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In-vitro mutagenicity assays have traditionally been used for first line identification of potential genotoxic hazard, purporting to chemical carcinogenesis and heritable genetic damage. The recent advances m combinatorial chemistry and high throughput screening technologies have led to a massive explosion in numbers of possible therapeutic candidates being produced at the early stages of drug discovery. This rapid increase in the number of chemicals to be classified results in a greater need for to acquire alternative methods for the prediction of toxicity. Quantitative StructureActivity Relationships (QSAR) can till this need for early hazard identifications by elucidating the physicochemical basis of biological activity. The assumption with predictive QSARs for toxicity is that "biological activity may be described as a function of chemical constitution". This thesis focuses on the Ames mutagenicity assay data for two compound sets; one of 90 compounds, with limited structural flexibility, comprising a range of chemical classes (non-congeneric series), the second, a set of 30 flavonoid compounds. Three physicochemical descriptor sets were generated: EV A, a theoretical molecular descriptor based on the normal co-ordinate modes of vibration; WHIM, derived from weighting functions applied to the 3D-structural molecular co-ordinates; and TSAR, a series of hydrophobic, electronic and steric parameters traditionally associated with the production of biological QSARs. Various "unsupervised" data pre-treatment methods were adopted, to reduce the level of degeneracy within the individual descriptor sets, prior to the calculation of stepwise linear discriminant classification functions. Good predictive models for Ames mutagenicity, as determined by leave-one-out (jackknife) cross-validation, were obtained with each of the three physicochemical descriptor sets. An increase in the predictive ability was observed following the combination of variables from the individual descriptor sets, inferring that some unique information associated with mutagenic activity is contained within each descriptor set. The predictive stability of the models produced was assessed via independent compound predictions, with a poor overall success rate determined. This failure in external prediction was investigated and fundamental differences in physicochemical data space occupancy revealed. Conclusions on training set composition and general model applicability are made with consideration to individual model physicochemical data space coverage.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Subpopulations of tumor infiltrating leukocytes in immunogenic skin tumors were identified with monoclonal antibodies. The tumors studied included primary UV-induced tumors and JB/MS melanomas, which survive in the host by immunosuppression of the immune response. The proportions of nucleated cells in primary UV-induced tumor cell suspensions which reacted with monoclonal antibodies were: 52% Mac-1+, 21% Lyt-1+, 13% Lyt-2+, 7% L3T4+, and 8% IL-2R+. Thus there was a high proportion of cells of the macrophage lineage in the growing UV-induced tumors. In JB/MS melanoma cell suspensions the mean proportion of macrophages was 6.4%, and total T lymphocytes (Lyt-1) averaged only 5.5%. Thus, there was little leukocytes infiltration into JB/MS melanoma, suggesting that chemotaxis was defective. The high level of macrophages and T cells in the primary UV-induced tumors indicates that chemotaxis was intact. Therefore, either the tumorcidal capacities of the macrophages and Tc were insensitive to activated macrophages and to Tc cells.

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Arsenic is a human bladder carcinogen. Inorganic arsenic and methylated metabolites are excreted from the human body in urine. This study investigates the effects of arsenite [As(III)] and monomethylarsonous acid [MMA(III)] on human urothelial cells (UROtsa). Cytotoxicity studies found that MMA(III) was 20 times more toxic than As(III). In addition, UROtsa cells have the ability to biotransform As(III) to pentavalent and trivalent mono-methylated metabolites.To understand the mechanism of arsenic carcinogenesis, it is necessary to know which arsenicals are carcinogenic. Therefore, non-tumorigenic UROtsa cells were chronically exposed to 0.05 uM MMA(III) and monitored for signs of transformation. MMA(III)-treated cells (URO-MSC) became hyperproliferative after 12 weeks of exposure. Anchorage-independent growth was detected after 24 weeks of exposure to MMA(III). Gene array analysis conducted in URO-MSC cells after 52 weeks of exposure detected expression changes consistent with malignant transformation. Enhanced tumorigenicity in SCID mouse xenografts was also observed after 52 weeks of treatment.URO-MSC cells form squamous cell carcinoma, a histology associated with inflammation, when injected into SCID mice. Induction of cycolooxygenase-2 (COX-2) promotes proliferation, angiogenesis, and survival in cancer cells. To identify a potential mechanism of MMA(III) carcinogenesis, the effects of chronic and acute MMA(III) treatment on COX-2 expression were investigated. Western blot analysis revealed that COX-2 was induced in a time-dependent manner in URO-MSC cells. Acute MMA(III) exposure also increased COX-2 protein. To identify signal transduction pathways responsible for COX-2 induction, pharmacological inhibitors of various signaling pathways were co-administered with 0.05 uM MMA(III) and identified src and extracellular signal regulated protein kinase (ERK) activation to be responsible for COX-2 induction. Thus, MMA(III) causes ligand-independent activation of epidermal growth factor receptor (EGFR), which activates the signal cascade responsible for COX-2 expression. EGFR is elevated in URO-MSC cells. To determine if EGFR is a key mediator of URO-MSC cell tumorigenicity, inhibitors of downstream signal transduction (src, PI3K, and COX-1/-2) were found to reduce URO-MSC cell viability and growth in soft agar. Results from this work not only identify that MMA(III) can induce malignant transformation in human cells but also provides insight into the mechanism of arsenic-induced bladder cancer.

