Examination of the swimming capabilities of fish is increasingly being considered as an effective method for determining sublethal toxicity. Acute toxicant exposure is known to cause decreases in swim performance in fish but less is known about how developmental exposure can cause persistent effects that hinder swimming. In addition, little is known about how triglyceride levels fluctuate during fish swimming upon both acute and developmental exposure to toxicant. In this thesis, two studies, one acute and one developmental, were carried out using two different toxicants in order to address these issues.<p> In order to examine acute effects, adult zebrafish (Danio rerio) were exposed to ethanol vehicle or increasing concentrations of 2,4-dinitrophenol (DNP), a mitochondrial electron transport chain uncoupler, for a 24 h period. Following exposure, fish were placed in a swim tunnel for critical swimming speed (Ucrit) determination and swim motion analysis. Whole body triglyceride levels were then determined. Ucrit was decreased in a concentration dependent manner in both the 6 mg/L and 12 mg/L DNP exposure groups, with 6 mg/L DNP being considered sublethal and 12 mg/L approaching the LC50. A decrease in tail beat frequency was observed and is likely the main cause for the decrease in Ucrit in the DNP exposure groups. Triglyceride levels were elevated in a concentration dependent manner in the DNP exposure groups. This increase in triglyceride stores may be due to a behavioral adaption limiting swimming capabilities or due to a direct toxic action of DNP on lipid catabolism.<p> The second study examined whether developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure would cause persistent toxic effects. Zebrafish embryos were exposed to dimethyl sulfoxide control or increasing concentrations of TCDD between 2-4 days post fertilization (dpf). At 5 dpf, cytochrome P450 1A (CYP1A) activity was determined. Fish were raised to 90 dpf with mortalities and deformities being recorded at 5 dpf, 10 dpf, and 90 dpf. At 90 dpf, fish were placed in swim tunnel and Ucrit , swimming motion, and aerobic scope (oxygen consumption rate during exercise minus oxygen consumption rate during rest) were determined. Following swimming, some fish were used for whole body triglyceride analysis while others were used for histological examination. Ucrit was shown to be decreased in the two highest sublethal TCDD exposure groups (0.1 and 1 ng/L) but not in the lowest TCDD exposure group (0.01 ng/L). The exact cause of the decrease in Ucrit is not known, but may be linked to the observed decrease in dorsal aorta diameter, an inability to mobilize triglyceride stores, behavioral adaptations limiting swimming, decreased body length, or a combination of these factors. This TCDD related defect in swimming ability is not due to any increases in gross deformity or mortality rates, nor does it appear that CYP1A induction is required to mediate the toxic effects. Thus, it appears that examination of swim performance may serve as an effective measure of both sublethal acute and developmental toxicities.

Examination of the swimming capabilities of fish is increasingly being considered as an effective method for determining sublethal toxicity. Acute toxicant exposure is known to cause decreases in swim performance in fish but less is known about how developmental exposure can cause persistent effects that hinder swimming. In addition, little is known about how triglyceride levels fluctuate during fish swimming upon both acute and developmental exposure to toxicant. In this thesis, two studies, one acute and one developmental, were carried out using two different toxicants in order to address these issues.<p> In order to examine acute effects, adult zebrafish (Danio rerio) were exposed to ethanol vehicle or increasing concentrations of 2,4-dinitrophenol (DNP), a mitochondrial electron transport chain uncoupler, for a 24 h period. Following exposure, fish were placed in a swim tunnel for critical swimming speed (Ucrit) determination and swim motion analysis. Whole body triglyceride levels were then determined. Ucrit was decreased in a concentration dependent manner in both the 6 mg/L and 12 mg/L DNP exposure groups, with 6 mg/L DNP being considered sublethal and 12 mg/L approaching the LC50. A decrease in tail beat frequency was observed and is likely the main cause for the decrease in Ucrit in the DNP exposure groups. Triglyceride levels were elevated in a concentration dependent manner in the DNP exposure groups. This increase in triglyceride stores may be due to a behavioral adaption limiting swimming capabilities or due to a direct toxic action of DNP on lipid catabolism.<p> The second study examined whether developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure would cause persistent toxic effects. Zebrafish embryos were exposed to dimethyl sulfoxide control or increasing concentrations of TCDD between 2-4 days post fertilization (dpf). At 5 dpf, cytochrome P450 1A (CYP1A) activity was determined. Fish were raised to 90 dpf with mortalities and deformities being recorded at 5 dpf, 10 dpf, and 90 dpf. At 90 dpf, fish were placed in swim tunnel and Ucrit , swimming motion, and aerobic scope (oxygen consumption rate during exercise minus oxygen consumption rate during rest) were determined. Following swimming, some fish were used for whole body triglyceride analysis while others were used for histological examination. Ucrit was shown to be decreased in the two highest sublethal TCDD exposure groups (0.1 and 1 ng/L) but not in the lowest TCDD exposure group (0.01 ng/L). The exact cause of the decrease in Ucrit is not known, but may be linked to the observed decrease in dorsal aorta diameter, an inability to mobilize triglyceride stores, behavioral adaptations limiting swimming, decreased body length, or a combination of these factors. This TCDD related defect in swimming ability is not due to any increases in gross deformity or mortality rates, nor does it appear that CYP1A induction is required to mediate the toxic effects. Thus, it appears that examination of swim performance may serve as an effective measure of both sublethal acute and developmental toxicities.

