Dear Ponpon,

   Cytotoxicity can be done using MTT assay and ATP content analysis. Protocols for these two methods are available in google literature.

  Secondly, evaluation of cyto toxicity in plants by certain materials is getting its importance in recent times, since plants are in the base of food pyramid. An intoxicated plant effects every predator in the food pyramid that consumes it, directly or indirectly. Heavy metal contamination from inductrial effluents into agricultural field and delta region is a far cry these days.

Hence a careful study and appropriate solution for the cyto toxicity is of prime importance.

All the Best

Dear Abdul,

Cytotoxicity cannot be determined by the MTT assay for the reasons detailed below ...

Cytotoxic means direct "cell killing effects" induced by the drug of interest.

Cytostatic means that the compound of interested lowers the growth rate of a given cell population without direct cell killing effects. Cell death will occur as a consequence of a too long cytostatic effect.

A colorimetric assay can only bring "relative global growth inhibition information" because it is a relative test in which you compare the ODs of a treated cell population to the ODs of a control condition (untreated cells) arbitrarily scaled at 100%.

Thus, the IC50 / GI50-related values obtained by means of a colorimetric assay do actually not translate “cytotoxic” effects.

If one wants to determine actual cytotoxic effects for a compound of interest, the MTT colorimetric assay can be completed with the lactate dehydrogenase (LDH) test.

LDH is a soluble cytosolic enzyme that is released into surrounding culture medium upon cell damage or lysis during apoptosis or necrosis for example. The quantitative determination of LDH in the cell culture medium can be used as a marker for cytotoxicity.

Coming back to a colorimetric assay such as the MTT one, when one obtains a concentration (for a given compound) decreasing by 50% the global growth (after x days (usually 2 or 3)), i.e. the GI50 concentration (or the IC50 as commonly used in the literature) you do not know whether your compound of interest killed 50% of the cells (cytotoxic effects), whether it inhibited 50% of the cell proliferation (cytostatic effects), whether it detached 50% of the cells (anti-adhesive, i.e. "in vitro antimetastatic" effects), etc..., etc...

Once you have determined the GI50 / IC50 concentration for a given compound on a given cell line, you must use complementary biochemical and/or morphological techniques to determine whether your compound is cytotoxic, cytostatic, anti-adhesive, etc..., etc...

The two attached articles by Galluzzi and colleagues (2012, 2015; Appendix-1 and Appendix-2) are of great help in this domain.

The attached article by Kornienko et al. (2013; Appendix-3) reviewed various chemicals that are able to induce non-apoptotic cell deaths in cancer cells.

Coming back to the IC50 / GI50 values obtained by means of a colorimetric assay (as for example the MTT one):

in the Mathieu et al. (2009 (Appendix-4) and 2015 (Appendix-5)) articles, the MTT test-related GI50 concentrations relate to actual cytotoxic effects.

In the Lefranc – Nunzo et al. (2013; Appendix-6) article, the MTT test-related GI50 concentrations relate to cytotoxic effects that in turn do not relate to apoptosis … This means that each cytotoxic effect does not “universally” translate into pro-apoptotic ones.

In the Van Goietsenoven et al. (2010; Appendix-7) article, the MTT test-related GI50 concentrations relate to cytostatic effects, neither to cytotoxic nor to pro-apoptotic ones.

Be aware that you cannot always translate the MTT test-related growth inhibition of a given compound into a precise GI50 value. Some compounds reach a “plateau” of inhibition (see Lefranc – Nunzo et al., 2013; Appendix-6).

Lastly, you can also have "false" data generated with colorimetric assays (see the attached article by Chan et al. (2013; Appendix-8) and the NCI-60-cell line-related article (Shoemaker, 2006 (Appendix-9)).

The US NCI set up a fantastic tool to characterize the effects of a given drug in terms of growth inhibition in a panel of 60 cancer cell lines belonging to >10 histopathological types (Shoemaker, 2006; Appendix-9).

The US NCI clearly defined by means of the combination of the GI50 (growth inhibition), the LD50 (lethal dose by 50%) and the TGI (total growth inhibition) how to make the difference between a cytotoxic and a cytostatic compound:

The US NCI-related GI50 value corresponds to a global growth decrease by 50% induced by a compound on a given cell line “x” days after having cultured the cells with the drug and in comparison to an untreated control condition (= 100%) grown during the same time;

The US NCI-related LD50 value corresponds to the a global growth decrease by 50% induced by a compound on a given cell line “x” days after having cultured the cells with the drug and in comparison to the initial number of cells in the untreated control condition;

The TGI is the US NCI-related parameter to determine the concentration needed to kill 100% of the treated cells.

It is by comparing the GI50 to the LD50 value that one can determine whether a compound is cytotoxic or cytostatic, and not at all with the sole GI50 value.

We are using morphological approaches in the research unit to which I belong for determining whether a compound is cytotoxic or cytostatic (see Lefranc-Nunzo et al., 2013 (Appendix-6); Mathieu et al. 2009 (Appendix-4), 2015 (Appendix-5); Van Goeitsenoven et al., 2010 (Appendix-7)).

The US NCI is not so far for having tested about 800,000 anticancer drugs, whose data are publicly available on the NCI website https://dtp.cancer.gov/databases_tools/data_search.htm

I actually benefited several times from the amazing help of the NCI in identifying the mechanism of action of an innovative anticancer compound (see for example Frederick et al. JMC 2011 (Appendix-10)).

Hoping that this long explanation would not be too boring,

Best regards

Robert

Thanks Sir Robert Kiss. I'm very satisfied with your answer :)

Thanks Sir Abdul Azeez for sharing your ideas with regards to my question.