Cellular toxicity can result in multiple organ dysfunction and can cause severe health problems. Various studies have shown that toxicants may induce the over production of nitric oxide and reactive oxygen species and their subsequent accumulation can lead to cell death. While developing new therapeutics, it is essential to test the toxicity of the therapeutics on cells and their mechanisms before they are used for treating patients in clinical settings.
Researchers at Visikol have developed various 2D and 3D in vitro cell-based assay models to better understand the cytotoxicity of various compounds on different cell lines. These cytotoxicity assays determine the effectiveness of the compounds via IC50 measurements on the cells and the highest dose which could kill the cells. Visikol offers various assays such as Cell Titre-Glo, LDH assay, ToxiLight bioassays to measure cell viability/cell toxicity after compound treatment. All these assays use bioluminescence method as the end points detection. ToxiLight bioassay is a one of the rapid and most effective assays to measure cellular toxicity. It measures the concentration of Adenylate kinase (AK) enzyme released from the damaged cells. AK is found ubiquitously in different tissues of all living systems and plays a fundamental role in cellular energy and nucleotide homeostasis. But as there is an increase in the lysed/damaged cells with the treatment of the compound, AK enzyme accumulates in the cell’s supernatant. Unlike many other cytotoxicity assays, there is no need of cell lysis for this assay. The concentration of AK from the cell’s supernatant is measured via two steps of reaction. The first step involves addition of ADP as a substrate for the AK. In the presence of the enzyme, AK, ADP is converted to ATP. The bioluminescent method utilizes an enzyme luciferase, which catalyzes the formation of light from ATP and luciferin. By combining the two reactions, the emitted light intensity is linearly proportional to adenylate kinase concentration which is measured using a luminometer or beta counter.
Scientists at Visikol have successfully developed cytotoxicity assay for 2D A549 lung cancer cell lines using ToxiLight bioassay. The cells were treated in triplicate with three different compounds Chlorpromazine, Diclofenac, and Paclitaxel. Paclitaxel is an anti-cancer chemotherapy drug. Chlorpromazine is a medicine for schizophrenia disease and Diclofenac is an anti-inflammatory drug. A549 cells were seeded, dosed after twenty-four hours with seven doses along with vehicle and media only controls, and the ToxiLight bioassay (adenylate kinase assay) was performed after seventy-two hours of treatment. The luminescence value corresponds with the amount of adenylate kinase released from the cells with the compound treatment. As shown in Figure1., for chlorpromazine and Diclofenac, there was an increase in luminescence (RLU) with increase dose of the compounds suggesting an increase in cellular toxicity with higher dose. Interestingly for Paclitaxel, there was an increase in the cell toxicity as the dose increased, however the lower luminescence values at higher doses suggest that there is cell death with the compound treatment.
Figure 1. A549 cells were treated with three compounds and ToxiLight bioassay was performed at 72 hours after dosing. A. Chloropromazine B. Diclofenac C. Paclitaxel
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