Volume No. 6 Issue No.: 3A Page No.: 645-652 Jan-Mar 12

 

STANDARDIZATION OF A RAPID AND INEXPENSIVE IN-VITRO CYTOTOXICITY ASSAY

 

A. V. Pethkar*1, Pallavi Lele1, Sucheta N. Patil1, Anuja Ramdasi

1. Deptartment of Biotechnology, KTHM College, Gangapur Road, Nashik, Maharashtra State, (INDIA)
2. National Center for Cell Science, NCCS, Pune University campus, Pune (INDIA)
3. National Institute for Research in Reproductive Health, NIRRH, Mumbai (INDIA)

 

Received on : November 25, 2011

 

ABSTRACT

 

A large number of chemicals in our environment and in day-to-day life are not adequately tested for toxicity. Currently used toxicity tests have limitations such as, (a) in-vitro cell based assays depend on costly and specialized animal cell lines, (b) most assays are growth dependent and require chemicals, media and inventory for aseptic conditions and growth of cells (biosafety cabinets, CO2 incubators, etc.), (c) in-vivo assays require laboratory animals leading to additional financial burden and ethical issues. In order to encourage even the smalltime manufacturers of harmful chemicals to test their products, preferably in-house, there is a need to develop rapid, reliable and low-cost cytotoxicity assay. In the present work, metabolically active primary cells from a potential target organ (liver) were obtained by macerating liver tissue taken from a local chicken/mutton shop. Interestingly, tissue preserved at 2-8° C for up to 48 hr could be used in the assay. After adjusting cell density (OD576= 0.5), cells were exposed to different test compounds such as textile dyes, festival color and other chemicals for up to 3 hr under ambient conditions. Cells were kept in plain DMEM medium (without fetal calf serum) to maintain controlled conditions for the cells. After incubation, viability of cells was determined by dye exclusion test using Trypan Blue. The assay was standardized by varying different factors viz. concentration of test chemicals, exposure time, etc., one at a time while keeping other parameters constant. Test compounds were classified as highly toxic if the LC50 values were below 2μg/ml, toxic if the LC50 values were between 2-10μg/ml, low toxic for LC50 values between 10-50μg/ml and non-toxic for LC50 values above 50μg/ml. It was found that viability of cells exposed to festival color and orange2 was similar to that of the control (84.7%) and LC50 values for these chemicals were more than 50μg/ml (non-toxic). On the other hand, lead acetate and mercuric chloride, which are well known for toxicity, could be classified as highly toxic (LC50 values <2μg/ml) with viability reduced to almost non-detectable levels. The statistical significance of these observations was evaluated by applying the student t-test. The proposed liver cell-based assay could be potentially useful for high throughput screening of samples and development of a toxicological database for risk assessment and protection of public. It must be highlighted that inclusion of different animal organs which are usually thrown away (not consumed) might be useful in toxicity studies; especially for studying mechanism of toxicity. In addition to less time duration, the proposed assay will result in reduced numbers of animals for confirmatory in-vivo tests.

 

Keywords : Chemicals, In-vitro, Cytotoxicity assay, Taxicological, Dyes, Chemicals

 

 

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