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Introduction to Mammalian Cell Mutation Test - Mouse Lymphoma Assay The system recommended by ICH and others emplys L5178Y cells and the TK (thymidine kinase) locus. Mammalian cells generally have two copies of each gene, but this mouse lymphoma cell line has only one functional copy of the gene, and is referred to as TK+/TK-.Thymidine kinase is not an essential enzyme. It is part of a system that recycles free thymidine that is eventually incorporated into DNA. Trifluorothymidine (TFT) is a toxic analogue of thymidine and intereferes with DNA metabolism, killing the cell. However, if the functional copy of the TK gene is lost through mutation, the TFT is not metabolized and is no longer toxic. L5178Y cell cultures are treated with various concentrations of the test compund in both the presence and the absence of S9 mix for 3 hours. Duplicate cultures are used at each experimental point. At least four dose levels of the compound are assessed with the highest dose level being either that expected to reduced survival by 80-90% based on a scaled down preliminary test or, for non-toxic compounds, the sandard limit of 5 mg/ml or to the limit of solubility. The terated cells are washed free of compound and the cells are grown for an expression period of 48 hours hours to allow fixation of mutations. S9 Mix Many chemicals are not mutagenic or carcinogenic, but their metabolites are. These metabolites are usually generated by liver enzymes. In order to test the metabolites of the chemical under investigation, a crude preparation of enzymes is obtained from the homogenized liver of rats previoiusly treated with Aroclor to enhance liver enzyme levels and activity. This preparation is known as the S9 fraction and contains a wide range of enzymes, to which enzyme cofactors are added. Typically, S9 mix is added to the cells just prior to addition of the test substance solution. Agar Method The cells are counted, then their density decreased appropriately in selective medium containing agar and TFT. A second set of plates is set up without TFT to assess viability of cells following treatment. The cultures are incubated at 37°C for approximately 13 days. At least four dose levels are assessed and the test is normally performed twice to determine the reporducibility of any response. Colonies are counted as in the bacterial mutation test. Mutation frequencies are calculated in terms of mutant colony numbers per 106 viable cells.Microwell Method This method is based on the same principles as the soft agar method, except that after the treatment and expression period, cells are diluted to a standard density in selective TFT medium without agar. The cells are dispensed in round-bottom 96-well plates and incubated at 37°C for approximately 13 days. A parallel set of plates is set up using cells diluted in medium without TFT to measure viability. At the end of the culture period, each plate is examined to determine the number of wells with growing cells. The mutation frequency is expressed in terms of the cloning efficiency in selective medium divided by the cloning efficiency in mon-selective medium. In the microwell method, the "colonies" are in fact the accumulation of growing cells at the bottom of the round bottom wells.This feature of the "colonies" means that any given well will show only a single colony, no matter the number of initial colony-forming units. This also means that a large cfu will obscure the growth of a small cfu. In calculating mutation frequencies for small colonies, one must discard wells containing large colonies from the calculations. The calculations are detailed in Mouse Lymphoma Assay Calculations. Large and Small Colonies When the TK gene is lost by mutation, cells grow relatively well and form normal, large colonies. When the TK gene is lost to a greater genetic injury such as chromosome breakage, other genes are also negatively affected and the cells will grow with difficulty, often resulting in abnormally small colonies. While virtually all DNA-damaging agens produce a mixture of the two colony types, in theory, the proportions of large and small colonies may be indicative of the compound's mode of action.
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