With a view to establishing a model system for examining toxicity in skin, primary lingual keratinocyte cultures , a keratinocyte cell line and freshly isolated keratinocytes all derived from rat sub-lingual epithelium, were partially characterized both morphologically and enzymically and the toxicity and metabolism of the mycotoxin T-2, studied therein. A number of techniques for obtaining pure suspensions of sublingual keratinocytes (NCK) were examined and dispase is recommended for completely separating the epithelium from its dermis prior to trypsinization. Existing methods for the culture of PLK cells were improved to reduce the number of rats used and minimize the fibroblast contamination and a technique for culturing the epithelium associated with human hair follicles was also examined. The follicle technique was found to produce primary cultures which were 100% epithelial but considerable time and resources were required to generate relatively few cells. The keratinocyte cultures, PLK and RTE5, were shown to produce keratin and undergo stratification like the epithelium in vivo, Using a series of specific enzyme inhibitors, both the cultured and non-cultured epithelial cells were found to possess the same characteristic forms of acetylcholinesterase, butyrylcholinesterase and carboxylesterase. The effect of T-2 on protein synthesis in the keratinocyte cells was examined and a dose-related inhibition was evident. The primary and freshly isolated cells appeared to be the most resistant to synthesis inhibition. The rate of recovery from this inhibition was greatest in the cell line which also lost T-2 at the fastest rate. The uptake into and loss of T-2 from the keratinocyte cells was chiefly by simple diffusion, but studies using rotenone demonstrated that an active process may have been involved in reducing the rates of uptake and loss. The total amount of T-2 absorbed was likely to be dependent on the number of binding sites while the overall rate of loss was dependent on the strength of that binding. The possible nature of these binding sites is discussed. T-2 was metabolized in all the keratinocyte preparations to the same products found in mammalian skin, in vivo, and studies with specific enzyme inhibitors showed that a carboxylesterase was most likely to be involved in its hydrolysis. The greater metabolism of T-2 in the primary cells, which contained the most carboxylesterase, is likely to have been one of the factors involved in reducing the levels of protein synthesis inhibition in these cells. Rat sublingual epithelium and the cultures derived from it have a similar morphology and esterolytic capability to that of skin in viva. The PLK cells were most similar to NCK cells in terms of protein synthesis and esterolytic capability, so if it is assumed that NCK cells are representative of the sublingual epithelial cells in viva and hence skin, then the primary cultures might prove to be the best model culture system for studying toxicity in skin. Nevertheless, the RTE5 cell line was most similar to the NCK cells in respect of T-2 metabolism, and if it shown to have other characteristic skin metabolism systems then it too might prove useful for studying skin toxicity. In addition, the cell line would probably prove to be a more convenient, simpler, reproducible and cheaper means of examining skin toxicity on a large scale.

Background: The synthetic insecticide DDT (dichlorodiphenyltrichloroethane) has been speculated to increase breast cancer risk due to its environmental persistence, levels of bioaccumulation in breast adipose tissue, and endocrine disrupting actions. Epidemiological studies have had inconsistent findings, however a study in MMTV-neu mice determined that localized, developmental exposure to the reported anti-androgen p,p' DDE accelerated mammary tumor development. This study tested the potential cancer-promoting actions of p,p' DDE, the most prevalent and persistent DDT metabolite. <br>Objectives: To identify and characterize the expression of p,p' DDE -regulated genes to determine how developmental exposure may influence mammary tissue to promote tumor formation. Methods: For localized delivery, ELVAX 40P pellets containing various doses of p,p' DDE, hydroxyflutamide (another anti-androgen), and mixtures of p,p' DDE with other congeners like o,p' DDE and p,p' DDT were implanted into the mammary fatpads of prepubertal female mice. p,p' DDE-regulated genes were identified by microarray analysis and analyzed by real time RT-PCR. <br>Results: Lipid-adjusted levels of p,p' DDE in mammary adipose tissue and serum in young mice were within the ranges of human exposure. p,p' DDE significantly upregulated casein gamma (csn1s2a ), keratin 18 (krt18) and interferon-induced protein 44 (ifi44) genes in mammary tissue. These genes were similarly, but not significantly regulated by hydroxyflutamide. The dose of p,p' DDE that caused early tumor onset in a previous study resulted in unique expression for all three genes and concentrations of p,p' DDE also influenced gene responses for the mixtures. However, no qualitative changes were observed in gland morphology. Significant upregulation of transforming growth factor beta (tgfb1) and downregulation of interleukin 10 (il10) in splenic leukocytes indicated that localized delivery of p,p' DDE to the mammary gland also influences systemic immune responses. Significant upregulation of il10 by p,p' DDE and hydroxyflutamide suggest that some of p,p' DDE actions may be through its anti-androgenic activity. <br>Conclusions: Relevant human exposure levels of p,p' DDE induce significant increases in expression of csn1s2a, krt18 and ifi44. This activity as well as those induced by other doses, ratios and hydroxyflutamide suggest p,p' DDE actions may involve anti-androgenic activity and influence local and systemic effects in a HER2+ breast cancer mouse model. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences / Pharmacology-Toxicology / PhD / Dissertation

