The FRAME Alternatives Laboratory Database. 1. In Vitro Basal Cytotoxicity determined by the Kenacid Blue Total Protein Assay

Abstract

A database of over 280 chemicals has been compiled by using a mouse 3T3-L1 fibroblast-like cell line in exponential growth, exposed to chemicals for 72 hours in a 96-well tissue culture plate format, and determining cell number via the Kenacid blue (KB) assay for total protein. Ranking the chemicals according to their basal cytotoxicity, expressed as the concentration (mM) that inhibits increase in total cellular protein over 72 hours by 50% (the ID50 value) shows a wide range of ID50 values, from 0.00003mM to 10,096mM. This information includes the results for MEIC chemicals 1–50, and we have now added basal cytotoxicity data for 23 of the next 25 MEIC chemicals. When the neutral red uptake (NRU) assay was performed with the same cell cultures, before the KB assay, very similar indications of basal cytotoxicity were obtained. Comparisons between the results with 3T3-L1 cells and with a human fibroblast-like cell line, BCL-D1 showed a significant difference in order of magnitude of the ID50 value for only 5 of 52 chemicals. However, there was a difference in ID50 value of more than one order of magnitude for 8 of 24 chemicals tested with an undifferentiated teratocarcinoma cell line, F9.

Cytotoxicity as a Fundamental Response to Xenobiotics

“…Kenacid blue binding assay Through this assay, the change in total protein content is measured, which is a reflection of cell proliferation. If a compound is cytotoxic to the cells, it must affect at least one or more processes involved in cell proliferation, such as DNA synthesis, the proper functioning of organelles such as mitochondria and lysosomes or affect the integrity of the membrane or protein synthesis [51]. When the cell growth is affected, the number of cells present in the treated culture must be reduced with respect to the control, such that the measurement of the concentration of proteins present in the culture constitutes an index of toxicity [51].…”

“…If a compound is cytotoxic to the cells, it must affect at least one or more processes involved in cell proliferation, such as DNA synthesis, the proper functioning of organelles such as mitochondria and lysosomes or affect the integrity of the membrane or protein synthesis [51]. When the cell growth is affected, the number of cells present in the treated culture must be reduced with respect to the control, such that the measurement of the concentration of proteins present in the culture constitutes an index of toxicity [51]. Generally, the cells are exposed to the product for evaluation for a time period of 72 h, and the product is then removed and the cells are exposed to the dye, which then binds to the cellular proteins.…”

“…Generally, the cells are exposed to the product for evaluation for a time period of 72 h, and the product is then removed and the cells are exposed to the dye, which then binds to the cellular proteins. Finally, the amount of kenacid blue retained by the cells is determined and the percent of inhibition of cell growth is quantified [51,52].…”

Ethical Issues in the Production of Human Cell Lines and Stem Cell Lines

“…However, a few years after we had successfully developed the kenacid blue cytotoxicity test, 3 the human cell line was withdrawn from sale, because of adverse publicity due to its fetal origin, and we switched to a mouse cell line, 3T3-L1. 4 It must not be forgotten that, 30 years ago, most normal cell lines were obtained from embryonic tissues, as the procedures for producing cell lines from post-natal tissues had not yet been developed.…”

FRAME and the Validation Process

“…A database of over 280 chemicals has been compiled, involving the use of the mouse 3T3-L1 fibroblast-like cell line in exponential growth, exposed to chemicals for 72 hours in a 96-well tissue culture plate format, and determining cell number via the KB assay for total protein (38). Ranking the chemicals according to their basal cytotoxicities, expressed as the concentration (mM) that inhibits the increase in total cellular protein over 72 hours by 50% (the ID50 value), shows a wide range of values, from 0.00003mM to 10,000mM.…”