Typescript (photocopy). / Digitized by Kansas Correctional Industries

Typescript (photocopy). / Digitized by Kansas Correctional Industries

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Leung, Chun Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 155-169). / Abstracts in English and Chinese. / Title Page --- p.i / Acknowledgements --- p.ii / Table of Content --- p.iii / List of Figures --- p.viii / List of Graphs --- p.x / List of Tables x --- p.iv / Abbreviations --- p.xvii / Abstract --- p.xviii / Abstract (Chinese) --- p.xx / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Introduction to retinoids --- p.2 / Chapter 1.2 --- Role of endogenous retinoic acid in embryonic development --- p.3 / Chapter 1.3 --- Regulation of retinoic acid in embryonic development --- p.5 / Chapter 1.3.1 --- Retinoic acid synthesis and degradation --- p.5 / Chapter 1.3.2 --- Retinoic acid signaling --- p.8 / Chapter 1.4 --- Effect of excess vitamin AJ RA on embryogenesis --- p.8 / Chapter 1.4.1 --- Examples of human animal studies --- p.9 / Chapter 1.4.2 --- Mechanisms of retinoid teratogenesis --- p.11 / Chapter 1.4.2.1 --- Apoptosis --- p.11 / Chapter 1.4.2.2 --- Altered proliferation --- p.12 / Chapter 1.4.2.3 --- Altered cell migration --- p.12 / Chapter 1.4.2.4 --- Altered differentiation --- p.13 / Chapter 1.4.3 --- Critical period of RA administration caused specific Malformations --- p.14 / Chapter 1.5 --- Effect of vitamin A/ RA deficiency on embryogenesis --- p.15 / Chapter 1.6 --- Excess and deficiency of RA cause similar types of malformations --- p.17 / Chapter 1.6.1 --- Retinoic acid-induced renal malformations mouse model --- p.18 / Chapter 1.7 --- Strategy of thesis --- p.19 / Chapter Chapter 2: --- General Materials and Methods / Chapter 2.1 --- Mouse maintenance and mating methods --- p.23 / Chapter 2.2 --- All-trans retinoic acid preparation and injection --- p.23 / Chapter 2.3 --- Whole mount in situ hybridization --- p.24 / Chapter 2.3.1 --- Riboprobe synthesis --- p.24 / Chapter 2.3.1.1 --- Bacterial culture --- p.24 / Chapter 2.3.1.2 --- DNA plasmids extraction --- p.24 / Chapter 2.3.1.3 --- Linearization of plasmid --- p.25 / Chapter 2.3.1.4 --- Purification of linearized plasmid --- p.26 / Chapter 2.3.1.5 --- In vitro transcription and labeling --- p.26 / Chapter 2.3.2 --- Sample collection --- p.27 / Chapter 2.3.3 --- Hybridization --- p.28 / Chapter 2.3.4 --- Post hybridization wash and antibody development --- p.29 / Chapter 2.3.4.1 --- Embryo powder preparation --- p.30 / Chapter 2.3.4.2 --- Pre-absorption of antibody --- p.30 / Chapter 2.3.5 --- Post-antibody and staining --- p.31 / Chapter 2.4 --- Real-time quantitative reverse transcription -polymerase chain reaction (RT-PCR) --- p.32 / Chapter 2.4.1 --- Sample collection --- p.32 / Chapter 2.4.2 --- RNA extraction --- p.32 / Chapter 2.4.3 --- Reverse transcription into cDNA --- p.33 / Chapter 2.4.4 --- Quantitative real-time PCR --- p.33 / Chapter 2.4.5 --- Preparation of cDNA standards --- p.34 / Chapter 2.5 --- High pressure liquid chromatography (HPLC) --- p.35 / Chapter 2.5.1 --- Chromatographic system --- p.35 / Chapter 2.5.2 --- Standards preparation --- p.35 / Chapter 2.5.3 --- Embryo sample collection and preparation --- p.36 / Chapter 2.5.4 --- HPLC conditions --- p.36 / Chapter 2.5.5 --- Sample recovery --- p.37 / Chapter 2.5.6 --- Bradford assay --- p.38 / Chapter 2.6 --- RA-responsive cell line --- p.38 / Chapter 2.6.1 --- Cell culture --- p.39 / Chapter 2.6.2 --- Seeding and loading sample to 96-well plate --- p.40 / Chapter 2.6.3 --- X-gal staining --- p.41 / Chapter Chapter 3: --- Time and Dose Responses to RA / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.1.1 --- Time response to RA --- p.43 / Chapter 3.1.2 --- Dose response to RA --- p.45 / Chapter 3.1.3 --- Other factors affecting susceptibilities to RA --- p.46 / Chapter 3.2 --- Experimental design --- p.48 / Chapter 3.3 --- Materials and methods --- p.50 / Chapter 3.3.1 --- Time response to RA --- p.50 / Chapter 3.3.2 --- Dose response to RA --- p.50 / Chapter 3.3.3 --- Examination of fetuses --- p.51 / Chapter 3.3.4 --- Statistical analysis --- p.51 / Chapter 