Along with an increasing understanding of the harmful effects on the environment of a wide range of pollutants has come the need for more sensitive, faster and less expensive detection methods of identification and quantitation. Many environmental pollutants occur in low levels and often in complex matrices thus analysis can be difficult, time consuming and costly. Because of the availability and easy cultivation of the microorganisms with potentially high specificity, there is considerable interest in the use of living microorganisms as the analytical component (the biocomponent) of sensors for pollutants. While a number of biosensors using bacteria have been developed, yeast has been comparatively rarely used as the biocomponent. Yeast are attractive because they are easy to culture and they are eukaryotes which means their biochemistry is in many respects closer to that of higher organisms. This thesis describes the development of whole cell bioassays that use yeast cells as a sensing element and redox mediators to probe the intracellular redox reactions to monitor the catabolic activity of the yeast resulting from the external substrate, steady-state voltammetry is utilised as the electrochemical detection technique. The isogenic differential enzyme analysis (IDEA) concept of Lincoln Ventures Limited, lead NERF funded research consortium uses bacteria that have been cultured using specific organic pollutants as the carbon source which are the biocomponent in sensors. The use of wild type yeast Arxula adeninivorans that has the ability to use a very wide variety of substrates as sources of carbon and nitrogen was used as an alternative to bacteria to validate the “IDEA” concept. Naphthalene and di-butyl phthalate were chosen as model target contaminant molecules. The performance, detection limits and the usefulness of yeast based biosensor applications for environmental analysis are discussed. This thesis also describes the development and optimisation of a simple, cost effective in vivo estrogens bioassay for the detection of estrogens using either genetically modified or a wild type yeast Saccharomyces cerevisiae. In this study, catabolic repression by glucose was exploited to achieve specificity to estrogens in complex environmental samples that eliminates the requirement for conventional sample preparation. This is the first time that the use of wild type yeast to quantify estrogens has been reported. The attractive features of the bioassay are its use of a non-GMO organism, its speed, its high specificity and sensitivity with a detection limit of 10-15 M. The similarity of binding affinities for major estrogens to those of human estrogens receptors makes this in vivo estrogen bioassay very useful for analytical/screening procedures. The electrochemical detection method also makes it easy to interface with a variety of electronic devices.

