Skip to main content
Top
Published in: Clinical Oral Investigations 5/2013

01-06-2013 | Original Article

Assessment of genotoxic effect of maleic acid and EDTA: a comparative in vitro experimental study

Authors: Nidambur Vasudev Ballal, Bhuvanagiri Nageshwar Rao, Kundabala Mala, Kadengodlu Seetharama Bhat, Bola Sadashiva Satish Rao

Published in: Clinical Oral Investigations | Issue 5/2013

Login to get access

Abstract

Objectives

This study aims to evaluate and compare the genotoxic and apoptotic effect of aqueous solutions of ethylenediaminetetraacetic acid (EDTA) with that of maleic acid (MA) using Chinese hamster lung fibroblast (V79) cells growing in vitro.

Materials and methods

Exponentially growing V79 cells were treated with various concentrations of EDTA or MA alone for 30 min, and genotoxic effect was analyzed by micronucleus as well as comet assays and the type of cell death by apoptotic cell measurements using microscopic and flow cytometric methods. For all the experiments, H2O2 was used as a positive control.

Results

Treatment of V79 cells with H2O2 resulted in significantly (P < 0.001) increased micronuclei and levels of DNA damage, whereas, EDTA/MA alone treated cells did not show significant increase of MN frequencies and comet parameters even at their higher concentrations when compared with that of untreated control. V79 cells treated with EDTA/MA for 30 min showed a nonsignificant increase in the percentage of apoptotic and necrotic cells at their lower concentrations (0.025 and 0.05 % for EDTA and MA, respectively). However, at higher concentrations, i.e., >IC50 (0.1 and 0.5 %) for EDTA and MA resulted in increased number of apoptotic and necrotic cells when compared with the untreated group.

Conclusions

This study clearly demonstrates that MA and EDTA are not potentially genotoxic agents and MA induced lesser apoptotic/necrotic death than that of EDTA at their clinically relevant doses.

