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.

JC-10 probe as a novel method for analyzing the mitochondrial membrane potential and cell stress in whole zebrafish embryos / Younes, N.; Alsahan, B. S.; Al-Mesaifri, A. J.; Da'As, S. I.; Pintus, G.; Majdalawieh, A. F.; Nasrallah, G. K.. - In: TOXICOLOGY RESEARCH. - ISSN 2045-452X. - 11:1(2022), pp. 77-87. [10.1093/toxres/tfab114]

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

Pintus G.;
2022

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.
JC-10 probe as a novel method for analyzing the mitochondrial membrane potential and cell stress in whole zebrafish embryos / Younes, N.; Alsahan, B. S.; Al-Mesaifri, A. J.; Da'As, S. I.; Pintus, G.; Majdalawieh, A. F.; Nasrallah, G. K.. - In: TOXICOLOGY RESEARCH. - ISSN 2045-452X. - 11:1(2022), pp. 77-87. [10.1093/toxres/tfab114]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11388/283923
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