The development of real time, label-free biosensors based on ligand-induced nanomechanical responses of microcantilevers (MCs) allows for sensitive and selective detection. High sensitivity is afforded by the MCs small dimensions. Immobilizing biomolecular recognition phases imparts selectivity from bioaffinity interactions. Biological sensors on a MC platform utilize various proteins, such as antibodies and nuclear receptors, which can be used to detect and screen for potential environmental contaminants. The interaction between contaminants and immobilized receptors induces an apparent surface stress that leads to static bending of the MC, which is monitored by an optical beam bending technique. Biofunctionalized MCs can provide high sensitivity and selectivity on a relatively inexpensive platform that requires small amounts of analyte. The goal of this research is to develop and optimize MCs as biosensors to detect low concentrations of contaminants. Initially, the research utilized specific receptors and antibodies to detect and screen for contaminants that are deemed endocrine disrupting chemicals (EDCs). Immobilizing estrogen receptors and specific antibodies on the MC surface may provide information on the ever expanding list of EDCs, along with fundamental endocrine studies. Then, the MC surface was morphologically and chemically optimized. This optimization included the thickness and metal ratio of the dealloyed surface. The concentration, reaction time, and pH of chemical immobilization reagents, which include aminoethanethiol and glutaraldehyde, were optimized by using an anti-body test system. Antibody and protein functionalization conditions, which are incubation time and concentration, were optimized using the anti-immunoglobulin G (anti-IgG) receptor: IgG and an anti-biotin:biotin test systems. The optimized immobilization conditions were applied to the detection of thyroid disrupting chemicals (TDCs) using MCs functionalized with the transport protein thyroxine-binding globulin. The final project involved developing a nanomechanical transducer to study xenobiotic and EDC interactions with the bioreceptor PXR’s ligand binding domain (LBD). The combination of immobilized LBD PXR with a nanostructured microcantilever (MC) platform allows for the study of ligand interaction with the receptor’s binding domain. PXR shows real-time, reversible responses when exposed to specific pharmaceutical, EDC, and xenobiotic ligands. Three binding interactions that involve EDCs are tested, which include phthalic acid, nonylphenol, and bisphenol A, with PXR.

The Swedish otter (Lutra lutra) population decreased markedly after the 1950’s and was considered to be seriously threatened for decades. Pollution from industries and other anthropogenic activities were suspected to be the major cause of the decline. The otter is a top-predator in aquatic systems and therefore particularly susceptible to bioaccumulative and toxic substances. For this reason, the species can be regarded as a valuable indicator of environmental health. Today, the Swedish otter population is increasing and has been reestablished in many areas. However, the otter is still missing in some regions of Sweden, especially in the southeast. The aim of this study was to investigate past and present levels, and temporal trends, of environmental contaminants in otters from Småland.The study is based on data of environmental contaminants in liver and muscle tissue from dead otters found in Småland during 1966-2014. I found that the perfluorinated compounds PFOS and PFOA have increased in otters since the 1970’s which is alarming and should be given special concern in future monitoring. Also, while mercury (Hg) is rather stable, selenium (Se) concentrations are decreasing, resulting in Hg exceeding Se on molar mass basis in several otters. This indicates that all of the available Se is bound to mercury, which may imply that the otters are vulnerable to additional Hg contamination. My conclusions are that the present concentrations of the studied environmental contaminants have probably not affected the otter population in general. However, otters may be exposed to toxic levels of some contaminants in certain areas that are more polluted from anthropogenic activities. This could be harmful to individual otters or their reproduction, and therefore, continued monitoring is highly relevant.

Chemical exposure in humans rarely consists of a single chemical. The everyday exposure is characterized by thousands of chemicals mainly present at low levels. Despite that fact, risk assessment of chemicals is carried out on a chemical-by-chemical basis although there is a consensus that this view is too simplistic. This thesis aims to validate a statistical method to study the impact of mixtures of contaminants and to use that method to investigate the associations between circulating levels of a large number of environmental contaminants and atherosclerosis and the metabolic syndrome in an elderly population. Contaminants measured in the circulation represented various classes, such as persistent organic pollutants, plastic-associated chemicals and metals. There was little co-variation among the contaminants and only two clusters of PCBs could be discerned. Gradient boosted CARTs were used to assess additive and multiplicative associations between atherosclerosis, as measured by the intima-media thickness (IMT) and the echogenicity of the intima-media complex (IM-GSM), and prevalent metabolic syndrome. Systolic blood pressure was the most important predictor of IMT while the influence of the contaminants was marginal. Three phthalate metabolites; MMP, MEHP and MIBP were strongly related to IM-GSM. A synergistic interaction was found for MMP and MIBP, and a small antagonistic interaction was found for MIBP and MEHP. Associations between the contaminants and prevalent metabolic syndrome were modest, but three pesticides; p,p’-DDE, hexachlorbenzene and trans-nonachlor along with PCBs 118 and 209 and mercury were the strongest predictors of prevalent metabolic syndrome. This thesis concludes that many contaminants need to be measured to get a clear picture of the exposure. Boosted CARTs are useful for uncovering interactions. Multiplicative and/or additive effects of certain contaminant mixtures were found for atherosclerosis or the metabolic syndrome.