The century poison ¡§dioxins¡¨ are hydrophobic compounds that can combine with many organic matters and persist in the environment as well as to accumulate in living organisms. Dioxins caused great risk to the health of living organisms and to the entire ecological environment. We had isolated previously one bacterial species, Pseudomonas mendocina NSYSU, which can use pentachlorophenol (PCP) as its sole carbon source and degrade dioxin compounds. In order to study the feasibility of using this bacterial strain to bioremediate an PCDD/Fs polluted site, four microcosm experiment groups were designed to test the degradation efficiency of this strain: sterile soil group, non-sterile soil group, soya lecithin group and non-sterile soil with soya lecithin group. In addition, we also analyzed the shift of community structure of each microcosm by PCR-DGGE. The results show that the soya lecithin group has the highest efficiency to degrade OCDD/OCDF. After fifty days of reaction, the degradation rates of OCDD/OCDF were 62% and 47% respectively. The microbial diversity analysis indicated that the soya lecithin group presented less abundant from the initial stage, but increasing gradually over time. This might related to the formation of micelles in water phase which contained higher concentration of PCDD/Fs dissolved from the soil particles. Therefore, soya lecithin not only can reduce the toxicity of PCDD/Fs, but also can enhance the bioavailability of the organic pollutants to the microorganisms. In conclusion, monitoring the transition of P. mendocina NSYSU as well as the microbial diversity can provide valuable information during the bioremediation process by applying soya lecithin.

No description available.

The American eel (Anguilla rostrata) has suffered a serious population decline in Lake Ontario since the early 1980s due to a decline in recruitment of juveniles migrating from the Sargasso Sea. This has resulted in the closure of the Lake Ontario fishery in 2004 and its listing as endangered under the Ontario Species at Risk act in June of 2008 in Ontario. Due to their complex life cycle, little is known about eels once they leave their freshwater habitats and migrate to the Sargasso Sea to reproduce. Ocean conditions, habitat destruction, disease, reduced lipid content, over-fishing, physical barriers (hydroelectric dams), and chemical contamination are all possible reasons for recruitment decline and may be acting cumulatively. Maternally derived dioxin-like contaminants (DLCs) accumulated during the growth phase of eels in Lake Ontario are toxic to fish embryos, and embryotoxicity is expressed as a series of malformations known as blue sac disease (BSD). I assessed whether these toxicants are in high enough concentrations in sexually maturing, eels to be embryotoxic to their offspring, as assessed by using Japanese medaka (Oryzias latipes), a surrogate species. Medaka embryos were first injected with 2,3,7,8-tetracholordibenzo-p-dioxin (TCDD) to establish their sensitivity to this test chemical expressed as an 11-day EC50 of 3.79 pg/mg, for the induction of BSD. Medaka embryos were injected with eel extracts and their response compared to the TCDD toxicity curve to assess whether extracts caused developmental problems and to estimate the relative concentration of DLCs. Eel extracts from all collection sites caused no dioxin-like toxicity to Japanese medaka embryos. However, significantly higher toxicity at 10 eeq relative to triolein was found for all extracts with no differences among sites, suggesting the presence of non-dioxin-like toxicants. The low level of maternal tissue contamination by DLCs implied by this bioassay is mirrored in chemical monitoring of persistent organic pollutants in Lake Ontario fish. If correct, the low levels of toxicity of extracts to embryos could contribute to the observed increase of eels entering L. Ontario from 2003 to 2008. / Thesis (Master, Biology) -- Queen's University, 2010-09-01 09:31:19.466

