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Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay
Published on: February 9, 2021
Cytotoxicity of novel derivatives of the spin trap EMPO
Nataliya Rohr-Udilova1, Klaus Stolze, Brigitte Marian
1Research Institute of Biochemical Pharmacology and Molecular Toxicology, Veterinary University of Vienna, Veterinaerplatz 1, A-1210 Vienna, Austria.
Abstract:
Free radicals are involved in different regulatory and pathological processes. The formation of superoxide in living cells or whole organisms is of major interest. ESR spin trapping allows identification of the radicals if proper spin traps are available. Our study investigates the toxicity of novel derivatives of the spin trap EMPO to cultured human lung carcinoma cells (A549), breast carcinoma cells (SKBR3), colon carcinoma cells (SW480) as well as to human fibroblasts (F2000). A dose-dependent decrease of the cell number was observed for all spin traps. At 100mM BuMPO, t-BuMPO and s-BuMPO caused pronounced cell loss (>90%) and increased LDH-release, while DEPMPO, EMPO, PrMPO and i-PrMPO caused only moderate cell loss (<60%) without any effect on the LDH-release after 24h. At 10mM and 50mM the latter agents even decreased LDH-release. 10mM and 50mM of i-PrMPO as well as 10mM PrMPO increased intracellular GSH content acting like antioxidants, whereas 50mM s-BuMPO and PrMPO decreased GSH content by 67% and 38%, respectively. Staining for apoptotic nuclei did not reveal any differences between controls and treated cultures indicating necrotic cell death possibly due to membrane toxicity. The following toxicity ranking was obtained: t-BuMPO>BuMPO>s-BuMPO>PrMPO>i-PrMPO approximately DEPMPO approximately EMPO. The least toxic compounds were DEPMPO (LD(50)=143 mM for SW480, 117 mM for A549 or 277 mM for F2000) and i-PrMPO (LD(50)=114 mM for SKBR3), the most toxic one was t-BuMPO (LD(50)=5-6mM for all cell types). In conclusion, up to 50mM i-PrMPO (t(1/2)=18.8 min) and up to 10 mM s-BuMPO (t(1/2)=26.3 min) can be recommended for further investigation of superoxide in biological systems.
Insights
Novel spin traps were tested for toxicity in human cell lines. DEPMPO and i-PrMPO showed low toxicity, making them suitable for superoxide detection in biological systems.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- Free radicals, particularly superoxide, play critical roles in cellular regulation and pathology.
- Electron Spin Resonance (ESR) spin trapping is a key technique for identifying free radicals.
- Novel spin trap derivatives are needed for effective superoxide detection in biological systems.
Purpose of the Study:
- To evaluate the cytotoxicity of new EMPO spin trap derivatives.
- To determine the toxicity ranking of these novel spin traps across various human cell lines.
- To identify spin traps suitable for further investigation of superoxide in biological systems.
Main Methods:
- Cultured human lung carcinoma (A549), breast carcinoma (SKBR3), colon carcinoma (SW480) cells, and human fibroblasts (F2000) were exposed to varying concentrations of spin traps.
- Cell number, lactate dehydrogenase (LDH) release, and intracellular glutathione (GSH) content were measured.
- Apoptotic nuclei staining was performed to differentiate cell death mechanisms.
Main Results:
- A dose-dependent decrease in cell number was observed for all tested spin traps.
- t-BuMPO, BuMPO, and s-BuMPO exhibited significant cytotoxicity and increased LDH release at 100mM.
- DEPMPO and i-PrMPO demonstrated the lowest toxicity, with high LD(50) values, and some concentrations even reduced LDH release and increased GSH content, suggesting antioxidant properties.
Conclusions:
- The toxicity ranking of the spin traps was determined as t-BuMPO > BuMPO > s-BuMPO > PrMPO > i-PrMPO ≈ DEPMPO ≈ EMPO.
- DEPMPO and i-PrMPO are recommended for further studies of superoxide in biological systems due to their favorable toxicity profiles.
- Necrotic cell death, likely due to membrane toxicity, was indicated by the absence of apoptotic nuclei.