Clinical relevance

MA may have a better clinical acceptability with comparable smear layer removal ability. Hence, the results presented here might be an additional supporting evidence for the use of MA in endodontic practice.
Literature
1.
go back to reference Haapasalo M, Endal U, Zandi H, Coil JM (2005) Eradication of endodontic infection by instrumentation and irrigation solutions. Endod Top 10:77–102CrossRef Haapasalo M, Endal U, Zandi H, Coil JM (2005) Eradication of endodontic infection by instrumentation and irrigation solutions. Endod Top 10:77–102CrossRef
2.
go back to reference McComb D, Smith DC (1975) A preliminary scanning electron microscopic study of root canals after endodontic procedures. J Endod 1:238–242PubMedCrossRef McComb D, Smith DC (1975) A preliminary scanning electron microscopic study of root canals after endodontic procedures. J Endod 1:238–242PubMedCrossRef
3.
go back to reference Khedmat S, Shokouhinejad N (2008) Comparison of the efficacy of three chelating agents in smear layer removal. J Endod 34:599–602PubMedCrossRef Khedmat S, Shokouhinejad N (2008) Comparison of the efficacy of three chelating agents in smear layer removal. J Endod 34:599–602PubMedCrossRef
4.
go back to reference Shahravan A, Haghdoost AA, Adl A, Rahimi H, Shadifar F (2007) Effect of smear layer on sealing ability of canal obturation: a systematic review and meta-analysis. J Endod 33:96–105PubMedCrossRef Shahravan A, Haghdoost AA, Adl A, Rahimi H, Shadifar F (2007) Effect of smear layer on sealing ability of canal obturation: a systematic review and meta-analysis. J Endod 33:96–105PubMedCrossRef
5.
go back to reference Ciucchi B, Khettabi M, Holz J (1989) The effectiveness of different endodontic irrigation procedures on the removal of the smear layer: a scanning electron microscopic study. Int Endod J 22:21–28PubMedCrossRef Ciucchi B, Khettabi M, Holz J (1989) The effectiveness of different endodontic irrigation procedures on the removal of the smear layer: a scanning electron microscopic study. Int Endod J 22:21–28PubMedCrossRef
6.
go back to reference Peters OA, Barbakow F (2000) Effects of irrigation on debris and smear layer on canal walls prepared by two rotary techniques: a scanning electron microscopic study. J Endod 26:6–10PubMedCrossRef Peters OA, Barbakow F (2000) Effects of irrigation on debris and smear layer on canal walls prepared by two rotary techniques: a scanning electron microscopic study. J Endod 26:6–10PubMedCrossRef
7.
go back to reference Silveria N, Tavares T, Soares I (1994) Potencial irretativo de solucoes a base de EDTA. Revista da Associação Paulista de Cirurgiões Dentistas 48:1489–1493 Silveria N, Tavares T, Soares I (1994) Potencial irretativo de solucoes a base de EDTA. Revista da Associação Paulista de Cirurgiões Dentistas 48:1489–1493
8.
go back to reference Segura JJ, Calvo JR, Guerrero JM, Jimenez-Planas A, Sampedro C, Llamas R (1997) EDTA inhibits in vitro substrate adherence capacity of macrophages: endodontic implications. J Endod 23:205–208PubMedCrossRef Segura JJ, Calvo JR, Guerrero JM, Jimenez-Planas A, Sampedro C, Llamas R (1997) EDTA inhibits in vitro substrate adherence capacity of macrophages: endodontic implications. J Endod 23:205–208PubMedCrossRef
9.
go back to reference Koulaouzidou EA, Margelos J, Beltes P, Kortsaris AH (1999) Cytotoxic effects of different concentrations of neutral and alkaline EDTA solutions used as root canal irrigants. J Endod 25:21–23PubMed Koulaouzidou EA, Margelos J, Beltes P, Kortsaris AH (1999) Cytotoxic effects of different concentrations of neutral and alkaline EDTA solutions used as root canal irrigants. J Endod 25:21–23PubMed
10.
go back to reference Serper A, Calt S, Dogan AL, Guc D, Ozcelik B, Kuraner T (2001) Comparison of the cytotoxic effects and smear layer removing capacity of oxidative potential water, NaOCl and EDTA. J Oral Sci 43:233–238PubMedCrossRef Serper A, Calt S, Dogan AL, Guc D, Ozcelik B, Kuraner T (2001) Comparison of the cytotoxic effects and smear layer removing capacity of oxidative potential water, NaOCl and EDTA. J Oral Sci 43:233–238PubMedCrossRef
11.
go back to reference Wieczkowski G Jr, Yu XY, Davis EL, Joynt RB (1992) Microleakage in various dentin bonding agent/composite resin systems. Oper Dent 5:62–67PubMed Wieczkowski G Jr, Yu XY, Davis EL, Joynt RB (1992) Microleakage in various dentin bonding agent/composite resin systems. Oper Dent 5:62–67PubMed
12.
go back to reference Ballal NV, Kandian S, Mala K, Bhat KS, Acharya S (2009) Comparison of the efficacy of maleic acid and ethylenediaminetetraacetic acid in smear layer removal from instrumented human root canal: a scanning electron microscopic study. J Endod 35:1573–1576PubMedCrossRef Ballal NV, Kandian S, Mala K, Bhat KS, Acharya S (2009) Comparison of the efficacy of maleic acid and ethylenediaminetetraacetic acid in smear layer removal from instrumented human root canal: a scanning electron microscopic study. J Endod 35:1573–1576PubMedCrossRef
13.
go back to reference Goracci G, Mori G, Bazzucchi M (1995) Marginal seal and biocompatibility of a fourth-generation bonding agent. Dent Mater 11:343–347PubMedCrossRef Goracci G, Mori G, Bazzucchi M (1995) Marginal seal and biocompatibility of a fourth-generation bonding agent. Dent Mater 11:343–347PubMedCrossRef
14.
go back to reference Ballal NV, Kundabala M, Bhat S, Rao N, Rao BS (2009) A comparative in vitro evaluation of cytotoxic effects of EDTA and maleic acid: root canal irrigants. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108:633–638PubMedCrossRef Ballal NV, Kundabala M, Bhat S, Rao N, Rao BS (2009) A comparative in vitro evaluation of cytotoxic effects of EDTA and maleic acid: root canal irrigants. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108:633–638PubMedCrossRef
15.
16.
go back to reference Rao BS, Shanbhoge R, Upadhya D, Jagetia GC, Adiga SK, Kumar P (2006) Antioxidant, anticlastogenic and radioprotective effect of Coleus aromaticus on Chinese hamster fibroblast cells (V79) exposed to gamma radiation. Mutagenesis 21:237–242PubMedCrossRef Rao BS, Shanbhoge R, Upadhya D, Jagetia GC, Adiga SK, Kumar P (2006) Antioxidant, anticlastogenic and radioprotective effect of Coleus aromaticus on Chinese hamster fibroblast cells (V79) exposed to gamma radiation. Mutagenesis 21:237–242PubMedCrossRef
