Mutant frequency and mutational spectra in the Tk and Hprt genes of N‐ethyl‐N‐nitrosourea‐treated mouse lymphoma cells†

Abstract

The mouse lymphoma assay (MLA) utilizing the Tk gene is widely used to identify chemical mutagens. The autosomal location of the Tk gene allows for the detection of a wide range of mutational events, from point mutations to chromosome alterations. However, chemically induced point mutation spectra in the Tk gene of mouse lymphoma cells have not been characterized. In this study, we determined and compared the mutagenicity and mutational spectra of N-ethyl-N-nitrosourea (ENU) in the Tk and Hprt genes of mouse lymphoma cells. Treatment of L5178Y mouse lymphoma cells with 100 microg/ml ENU induced a Tk mutant frequency of 756 x 10(-6) and an Hprt mutant frequency of 311 x 10(-6). Sequence analysis of Tk and Hprt mutant cDNAs showed a similar overall mutation pattern in the two genes with base-pair substitutions accounting for 83% of non-loss of heterozygosity mutations in the Tk gene and 75% of all mutations in the Hprt gene. The most common point mutation induced by ENU was G:C --> A:T transition (36 and 28% of independent mutations detected in the Tk and Hprt genes, respectively). The mutation spectra induced by ENU in both the Tk and Hprt genes were different from the respective patterns produced in mutants from untreated cells. About 9% of Tk and 7% of Hprt mutations from control cells were in-frame deletions, whereas no such mutations were found among the ENU-induced Tk and Hprt mutations. Our results indicate that ENU produces a chemical-specific point mutational profile in the Tk gene of mouse lymphoma cells that is remarkably similar to that found in the X-linked Hprt gene. This study provides evidence that the MLA can be used not only to detect point mutagens but also for analysis of mutational spectra.

Spectrum of ENU‐induced mutations in phenotype‐driven and gene‐driven screens in the mouse

“…Analysis of sequence composition in sites neighboring ENUinduced mutations identified in mouse phenotype-driven screens (above) and in mouse cell-culture-based selection systems (below). The data for the latter system were obtained from the following: [Skopek et al, 1992;Walker et al, 1996;Dobrovolsky et al, 1999;Nishimura et al, 2001;Chen et al, 2002;Kakinuma et al, 2005].…”

Genotoxic effects of acrylamide and glycidamide in mouse lymphoma cells

“…The L5178Y mouse lymphoma assay (MLA) is widely used as a short-term mutagenicity assay (Chen and Moore, 2004;Clements, 2000). The MLA is particularly useful for evaluating the ability of mutagens to induce a wide variety of mutational events, because it detects not only intragenic events (mainly point mutations) but also loss of heterozygosity (LOH) including Tk gene loss and karyotypically visible alterations of the Tk + -bearing chromosome 11b (Applegate et al, 1990;Chen et al, 2002;Hozier et al, 1982;Moore et al, 1985). AA has been found to be both mutagenic and clastogenic in mouse lymphoma cells in the absence of an exogenous activation system (Moore et al, 1987).…”

<i>In vitro</i> genotoxicity test package of antibiotics for human use submitted to the Japanese regulatory agency during 2004–2015

“…On the contrary, the HgPRT assay was performed in 7 quinolones including 2 MLA-positive ones, and only one quinolone showed positive result. Interestingly, chemical-specific point mutational profiles in the HgPRT assay were considered to be similar to those in the MLA (Chen et al, 2002), while positive results can definitely be found in the MLA assay more than the HgPRT assay because of the characteristics of the MLA (Johnson et al, 2012).…”