Fish are exposed to multiple stressors in their environment. The interactive effects of pesticide exposure and increased temperature on the physiological stress response were investigated in a comparative field study with cold-water (whitefish, Prosopium williamsoni) and cool-water (sucker, Catostomus) fish from the Oldman River, Alberta, Canada, and in a laboratory study with rainbow trout, Oncorhynchus mykiss. Physiogical stress indicators were measured, and exposure to pesticides was estimated using acetylcholinesterase (AChE) inhibition. Species-specific differences in AChE activities and responses of the physiological stress axis were detected in whitefish and suckers, suggesting that whitefish are a more sensitive species to temperature and pesticide stress. In vivo Dimethoate exposure inhibited AChE activity in various tissues and disrupted the physiogical stress response. Commercial Dimethoate, in vitro, caused a decrease in viability and cortisol secretion while pure grade Dimethoate did not. The results from this study can be used in predictions of fish vulnerability to stress. / ix, 137 leaves : ill. ; 29 cm.

Two factorial experiments were conducted to determine if high dietary fluoride would inhibit selenite toxicity in rats. In each study, two levels of selenite (0.05 and 5 mg/kg diet) were matched against two levels of fluoride (1 and 150 mg/kg diet) for either 6 or 8 weeks. Fluoride failed to prevent the depressive effect of selenite on food intake and body weight gain in either study. Although liver selenium concentration was slightly (15%) but significantly (P < 0.005) reduced when the highest fluoride and selenium level were combined in the first study, this effect could not be repeated. These three measures therefore failed to provide evidence for a fluoride and selenium interaction. Fluoride, however, prevented hepatic necrosis seen in most of the selenite-toxic rats. Hepatic lesions seen histologically in selenite-toxic rats were not observed for either kidney or heart. With regard to a possible mechanism for the fluoride effect upon selenite liver pathology, fluoride partially (26%) but significantly (P < 0.025) reduced thiobarbituricreactive substances (an indicator of peroxidative cell membrane damage) in selenite-toxic rats, but there was no fluoride effect on an enzyme system (liver xanthine oxidase) that potentially could generate an initiator of lipid peroxidation. In agreement with results of others, fluoride deposition into bone was inconsistently affected by selenite, Overall, the protective effect of fluoride on selenite toxicity appears to be confined to liver pathology. The exact mechanism for this effect, however, remains unclear. / Graduation date: 1992

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1985. / Bibliography: leaves 210-235. / Photocopy. / Microfilm. / xiv, 235 leaves, bound 29 cm

Made available in DSpace on 2016-05-17T16:51:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-06-29. Added 1 bitstream(s) on 2016-05-17T16:54:46Z : No. of bitstreams: 1 000863477.pdf: 1608655 bytes, checksum: b6c6f0d441edc838abf3acc4f855cf67 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / FAPESP: 11/09870-2

Orientador: João Lauro Viana de Carvalho / Coorientador: Carla Adriane da Silva Franchi / Banca: Patrícia Garcia / Banca: Juliana Perobelli / Banca: Antonio Francisco Godinho / Banca: Maria Lucia Zaidan Dagli / Resumo: Não disponível / Abstract: Not available / Doutor

Investigations of three low-water requiring landscape species produced the following results: (1) Although Calliandra californica flowered under photoperiods from 12 to 16 hours, plants produced more elongation under 16 hour days. Plants grew taller and developed greener foliage under irradiances of 600 μmol·m⁻²·s⁻¹. Branching was not stimulated by foliar sprays of BA, PBA, or BA + GA₄₊₇. (2) Caesalpinia pulcherrima increased biomass under 16 hour days, but were stunted and chlorotic under irradiances of 1950 μmol·m⁻²·s⁻¹. Internodal lengths were restricted with drenches of 3.75 mg · pot-1 uniconazole, and plants sprayed with 500 mg·liter⁻¹ PBA developed more axillary branching without negative elongating effects. (3) Justicia specigera gained more height under 12 hour days, and produced greener foliage, more elongation, and faster flowering under irradiances of 600 μmol·m⁻²·s⁻¹. Plants showed restricted internodal elongation and fewer flowers when drenched with 5.0 mg·pot⁻¹ uniconazole, and developed more axillary branching with no detrimental elongation effects when sprayed with 100 mg·liter⁻¹ BA + GA₄₊₇.

