Differential mutant quantitation at the mouse lymphoma tk and CHO hgprt loci
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M M Moore1, K Harrington-Brock, C L Doerr
1Genetic Toxicology Division, US Environmental Protection Agency Research Triangle Park, NC 27711.
Abstract:
Recent reports by several laboratories indicate that not all non-essential target loci are equally capable of detecting chromosomal mutations. The present study was undertaken to determine if both the tk locus in mouse lymphoma cells and the hgprt locus in Chinese hamster ovary (CHO) cells can be used to quantitate chromosomal mutations. Seven known mutagens for the tk locus were selected. These compounds were evaluated in the mouse lymphoma assay and in a suspension adapted CHO assay for their mutagenicity. In addition to the specific locus mutagenesis analysis, mouse lymphoma and CHO cells were evaluated for the frequency of gross chromosome aberrations. From these investigations, it appears that only those compounds [2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl] aminopropylamino)-acridine-dihydrochloride (ICR 170), ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS)] that induce significant numbers of large-colony thymidine kinase (TK) mutants also induce significant numbers of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) mutants. The four acrylates evaluated (methyl acrylate, ethyl acrylate, trimethylolpropane triacrylate and tetraethyleneglycol diacrylate) induced almost exclusively small-colony TK mutants and very few if any HGPRT mutants. Aberration analysis revealed that both the mouse lymphoma and CHO cells responded to the clastogenicity of the compounds (except for ICR 170 which was not positive in CHO cells) and that neither cell line was clearly more sensitive than the other to the clastogens tested. It is significant that the four acrylates give little or no evidence of genotoxicity when evaluated using selection for HGPRT-deficient mutants, yet are clearly clastogenic to the same cells in the same experiment. These results are consistent with the hypothesis that the hgprt locus may not be useful as a marker to evaluate the clastogenic component of a genotoxic compound. The present study adds to the increasing number of studies that support the view that the hemizygous nature of the hgprt locus permits the recovery of mutations primarily affecting the function of a single gene; whereas the heterozygous nature of the tk locus permits the recovery of both single gene and chromosomal mutations.
Insights
The thymidine kinase (TK) locus in mouse cells detects both gene and chromosomal mutations, while the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus in CHO cells primarily detects gene mutations. This suggests HGPRT is less useful for evaluating clastogenic genotoxicity.
Area of Science:
- Toxicology
- Genetics
- Cell Biology
Background:
- Different non-essential target loci exhibit varying sensitivities in detecting chromosomal mutations.
- The utility of the thymidine kinase (TK) locus in mouse lymphoma cells and the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus in Chinese hamster ovary (CHO) cells for quantifying chromosomal mutations requires further investigation.
Purpose of the Study:
- To determine if both the TK and HGPRT loci can be reliably used to quantify chromosomal mutations.
- To compare the mutagenicity and clastogenicity of selected compounds in mouse lymphoma and CHO cells.
Main Methods:
- Seven known TK mutagens were evaluated for mutagenicity in mouse lymphoma and CHO cell assays.
- Specific locus mutagenesis analysis was performed for both TK and HGPRT loci.
- Gross chromosome aberrations were assessed in both cell lines.
Main Results:
- Compounds like ICR 170, ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS) induced significant TK and HGPRT mutants.
- Four acrylates induced primarily small-colony TK mutants and few HGPRT mutants, despite being clastogenic.
- Both cell lines responded to clastogens, with no clear difference in sensitivity, but HGPRT assays showed limited genotoxicity for acrylates.
Conclusions:
- The HGPRT locus may not be suitable for evaluating the clastogenic effects of genotoxic compounds.
- The hemizygous HGPRT locus favors the recovery of single gene mutations, whereas the heterozygous TK locus allows recovery of both single gene and chromosomal mutations.