Development of a Yeast Inhibitive Assay for Anionic Heavy Metals Using the 1-(4,5-Dimethylthiazol-2-Yl)-3,5-Diphenyltetrazolium Bromide (MTT) Assay
DOI:
https://doi.org/10.54987/bessm.v6i1.708Keywords:
Saccharomyces cerevisiae, MTT assay, Biomonitoring, Bioassay, Anionic heavy metalsAbstract
Currently, heavy metals pollution is significantly becoming a global concern as it causes severe toxicity towards human health and the environmental condition. Intensive efforts to develop a highly sensitive, rapid, and simple toxicity assay to assess the toxic effects of heavy metals in aquatic bodies have been done. An emerging tool to solve this matter is by using microorganisms and one of them is yeast. In this study, a rapid, simple, and cost-efficient toxicity assay by using the Baker’s yeast (Saccharomyces cerevisiae) respiration assay with a tetrazolium dye (MTT) is developed as potential environmental biomonitoring tool. To achieve high sensitivity, optimization using one-factor-at-a-time (OFAT) was first carried out. The best conditions giving optimum response occurred at pH 5.8 and 30 min contact time. Molybdate and chromate exhibited exponential decay type inhibition curves with calculated half maximal inhibitory concentration, IC50 of 1.137 mg/L for molybdate and 1.247 mg/L for chromate. The Limits of detection (LOD) were 0.313 mg/L and 1.247 mg/L for molybdate and chromate, respectively. The newly developed assay can help in monitoring heavy metals pollution in rivers and agricultural areas.
References
Ranjbar L, Yamini Y, Saleh A, Seidi S, Faraji M. Ionic liquid based dispersive liquid-liquid microextraction combined with ICP-OES for the determination of trace quantities of cobalt, copper, manganese, nickel and zinc in environmental water samples. 2012;
Tian M, Fang L, Yan X, Xiao W, Row KH. Determination of Heavy Metal Ions and Organic Pollutants in Water Samples Using Ionic Liquids and Ionic Liquid-Modified Sorbents. J Anal Methods Chem. 2019;2019.
Vodyanitskii YN. Determination of the oxidation states of metals and metalloids: An analytical review. Eurasian Soil Sci 2013 4612. 2014 Jan 5;46(12):1139-49.
Gidlow DA. Lead toxicity. Occup Med. 2015 Jul 1;65(5):348-56.
Jung K, Bitton G, Koopman B. Assessment of urease inhibition assays for measuring toxicity of environmental samples. Water Res. 1995;29(8):1929-33.
Shukor Y, Baharom NA, Rahman FA, Abdullah MP, Shamaan NA, Syed MA. Development of a heavy metals enzymatic-based assay using papain. Anal Chim Acta. 2006;566(2):283-9.
Malara A, Oleszczuk P. Application of a battery of biotests for the determination of leachate toxicity to bacteria and invertebrates from sewage sludge-amended soil. Environ Sci Pollut Res. 2013;20(5):3435-46.
Monarca S, Zani C, Richardson SD, Thruston Jr. AD, Moretti M, Feretti D, et al. A new approach to evaluating the toxicity and genotoxicity of disinfected drinking water. Water Res. 2004;38(17):3809-19.
Sirisattha S, Momose Y, Kitagawa E, Iwahashi H. Toxicity of anionic detergents determined by Saccharomyces cerevisiae microarray analysis. Water Res. 2004;38(1):61-70.
Adilah AN, Marhidayu MSSN. Study on characteristics of sediment and bed load discharge in Sungai Jemberau at Tasik Chini. IOP Conf Ser Earth Environ Sci. 2019 Mar;244:012046.
Elias M, Ibrahim S, Samudin K, Kantasamy N, Rahman S, Hashim A. Rare earth elements (REEs) as pollution indicator in sediment of Linggi River, Malaysia. Appl Radiat Isot. 2019 May 1;151.
DOE. Environmental Quality Report [Internet]. 2017 [cited 2022 Jan 22]. Available from: https://enviro2.doe.gov.my/ekmc/wp-content/uploads/2018/10/EQR-2017.pdf
Botsford JL. A simple, rapid, inexpensive assay for toxic chemicals using a bacterial indicator. Stud Environ Sci. 1997 Jan 1;66(C):429-43.
