Original Article. Toxicity of Glandularia selloi (Spreng.) Tronc. leave extract by MTT and neutral red assays: influence of the test medium procedure

Luciana Rizzieri Figueiró; Luana Christine Comerlato; Marcia Vignoli Da Silva; José Ângelo Silveira Zuanazzi; Gilsane Lino Von Poser; Ana Luiza Ziulkoski

doi:10.1515/intox-2016-0004

Abstract

Cytotoxicity assays using cell cultures may be an alternative to assess biological toxicity of plant extracts with potential phytotherapeutic properties. This study compared three methods to prepare culture media for the exposure of Vero cells to plant extracts. Leaves of Glandularia selloi (Spreng.) Tronc. were used to prepare culture medium with aqueous extract, extract in culture medium and methanol extract. Toxicity was assessed using the MTT and neutral red (NR) assays. In general, alterations in the cellular functions were found in all extracts and assays. Cytotoxic effect occurred at lower doses in aqueous extract and the range of effect of the methanol extract was small. The procedure of preparing the test medium has an effect on the outcome of the assay. Cytotoxicity of plant extract can be assessed by MTT and NR assays. Aqueous extract added to the culture medium presented the best profile to assess cytotoxicity.

References

Agencia Nacional de Vigilância Sanitária. (2010). Resolução-RDC nº 14, de 31 de março de 2010. Brasília, Brasil.
Search in Google Scholar Back to article
Agencia Nacional de Vigilância Sanitária. (2004). Resolução nº 90, de 16 de março de 2004. Brasília, Brasil.
Search in Google Scholar Back to article
Akerreta S, Cavero RY, Calvo MI. (2007). First comprehesive contribution to medical ethnobotany of wester pyrenes. J Ethnobiol Ethnomed, 3 26.10.1186/1746-4269-3-26
Search in Google Scholar Back to article
Arechabala B, Coiffard C, Rivalland P, Coiffard LJ, de Roeck-Holtzhauer Y. (1999). Comparison of cytotoxicity of various surfactants tested on normal human fi broblast cultures using the neutral red test, MTT assay and LDH release. J Appl Toxicol, 19, 163-65.10.1002/(SICI)1099-1263(199905/06)19:3<163::AID-JAT561>3.0.CO;2-H
Search in Google Scholar Back to article
Bent S. (2008). Herbal medicine in the United States: review of effi cacy, safety, and regulation: grand rounds at University of California, San Francisco Medical Center. J Gen Intern Med, 23, 854-59.10.1007/s11606-008-0632-y
Search in Google Scholar Back to article
Berridge MV, Herst PM, Tan AS. (2005). Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev, 11, 127-52.10.1016/S1387-2656(05)11004-7
Search in Google Scholar Back to article
Borenfreund E, Puerner JA. (1985). Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lett, 24, 119-24.10.1016/0378-4274(85)90046-3
Search in Google Scholar Back to article
Borenfreund E, Babich H, Martin-Alguacil N. (1988). Comparisons of two in vitro cytotoxicity assays-the neutral red (nr) and tetrazolium mtt tests. Toxicol In Vitro, 2, 1-6.10.1016/0887-2333(88)90030-6
Search in Google Scholar Back to article
Bruggisser R, von Daeniken K, Jundt G, Schaffner W, Tullberg-Reinert H. (2002). Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay. Planta Medica, 68, 445-8.10.1055/s-2002-32073
Search in Google Scholar Back to article
Bunel V, Ouedraogo M, Nguyen AT, Stévigny C, Duez P. (2014). Methods applied to the in vitro primary toxicology testing of natural products: State of the art, strengths, and limits. Planta Med [Epub in print]10.1055/s-0033-1360273
Search in Google Scholar Back to article
Butler M. (2004). Animal cell culture and technology. 2a Ed. London, England: Taylor & Francis.10.4324/9780203427835
Search in Google Scholar Back to article
Česen MH, Pegan K, Spes A, Turk B. (2012). Lysosomal pathways to cell death and their therapeutic applications. Exp Cell Res, 318, 1245-51.10.1016/j.yexcr.2012.03.005
Search in Google Scholar Back to article
Fotakis G, Timbrell JA. (2006). In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol Lett, 160, 171-7.10.1016/j.toxlet.2005.07.001
Search in Google Scholar Back to article
Freshney RI. (2005). Culture of animal cells: a manual of basic technique. 5a. Ed. New Jersey, U.S.: Wiley-Blackwell.
Search in Google Scholar Back to article
Khatibisepehr S, Huang B, Ibrahim F, Xing JZ, Roa W. (2011). Data-based modeling and prediction of cytotoxicity induced by contaminants in water resources. Comput Biol Chem, 35, 69-80.10.1016/j.compbiolchem.2011.02.001
Search in Google Scholar Back to article
Kim H, Yoon SC, Lee TY, Jeong D. (2009). Discriminative cytotoxicity assessment based on various cellular damages. Toxicol Lett, 184, 13-7.10.1016/j.toxlet.2008.10.006
Search in Google Scholar Back to article
Lupu AR, Popescu T. (2013). The noncellular reduction of MTT tetrazolium salt by TiO2 nanoparticles and its implications for cytotoxicity assays. Toxicol In Vitro, 27, 1445-50.10.1016/j.tiv.2013.03.006
Search in Google Scholar Back to article
Malich G, Markovic B, Winder C. (1997). The sensitivity and specifi city of the MTS tetrazolium assay for detecting the in vitro cytotoxicity of 20 chemicals using human cell lines. Toxicology, 124, 179-92.10.1016/S0300-483X(97)00151-0
