Figure 1. (Continued)

Material and Methods Chemical Reagents The chemical reagents used in this study were 20% CHX (standard substance; Sigma-Aldrich Corp, Steinheim am Albuch, BW, Germany), 95% Ca(OH)2 (Sigma-Aldrich), and PCA (SigmaAldrich). 1510

Barbin et al.

Sample Preparation PCA solution was prepared by dilution of 2.5 mg/mL (1.96 ⫻ 10–5 mol) of PCA in 1.0 mL of deionized water (1.96 ⫻ 10–2 mol ⫻ L–1). A 0.2% CHX solution was prepared by dilution of 20 ␮L of 20% CHX in 2.0 mL of deionized water. Ca(OH)2 (116 mg) was mixed with 150 ␮L of 0.2% CHX. JOE — Volume 34, Number 12, December 2008

Basic Research—Technology

Figure 1. (Continued)

Electrospray ionization time-of-flight mass spectrometry (ESITOF-MS) was performed in an ultrOToF-Q/ESI-Qq-ToF equipment (Bruker Daltonics, Billerica, MA) to detect the presence of PCA in 0.2% CHX and Ca(OH)2 ⫹ 0.2% CHX mixture. High-performance liquid chromatography (HPLC) was performed in a diode array detector (SPD-M 10 VP; Shimadzu, Kyoto, Japan) to confirm the presence of CHX in the Ca(OH)2 ⫹ 0.2% CHX mixture. The samples were analyzed immediately after preparation of the substances (T1), after 7 days (T2), and after 14 days (T3). During the intervals of the experiment, the samples were maintained at 36.5°C and 95% relative humidity.

Preparation of the Reference Solution: Chromatographic System The method was carried out according to the United States Pharmacopoeia 29/National Formulary 24 (USP 29/NF 24; United States Pharmacopeial Convention, 2006, Rockville, MD). Separation of CHX from the sub-products originated from the Ca(OH)2 ⫹ 0.2% CHX mixture was achieved by using a HPLC LiChrospher 100RP-C18 analytical column (250 ⫻ 4.5 mm, particle size 5 ␮m; Merck KGaA, Darmstadt, JOE — Volume 34, Number 12, December 2008

Germany) protected by a LiChrospher 100RP-C18 precolumn (4 mm ⫻ 4 mm). The mobile phase was composed of a solution of acetonitrile (ACN) with 0.2% trifluoroacetic acid (TFA). The ACN/TFA ratio was 65:35 (v/v) and was maintained in buffer solution, pH 3.2, at a constant flow rate of 0.2 mL/min. A detection wavelength of 239 nm was chosen for chromatography according to absorption spectra of separated components. The isocratic system was maintained at room temperature. At those conditions, duration of the chromatographic analysis of the reference standard solution was less than 20 minutes. All assays were performed in triplicate under aseptic conditions.

Results A mass spectrometry analysis of a stock solution of PCA was inititally performed to establish a standard reference for detecting this substance. The mass spectrum showed a relation between mass and load (m/z) of ionized molecules in the PCA standard, with peaks at 149 m/z and 167 m/z immediately after preparation of the PCA solution. These

Detection of P-Chloroaniline in Chlorhexidine

1511

Basic Research—Technology CHX 6.689 70000

A – 0.2% Chlorhexidine

Absorption (mAu)

60000

50000

40000

30000

20000

10000

0 0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

Time (min)

Figure 2. HPLC analysis of a stock solution of CHX immediately after preparation. Note that CHX has a 6.7-minute retention time before eluting from the column.

1512

Barbin et al.

result confirms the mass spectrometry findings for the degradation of CHX after preparation of the mixture.