Isa HWM, Mustafa M, Wan Johari WL, Syahir A, Shukor MY, Nor Azwady AA, et al. Development of a Bacterial-based Tetrazolium Dye (MTT) Assay for Monitoring of Heavy Metals. Artic Int J Agric Biol. 2014;16:1123-8.
Sánchez NS, Königsberg M. Using yeast to easily determine mitochondrial functionality with 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT) assay. Biochem Mol Biol Educ. 2006 May;34(3):209-12.
Hayon T, Dvilansky A, Shpilberg O, Nathan I. Appraisal of the MTT-based assay as a useful tool for predicting drug chemosensitivity in leukemia. Leuk Lymphoma. 2003 Nov;44(11):1957-62.
Rumlova L, Dolezalova J. A new biological test utilising the yeast Saccharomyces cerevisiae for the rapid detection of toxic substances in water. Environ Toxicol Pharmacol. 2012 May 1;33(3):459-64.
Riss TL, Moravec RA, Niles AL, Duellman S, Benink HA, Worzella TJ, et al. Cell Viability Assays. Assay Guidance Manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004.
Ahmad F, Halmi MIE, Baskaran G, Johari WLW, Shukor MY, Syed MA. Inhibitive bacterial MTT assay for river monitoring of heavy metals. Bioremediation Sci Technol Res. 2013;1(1):1-7.
Salari R, Salari R. Investigation of the Best Saccharomyces cerevisiae Growth Condition. Electron Physician. 2017 Jan 25;9(1):3592-7.
Vicent I, Navarro A, Mulet JM, Sharma S, Serrano R. Uptake of inorganic phosphate is a limiting factor for Saccharomyces cerevisiae during growth at low temperatures. FEMS Yeast Res. 2015 May 1;15(3):1-13.
Mazloum-Ardakani M, Barazesh B, Moshtaghioun SM, Sheikhha MH. Designing and optimization of an electrochemical substitute for the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay. Sci Rep. 2019 Dec 1;9(1).
Gong L, Yang G, Yang B, Gu J. Development of the yeast Saccharomyces cerevisiae as a biosensor for the toxicity detection of toxic substances. bioRxiv. 2020;1-18.
Fai PB, Grant A. Chemosphere A comparative study of Saccharomyces cerevisiae sensitivity against eight yeast species sensitivities to a range of toxicants. Chemosphere. 2009;75(3):289-96.
Diamantino TC, Guilhermino L, Almeida E, Soares AMVM. Toxicity of sodium molybdate and sodium dichromate to Daphnia magna Straus evaluated in acute, chronic, and acetylcholinesterase inhibition tests. Ecotoxicol Environ Saf. 2000;45(3):253-9.
Hsieh CY, Tsai MH, Ryan DK, Pancorbo OC. Toxicity of the 13 priority pollutant metals to Vibrio fisheri in the Microtox® chronic toxicity test. Sci Total Environ. 2004 Mar 5;320(1):37-50.
Wang CW, Liang C, Yeh HJ. Aquatic acute toxicity assessments of molybdenum (+VI) to Daphnia magna. Chemosphere. 2016 Mar 1;147:82-7.
Aidil MS, Sabullah MK, Halmi MIE, Sulaiman R, Shukor MS, Shukor MY, et al. Assay for heavy metals using an inhibitive assay based on the acetylcholinesterase from pangasius hypophthalmus (Sauvage, 1878). Fresenius Environ Bull. 2013;22(12):3572-6.
DOE. National Water Quality Standards For Malaysia [Internet]. 2019 [cited 2022 Jan 22]. Available from: https://www.doe.gov.my/portalv1/wp-content/uploads/2019/05/Standard-Kualiti-Air-Kebangsaan.pdf
Witeska M. The effect of toxic chemicals on blood cell morphology in fish | Request PDF. Fresenius Environ Bull. 2004;13(12):1379-84.
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2022 Bulletin of Environmental Science and Sustainable Management (e-ISSN 2716-5353)

This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).