This dissertation examines the ability of in vitro biotransformation assays to provide an indication of metabolic potential. The potential for xenobiotic compounds to bioconcentrate in aquatic organisms is expressed through the bioconcentration factor (BCF). The metabolic loss of ibuprofen, norethindrone and propranolol was measured using rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) gill and liver S9 fractions, microsomes and cell suspensions. Metabolic transformation rates (kM) were extrapolated from in vitro intrinsic clearance of parent compound (CLm) and integrated into a refined BCF model. In general, CLm of test compounds was greater in liver S9 fractions and hepatocytes. However, the influence of hepatic metabolism on kM and BCF was limited by hepatic blood flow (20-25%) compared to gill blood flow (~100%). A significant difference was noted between BCF solely based on KOW and BCF including kM. These studies indicate that the inclusion of kM in BCF models can bring predicted bioconcentration estimates closer to in vivo values. Primary cell suspensions are preferred over subcellular fractions as cell suspensions possess both phase I and phase II enzyme activity. Further study was conducted on ibuprofen biotransformation pathways. As fish do not contain the same cytochrome P450 (CYP) 2C homologs known to metabolize ibuprofen in mammals, it cannot be assumed that piscine biotransformation is similar. Metabolite analysis found 2-hydroxy-ibuprofen as the major metabolite in S9 and microsomal fractions. Additional assays involving the induction and inhibition of specific CYP isozymes support CYP1A2 as an alternative metabolic pathway.