Discussion Most studies have evaluated the interaction of endodontic medications from the perspective of increased efficacy. This study evaluated the production of 2 potentially toxic factors, PCA and ROS, from either CHX alone or after CHX was mixed with Ca(OH)2. The preparation of an aqueous solution of 0.2% CHX was found to generate detectable PCA after 14 days. ROS was detected in 0.2% CHX after 7 days and in the Ca(OH)2 ⫹ 0.2% CHX mixture immediately after mixture and 7 and 14 days later. In addition, CHX could not be detected under these conditions with either mass spectrometry or HPLC analytical methods. This finding suggests that the addition of Ca(OH)2 leads to an immediate and total degradation of CHX. The methodology used to detect PCA and ROS has been used elsewhere (3, 17, 18, 20). In vitro studies have demonstrated that CHX might induce ROS production in an alkaline environment (17, 18). The association of 4.193

B– - Clorexidina 0,2 % + Ca(OH) B 0.2% Chlorhexidine + Ca(OH) 2 2

8000

Absorption (mAu) Absorção (mAu)

values were used in the subsequent experiments as indicators for the presence of PCA. We note that prior studies have reported PCA values between 127 and 129 m/z (9, 18). These differences might be due to the presence of sodium. The mass spectrometry analysis of 0.2% CHX immediately after its preparation presented peaks at 505 and 701 m/z, which confirms the high degree of purity of this sample; interestingly, PCA was not detected at this time point (Fig. 1A). The chromatographic analysis of 0.2% CHX after 7 days showed material decomposition into several sub-products with different mass spectrometry values (101, 125, 152, 170, 177, 201, 319, 336, 353, and 505 m/z) (Fig. 1B). After 14 days (Fig. 1C), there was total degradation of CHX because the reference peaks (505 and 701 m/z) were not observed. However, the mass spectrometry detected peaks of 129 m/z (a literature-based indicator for PCA [9, 18]) and 149 and 167 m/z (indicators for PCA under these experimental conditions). These findings suggest that PCA is present after 14 days of 0.2% CHX at a temperature of 36.5°C (Fig. 1C). We next evaluated the mixture of CHX with Ca(OH)2. The mass spectrometry analysis of the mixture of 0.2% CHX with Ca(OH)2 immediately after its preparation showed peaks of 169, 195, 256, and 351 m/z. The peak of 169 m/z corresponds to chlorophenyl guanide. These results explain the ability of Ca(OH)2 to separate the CHX molecule in positions containing groups NHn. The peak of 195 m/z was probably originated by production of reactive compounds (ROS), as a result of the high concentration of hydroxyl ions (alkaline environment) in the presence of CHX. Indicators for PCA were not found (Fig. 1D). At 7 days after preparation of the Ca(OH)2 ⫹ 0.2% CHX mixture, the mass spectrometry analysis demonstrated an absence of peaks at 505 and 701 m/z, which suggests a total degradation of CHX (Fig. 1E). ROS were observed, but indicators for PCA were not detected (Fig. 1E). At 14 days after preparation of the mixture (Fig. 1F), there was no evidence for the presence of either CHX (ie, an absence of peaks at 505 and 701 m/z) or PCA (no peaks at 505 or 701 m/z). The chromatographic analysis of compounds in the standard solution of CHX immediately after preparation showed that CHX eluted from the HPLC column with a retention time of 6.7 minutes (Fig. 2). However, when 0.2% CHX was mixed with Ca(OH)2, no CHX was detectable when loaded immediately onto the HPLC column (Fig. 3). This

6000

2.051 3.574 5.067

2.325

4000

5.707

8.289

2000

15.928 0

0

2

4

6

8

10

12

14

16

18

Tempo (min) Time (min)

Figure 3. HPLC analysis immediately after a mixture of Ca(OH)2 with 0.2% CHX. No CHX is detected under these conditions (ie, no peak is observed at the 6.7-minute retention time).