3.4 --- Results --- p.53 / Chapter 3.4.1 --- Time response --- p.53 / Chapter 3.4.1.1 --- Time response to RA-induced resorption --- p.53 / Chapter 3.4.1.2 --- Time response to RA-induced renal malformations --- p.54 / Chapter 3.4.1.3 --- Time response to RA-induced changes in growth parameters --- p.57 / Chapter 3.4.1.4 --- Time response to RA-induced non-renal malformations --- p.60 / Chapter 3.4.2 --- Dose response --- p.64 / Chapter 3.4.2.1 --- Dose response to RA-induced resorption --- p.64 / Chapter 3.4.2.2 --- Dose response to RA-induced renal malformations --- p.65 / Chapter 3.4.2.3 --- Dose response to RA-induced changes in growth parameters --- p.68 / Chapter 3.4.2.4 --- Dose response to RA-induced non-renal malformations --- p.71 / Chapter 3.5 --- Discussion --- p.74 / Chapter Chapter 4: --- Effect of Teratogenic Dose of RA on RA Synthesis and Endogenous RA Levels in the Embryo / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.1.1 --- RA synthesis in embryo --- p.79 / Chapter 4.1.2 --- Detection of endogenous RA in embryo --- p.81 / Chapter 4.2 --- Experimental design --- p.83 / Chapter 4.3 --- Materials and methods --- p.84 / Chapter 4.3.1 --- Localization of mRNA transcripts in whole embryo by in situ hybridization --- p.84 / Chapter 4.3.2 --- Vibratome sectioning --- p.85 / Chapter 4.3.2.1 --- Preparation of Gloop --- p.85 / Chapter 4.3.2.2 --- Sample preparation and sectioning --- p.85 / Chapter 4.3.3 --- Quantification of mRNA expression levels in whole embryo and in metanephros by real-time RT-PCR --- p.86 / Chapter 4.3.4 --- Detection of RA levels in whole embryo by HPLC --- p.87 / Chapter 4.3.5 --- Detection of RA levels in metanephros by RA-responsive cell line --- p.87 / Chapter 4.3.6 --- Statistical analysis --- p.88 / Chapter 4.4 --- Results --- p.89 / Chapter 4.4.1 --- Comparison of mRNA expression levels of different iso forms of RA synthesizing enzymes Raldh and RA catabolizing enzymes Cyp26 between embryos of RA-treated and vehicle-treated control mice at various time points after treatment --- p.89 / Chapter 4.4.2 --- Comparison of mRNA expression levels of different iso forms of RA synthesizing enzymes Raldh and RA catabolizing enzymes Cyp26 between metanephroi of embryos of RA-treated and vehicle-treated control mice at various time points after treatment --- p.93 / Chapter 4.4.3 --- Comparison of the in situ hybridization pattern of different iso forms of Raldh between embryos of RA-treated and vehicle-treated control mice at different time points after treatment --- p.95 / Chapter 4.4.3.1 --- In situ hybridization pattern of Raldh 1 --- p.96 / Chapter 4.4.3.2 --- In situ hybridization pattern of Raldh2 --- p.97 / Chapter 4.4.3.3 --- In situ hybridization pattern of Raldh3 --- p.100 / Chapter 4.4.4 --- Comparison of the in situ hybridization pattern of Cyp26al and Cyp26bl between embryos of RA-treated and vehicletreated control mice at different time points after treatment --- p.101 / Chapter 4.4.4.1 --- In situ hybridization pattern of Cyp26al --- p.101 / Chapter 4.4.4.2 --- In situ hybridization pattern of Cyp26bl --- p.102 / Chapter 4.4.5 --- Comparison of RA levels between embryos of RA-treated and vehicle-treated control mice at different time points after treatment --- p.103 / Chapter 4.4.6 --- Comparison of RA levels between metanephroi of embryos of RA-treated and vehicle-treated control mice at different time points after treatment --- p.105 / Chapter 4.5 --- Discussion --- p.106 / Chapter Chapter 5: --- Effect of Supplementation with Low Doses of RA on RA Teratogenesis / Chapter 5.1 --- Introduction --- p.111 / Chapter 5.1.1 --- RA supplementation --- p.111 / Chapter 5.1.2 --- Wilms' tumor suppressor gene Wtl --- p.112 / Chapter 5.1.3 --- Apoptosis --- p.113 / Chapter 5.2 --- Experimental design --- p.115 / Chapter 5.3 --- Materials and methods --- p.117 / Chapter 5.3.1 --- Oral gavage of low dose of RA --- p.117 / Chapter 5.3.2 --- Determination of Wtl expression level by real-time quantitative RT-PCR --- p.117 / Chapter 5.3.3 --- Preparation of paraffin sections and TUNEL staining --- p.118 / Chapter 5.3.3.1 --- Sample collection --- p.118 / Chapter 5.3.3.2 --- "Dehydration, embedding and sectioning" --- p.118 / Chapter 5.3.3.3 --- TUNEL staining --- p.119 / Chapter 5.3.4 --- Statistical analysis --- p.121 / Chapter 5.4 --- Results --- p.122 / Chapter 5.4.1 --- Time response to RA supplementation in rescuing kidney development --- p.122 / Chapter 5.4.2 --- Dose response to RA supplementation in rescuing kidney development --- p.127 / Chapter 5.4.3 --- RA supplementation restored various growth parameters --- p.132 / Chapter 5.4.4 --- RA supplementation rescued non-renal malformations --- p.134 / Chapter 5.4.5 --- Wtl expression in the metanephros after RA supplementation --- p.142 / Chapter 5.4.6 --- Apoptotic cell death in the metanephros after RA supplementation --- p.143 / Chapter 5.5 --- Discussion --- p.145 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.150 / References --- p.155 / Figures / Graphs

Detection of both biological and chemical environmental toxicants is essential in the assessment of risk to human health. Cell-based biosensors are capable of activity- based detection of toxicity. Chromatophore cells, responsible for the pigmentation of poikilothermic animal, have shown immense potential as cell-based biosensors in the detection of a broad range of environmental toxicants. Chromatophore cells possess the motile pigment granules that intracellularly aggregate or disperse in response to external stimuli. Previous studies have assessed chromatophore cells isolated from red Betta splendens and grey Oncorhynchus tschawytscha fish for use as a biosensor. The objective of this study was to describe blue B. splendens chromatophore cells in tissue culture. Blue B. splendens chromatophore cells were assessed for their longevity in tissue culture and their responses to previously established control agents. Blue B. splendens chromatophore cells were exposed to select chemicals and pathogenic bacteria to assess their ability to respond to environmental toxicants. Three concentrations of mercuric chloride, methyl mercuric chloride, paraquat, sodium arsenite, sodium cyanide chemicals were tested. Bacillus cereus, Bacillus subtilis, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Typhimurium were tested. Red B. splendens chromatophore cells were subjected to the select chemical and bacterial toxicants, and observed for their responses. The data collected in this and previous studies were compiled to compare chromatophore cell responses to a broad range of environmental toxicants. Chromatophore cells isolated from both blue and red B. splendens were responsive to methyl mercuric chloride and sodium arsenite. Grey O. tschawytscha chromatophore cells have shown responsiveness to mercuric chloride and sodium arsenite. Blue and red B. splendens chromatophore cells were both responsive to B. cereus and both Salmonella serovars. Grey O. tschawytscha have previously been shown to respond to B. cereus as well. In conclusion, this study reports the chromatophore cells isolated from blue B. splendens in tissue culture and showed similar responsiveness to the selected chemical and bacterial environmental toxicants as chromatophore cells isolated from red and grey colored fish. This study provides compelling evidence that the chromatophore response is not dependent on fish color and that chromatophore cells used for a cell-based detection system may be isolated from different colored fish. / Graduation date: 2012

Lymphomas and leukemias are the most common cancer in children and young adults and in utero exposure to carcinogens may contribute to the etiology of these cancers. A polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP), was administered to pregnant mice (15 mg/Kg b.w., gavage) on gestation day 17. Significant mortalities in young offspring were observed due to T-cell lymphoma. Lung and liver tumors also were observed in survivors at 10 months of age. To assess the role of the Aryl Hydrocarbon Receptor (AHR), we utilized crosses of B6129SF1/J (responsive) mice with strain 129S1/SvImJ (non-responsive). Offspring born to AHR non-responsive mothers had greater susceptibility to lymphoma, irrespective of offspring genotype. Responsive offspring displayed