JC-10 probe as a novel method for analyzing the mitochondrial membrane potential and cell stress in whole zebrafish embryos

Nadin Younes; Bana S Alsahan; Asmaa J Al-Mesaifri; Sahar I Da'as; Gianfranco Pintus; Amin F Majdalawieh; Gheyath K Nasrallah

doi:10.1093/toxres/tfab114

JC-10 probe as a novel method for analyzing the mitochondrial membrane potential and cell stress in whole zebrafish embryos

Toxicol Res (Camb). 2021 Dec 21;11(1):77-87. doi: 10.1093/toxres/tfab114. eCollection 2022 Feb.

Authors

Nadin Younes¹, Bana S Alsahan², Asmaa J Al-Mesaifri², Sahar I Da'as³, Gianfranco Pintus⁴, Amin F Majdalawieh⁵, Gheyath K Nasrallah¹

Affiliations

¹ Biomedical Research Center, QU Health, Qatar University, P.O. Box 2713 Doha, Qatar.
² Department of Biomedical Science, College of Health Sciences, QU Health, Qatar University, P.O. Box 2713 Doha, Qatar.
³ Department of Human Genetics, Sidra Medicine, P.O. Box 26999, Doha, Qatar.
⁴ Department of Medical Laboratory Sciences, University of Sharjah, P.O. Box 27272 Sharjah, United Arab Emirates.
⁵ Department of Biology, Chemistry, and Environmental Sciences, College of Arts and Sciences, American University of Sharjah, P.O. Box 26666 Sharjah, United Arab Emirates.

Abstract

Background: A sensitive method to investigate cellular stress and cytotoxicity is based on measuring mitochondrial membrane potential. Recently, JC-10, was developed to measure mitochondrial membrane potential in vitro and used as an indicator for cytotoxicity. Yet, JC-10 has never been used in vivo (whole organism). In normal cells, JC-10 concentrates in the mitochondrial matrix, where it forms red fluorescent aggregates. However, in apoptotic/necrotic cells, JC-10 diffuses out of the mitochondria, changes to monomeric form, and stains cells in green. Here, we aimed to develop and optimize a JC-10 assay to measure cytotoxicity in zebrafish embryo. We also investigated the effectiveness of JC-10 assay by comparing it to common cytotoxicity assays.

Methods: Zebrafish embryos were exposed to a toxic surfactant AEO-7 at no observed effect concentration (6.4 μg/L), and then cytotoxicity was measured using (i) JC-10 mitochondrial assay, (ii) acridine orange (AO), (iii) TUNEL assay, and (iv) measuring the level of Hsp70 by western blotting.

Results: As compared to the negative control, embryos treated with NOEC of AEO-7 did not show significant cytotoxicity when assessed by AO, TUNEL or western blotting. However, when JC-10 was used under the same experimental conditions, a significant increase of green:red fluorescent ratio signal was detected in the AEO-7 treated embryos, indicating mitochondrial damage and cellular cytotoxicity. Noteworthy, the observed green: red ratio increase was dose dependent, suggesting specificity of the JC-10 assay.

Conclusion: JC-10 is a sensitive in vivo method, thus, can be used as surrogate assay to measure cytotoxicity in whole zebrafish embryos.

Keywords: AEO-7; JC-10 probe; cytotoxicity; mitochondria; zebrafish.