JOE — Volume 34, Number 12, December 2008

Basic Research—Technology Ca(OH)2 with CHX might produce more ROS, which is another factor to explain the antimicrobial activity of these substances (17). The implications and role of PCA and ROS on human health must be emphasized. According to the IARC (21), PCA is used as an intermediate in the manufacture of dyes, pigments, agricultural chemicals, and pharmaceuticals. PCA is a persistent environmental degradation product of some herbicides and fungicides, presents potential long-term toxicity and carcinogenicity in male rats and male mice, and can cause methemoglobinemia. In addition, PCA induces DNA damage in bacteria. However, the results for gene mutation are still inconclusive. Gene mutation, but not mitotic recombination, has been induced in fungi. Gene mutation, sister chromatid exchange, and chromosomal aberrations have been induced in cultured mammalian cells, but conflicting data were obtained for cell transformation. There is inadequate evidence in humans for the carcinogenicity of PCA, but there is sufficient evidence in experimental animals for the carcinogenicity of PCA. Thus, PCA is possibly carcinogenic to humans (IARC Group 2B) (16, 21). Kacmár et al. (22) evaluated the toxic and immunotoxic effects of PCA metabolite of herbicide monolinuron in peripheral blood leukocyte suspensions of 5 sheep by using a migration-inhibition test. The toxic effects of PCA were recorded at concentrations from 1.0 – 0.1 mg/mL–1, and the immunotoxic effects were recorded at concentrations from 0.01– 0.001 mg/mL–1. The toxic effects were demonstrated by total inhibition of leukocyte migration. The immunotoxic effects, determined as mitogenic activation of leukocytes by phytohemagglutinin, concanavalin A, and LPS, were detected at 10-fold to 100-fold lower concentrations of PCA than those that resulted in toxic effects. Although the present study evaluated the production of PCA, future studies should determine the concentrations of PCA generated from 0.1%–2% concentrations of CHX. ROS are generally small, short-lived, and highly reactive molecules formed by incomplete one-electron reduction of oxygen. These species (superoxide radical, hydrogen peroxide, singlet oxygen, and hydroxyl radical) are well-known to be cytotoxic and have been implicated in the etiology of a wide array of human diseases. They are constantly formed in the human body and have been shown to inactivate bacteria and proteins. They contribute to the microbicidal activity of phagocytes, regulation of signal transduction and gene expression, and cause oxidative damage to nucleic acids, proteins, and lipids. Generally, harmful effects of ROS on the cell are most often damage of DNA, oxidations of polydesaturated fatty acids in lipids, oxidations of amino acids in proteins, and oxidative inactivation of specific enzymes by oxidation of co-factors (23, 24). Waris and Ahsan (19) have reported that elevated levels of ROS and down-regulation of ROS scavengers and antioxidant enzymes are associated with various human diseases including different types of cancer. ROS are also implicated in diabetes and neurodegenerative diseases and influence cellular processes such as proliferation, apoptosis, and senescence, which are involved in cancer development. Understanding the role of ROS as key mediators in signaling cascades might provide various opportunities for pharmacologic intervention. Basrani et al. (18) have recently demonstrated that NaOCl mixed with CHX formed PCA, and the amount of PCA directly increased with increasing concentrations of NaOCl. Although the insoluble precipitate raises questions about the potential leaching of PCA, their findings might be clinically relevant because PCA has been shown to be toxic. Yeung et al. (17) evaluated the scavenging and generation of ROS by CHX in the presence or absence of saturated Ca(OH)2 solutions. CHX exhibited both antioxidant and pro-oxidant properties under different conditions. CHX induced ROS production including H2O2 and superoxide radicals in 0.1 N NaOH (pH 12.7) or Ca(OH)2 (pH 12.5) solutions. Combined use of CHX and Ca(OH)2 in the root canal might generate excessive ROS, which might potentially kill various root canal pathogens. Collec-