increased mortality if the mother was responsive. Lung adenomas showed Ki-ras mutations and exhibited a 50% decrease and a 35-fold increase in expression of Rb and p19/ARF mRNA, respectively. To examine the risk/benefit of maternal dietary phytochemical treatment against transplacental cancer, 2000 ppm indole-3-carbinol (I3C) was given to pregnant mice through diet from gestation day 9 till weaning. I3C significantly lowered mortality caused by lymphomas regardless of the maternal genotype, and also reduced lung tumor multiplicity in offspring born to AHR [superscript b-l/d] dams. Distribution of I3C in most maternal and fetal tissues was quantified following a single gavage of [¹⁴C]-I3C to the pregnant mice. DBP-DNA adducts were observed in both maternal and fetal tissues by ³³P postlabeling and HPLC analysis and were modulated by I3C and AHR genotype. I3C also modulated phase I and phase II enzyme protein expression in dams and gene expression in newborn thymus. I3C chemoprotection may involve modification of the bioavailability of DBP to the fetus and/or modulation of gene expression in the fetus as well. This is the first demonstration that transplacental exposure to an environmental PAH can induce a highly aggressive lymphoma in mice. These results raise the possibility that PAH exposures to pregnant women could contribute to similar cancers in children and young adults and, that the addition of chemoprotective agents to the maternal diet may reduce cancer risk among offspring. / Graduation date: 2006

During the past 30 years, numerous studies have focused on the toxicities of polycyclic aromatic hydrocarbons (PAH). Laboratory and field studies have helped elucidate the detrimental effects of these chemicals on growth, reproduction and immune response. Polycyclic aromatic hydrocarbons are in the priority list of chemicals to be studied by different governmental agencies and universities and understanding their mechanisms of action is the focus of the current research. The manuscripts presented in this dissertation are focused on the effects and mechanism of action of PAH on disease susceptibility. After a dietary exposure to PAH for up to 50 days (chapter II) and samplings after 3, 7, 14, 28 and 50 days, a number of biomarkers of PAH exposure were measured: Fluorescent aromatic compounds (FACs) in bile, ethoxyresorufin-o-deethylase (EROD) in liver microsomes, cytochrome P450 1A immunohistochemistry in liver and kidney and adduct formation in liver. Additionally markers of oxidative stress were measured: comet assay in blood, protein nitration in kidney and F2-isoprostanes in kidney. Oxidative stress was a probable factor in PAH induced responses in fish adapted to long-term PAH exposures and aryl hydrocarbon activation was not necessarily involved in this process. Disease challenge with Aeromonas salmonicida (chapter III) resulted in differences in mortalities that demonstrated that fish exposed to PAH were more susceptible to disease than fish not exposed to PAH. Determination of gene expression in head kidney of fish exposed and not exposed to PAH challenged with A. salmonicida using microarray and RT-PCR technologies 2, 4, 10 and 20 days after challenge (chapter IV), suggested that PAH exposure was associated with down regulation of interleukin 8, transport associated protein 1, NF-kB modulator, recombination activating gene and major histocompatibility complex II two days after challenge in fish exposed to PAH. The transcript levels were closer to control levels 20 days after challenge, this indicated a recovery from the effect of PAH exposure. / Graduation date: 2006

Foliar application of the phytotoxic herbicide diquat dibromide was made to twenty tuliptree seedlings actively growing under field conditions on October 6, 1973, at the Purdue Davis farm in Randolph County, Indiana. Herbicidal extraction was accomplished by two extraction procedures: Langlois, et. al., (1963), and a method described in the Pesticide Analytical Manual (9/1/67). Herbicidal application methods utilized two concentrations and three time periods for translocation to occur.Results from the herbicidal application to the tuliptree seedlings indicate that diquat dibromide is present in all parts of the seedling trees within a two hour time period, and that relative amounts of diquat per plant organ are time dependent.Ball State UniversityMuncie, IN 47306