The main goal of this thesis was to develop new methods and knowledge that will explain and predict species differences in sensitivity to dioxin-like compounds (DLCs) in birds. The important achievements and results obtained from the four experimental chapters of this thesis are summarized as follow: (1) an efficient luciferase reporter gene (LRG) assay was developed for use with 96-well cell culture plates; (2) the results obtained from LRG assay were shown to be highly correlated to available in ovo toxicity data; (3) amino acids at positions 324 and 380 within the aryl hydrocarbon receptor 1 ligand binding domain (AHR1 LBD) were shown to be responsible for reduced Japanese quail (Coturnix japonica) AHR1 activity to induce a dioxin-responsive reporter gene in comparison to chicken (Gallus gallus domesticus), and ring-necked pheasant (Phasianus colchicus) AHR1 in response to different DLCs; (4) AHR1 LBD sequences of 86 avian species were studied and differences at amino acid sites 256, 257, 297, 324, 337 and 380 were identified. It was discovered that only positions 324 and 380 play a role in AHR1 activity to induce a dioxin-responsive gene; (5) in COS-7 cells expressing chicken AHR1, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) are equipotent inducers of the reporter gene and bind with similar affinity to chicken AHR1, however, in the cells expressing pheasant, Japanese quail and common tern (Sterna hirundo) AHR1, PeCDF is a stronger inducer than TCDD. PeCDF also binds with higher affinity to pheasant and quail AHR1 than TCDD. The results of this thesis show that embryo lethal effect of DLCs in avian species can be predicted by use of two new non-lethal methods: (1) the LRG assay and (2) determination of the identity of the amino acids at positions 324 and 380. The findings and methods described in this thesis will be of use for environmental risk assessments of DLCs.

The circadian clock system is essential for mammals to adapt to environmental conditions such as light-dark cycles and to manage the optimal timing for cyclical physiological processes, including sleep-wakefulness, fasting-feeding and multiple aspects of metabolism. The circadian timing system is arranged in hierarchical fashion, with the master clock in the suprachiasmatic nucleus (SCN) of the hypothalamus, acting as the pace-maker and maintaining synchrony among clocks found in every organ system throughout the body. The core molecular clock consists of two interconnected transcriptional-translational feedback loops comprising core clock components: Brain and Muscle Arnt-Like protein1 (BMAL1), Circadian Locomoter Output Cycles Kaput (CLOCK), Period (PER), Cryptochrome (CRY), Nuclear Receptor family1 D1 (REV-ERB) and Retinoic acid-related Orphan Receptor (ROR). Circadian clock disruptions, through environmental changes to light-dark cycles or through genetic modification of core clock genes cause metabolic disturbances. Aryl hydrocarbon receptor (AhR), also known as the dioxin receptor, mediates systemic metabolism and toxicity of a range of environmental contaminants. Epidemiological studies have established a positive correlation between exposure to dioxins and other synthetic organic chemicals and metabolic diseases such as diabetes and dyslipidemia. Animal research have supported these findings by showing that AhR activation has detrimental effects on glucose and lipid homeostasis. Mechanisms for AhR-mediated metabolic dysfunction remain unknown. Coincidently, both AhR and many core clock components, for example BMAL1 and CLOCK, belong to the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) domain family. Previous studies have linked AhR signaling to circadian rhythm. Importantly, activation of the AhR can impair transcriptional activity of the CLOCK: BMAL1 heterodimer in cultured cells. However, because the AhR is differentially expressed among the body’s tissues, its activation may have distinctive, tissue-specific effects on the hierarchical circadian clock oscillators in vivo, which have not been investigated. Therefore, this dissertation is designed to examine the short-term and long-term effects of AhR activation on circadian clocks and downstream clock-regulated metabolic pathways. Specifically, this dissertation is aimed to explore how acute and chronic activation of AhR affects rhythmic aspects of behavior, as well as clock-controlled glucose and lipid metabolism. In the acute AhR activation model, a single dose of the synthetic AhR agonist, β-Naphthoflavone (BNF), was administered to C57Bl/6J wild type mice. Circadian behavior was monitored before and after acute AhR activation. Circadian expression of core clock genes, as well as key metabolic genes in the liver, skeletal muscle and adipose tissue were examined. Compared to the vehicle group, BNF-treated mice displayed a transient loss of behavioral rhythmicity and delayed activity onset, which suggest that acute activation of AhR acts directly on the central clock, the suprachiasmatic nucleus of the hypothalamus. In contrast, circadian oscillations of core clock genes were not eliminated in the peripheral tissues (liver, skeletal muscle and adipose tissue), but changes were observed in their rhythmic amplitude or phase. Rhythms of key enzymes related to glucose and lipid metabolic pathways in the liver and adipose were decreased while those in the skeletal muscle were increased. These results indicate that acute AhR activation affects the central clock and peripheral clock differently. Moreover, acute AhR activation significantly dampened the rhythm of genes involved in lipogenesis, lipolysis and lipid storage. In the chronic AhR activation model, C57Bl6/J mice were exposed to BNF for a month to explore whether long-term AhR activation can cause bigger disruption of circadian clocks and lead to metabolic dysfunction in vivo. Unexpectedly, general circadian behavior was maintained although after each dose of BNF there was a consistent, transient loss of behavioral rhythmicity and significant phase delay (about 30 minutes) in BNF-treated mice. Liver and skeletal muscle clocks were not significantly altered after 4 doses of BNF, and the in-phase oscillations of core clock genes in liver and skeletal muscle suggested a functional SCN as well as the two peripheral clocks. However, the adipose clock was significantly disrupted. Altered clock-regulated rhythms in lipid metabolism genes are associated with impaired lipid storage functions in white adipose tissues and deregulated plasma lipids in BNF-treated mice. The results of acute and chronic AhR activation support a significant interaction of AhR with the circadian clock system. Although future studies are needed to elucidate how AhR signaling specifically interacts with the clock in different cell types, the current research establishes a model for studying the crosstalk between AhR and circadian rhythm and provides new perspectives into the mechanisms of metabolic diseases correlated with exposure to synthetic organic chemicals.

