Big Blue® lacI transgenic mice and rats carry the E. coli lacI gene integrated as a tandem array of approximately 40 copies integrated at a single site in chromosome 4. This mutationally well-characterized gene is highly sensitive to base substitution mutations and is readily recovered from virtually any tissue of the transgenic host, facilitating the in vivo study of mutation. The Big Blue® assay was used to investigate spontaneous and induced mutation, with an emphasis on dietary influences on mutational frequency and specificity. The effects of ageing and dietary restriction on spontaneous mutation in the lacI transgene were determined in mice, permitting evaluation of several well established theories of ageing. Mutation frequencies were found to increase with age in tissues that proliferate (bladder and liver, but not brain), validating a principle tenet of the somatic ageing theory. However, the unexpected lack of a change in mutational specificity in ageing mice suggests that theories of ageing based on oxidative damage, or a reduction in DNA repair efficiency, may not be seminal to the ageing process, at least until more advanced age. Similarly, dietary restriction, which is known to extend lifespan in rodents and was predicted to decrease oxidative DNA-damage, had no appreciable effect on either the frequency or the specificity of spontaneous mutation in liver of younger (6 month old) and older (12 month old) mice.
Dietary influences on induced mutation were examined following treatment with aflatoxin B₁ (AFB₁) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) powerful animal carcinogens which demonstrate tissue, species and sex-linked differences in rats and mice. As expected, AFB₁ was found to be potently mutagenic in rat but not mouse liver, in agreement with rodent carcinogenicity studies that found F344 rats to be highly susceptible to AFB₁-induced hepatocarcinogenesis, while C57BL/6 mice are highly resistant. PhIP was found to be potently and equally mutagenic in colon of both male and female rats. The result in female colon was surprising since PhIP predominantly induces colon cancer in male rats but mammary tumors in female rats. Therefore, the progression of PhIP-induced colon cancer in the rat colon is likely due to factors acting at a later stage in the tumorigenic process, following the damage and mutation of DNA. Rat prostate tissue, another tumor target tissue in PhIP-treated rats, was also found to be highly susceptible to PhIP-induced mutagenesis. Lastly, the PhIP studies were extended to an additional transgene target located in the shuttle vector construct from Big Blue® rodents, the bacteriophage λ-derived cII gene. These studies validated the use of the λ cII gene as an alternative mutational target for use in the Big Blue® assay, while the analyses of mutation in the lacI and the λcII transgenes serves as a paradigm for mutational studies which compare mutational responses in different genes. Collectively, these studies demonstrate the utility of the lacI (λ cII) transgenic mutagenicity assay for the in vivo investigation of mutational processes as a function of age, diet, sex, species, and target tissue specificity with respect to sites of mutation and cancer. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/8991 |
| Date | 19 January 2018 |
| Creators | Stuart, Gregory Roy |
| Contributors | Glickman, Barry W. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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