Along with an increasing understanding of the harmful effects on the environment of a wide range of pollutants has come the need for more sensitive, faster and less expensive detection methods of identification and quantitation. Many environmental pollutants occur in low levels and often in complex matrices thus analysis can be difficult, time consuming and costly. Because of the availability and easy cultivation of the microorganisms with potentially high specificity, there is considerable interest in the use of living microorganisms as the analytical component (the biocomponent) of sensors for pollutants. While a number of biosensors using bacteria have been developed, yeast has been comparatively rarely used as the biocomponent. Yeast are attractive because they are easy to culture and they are eukaryotes which means their biochemistry is in many respects closer to that of higher organisms. This thesis describes the development of whole cell bioassays that use yeast cells as a sensing element and redox mediators to probe the intracellular redox reactions to monitor the catabolic activity of the yeast resulting from the external substrate, steady-state voltammetry is utilised as the electrochemical detection technique. The isogenic differential enzyme analysis (IDEA) concept of Lincoln Ventures Limited, lead NERF funded research consortium uses bacteria that have been cultured using specific organic pollutants as the carbon source which are the biocomponent in sensors. The use of wild type yeast Arxula adeninivorans that has the ability to use a very wide variety of substrates as sources of carbon and nitrogen was used as an alternative to bacteria to validate the “IDEA” concept. Naphthalene and di-butyl phthalate were chosen as model target contaminant molecules. The performance, detection limits and the usefulness of yeast based biosensor applications for environmental analysis are discussed. This thesis also describes the development and optimisation of a simple, cost effective in vivo estrogens bioassay for the detection of estrogens using either genetically modified or a wild type yeast Saccharomyces cerevisiae. In this study, catabolic repression by glucose was exploited to achieve specificity to estrogens in complex environmental samples that eliminates the requirement for conventional sample preparation. This is the first time that the use of wild type yeast to quantify estrogens has been reported. The attractive features of the bioassay are its use of a non-GMO organism, its speed, its high specificity and sensitivity with a detection limit of 10-15 M. The similarity of binding affinities for major estrogens to those of human estrogens receptors makes this in vivo estrogen bioassay very useful for analytical/screening procedures. The electrochemical detection method also makes it easy to interface with a variety of electronic devices.

Environmental contamination has become a significant threat to the health and well-being of mankind as well as to the environment, prompting the establishment and implementation of stringent environmental regulations. The ability to accurately detect and quantify contaminants, such as mercury (Hg), uranium (U), and volatile organic compounds (VOCs), in real-time, in situ is of significant importance to monitoring and remediation efforts. In an effort to develop a real-time, fast-response detector that is portable, highly sensitive, and cost efficient, this research explored the feasibility of utilizing cavity ringdown spectroscopy (CRDS) in conjunction with various plasma sources and vacuum cavities to accurately detect trace quantities of contaminants. The feasibility of detecting Hg with a low power, low temperature candle-shaped microwave-induced plasma (MIP) and a copper surfatron microwave cavity with various plasma discharge tube configurations in conjunction with cavity ringdown spectroscopy (MIP-CRDS) is discussed. Detection limits were on the order of 221 ppt Hg in the vapor phase for the candle-shaped MIP and improved by a factor of 10 with the tube-shaped plasma. The ability to detect elemental Hg naturally-evaporating from contaminated soils and solutions was evaluated, and 10’s of ppt were consistently obtained. Additionally, the fine structure of the Hg 253.65 nm transition was observed with each iteration of this approach. The potential of effectively generating uranium atoms and ions with a low-power, lowlow rate microwave-induced plasma was evaluated. Uranium emission spectra covering 320 – 430 nm were obtained, labeled, and compared to the available literature values. Calibration curves were generated, and the detection limits were determined to be ~0.4 ppm. The feasibility of measuring U incorporating diode laser-plasma-CRDS was explored. The preliminary studies clearly show the ability to detect U vapor with this technique and sub-ppm detection limits were obtained. A continuous wave cavity ringdown spectroscopy system (CW-CRDS) incorporating commercially available telecommunications diode lasers was constructed, and the overall sensitivity of this system was evaluated by utilizing the absorption of the asymmetric C-H stretch overtones of several VOCs, including benzene, chlorobenzene, 1,2-dichlorobenzene, toluene, and acetone. Detection limits are determined to be in the ppb’s for each of the organics examined.

