Conazoles are a class of azole fungicides which are used for agricultural and pharmaceutical applications. This study examined two conazoles that possess a range of reproductive, carcinogenic, and neurodevelopmental effects. Sprague-Dawley rats were dosed daily by oral gavage for 14 days with triadimefon (5 or 115 mg/kg/day) or myclobutanil (10 or 150 mg/kg/day). Relative liver weight increased following high dose triadimefon and myclobutanil exposure. Weight changes were not observed in testes, nor were there significant changes in serum testosterone levels. Microarray analysis of 4,370 genes yielded forty-eight differentially expressed genes in blood. Of these, nine genes were found to be concordant with liver and testis gene expression data.

Conazoles are a class of azole fungicides used in both pharmaceutical and agricultural applications. This study focused on 4 conazoles that exhibit a range of carcinogenic and reproductive effects, in order to identify common and unique modes of action. Conazoles target cytochrome P450s (CYPs), and the inhibition and induction of various CYP activities may be part of the toxic modes of action in liver and testis. We used gene expression profiling to characterize a broader range of conazole effects and to identify additional modes of action. Adult male CD-1 mice were dosed daily by gavage for 14 days with fluconazole, propiconazole, myclobutanil or triadimefon (three doses each). Relative liver weight increased following fluconazole and propiconazole exposure, and histological analysis revealed centrilobular hepatocyte hypertrophy in response to all 4 conazoles. No weight or histological changes were observed in testis, and serum testosterone and luteinizing hormone were also unchanged. Microarrays queried expression of 16,475 genes, and identified 2,081 and 1,424 differentially expressed genes in liver and testis, respectively, following conazole exposure. Of these genes, 118 in the liver and 94 in the testis were common to two or more conazoles. The majority of differentially expressed genes related to stress response, oxidative stress, xenobiotic metabolizing enzymes, steroidogenesis or carcinogenesis. Expression profiles between conazoles and between liver and testis affected similar biological pathways, suggesting the potential for common modes of action.

Estuaries are complex ecosystems that are composed of a number of sensitive and inter-dependent environments. An abundance of nutrients combine with the dynamic conditions to create some of the most productive environments on the planet. Almost 85% of the commercially harvested fish in the United States depend on estuaries and the surrounding coastal waters at some stage in their life history. Many estuarine drainage basins contain large quantities of agricultural acreage. Over 29 million pounds of active ingredient of pesticides are applied in coastal drainage basins in the United States each year. Studies have shown that the overall condition of the nation's estuaries is fair, while benthic condition is poor. Seventy five percent of estuarine sediments are contaminated with pesticides. Concentrations of pesticides in 30% of the estuaries exceed the levels that are known to result in ecological effects at least 10% of the time. There have been few comprehensive pesticide studies in estuaries to date. The Albemarle-Pamlico (A-P) Drainage Basin of North Carolina forms the second largest estuarine system in the United States and supports heavy agricultural production with high pesticide use. I evaluated measurement and modeling strategies to assess exposure in the A-P drainage basin. Atrazine and metolachlor were the most frequently detected pesticides in water samples that were collected in the A-P drainage basin in 2000 and 2001. Concentrations of these compounds exceeded both human health and aquatic life criteria in 2000. No toxicity thresholds were exceeded in samples collected in 2001. Due to the expense associated with field sampling and pesticide analysis alternative methods for estimating pesticide exposure have been developed, including fate models. The Exposure Analysis Modeling System (EXAMS) was modified to model the fate of pesticides, specifically atrazine and metolachlor, in a small tidal estuary of the larger A-P estuarine system. Based on the estimate that 10% of the total amount of atrazine and metolachlor applied in the Bath Creek drainge basin would enter the estuary the EXAMS steady state model predicts concentrations of both atrazine and metolachlor that fall between the mean and maximum values that were measured in Bath Creek in 2002. Concentrations of pesticides in the A-P drainage basin, both measured and modeled, are significantly less than acute toxicity thresholds for even the most sensitive aquatic species. Due to the short lived nature of these compounds it is unlikely that organisms in the region would experience adverse health effects due to exposure to the existing concentrations.