Aflatoxin B1 (AFB1) and Fumonisin B1 (FB1), potentially cytotoxic and carcinogenic mycotoxins are common contaminants of agricultural commodities in South Africa and thus could be detrimental to the human immune system. Many of the cytotoxic effects of AFB1 require its bioactivation to an epoxide, which will bind covalently to macromolecules to form protein and DNA adducts. Fumonisin B1 is a competitive inhibitor of sphingosine and sphinganine N aceyltransferase, which are key components in the pathways for sphingolipid biosynthesis. Accumulation of free sphingoid bases, which are both cytotoxic and mitogenic, could provide a plausible explanation for the toxicity and carcinogenicity of FB1. The cytotoxic effects of AFB1 and FB1 on normal human lymphocytes, individually and in combination were assessed using the methylthiazol tetrazolium (MTT) bioassay. Two different methods of treatment were used, the treatment of isolated normal human lymphocytes for 12, 24, 48, 72 and 96 hours and whole blood treated for 12 hours. Flow cytometry and fluorescent microscopy were used to determine whether AFB1 and FB1 (5uM and 50uM), individually or in combination, were capable of inducing apoptosis, necrosis or nuclear fragmentation in isolated lymphocytes and whole blood treated for 12 hours. DNA damage was evaluated using the comet assay. The results showed that AFB1routinely induced higher levels of cytotoxicity in isolated lymphocytes than FB1. In the combination treatment, the mitogenic properties of FB1 appeared to partially counteract the cytotoxic effect exerted by AFB1. When whole blood was treated with the same concentration and ratio of toxin, FB1 was shown to be more cytotoxic than AFB1. The combination treatment of whole blood was shown to be cytotoxic in a dose dependent manner. The toxins appeared to exert a greater cytotoxic effect, when treated in combination than individually at higher concentrations. Aflatoxin B1 induced increased levels of apoptosis and necrosis in isolated lymphocytes while treatment with the FB1 resulted in increased levels of apoptosis at both concentrations. Treatment with the combination also resulted in increased levels of apoptosis. The levels of apoptosis were reduced in whole blood lymphocytes when compared to isolated lymphocytes. However, treatment with AFB1 and FB1 resulted in increased levels of apoptosis. Both AFB1 and FB1 are capable of inducing nuclear fragmentation. Treatment with FB1 (5uM and 50uM) resulted in greater degree of fragmentation than AFB1. The most nuclear fragmentation was induced by the 5uM combination treatment. The 50uM combination treatment of isolated lymphocytes induced the most DNA damage. As both toxins are common contaminants and have been known to coexist, this could be a potential area of concern for public health. / Thesis (M.Med.)-University of KwaZulu-Natal, 2004.

Selenium (Se), an essential element, can bioaccumulate and become toxic. The main toxicity symptom in fish, teratogenicity, is mediated by oxidative stress; however, little is known about the effect of Se on the physiological stress response (PSR). The effects of Se on the PSR and oxidative stress parameters were investigated in rainbow trout, brook trout, and white suckers. The PSR was activated by acute and sub-chronic laboratory exposures to NaSeO3, but not exposure to environmental levels of Se. Species-specific sensitivity to Se may be explained by biochemical differences. Rainbow trout (exhibit oxidative damage with Se exposure) have greater GSH reserves (cycles with Se causing oxidative stress) than brook trout. Selenium in agricultural drain water did not accumulate to dangerous levels in white suckers, but additional stressors (e.g. pesticides) were present. Differences in sensitivity to Se may alter competitive interactions between species, changing community compositions and putting additional pressure on threatened species. / xiii, 150 leaves ; 29 cm.