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The in vitro mouse lymphoma assay (MLA) is one of the most widely practiced assays in genetic toxicology. MLA detects forward mutations at the thymidine kinase (Tk) locus of the L5178Y (Tk (+/-) -3.7.2C) cell line derived from a mouse thymic lymphoma. This assay is capable of detecting a wide range of genetic events including point mutations, deletions (intragenic) and multilocus, chromosomal rearrangements, mitotic recombination, and nondisjunction. There are two equally accepted versions of the assay, one using soft agar cloning and the second method using liquid media cloning in 96-microwell plates. There are two morphologically distinct types of mutant colonies recovered in the MLA: small- and large-colony mutants. The induction of small-colony mutants is associated with chemicals inducing gross chromosomal aberrations whereas the induction of large mutant colonies is generally associated with chemicals inducing point mutations. The source and karyotype of the cell line as well as the culture conditions are important variables that could influence the assay performance. The assay when performed according to the standards recommended by the International Workshops on Genotoxicity Testing is capable of providing valuable genotoxicity hazard information as part of the overall safety assessment process of various classes of test substances.

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Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. It is present in practically all cells and has several important roles, such as preventing the oxidation of the sulfhydryl groups of proteins within a cell. Evidence for GSH deficiency or depletion has been found in a variety of diseases and toxicity-related studies, including diabetes and induction of oxidative stress to form reactive oxygen species which cause DNA, lipid, and protein oxidations. A simple, selective, and sensitive analytical method for measuring low levels of GSH in biological fluids would therefore be desirable to conduct GSH deficiency or depletion-related mechanistic toxicity studies. Here a method for both low- and high-level quantitation of GSH from cultured cells and rat liver tissues via liquid chromatography/positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed. The lower limit of quantitation (LOQ) of the method was 5 ng/mL. The method is linear over a wide dynamic concentration range of 5.0 to 5000.0 ng/mL, with a correlation coefficient R2 > 0.99. The intra-day assay precision relative standard deviation (RSD) values for all quality control (QC) samples were < or =16.31%, with accuracy values ranging from 94.13 to 97.80%. The inter-day assay precision RSD values for all QC samples were < or =15.94%, with accuracy values ranging from 94.51 to 100.29%. With this method, low levels of GSH from diethyl maleate (DEM)-treated mouse lymphoma cells, and GSH in rat liver tissues, were quantified.