JOE — Volume 34, Number 12, December 2008

tively, recent studies (17, 18) have demonstrated that 2 different medicaments associated with CHX, namely NaOCl and Ca(OH)2, were able to induce PCA and ROS. It is important to consider that the production of these substances occurred in an alkaline environment. In the present study when CHX was combined with Ca(OH)2, we detected ROS in all time points. Yeung et al. (17) believe that ROS might destroy root canal microorganisms, and this is consistent with the demonstration that the mixture of CHX and Ca(OH)2 has a greater antimicrobial action than CHX alone (6). In view of these sub-products found in CHX and in CHX associated with Ca(OH)2, it is interesting to analyze the potential toxicity of CHX. Grassia et al. (25) have investigated whether CHX is able to cause, in terms of DNA damage, alterations in leukocytes, liver, kidney, and urinary bladder by the single cell gel (comet) assay. The results indicated that leukocytes and kidney cells are potential targets for primary DNA damage after oral exposure to CHX. Faria et al. (26) have reported that CHX injected in the subplantar space of the hind paw of mice induced severe toxic effects, as evidenced by necrotic changes in the epidermis, dermis, and subcutaneous tissue in association with reactive inflammatory response, particularly at higher concentrations. In addition, in cultured fibroblasts, CHX induced apoptosis at lower concentrations and necrosis at higher concentrations and increased expression of heatshock protein 70, an indicator of cellular stress. The residual effect of CHX has been demonstrated (27), mainly when this substance is applied for a period of 7 days or longer (28). However, the present study demonstrated the immediate loss of detectable CHX when added to a solution of Ca(OH)2. It should be stressed that experimental methods, biologic indicators, concentration, and exposure time can influence the magnitude of the measured antimicrobial effects of various substances. The minimum inhibitory concentration (MIC) of an antimicrobial agent being tested must be known. In a previous study of our research group, CHX has been shown to have MIC equal to 0.000002% for Staphylococcus aureus, 0.02% for E. faecalis, Bacillus subtilis, Candida albicans, and the mixed culture and 0.002% for Pseudomonas aeruginosa (29). This measurement must be taken into account to choose an antimicrobial agent at a concentration that can be effective against the target microorganisms, although without causing systemic damage to the patient. In direct contact, CHX and Ca(OH)2 are capable to inactivate bacteria, but similar efficacy was not observed on root canal biofilm or in infected dentinal tubules (30). In view of the cytotoxic nature of CHX, the risks and benefits of this substance must be weighed against those of other substances with antimicrobial properties, such as sodium hypochlorite and Ca(OH)2. Further research is required to evaluate the CHX concentration that is safe for human use and contemplates most ideal properties.

References 1. Paquette L, Legner M, Fillery ED, Friedman S. Antibacterial efficacy of chlorhexidine gluconate intracanal medication in vivo. J Endod 2007;33:788 –95. 2. Bui TB, Baumgartner JC, Mitchell JC. Evaluation of the interaction between sodium hypochlorite and chlorhexidine gluconate and its effect on root dentin. J Endod 2008;34:181–5. 3. Wang CS, Arnold RR, Trope M, Teixeira FB. Clinical efficiency of 2% chlorhexidine gel in reducing intracanal bacteria. J Endod 2007;33:1283–9. 4. Kishen A, Sum CP, Mathew S, Lim CT. Influence of irrigation regimens on the adherence of Enterococcus faecalis to root canal dentin. J Endod 2008;34:850 – 4. 5. Estrela C, Pimenta FC, Ito IY, Bammann LL. Antimicrobial evaluation of calcium hydroxide in infected dentinal tubules. J Endod 1999;26:416 – 8. 6. Siqueira JF Jr, Paiva SS, Roças IN. Reduction in the cultivable bacterial populations in infected root canals by a chlorhexidine-based antimicrobial protocol. J Endod 2007;33:541–7.

