Quantitative analyses of forward and reverse mutations at the ẖg̲p̲ṟṯ locus in CHO cells
Date Issued
December 1, 1980
Author(s)
Fuscoe, James Charles
Advisor(s)
A. W. Hsie
Additional Advisor(s)
J. L. Epler
F. H. Gaertner
R. J. Preston
Abstract
This thesis describes (i) the relationship between the structures of 19 heterocyclic mustards (ICR compounds) and mutagenicity and cytotoxicity and (ii) the development of a reverse mutation assay to further characterized the types of mutations induced by ICR 191 and 170. Forward mutations at the hypoxanthine-guanine phosphoribosyltransferase (HGpRT) locus in Chinese hamster ovary (CHo) cells were selected utilizing the purine analog 6-thioguanine. It was found that ICR 191 and 170 exhibit a concentration-independent phenotypic expression time of 7-9 days and that with pairs of compounds with the same heterocyclic nucleus, all secondary amine compounds (ICR 191, 371, 372, and 449) are less mutagenic than the tertiary amine compounds (ICR 170, 217, 292, 340, 355, and 368). All secondary amine compounds, but none of the tertiary amine compounds, showed a "plateau" in their concentration-dependent mutagenesis curves. When the 2-chloroethyl group of the side chain is replaced by a 2hydroxyethyl group (ICR 170-OH, 191-OH, 292-OH, 340-OH, and 372-OH) or the amine linkage of the side chain is replaced by an ether linkage (ICR 283), mutagenicity is lost. Shortening of the side chain by 1 carbon (ICR 171) reduces mutagenicity. Substitution of a sulfur atom for a nitrogen atom in the side (ICR 342) or the addition of a second 2-chloroethyl group (ICR 220) results in increased mutagenicity and cytotoxicity. The distinct differences in mutagenicity between the secondary amine compounds and their tertiary amine analogs suggested that these compounds may be inducing different types of mutations. Reversion analysis was used to investigate this possibility. An assay to quantify reverse mutations at the hgprt locus in CHO cells was defined in terms of optimal selective agent (azaserine) concentration, cell density, phenotypic expression time, and selective stringency. After treatment, replicate cultures of 106 cells are allowed a 48 hr phenotypic expression time in 100-mm plates. Azaserine (10µM) is then added directly to the growing culture and azaserine-resistant cells form visible colonies. It was found that all azaserine-resistant clones had re-expressed HGPRT enzyme and that two revertants contain altered HGPRT-indicating suppression. The results of reversion analysis with the mutagens ICR 191, ICR 170, and ethylnitrosourea suggest that these three agents may cause similar types of mutations in CHO cells.
Degree
Doctor of Philosophy
Major
Biomedical Sciences
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