The Potential of Phenothiazinium Dyes As Cytotoxicity Markers in Cisplatin-treated Cells

Overview

Journal Sci Rep

Specialty Science

Date 2023 Jun 23

PMID 37353536

Authors

Luiz Miguel Pereira

Gisele Bulhoes Portapilla

Guilherme Thomaz Pereira Brancini

Bruna Possato

Cassia Mariana Bronzon da Costa

Pericles Gama Abreu-Filho

Mark Wainwright

Ana Patricia Yatsuda

Gilberto Ubida Leite Braga

Affiliations

Soon will be listed here.

Abstract

Assessing the in vitro toxicity of compounds on cell cultures is an important step during the screening of candidate molecules for diverse applications. Among the strategies employed to determine cytotoxicity, MTT, neutral red, and resazurin are commonly used. Methylene blue (MB), a phenothiazinium salt, has several uses, such as dye, redox indicator, and even as treatment for human disease and health conditions, such as malaria and methemoglobinemia. However, MB has only been sparsely used as a cellular toxicity indicator. As a viability indicator, MB is mostly applied to fixed cultures at high concentrations, especially when compared to MTT or neutral red. Here we show that MB and its related compounds new methylene blue (NMB), toluidine blue O (TBO), and dimethylmethylene blue (DMMB) can be used as cytotoxicity indicators in live (non-fixed) cells treated for 72 h with DMSO and cisplatin. We compared dye uptake between phenothiazinium dyes and neutral red by analyzing supernatant and cell content via visible spectra scanning and microscopy. All dyes showed a similar ability to assess cell toxicity compared to either MTT or neutral red. Our method represents a cost-effective alternative to in vitro cytotoxicity assays using cisplatin or DMSO, indicating the potential of phenothiazinium dyes for the screening of candidate drugs and other applications.

References

Paunescu E, Boubaker G, Desiatkina O, Anghel N, Amdouni Y, Hemphill A . The quest of the best - A SAR study of trithiolato-bridged dinuclear Ruthenium(II)-Arene compounds presenting antiparasitic properties. Eur J Med Chem. 2021; 222:113610. DOI: 10.1016/j.ejmech.2021.113610. View

Bopp S, Lettieri T . Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line. BMC Pharmacol. 2008; 8:8. PMC: 2438350. DOI: 10.1186/1471-2210-8-8. View

Portapilla G, Pereira L, Bronzon da Costa C, Providello M, Oliveira P, Goulart A . Phenothiazinium Dyes Are Active against In Vitro. Biomed Res Int. 2019; 2019:8301569. PMC: 6637691. DOI: 10.1155/2019/8301569. View

Rai Y, Pathak R, Kumari N, Sah D, Pandey S, Kalra N . Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition. Sci Rep. 2018; 8(1):1531. PMC: 5784148. DOI: 10.1038/s41598-018-19930-w. View

Fautz R, Husein B, Hechenberger C . Application of the neutral red assay (NR assay) to monolayer cultures of primary hepatocytes: rapid colorimetric viability determination for the unscheduled DNA synthesis test (UDS). Mutat Res. 1991; 253(2):173-9. DOI: 10.1016/0165-1161(91)90130-z. View

Yuan C, Gao J, Guo J, Bai L, Marshall C, Cai Z . Dimethyl sulfoxide damages mitochondrial integrity and membrane potential in cultured astrocytes. PLoS One. 2014; 9(9):e107447. PMC: 4169574. DOI: 10.1371/journal.pone.0107447. View

Dos Santos A, Terra L, Wailemann R, Oliveira T, de Morais Gomes V, Mineiro M . Methylene blue photodynamic therapy induces selective and massive cell death in human breast cancer cells. BMC Cancer. 2017; 17(1):194. PMC: 5353937. DOI: 10.1186/s12885-017-3179-7. View

Chen S, Chang J . New Insights into Mechanisms of Cisplatin Resistance: From Tumor Cell to Microenvironment. Int J Mol Sci. 2019; 20(17). PMC: 6747329. DOI: 10.3390/ijms20174136. View

Repetto G, Peso A, Zurita J . Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc. 2008; 3(7):1125-31. DOI: 10.1038/nprot.2008.75. View

10.

Arita M, Watanabe S, Aoki N, Kuwahara S, Suzuki R, Goto S . Combination therapy of cisplatin with cilastatin enables an increased dose of cisplatin, enhancing its antitumor effect by suppression of nephrotoxicity. Sci Rep. 2021; 11(1):750. PMC: 7804437. DOI: 10.1038/s41598-020-80853-6. View

11.

Borenfreund E, Puerner J . Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lett. 1985; 24(2-3):119-24. DOI: 10.1016/0378-4274(85)90046-3. View

12.

van Tonder A, Joubert A, Cromarty A . Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays. BMC Res Notes. 2015; 8:47. PMC: 4349615. DOI: 10.1186/s13104-015-1000-8. View

13.

Pereira L, Candido-Silva J, de Vries E, Yatsuda A . A new thrombospondin-related anonymous protein homologue in Neospora caninum (NcMIC2-like1). Parasitology. 2010; 138(3):287-97. DOI: 10.1017/S0031182010001290. View

14.

Lowik C, Alblas M, Van de Ruit M, Papapoulos S, van der Pluijm G . Quantification of adherent and nonadherent cells cultured in 96-well plates using the supravital stain neutral red. Anal Biochem. 1993; 213(2):426-33. DOI: 10.1006/abio.1993.1442. View

15.

Sousa P, da Silva Souza R, Tessarolo L, de Menezes R, Sampaio T, Canuto J . Betulinic acid induces cell death by necrosis in Trypanosoma cruzi. Acta Trop. 2017; 174:72-75. DOI: 10.1016/j.actatropica.2017.07.003. View

16.

Uzarski J, DiVito M, Wertheim J, Miller W . Essential design considerations for the resazurin reduction assay to noninvasively quantify cell expansion within perfused extracellular matrix scaffolds. Biomaterials. 2017; 129:163-175. PMC: 5765551. DOI: 10.1016/j.biomaterials.2017.02.015. View

17.

Hall J, Novakofski J, Beasley V . Neutral red assay modification to prevent cytotoxicity and improve reproducibility using E-63 rat skeletal muscle cells. Biotech Histochem. 1998; 73(4):211-21. DOI: 10.3109/10520299809141112. View

18.

Larsson P, Engqvist H, Biermann J, Ronnerman E, Forssell-Aronsson E, Kovacs A . Optimization of cell viability assays to improve replicability and reproducibility of cancer drug sensitivity screens. Sci Rep. 2020; 10(1):5798. PMC: 7118156. DOI: 10.1038/s41598-020-62848-5. View

19.

van de Loosdrecht A, Beelen R, Ossenkoppele G, Broekhoven M, Langenhuijsen M . A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J Immunol Methods. 1994; 174(1-2):311-20. DOI: 10.1016/0022-1759(94)90034-5. View

20.

Sliwka L, Wiktorska K, Suchocki P, Milczarek M, Mielczarek S, Lubelska K . The Comparison of MTT and CVS Assays for the Assessment of Anticancer Agent Interactions. PLoS One. 2016; 11(5):e0155772. PMC: 4873276. DOI: 10.1371/journal.pone.0155772. View