Background and objective: Major risk factors for congestive heart failure (CHF) are myocardial infarction, hypertension, diabetes, atrial fibrillation, smoking, left ventricular hypertrophy (LVH) and obesity. However, since these risk factors only explain part of the risk of CHF, we investigated whether persistent organic pollutants (POPs) might also play a role. Methods: In the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study, left ventricular ejection fraction, (EF), E/A-ratio and isovolumic relaxation time (IVRT), were determined by echocardiography and serum samples of 21 POPs were analyzed in serum measured by high-resolution chromatography coupled to high-resolution mass spectrometry (HRGC/HRMS) in 998 subjects all aged 70 years. Results: In this cross-sectional analysis, high levels of several of the polychlorinated biphenyls (PCB congeners 99, 118, 105, 138, 153, and 180) and octachlorodibenzo-p-dioxin (OCDD) were significantly related to a decreased EF. Some POPs were also related to a decreased E/A-ratio (PCBs 206 and 209). All the results were adjusted for gender, hypertension, diabetes, smoking, LVH and BMI, and subjects with myocardial infarction or atrial fibrillation were excluded from the analysis. Conclusions: Circulating levels of POPs were related to impairments in both left ventricular systolic and diastolic function independently of major congestive heart failure risk factors, suggesting a possible role of POPs in heart failure.

Traditional food is fundamental for the cultural identity, mental and spiritual well-being, and physical and nutritional health of First Nations in Canada. Rapid environment changes including environmental contamination and degradation, climate change, urbanization and industry growth reduce the availability and diversity of traditional foods. This is concomitant with changing lifestyle and an increased prevalence of malnutrition, obesity, diabetes, and cardiovascular diseases. The goal of this thesis is to investigate the roles of traditional fish consumption for First Nations’ health in the complex interplays between environmental contaminant exposure, climate change, and food and nutrition security. Data collected from the First Nations Food Nutrition and Environment Study were used. The thesis is comprised of seven chapters presented in three sections. Section 1 addressed the importance of traditional fish consumption for food and nutritional security among First Nations in Canada. With increased income-related food insecurity, First Nations rely more on traditional foods including fish and participate more in fishing and other traditional practices. Nevertheless, many factors such as climate change, governmental restrictions, hydro and forestry operations continue to reduce the availability of traditional fish and access to traditional food sources, land and waterways. Section 2 explored the associations between locally-harvested fish consumption, long chain omega-3 fatty acid (n-3 FA) intake and dietary exposure to persistent organic pollutants (POP) with type 2 diabetes in First Nations in Canada. Dietary POP exposure was positively associated with the prevalence of type 2 diabetes whereas fish consumption (n-3 FA) showed protective dose-response associations. Furthermore, we found that relatively high POP exposure from fish may outweigh the protective associations of fish on type 2 diabetes. Therefore, the balance of risks and benefits associated with fish consumption is highly dependent on the regional POP concentrations in fish. Section 3 entailed studies on the nutritional benefits of seafood consumption and modelling potential impacts of the climate-related decline in seafood abundance on the nutritional quality of adult diets and cardiovascular health among coastal First Nations in British Columbia. We estimated that projected climate change may reduce the intakes of essential nutrients by 21%– 31% by 2050 relative to 2000. Moreover, hypothetical substitution of seafood with alternative non-traditional foods would not provide adequate amounts of nutrients. Reduced fish consumption and consequent n-3 FAs intake may increase the risk of cardiovascular diseases in First Nations. Our findings provide important information for communities, fishery governance, local resource managers and public health professionals to promote traditional food systems, nutritional health, food security, and food sovereignty in Canadian First Nations.