We also investigated whether BDE-47 exposure could worsen PD situation by applying transgenic Drosophila (fly) model in which human α-synuclein (α-syn) was overexpressed in wide-type fly to simulate PD. BDE-47 (0, 2, 10 and 50 µM) was fed to flies continuously for 30 days. Integrated LC-MS and GC-MS profiling indicated metabolic changes in tryptophan, phenylalanine, purine, and alanine, aspartate and glutamate pathways, similar to those from mouse experiment. After quantified metabolites of interest by LC-triple quadrupole MS, we confirmed the slowed-down formation of KYNA (kynurenic acid, a neuro-protector) and speeded-up formation of 3HKYN (3-hydroxykynurenine, a neurotoxin) in all BDE-47 exposed groups on the 20th exposure day. The levels of SAM/SAH (methylation biomarker) and GSH/GSSG (oxidative stress biomarker) were found to decrease on the 30th exposure day. Collectively, we propose that BDE-47 could induce imbalance of kynurenine metabolism, insufficient methylation and oxidative stress, which might contribute to the PD progression. To further explore the underlying mechanism of 6-OH-BDE-47 induced neurotoxicity, we conducted omics study of metabolic changes induced by 6-OH-BDE-47 on N2a cells. Cells were exposed to 6-OH-BDE-47 (0, 0.5 and 1 μM) for 24 hours. Considerable metabolic changes in pyrimidine and purine metabolism were observed in high exposure condition while oxidative stress was appeared under low exposure condition. Moreover, 6-OH-BDE-47 was found to affect the dopamine production. iTRAQ proteomics was carried out and pinpointed the dysregulation of ribosome, proteasome, RNA metabolism, aminoacyl-tRNA biosynthesis, vesicular trafficking, purine pathway, and mitochondria electron transport. Immunocytochemistry and Western blot analysis further confirmed that 6-OH-BDE-47 could inhibit autophagy flux, which might result in the aberrant protein aggregation, a pathological hallmark of PD. We further investigated whether 6-OH-BDE-47 exposure could directly induce PD pathology in Sprague Dawley rat. 6-OH-BDE-47 (0.1, 1 and 10 µg) was stereotaxically injected into the right VTA and SNc regions in the midbrain of rat where there are abundant dopaminergic neurons. The apomorphine-induced rotation test indicated significant deterioration in motor function in the group receiving injection of 10 µg. Striatal dopamine was found to decline in a dose-dependent manner. Notably, 6-OH-BDE-47 also promoted the formation of α-syn aggregate, an important pathological hallmark of PD. Proteomics study revealed that protein degradation processes were crucial rather than oxidative stress in 6-OH-BDE-47 induced neurotoxicity in vivo. Mechanistic study based on Western blot further confirmed that 6-OH-BDE-47 could inhibit ubiquitination and autophagy. Collectively, the rat experiment demonstrated that 6-OH-BDE-47 administration could induce motor defect by impairing dopaminergic system and promote α-syn aggregation by inhibiting ubiquitination and autophagy, suggesting that 6-OH-BDE-47 could be a novel risk factor of PD.;Polybrominated diphenyl ethers (PBDEs), as one typical persistent organic pollutants (POPs), are widely spread in the environment and pose potential adverse impacts on human health. As a predominant congener of PBDEs, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) has been reported to affect habituation capability, synaptic plasticity, and vesicular neurotransmitter release. As an important in vivo metabolite derived from BDE-47, 6-hydroxy-BDE-47 (6-OH-BDE-47) was also reported as a neurotoxin. However, the possible linkages between BDE-47/6-OH-BDE-47 exposure and typical neurodegenerative diseases such as Parkinson's disease (PD) are still unclear. Mass spectrometry (MS) based omics integrated with bioinformatics is emerging as a powerful tool to evaluate metabolic changes occurred after different exposures. Here we developed non-targeted metabolomics, lipidomics, and isobaric tag for relative and absolute quantitation (iTRAQ) proteomics methods based on liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) to depict BDE-47/6-OH-BDE-47 induced metabolic changes and to explore the possible contribution of their exposure to PD pathology/pathogenesis. BDE-47 dissolved in corn oil (0, 1, 10 and 100 mg/kg bwt) was orally administered to adult male C57BL/6 mice for 30 consecutive days. Results of global metabolomics and lipidomics studies of PD-related brain regions based on LC-orbitrap MS revealed significant metabolite changes between the exposed and control groups in purine pathway, glutathione pathway, tryptophan pathway, phenylalanine pathway, alanine, aspartate and glutamate pathway, and lipid composition, mainly involved in oxidative stress and neurotransmitter production. By further quantifying metabolites involved in tryptophan and phenylalanine pathways in mice serum, colon and brain samples by using LC-triple quadrupole MS, dysregulation of PD linked neurotransmitters dopamine and serotonin were confirmed. iTRAQ proteomics study of the striatum, the part of the brain that is most intensively studied in PD pathogenesis, revealed that BDE-47 could induce neurotransmitter system disturbance, mitochondrial dysfunction, oxidative stress and abnormal phosphorylation. Oxygen consumption rate after BDE-47 treatment (0, 1 and 10 μM) in mouse neuroblastoma (N2a) cells was measured for the confirmation. BDE-47 was demonstrated to impair mitochondrial function.

