published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Vitamin A is known to have an important role in cellular differentiation and proliferation. In addition to regulating normal cellular processes vitamin A has also been shown to possess potent antineoplastic activity. The work in this dissertation characterizes the role of retinoic acid (RA) in cellular transformation and tumorigenesis with known oncogene involvement. These studies were initiated by examining the effects of RA on human carcinoma cell lines which express an activated c-ras gene. The bladder carcinoma, EJ/T24 (c-rasᴴ) and the two lung carcinoma cell lines, LXl (c-rasᴷ) and A2182 (c-rasᴷ), were not sensitive to RA. No inhibition of anchorage- or density-dependent growth was observed. Therefore, since these in vitro markers of transformation indicated a lack of effectiveness of RA on carcinomas containing a c-ras gene, retrovirally transformed cells were tested for RA sensitivity. Kirsten murine sarcoma, Balb/c murine sarcoma virus, and Simian sarcoma virus transformed NIH/3T3 and NRK cells were used in these studies. In contrast to the human carcinoma cell lines, anchorage-independent growth of some of the virally transformed cells was very sensitive to inhibition by RA. Anchorage-independent growth of KNRK and SSVNRK cells was sensitive to high concentrations (5 μM) of RA; whereas, Balb/cMSV3T3 and SSV3T3 were sensitive to 1-20 nM RA. BALB/cMSVNRK anchorage-independent growth was stimulated 3.5 fold by 1 μM RA. KNRK displayed a 60% reduction in anchorage-dependent growth at 10 μM RA while little inhibition was observed with the other retrovirally transformed cells. A high level of sensitivity to RA inhibition of anchorage-independent growth was correlated with the presence of cytoplasmic retinoic acid binding protein (CRABP). This indicated that CRABP may have some role in the inhibition of retrovirally induced cellular transformation. RA was shown to significantly reduce the incidence and size of Balb/cMSV3T3 cell tumors in nude mice. The inhibition of tumorigenesis in vivo therefore confirmed the results observed in vitro. To investigate the mechanism by which RA was acting to inhibit retroviral transformation, v-onc mRNA levels were examined. RA had no effect on v-onc mRNA levels in cell lines sensitive to the inhibition of transformation. The effect of RA on the relative rate of synthesis of p21, the transforming protein of KMSV and Balb/cMSV, was investigated. No effect of RA was observed in any of the cell lines. Also, GDP binding by p21 in KNRK cell was unchanged by RA treatment indicating that the functional activity of this transforming protein was not modified. RA does appear to be effective in inhibiting retrovirally induced cellular transformation and tumorigenesis. Evidence presented here indicates that this inhibition is not due to a direct effect of RA on the expression of the v-onc gene and/or gene product. Therefore, some other essential cooperating event(s) occurring within the cell are being acted upon by RA.

Barrett's esophagus (BE) is a premalignant disease associated with esophageal adenocarcinoma (EAC). This condition is highly associated with gastroesophageal reflux disease (GERD) which is characterized as chronic exposure of the esophagus to acid and bile acids. An understanding of the cytotoxic and tumorigenic capacity of bile acids and acid during a reflux episode will lead to the identification of markers for therapeutic intervention. The major goal of the following studies was to determine the mechanisms responsible for bile acid-induced alteration in intracellular pH (pHi) the effect on DNA damage, apoptosis and the adaptive resistance to reflux episodes in cells derived from normal esophagus (HET1A) or BE (CP-A) and EAC (JH-EsoAd1). In addition, I explored the therapeutic potential of UDCA oral therapy in BE cells.Here we show a novel mechanism of bile acid-induced, nitric oxide-mediated inhibition of the sodium-hydrogen exchanger (NHE) is a pathway bile acids utilize to induce acid-mediated DNA damage. This same mechanism can elicit apoptosis-resistance which we demonstrate by the complete inhibition of NHE with pharmacological inhibitor of NHE, EIPA. In addition, chronic exposure of bile acids and acid, in-vitro, confers resistance to cytotoxicity and makes cells derived from the squamous epithelium of the esophagus resemble BE and EAC. Finally, modifying the bile acid composition with glycol-Ursodeoxycholic acid (GUDCA) prevents many of the malignant effects of bile acids and acid and suggests a possible therapeutic strategy for those that suffer from GERD. The conclusion from this study suggest that bile acid reflux should be controlled in patients who suffer from GERD

Young children are particularly sensitive to environmental pollutants. They can directly ingest soil by putting dirty hands and objects in their mouths. The reliance on animal derived models for human health risk and exposure assessment has several limitations. In this investigation, a tool-kit was developed and optimised to facilitate more accurate, reliable and representative predictions of soil contaminants that might pose a significant hazard to young children. The tool-kit was developed and optimised using an in vitro human digestion bioassay. This procedure was followed by the optimisation of several mutagenicity bioassays to link to the bioaccessible fraction which quantified by the in vitro bioassay. The application of novel and sensitive environmental-based biosensors requires them to work in parallel with effective and proven extraction techniques. In this study, chemical analysis was used to quantify the bioaccessible (human assimilated portion) of pollutants in soils. Acute toxicity was measured using constitutively marked bioluminescent bacterial biosensors and these were indicative of the total contaminant burden. A range of mutagenic assays were applied and optimised. In the Ames assay, any compound exhibiting a greater than two-fold increase in the number of revertants colonies over the number of spontaneous revertants was considered as a mutagen. Mutagenic-responsive SOS-lux based microbial biosensors were compared to the Ames assay. Mutagenicity assessment of a broad range of environmental pollutants (i.e. B[a]P, DiB(a,h)A, B[a]A, Ni and Cu), was performed using four SOS-lux microbial biosensors; E. coli DPD1718, E. coli K12C600, S. aureus pAmiUmuC and S. aureus pAmiRecA. The results substantiated that the four biosensors were unable to be induced by these pollutants. Nevertheless, E. coli DPD1718 and E. coli K12C600 were successfully induced by Mitomycin C (MMC) in a dose response manner. The Ames assay was performed for the above pollutants in the absence and the presence of the metabolic activation S9 mix. The standard plate incorporation assay and a modification protocol for the Ames assay were applied. Results reported from the Ames assay confirmed mutagenicity responses of the tested pollutants except Cu and Ni. MMC was selected and introduced into soil samples as a case study to assess the performance of the developed tool-kit. Soils amended with MMC were extracted by the in vitro human digestion bioassay, and the mutagenicity of the bioaccessible fraction was measured using the Ames assay and the biosensors. A comparison was made between the permissible concentrations of MMC obtained from the developed tool-kit and RISC4 derived concentrations. The four microbial biosensors applied in this study were incapable to detect the mutagenicity of the tested pollutants. On the other hand, the Ames assay was more robust and sensitive to a broad range of environmental pollutants. The in vitro human digestion bioassay enabled the quantification of the human bioaccessible fraction of the tested pollutants. This fraction posed a concern due to its estimation of the doses that would reach the blood circulation and cause harm to human. While the permissible concentration of MMC measured by the developed tool-kit was less than 10 μg MMC/g, the RISC4 model calculated that it should be 40 μg MMC/g. This revealed that, in this situation, risk assessment model was less conservative than empirical study for human health risk assessment. This study enabled the assessment of the permissible concentrations of environmental pollutants that could remain in a soil and pose permissible harm to humans. This approach also enabled a comparison of modelled and empirical data to allow a measure of sensitivity to be judged. There is a need to develop bioassay techniques more able to assess the potency of hydrophobic compounds both in isolation and combination.