17.
go back to reference Fenech M, Chang WP, Kirsch-Volders M, Holland N, Bonassi S, Zeiger E (2003) HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures. Mutat Res 534:65–75PubMedCrossRef Fenech M, Chang WP, Kirsch-Volders M, Holland N, Bonassi S, Zeiger E (2003) HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures. Mutat Res 534:65–75PubMedCrossRef
18.
go back to reference Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191PubMedCrossRef Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191PubMedCrossRef
19.
go back to reference Collins A, Dusinska M, Franklin M, Somorovska M, Petrovska H, Duthie S et al (1997) Comet assay in human biomonitoring studies: reliability, validation, and applications. Environ Mol Mutagen 30:139–146PubMedCrossRef Collins A, Dusinska M, Franklin M, Somorovska M, Petrovska H, Duthie S et al (1997) Comet assay in human biomonitoring studies: reliability, validation, and applications. Environ Mol Mutagen 30:139–146PubMedCrossRef
20.
go back to reference Renvoize C, Biola A, Pallardy M, Breard J (1998) Apoptosis: identification of dying cells. Cell Biol Toxicol 14:111–120PubMedCrossRef Renvoize C, Biola A, Pallardy M, Breard J (1998) Apoptosis: identification of dying cells. Cell Biol Toxicol 14:111–120PubMedCrossRef
21.
go back to reference Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279PubMedCrossRef Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279PubMedCrossRef
22.
go back to reference Zeiger E (1998) Identification of rodent carcinogens and noncarcinogens using genetic toxicity tests: premises, promises, and performance. Regul Toxicol Pharmacol 28:85–95PubMedCrossRef Zeiger E (1998) Identification of rodent carcinogens and noncarcinogens using genetic toxicity tests: premises, promises, and performance. Regul Toxicol Pharmacol 28:85–95PubMedCrossRef
23.
go back to reference Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H et al (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221PubMedCrossRef Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H et al (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221PubMedCrossRef
24.
go back to reference Holz O, Jorres R, Kastner A, Magnussen H (1995) Differences in basal and induced DNA single-strand breaks between human peripheral monocytes and lymphocytes. Mutat Res 332:55–62PubMedCrossRef Holz O, Jorres R, Kastner A, Magnussen H (1995) Differences in basal and induced DNA single-strand breaks between human peripheral monocytes and lymphocytes. Mutat Res 332:55–62PubMedCrossRef
25.
go back to reference Noroozi M, Angerson WJ, Lean ME (1998) Effects of flavonoids and vitamin C on oxidative DNA damage to human lymphocytes. Am J Clin Nutr 67:1210–1218PubMed Noroozi M, Angerson WJ, Lean ME (1998) Effects of flavonoids and vitamin C on oxidative DNA damage to human lymphocytes. Am J Clin Nutr 67:1210–1218PubMed
27.
go back to reference Kirsch-Volders M, Elhajouji A, Cundari E, Van Hummelen P (1997) The in vitro micronucleus test: a multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction. Mutat Res 392:19–30PubMedCrossRef Kirsch-Volders M, Elhajouji A, Cundari E, Van Hummelen P (1997) The in vitro micronucleus test: a multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction. Mutat Res 392:19–30PubMedCrossRef
28.
go back to reference Burgaz S, Iscan A, Buyukbingol ZK, Bozkurt A, Karakaya AE (1995) Evaluation of micronuclei in exfoliated urothelial cells and urinary thioether excretion of smokers. Mutat Res 335:163–169PubMedCrossRef Burgaz S, Iscan A, Buyukbingol ZK, Bozkurt A, Karakaya AE (1995) Evaluation of micronuclei in exfoliated urothelial cells and urinary thioether excretion of smokers. Mutat Res 335:163–169PubMedCrossRef
29.
go back to reference Eguchi Y, Shimizu S, Tsujimoto Y (1997) Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res 57:1835–1840PubMed Eguchi Y, Shimizu S, Tsujimoto Y (1997) Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res 57:1835–1840PubMed
30.
go back to reference Leist M, Single B, Castoldi AF, Kuhnle S, Nicotera P (1997) Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med 185:1481–1486PubMedCrossRef Leist M, Single B, Castoldi AF, Kuhnle S, Nicotera P (1997) Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med 185:1481–1486PubMedCrossRef
31.
go back to reference Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148:2207–2216PubMed Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148:2207–2216PubMed
32.
go back to reference Wyllie AH, Kerr JF, Currie AR (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68:251–306PubMedCrossRef Wyllie AH, Kerr JF, Currie AR (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68:251–306PubMedCrossRef
33.
go back to reference Masillamoni CR, Kettering JD, Torabinejad M (1981) The biocompatibility of some root canal medicaments and irrigants. Int Endod J 14:115–120PubMedCrossRef Masillamoni CR, Kettering JD, Torabinejad M (1981) The biocompatibility of some root canal medicaments and irrigants. Int Endod J 14:115–120PubMedCrossRef
34.
go back to reference Patterson SS (1963) In vivo and in vitro studies of the effect of the disodium slat of ethylenediamine tetra-acetate on human dentine and its endodontic implications. Oral Surg Oral Med Oral Pathol 16:83–103PubMedCrossRef Patterson SS (1963) In vivo and in vitro studies of the effect of the disodium slat of ethylenediamine tetra-acetate on human dentine and its endodontic implications. Oral Surg Oral Med Oral Pathol 16:83–103PubMedCrossRef
Metadata
Title
Assessment of genotoxic effect of maleic acid and EDTA: a comparative in vitro experimental study
Authors
Nidambur Vasudev Ballal
Bhuvanagiri Nageshwar Rao
Kundabala Mala
Kadengodlu Seetharama Bhat
Bola Sadashiva Satish Rao
Publication date
01-06-2013
Publisher
Springer-Verlag
Published in
Clinical Oral Investigations / Issue 5/2013
Print ISSN: 1432-6981
Electronic ISSN: 1436-3771
DOI
https://doi.org/10.1007/s00784-012-0818-x

Other articles of this Issue 5/2013

Clinical Oral Investigations 5/2013 Go to the issue