Indiana University-Purdue University Indianapolis (IUPUI) / Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants suspected to act as endocrine disruptors. We tested the commercial PBDE mixture DE-71 and its in vivo metabolites for estrogenic activity. MCF-7 breast cancer cells culture, ERE-luciferase gene expression, 3H-β-estradiol displacement from recombinant ERα, and ovariectomized (OVX) mice served as bioassays. Although DE-71 did not bind ERα, it was able to increase MCF-7 cell proliferation and this was prevented by the antiestrogen fulvestrant. DE-71 co-treatment reduced the effect of estradiol in MCF-7 cells. In the OVX mouse (BALB/c) 3-day assay, DE-71 administered alone had no effect on uterine or vaginal tissues but when administered subcutaneously potentiated estradiol’s effect on uterine weight in a dose-dependent manner. DE-71 administered SQ to BALB/c mice for 34 days slightly increased uterine epithelial height (UEH), vaginal epithelial thickness (VET) and mammary ductal lumen area, and attenuated the estradiol-induced increase in UEH; these effects were not seen in C57BL/6 mice. DE-71 increased liver weight in BALB/c, C57BL/6 and estrogen receptor-alpha knockout (ERαKO) mice. Liver cytochrome P450 1A (CYP1A) and CYP2B activities increased 2.5-fold and 7-fold respectively when DE-71 was administered PO, but only CYP2B increased (5-fold) after SQ treatment. Six OH-PBDE metabolites were found in mice after 34-day DE-71 treatment and all were able to bind recombinant ERα. Para-hydroxylated metabolites displayed a 10- to 30-fold higher affinity for ERα compared to ortho-hydroxylated PBDEs. Para-OH-PBDEs induced ERE-luciferase and produced an additive effect when coadministered with β-estradiol. DE-71 was also additive with β-estradiol. At high concentrations (≥ 5x10-5 M), ortho-OH-PBDEs were antiestrogenic in the ERE-luciferase assay. In conclusion, DE-71 behaves as a weak estrogen in both MCF-7 breast cancer cells and ovariectomized adult mice. Mice strain, treatment route and duration determined if DE-71 was estrogenic. BALB/c mice are more susceptible to DE-71 effects in estrogen target tissues than C57BL/6 mice. DE-71 increased liver weight, 5%-51% depending on mouse strain and treatment regime, independently of ERα. The observations that the DE-71 mixture does not displace 3H-β-estradiol from ERα while the hydroxylated metabolites do, suggest that the cellular and tissue effects were due to a metabolic activation of individual congeners.

In the current studies the effects of administration of 0.1 to 1000 ppb of aldicarb, a carbamate pesticide, on the immune system of C3H mice were investigated. It was observed that aldicarb caused significant immunomodulation of macrophage functions analyzed in a variety of different systems. Initially, it was found that aldicarb decreased the stimulatory functions of the macrophages as studied by decreased capacity to stimulate normal autoreactive TF cells in the SMLR. This decreased stimulatory activity of the macrophages was found not to be due decrease in the expression of class II MHC-antigens (la molecules) nor was it due to the generation of any suppressor macrophages acting to down-regulate the immune response. Further investigations revealed that the decreased stimulatory activity of the macrophages correlated with decreased IL-1 production/signal to the T cells by the macrophages. It was also evident that aldicarb did not affect the T cell functions directly. Thus, T cells from aldicarb-treated mice when studied in the SMLR and AlloMLR or when stimulated with ConA or anti-CD3 mAbs, in the presence of normal macrophages, demonstrated normal responses. In contrast, normal T cells exhibited decreased responsiveness in the presence of aldicarb-treated macrophages. The fact that aldicarb did not affect the T cell functions directly was also evident by the fact that aidicarb-treated T cells could respond normally to stimulation with PMA + Ca²⁺ ionophore, a response which is independent of accessory cells. The aldicarb-treated macrophages also exhibited decreased capacity to process and present the antigen, conalbumin, to the T helper cell clone D10.G4. When the mechanism of aldicarb induced defect was investigated, it was observed that aldicarb-treated macrophages produced decreased amounts of IL-1 which was also confirmed by complete reconstitution of the response following addition of exogenous IL-1. With this in mind, macrophage functions in a number of other systems were examined and demonstrated that aldicarb-treatment also suppressed the macrophage-mediated cytotoxicity of tumor cells, but failed to inhibit the NK cell-mediated cytotoxicity of tumor cells. Together, these studies suggest that aldicarb selectively affects the macrophage but not NK or T ceil functions directly. However, since macrophages play an important role as accessory cells in T cell-mediated responses, it is likely that aldicarb indirectly will also affect the T cell responses. / Master of Science