Search in Google Scholar Back to article
Mosmann T. (1983). Rapid colorimetric assays for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 65, 55-63.10.1016/0022-1759(83)90303-4
Search in Google Scholar Back to article
National Institutes of Health. (2006). Background Review Document (BRD): Validation of Neutral Red Uptake Test Methods NIH/In Vitro Cytotoxicity Test Methods for Estimating Acute Oral Systemic Toxicity. Publication No. 07-4518, November 2006. Available at: http://ntp.niehs.nih.gov/iccvam/docs/acutetox_docs/BRD_TMER/BRDvol1_Nov2006.pdf Accessed on 16 April 2014.
Search in Google Scholar Back to article
National Institutes of Health. (2001). Report of the International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity. NIH Publication No. 01-4499. National Institute for Environmental Health Sciences, Research Triangle Park, NC. Available at: http://www.epa.gov/hpv/pubs/general/nih2001a.pdf Accessed on 16 April 2014.
Search in Google Scholar Back to article
Peng L, Wang B, Ren P. (2005). Reduction of MTT by fl avonoids in the absence of cells. Colloids Surf B Biointerfaces, 45, 108-11.10.1016/j.colsurfb.2005.07.01416150580
Search in Google Scholar Back to article
Piersma AH. (2006). Alternative Methods for Developmental Toxicity Testing. Basic Clin Pharmacol Toxicol, 98, 427-31.10.1111/j.1742-7843.2006.pto_373.x16635099
Search in Google Scholar Back to article
Repetto G, del Peso A, Zurita JL. (2008). Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc, 3, 1125-31.10.1038/nprot.2008.7518600217
Search in Google Scholar Back to article
Rogero SO, Lugão AB, Ikeda TI, Cruz AS. (2003). Teste in vitro de cytotoxicidade: estudo comparativo entre duas metodologias. Mat Res, 6, 317-20.10.1590/S1516-14392003000300003
Search in Google Scholar Back to article
Shoemaker M, Cohen I, Campbell M. (2004). Reduction of MTT by aqueous herbal extracts in the absence of cells. J Ethnopharmacol, 93, 381-4.10.1016/j.jep.2004.04.011
Search in Google Scholar Back to article
Suares Rocha P, Azab E, Schmidt B, Storch V, Hollert H, Braunbeck T. (2010). Changes in toxicity and dioxin-like activity of sediments from the Tiete River (Sao Paulo, Brazil). Ecotoxicol Environ Saf, 73, 550-8.10.1016/j.ecoenv.2009.12.017
Search in Google Scholar Back to article
Thode VA, Mentz LA. (2010). The genus Glandularia J.F. Gmel. (Verbenaceae) in Rio Grande do Sul, Brazil. Acta Bot Bras, 24. [On line]. Available at: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0102-33062010000200023. Accessed on 13 Aug 2014.
Search in Google Scholar Back to article
Triglia D, Sherard Braa S, Yonan C, Naughton GK. (1991). Cytotoxicity testing using neutral red and mtt assays on a three- dimensional human skin substrate. Toxicol In Vitro, 5, 573-8.10.1016/0887-2333(91)90096-V
Search in Google Scholar Back to article
Trintinaglia L, Bianchi E, Basso da Silva L, Nascimento CA, Spilki FR, Ziulkoski AL. 2015.Cytotoxicity assays as tools to assess water quality in the Sinos river basin. Braz J Biol, 75, 2 suppl, 75-80.
Search in Google Scholar Back to article
U.S. Congress Office of Technology Assessment. (1986). Alternatives to Animal Use in Research, Testing, and Education. Washington, DC, U.S: Government Printing Offi ce.
Search in Google Scholar Back to article
Vanden Berghe T, Grootjans S, Goossens V, Dondelinger Y, Krysko DV, Takahashi N, Vandenabeele P. (2013). Determination of apoptotic and necrotic cell death in vitro and in vivo. Methods, 61, 117-29.10.1016/j.ymeth.2013.02.011
Search in Google Scholar Back to article
Von Poser GL, Toff oli ME, Sobral M, Henriques AT. (1997). Iridoid glucosides substitution patterns in Verbenaceae and their taxonomic implication. Plant Syst Evol, 205, 265-87.10.1007/BF01464409
Search in Google Scholar Back to article
World Health Organization. (2005). National policy on traditional medicine and regulation of herbal medicines. Geneva, Switzerland: WHO Press.
Search in Google Scholar Back to article
World Health Organization. (2013). WHO traditional medicine strategy: 2014-2023. Geneva, Switzerland: WHO Press.
Search in Google Scholar Back to article
Zeni ALB, Zombowski ADE, Dal-Cim T, Maraschin M, Rodrigues ALS, Tasca CI. (2011). Antidepressant-like and neuroprotective eff ects of Aloysia gratissima: Investigation of involvement of L-arginina-nitric oxide-cyclic guanoside monophosphate pathway. J Ethnopharmacol, 37, 864-74.10.1016/j.jep.2011.07.009
Search in Google Scholar Back to article
Zhang SZ, Lipsky MM, Trump BF, Hsu IC. (1990). Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes. Cell Biology and Toxicology, 6, 219-34.10.1007/BF00249595
Search in Google Scholar Back to article
Zucco F, De Angelis I, Testai E, Stammati A. (2004). Toxicology investigations with cell culture systems: 20 years after. Toxicol In Vitro, 18, 153-63.10.1016/S0887-2333(03)00147-4
Search in Google Scholar Back to article

Original Article. Toxicity of Glandularia selloi (Spreng.) Tronc. leave extract by MTT and neutral red assays: influence of the test medium procedure

Abstract

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