Detection of P-Chloroaniline in Chlorhexidine

1513

Basic Research—Technology 7. Lee Y, Han SH, Hong SH, Lee JK, Ji H, Kum KY. Antimicrobial efficacy of a polymeric chlorhexidine release device using in vitro model of Enterococcus faecalis dentinal tubule infection. J Endod 2008;34:855– 8. 8. Kontakiotis EG, Tsatsoulis IN, Papanakou SI, Tzanetakis GN. Effect of 2% chlorhexidine gel mixed with calcium hydroxide as an intracanal medication on sealing ability of permanent root canal filling: a 6-month follow-up. J Endod 2008;34:866 –70. 9. Havlíyková L, Matysová L, Nováková L, Hájková R, Solich P. HPLC determination of chlorhexidine gluconate and p-chloroaniline in topical ointment. J Pharmaceutic Biomedic Anal 2007;43:1169 –73. 10. Denton GW. Chlorhexidine. In: Block SS, ed. Disinfection, sterilization and preservation. 4th ed. Philadelphia: Lea & Febiger, 1991:274 – 89. 11. Ohara P, Torabinejad M, Kettering JD. Antibacterial effects of various endodontic irrigants on selected anaerobic bacteria. Endod Dent Traumatol 1993;9:95–100. 12. Buck RA, Cai J, Eleazer PD, Staat RH, Hurst HE. Detoxification of endotoxin by endodontic irrigants and calcium hydroxide. J Endod 2000;27:325–7. 13. Safavi KE, Nichols FC. Effect of calcium hydroxide on bacterial lipopolysaccharide. J Endod 1993;19:76 – 8. 14. Estrela C, Bammann LL, Pimenta FC, Pécora JD. Control of microorganism in vitro by calcium hydroxide pastes. Int Endod J 2001;34:416 – 8. 15. Usui K, Hishinuma T, Yamaguchi H, Tachiiri N, Goto J. Determination of chlorhexidine (CHD) and nonylphenolethoxylates (NPEOn) using LC-ESI-MS method and application to hemolyzed blood. J Chromatogr B 2006;831:105–9. 16. World Health Organization. International Agency for Research on cancer: IARC monography on the evaluation of carcinogenic risks to human. Lyon, France, 2006; 86:1–25. 17. Yeung SY, Huang CS, Chan CP, et al. Antioxidant and pro-oxidant properties of chlorhexidine and its interaction with calcium hydroxide solutions. Int Endod J 2007;40:837– 44. 18. Basrani BR, Manek S, Sodhi RNS, Fillery E, Manzur A. Interaction between sodium hypochlorite andchlorhexidine gluconate. J Endod 2007;33:966 –9.

1514

Barbin et al.

19. Waris G, Ahsan H. Reactive oxygen species: role in the development of cancer and various chronic conditions. J Carcinogen 2006;5:14 –22. 20. Ciarlone CE, Gangarorosa LP, Fong BC. Detection of p-chloraniline in chlorhexidine solutions using thin-layer chromatography. J Dent Res 1976;55:918. 21. World Health Organization. International Agency for Research on cancer: IARC p-chloroaniline— occupational exposures of hairdressers and barbers and personal use of hair colourants; some hair dyes, cosmetic colourants, industrial dyestuffs and aromatic amines. Lyon, France, 1997;57:1– 43. 22. Kacmár P, Pistl J, Mikula I. The effect of p-chloroaniline on leukocytes of sheep peripheral blood under the migration-inhibition test conditions. Immunopharmacol Immunotoxicol 1995;17:577– 84. 23. Scandalios JG. The rise of ROS. Trends Biochem Scien 2002;27:483– 6. 24. Chapple IL. Reactive oxygen species and antioxidants in inflammatory diseases. J Clin Periodontol 1997;24:287–96. 25. Grassia TF, Camargoa EA, Salvadoria DMF, Marquesa MEA, Ribeiro DA. DNA damage in multiple organs after exposure to chlorhexidine in Wistar rats Int J Hyg Environ Health 2007;210:163–7. 26. Faria G, Celes MRN, Rossi A, Silva LAB, Silva JS, Rossi MA. Evaluation of chlorhexidine toxicity injected in the paw of mice and added to cultured L929 fibroblasts. J Endod 2007;33:715–22. 27. White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod 1997;23:229 –31. 28. Komorowski R, Grad H, Wu XY, Friedman S. Antimicrobial substantivity of chlorhexidine-treated bovine root dentin. J Endod 2000;26:315–7. 29. Estrela CRA, Estrela C, Reis, Bammann LL, Pecora JD. Control of microorganisms in vitro by endodontic irrigants. Braz Dent J 2003;14:187–92. 30. Estrela C, Estrela CRA, Decurcio DA, Hollanda ACB, Silva JA. Antimicrobial efficacy of ozonated water, gaseous ozone, sodium hypochlorite and chlorhexidine in infected human root canals. Int Endod J 2007;40:85–93.

JOE — Volume 34, Number 12, December 2008