<p>Potassium bromate (KBrO3) is a drinking water disinfection by-product and may represent a health hazard if found to be carcinogenic for humans, as was determined in rats (DeAngelo et al., 1998; Hayashi et al., 1986; Kurokawa, 1985; Kurokawa et al., 1985; Kurokawa et al., 1986a; Kurokawa et al., 1982; Kurokawa et al., 1983a; Kurokawa et al., 1987a; Kurokawa et al., 1983b; Kurokawa et al., 1986b; Ohno et al., 1982; Onodera et al., 1985). The purpose of this research was to determine the mechanism of toxicity. The peritoneal mesothelium is a (rat) target organ of potassium bromate carcinogenicity, but has not been studied due to the inherent difficulties of working with this one cell layer-thick tissue. Data from a two-year bioassay were used to more precisely map the location of origin, revealing that these tumors in the male F344 rat originate on the mesorchium of the tunica vaginalis testis, the mesosplenium, or at a point in between. An in vitro cell culture system was developed to study the mechanism of toxicity. It was demonstrated that KBrO3 caused cell cycle arrest and markedly increased the number of apoptotic cells. KBrO3 is a powerful oxidant and glutathione (GSH) is the major cellular antioxidant. Therefore, GSH-related responses were studied revealing mesothelial cells contained substantially less GSH than a human hepatocellular carcinoma cell line (Hep-G2). Studies employing GSH ester or N-acetyl cysteine (a GSH precursor) pre-treatment demonstrated abatement of toxicity in mesothelial, but not Hep-G2, cells. Experiments carried out to determine the chemical or enzymatic nature of the reaction between GSH and KBrO3 revealed differences between the reaction kinetics of the unbuffered and buffered chemical and cell-free/cell lysate reactions, probably due to reaction pH. Both chemical and cellular reactions exhibited a similar first step reaction between GSH and KBrO3; thus, enzyme participation is probably not required. Experiments using diethylmaleate, which depletes GSH by a reaction involving KBrO3, showed that GSH depletion greatly enhanced KBrO3 toxicity, indicating GSH glutathione S-transferase, buthionine sulfoximine (which prevents synthesis of GSH) and was protective. This does not support the hypothesis that the reaction of KBrO3 and GSH itself produces a radical/reactive species that oxidatively damages lipids, proteins and DNA. Rather, depletion of GSH likely precedes oxidative damage. Gene expression studies demonstrated that peritoneal mesothelial cells displayed expression changes in a discrete set of genes, including oxidative stress-responsive genes, after treatment with KBrO3 for four or 12 hours. Mesothelial cells severely damaged by five days KBrO3 treatment recovered from complete cell cycle arrest after four weeks and exhibited explosive growth, focus formation and altered morphology. The redox imbalance created by GSH depletion appears to mediate increased expression of known oxidative stress responsive genes (e.g., HO-1,GADD45, GADD153, QR), activation of transcription factors (AP-1 and NFkB) and down-regulation of cell cycle initiating cyclins (and up-regulation of the CDK inhibitor p21waf1/cip1) in KBrO3-mediated toxicity. These alterations may permit cell survival, as observed after severe toxicity, and may be accompanied by transforming mutations or clastogenic changes. Taken together, these data suggest that mesothelial cells represent a population susceptible to KBrO3-mediated toxicity in vitro, and suggest that tissue susceptibility in vivo plays a role in the nascence of mesotheliomas in the male F344 rat.<P>

<p>Skin is a complex, hormone responsive tissue that functions as a barrier against water loss and infection. Estrogens have been shown to influence dermal thickness, vasodilatation and hair growth in skin. Remarkably, cutaneous E2 levels and capacity for E2 synthesis have not been fully assessed. We have determined that cutaneous17b-estradiol (E2) levels average nine times greater than serum E2 levels in female mice and that skin E2 is independent of serum E2. Additionally, we determined that estrogens are a major metabolite of testosterone in mouse skin explants, indicating that skin is a major site of extraglandular estrogen biosynthesis. Earlier studies have shown that castration accelerates hair growth in mice, and we have determined that castration induces a greatly diminished telogen phase, of the hair cycle. Previously our laboratory has shown that E2 blocks telogen to anagen transition of the hair cycle. We observed that only twice weekly 1 nmol E2 treatment reversed the effects of castration while daily treatment with 100 nmol testosterone or 25 nmol DHT was required, indicating that E2 is up to 100 times more potent than androgens. Previous studies have shown that mirex, a non-phorbol ester skin tumor promoter, promotes three times more tumors in female mice than OVX mice. E2 implants were able to restore 80% of the intact female mirex promotion response to OVX mice, indicating that E2 is the primary ovarian hormone that regulates mirex promotion. Since mirex promotes three times more tumors in female mice than in male mice, we conducted a tumor promotion study on intact and castrate mice given empty or E2 containing implants, and found that intact mice develop three times more tumors than castrated mice and that E2 implants fully restore intact male response to castrate mice, indicating that E2 also regulates mirex tumor promotion sensitivity in male mice. Collectively, these data indicate that skin is an important extraglandular source of E2 and skin E2 influences the hair cycle and chemical carcinogenesis.<P>

In utero exposure to benzene, a known environmental contaminant, is associated with increased risk of childhood leukemia. We have previously shown that in utero benzene exposure can alter the redox sensitive transcription factor NF-κB, which is up-regulated in leukemia. We hypothesize that this is through benzene-induced reactive oxygen species (ROS) production interfering with the signalling pathway involving NF-κB, p38-MAPK and c-Myb. The objectives were to determine if ROS and p38-MAPK mediate benzoquinone (BQ) induced changes in NF-κB activity, and if downstream targets of NF-κB are altered after BQ exposure. HD3 chicken erythroblast cells were transfected with either a c-Myb or an NF-κB luciferase linked reporter plasmid and exposed to 25 μM BQ for 2-24 hours. C-Myb and NF-κB activities were determined using luciferase transcription factor activity assays, Western blotting was conducted to assess changes in protein levels in non-transfected cells, immunofluorescence was used to determine nuclear translocation of NF-κB and the presence of ROS was determined via DCFDA assays. A TaqMan qRT-PCR kit was used to assess mRNA changes of c-myc and bcl-2, two targets of NF-κB. NF-κB activity was significantly increased following 16 and 24 hours of BQ exposure in HD3 cells. DCFDA assays and pre-treatment with antioxidants indicated that BQ-mediated ROS production was responsible for increased NF-κB activity. Immunofluorescence and Western blotting indicated that NF-κB translocates into the nucleus after BQ exposure. P38-MAPK was activated through a ROS dependent pathway after 8-24 hours of BQ exposure, and pre-treatment with the p38-MAPK inhibitor, SB203580, attenuated BQ-mediated increased NF-κB activity, partially due to increased IκB-α protein expression. At this point, the chosen downstream targets were not significantly different compared to control. Future studies should continue to evaluate the role of p38-MAPK in this pathway as well as look at epigenetic changes in key signalling proteins. Evaluating the effects of toxicant exposure on cell signalling pathways is vital for understanding mechanisms of xenobiotic-induced toxicity. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2013-08-28 12:07:26.393

Thesis (Ph. D.)--University of Michigan.

Thesis (Ph. D.)--University of Michigan.

Thesis (Ph. D.)--University of Michigan.

Thesis--University of Wisconsin--Madison. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 125-129).