Cyclopropene fatty acids (CPFA) found in cottonseed oil (CT) have been shown to reduce production of progesterone, a precursor of estrogen. Estrogenic hormones have been implicated in enhancing growth of mammary tumors. In this study, the effect of dietary cottonseed oil on estrogen production by mature female mice was determined by measuring urinary estrogen using High Performance Liquid Chromatography.At four months of age, five groups of three Strain A/ST female mice were placed on 20% fat diets containing 0, 2.5, 5.0, 10, and 20% cottonseed oil. The remainder of the fat in the experimental diets was corn oil sufficient to provide the balance of the 20% fat content in conjunction with other nutrients of equal percentages in all diets. At five day intervals mice were housed in metabolic cages and twenty-four hour urine samples were collected. Urine was purified on C18 columns and eluted with 1% phosphoric acid: acetonitrile: methanol: (54:35:11). Estrogen was quantified by High Performance Liquid Chromatography using a 250 X 5mm C18 column, hydrocortisone as an internal standard, and variable wavelength recorder set at 242 nm. The level of urinary estrogens after day 35 of the study was lower in all diets containing cottonseed oil. This is in agreement with several authors who have reported instances of physiological abnormalities in mammals which were fed increasing but low levels of dietary cyclopropenes. Since elevated estrogen levels have been identified as a risk for breast cancer, this study examines the relationship between dietary cyclopropenes and estrogen hormone production in strain A/St mice. / Department of Biology

Dietary fatty acids are considered promoters of murine and human mammary tumors. The mechanism responsible is not known. Mammary adenocarcinomas in mice originate from preneoplastic cells (hyperplastic alveolar nodules (HAN)) which are derived from normal mammary epithelial cells. Diets rich in linoleic acid (18:2) have been associated with increased incidence of HAN and promotion of tumor growth. Diets rich in stearic acid (18:0) have been associated with decreased incidence of HAN and increased latency period for mammary tumor formation in mice.The effects of dietary 18:0 and 18:2 stages of murine mammary tumorigenesis were examined. The purpose of this study was to determine the effects of these dietary fatty acids on HAN production, mammary gland development, and fatty acid composition of mammary epithelial cells and adipocytes.Spontaneous mammary tumor producing strain A/ST mice were fed a high fat (15%) or low fat (5%) diet. High fat stock (ST) diet containing 1.5% 18:2 or a low fat corn oil (CO) diet containing 3% 18:2 were fed. Animals were sacrificed at 6 or 10 months of age. HAN, ductile and alveolar development were histologically determined in the left inguinal mammary gland. The contralateral gland was on the early diets rich in 18:2 (SF) or 18:0 (SA) were fed. A low*fat enzymatically dissociated and fatty acid compositions of adipocyte and epithelial cells were determined by GLC. Fatty acid profiles were examined for correlation to histologic findings.SA-fed mice had fewer HAN and less well developed mammary alveoli than the other dietary groups which exhibited moderate (ST) or high (CO, SF) HAN incidence. SF-fed mice had the earliest onset of any dietary group. CO-fed mice had later onset of HAN as compared to SF-fed mice but the HAN incidence was similarly high in both groups at 10 months of age.SA-fed mice were protected from development of expected numbers of HAN as compared to ST-fed mice. The reduction in HAN risk in this group was associated with reduced mammary alveolar development. Groups with high risk of HAN (SF and CO) exhibited increased amounts of 18:2 in their mammary epithelial cells and adipocytes. / Department of Biology

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