Requirement of a conservative cytotoxicity assay selection in cellular experiments

Abstract

Assessment of the cellular effects of natural and/or synthetic products is one of the major goals in biological experiments. This assessment may be conducted at different levels; cellular viability, cellular biological activity, and cellular structure and morphology. Different assays are available for any of above purposes. It is becoming of a routine exercise to apply any of these assays, and report the cyto-effect of natural or synthetic agents using the standard protocols on any different cell lines. A comparison of the application of different cytotoxicity assays for natural and synthetic materials, on different cell lines and at different conditions is presented here to proof that a very wise selection of the assay, as well as the method optimization is necessary for different purposes. IC50 of the essential oil and extracts of four different plants, four different synthetic chemical products, three different therapeutic drugs, and one biological fungal agent have been evaluated on seven different tumor or normal cell lines. A variety of cytotoxicity assays including the clonogenic, cell detachment, Neutral-red (NR) and MTT assays have been used after the exposure of different cell lines to any of above samples in the same condition. In some cases, cellular glutathione and DNA fragmentation assays have also been carried out. As is shown in our experiments, IC50 of different natural and synthetic agents changes significantly from assay to assay, or using the same assay in different cell lines and conditions. The survival curves shape is also variable when evaluating of the same agent using different methods. As an example, while NR was not able to demonstrate the 50% killing for the essential oil of M. pulegium on any of A375 or ACHN cell lines, clonogenic assay has resulted in IC50s of 9.1 and 46.9 ug/ml at the same condition, respectively. On A549 cell line, the application of same agent has resulted in the IC50s of 28.1 and 18.7 ug/ml using either NR or clonogenic assay, respectively. As an example of a therapeutic drug, the IC50 of cisplatin was found to be three times higher when measured suing NR (3 ug/ml) compared to the clonogenic assay (1 ug/ml) after one hour exposure to the SKOV3 cell line, and up to 20 fold different in different cell lines. A delay of the couple of hours for the MTT measurement of cisplatin cytotoxicity on LLCPK cell line has also resulted in up to 10 times differences in the calculation of IC50. We were not able to find any significant correlation between the amount of DNA fragmentation, total protein content and the GSH level with cytotoxicity in different cell lines for different agents. Detail of all results will be presented at the symposium. IC50 has widely been accepted as a standard measurement when evaluating potentially natural or synthetic cytotoxic agents. Different methods of IC50 measurements are available. Although many investigators are relying on IC50 to conclude the relative cytotoxicity of different natural or synthetic agents, one should be warned that it is dramatically influenced by the assay protocol, as well as the biology of different cell lines. Presently, approval or rejection of different natural or synthetic products in the screening investigations is not well standardized. Selection of the cell line, the nature of cytotoxicity assay, exposure duration and condition, and the interpretation of results should be precisely assigned and monitored based on the proposed research and purpose of evaluation. Using our results from the application of different assays for different agents and on different cell lines, a discussion on conservative decision will be presented at the meeting.