Abstract
Many in vitro assays have been developed for the identification of potential human carcinogens, with endpoints that range from point mutations to visible chromosomal aberrations involving many millions of base pairs. Molecular biology techniques have demonstrated that these two mechanisms and other DNA changes, including damage intermediate between the two, are important in tumorigenesis. Therefore, assays that can detect a wide range of mechanisms leading to DNA damage have more relevance for the detection of carcinogens than assays which detect only a single endpoint. In this thesis two mammalian cell mutation assays, the HPRT/V79 Chinese hamster and TK/mouse lymphoma assays, were compared for their ability to respond to genotoxic chemicals. Molecular analysis of the mutants was used to determine the nature of the DNA damage incurred. Two chemicals were studied, ethyl methanesulphonate (EMS) and mitomycin C (MMC), which are known to differ in their biological activity. EMS is a potent point-mutagen, whereas MMC induces a higher frequency of chromosome breakage events. The data presented in this thesis shows that the HPRT and TK assays differed in their ability to detect mutation induced by EMS and MMC. Whilst EMS was capable of inducing a high mutant frequency at both loci, MMC produced relatively few mutants at the hprt locus, but induced a large number of mutants at the tk locus. Furthermore, in the TK assay a higher proportion of small, slow-growing mutants were observed with MMC than with EMS. Chromosome aberration assays using V79 cells were used to demonstrate the greater chromosome breakage ability of MMC compared to EMS. Molecular analysis of HPRT and TK mutants showed that the difference in induced mutant frequency was due to the type of genetic damage incurred. Intragenic changes, ranging from point mutations to loss of the entire gene, were recovered as viable mutants at both the hprt and tk loci. EMS, which caused mainly intragenic damage, induced similar mutant frequencies at both loci. In contrast, MMC induced mainly large, multilocus deletions, in which the damage was assumed to extend into genes essential for normal growth, since most TK mutants were slow-growing. Mutants carrying large scale damage were recoverable at the heterozygous tk locus, but not at the hemizygous, hprt locus. A diverse range of genetic alterations have been associated with the cancer process, involving both the mutational activation of oncogenes and the mutational inactivation of tumour suppressor genes. The TK/mouse lymphoma assay appears to be a more appropriate test for the detection of chemicals capable of causing the types of DNA lesions important in human cancer. Study of the hprt gene of human T-lymphocytes showed an age-related increase the in in vivo mutant frequency. Life-style factors, such as smoking and alcohol intake had no effect on the mutational response, although the wide variability in mutant frequency suggested that a much higher number of individuals would have to be studied in order to detect differences between exposed and control groups. Publications resulting from the studies presented here are bound in the back of this thesis.