Des études épidémiologiques ont montré que l’exposition à certains xénobiotiques est associée à une augmentation de la prévalence des maladies métaboliques. L’Homme est exposé à des mélanges de xénobiotiques de manière chronique et inévitable. Nous avons étudié les effets de l’interaction de deux xénobiotiques sur le métabolisme du foie, organe majeur de détoxification de l’organisme. Nous avons choisi deux perturbateurs endocriniens et polluants organiques persistants, qui activent des voies de signalisation différentes: la 2, 3, 7, 8 tétrachlorodibenzo-p-dioxine (TCDD), agissant via le récepteur aux hydrocarbures aromatiques (AhR), et l’α-endosulfan, un pesticide organochloré, qui peut agir via la voie du récepteur aux oestrogènes (ER) ou du récepteur X aux pregnanes (PXR). Notre objectif est de déterminer l’effet du mélange de ces polluants par rapport à chaque polluant isolé sur la régulation de certaines voies du métabolisme hépatique in vitro dans la lignée hépatocytaire humaine, HepaRG. Dans une première publication, une étude du transcriptome de cellules HepaRG différenciées a été effectuée. Ces cellules ont été exposées pendant 30 heures à 25nM de TCDD, 10μM d’α-endosulfan, ou au mélange. Nous avons observé que le mélange inhibe fortement l’expression de certains gènes impliqués dans le métabolisme glucidique et dans celui des alcools. Dans une seconde étude, nous avons donc étudié le mécanisme d’action du mélange sur le métabolisme glucidique. L’expression de deux gènes de la néoglucogenèse hépatique, le transporteur de glucose 2 (Glut2) et la glucose 6 phosphatase (G6Pc), est réduite de plus de 80% par le mélange. L’expression d’autres gènes du métabolisme glucidique (pyruvate kinase, glycogène synthase, glycogène phosphorylase, pyruvate déhydrogénase 2) est aussi diminuée, suggérant que le mélange peut affecter ce métabolisme de manière significative. De plus, la production de glucose diminue de 80% avec le mélange dans des conditions néoglucogéniques. En condition glycolytique, l’oxydation du glucose en CO2 diminue de 30% après 72 heures d’exposition au mélange. Un traitement à plus long terme (8 jours) avec des doses plus faibles des polluants (0.2 à 5nM de TCDD, 3μM d’α-endosulfan) diminue aussi l’expression de la G6Pc et de Glut2. Nous avons montré que la TCDD active bien la voie du AhR, et que le ER est impliqué dans l’action de l’α-endosulfan. Dans la troisième partie de cette thèse, nous avons étudié la régulation de plusieurs enzymes impliquées dans le métabolisme de l’alcool (alcool déshydrogénases, ADHs, cytochrome P450 2E1, CYP2E1) après l’activation du AhR. Les agonistes du AhR entrainent la diminution de l’expression des ARNm des ADH1, 4, 6 et du CYP2E1 et des protéines correspondantes. Nous avons montré que cette régulation utilise la voie génomique du AhR. De plus, cet effet est également observé après traitement de 8 jours par de faibles doses de TCDD. L’exposition chronique de l’Homme à de faibles doses de xénobiotiques en mélange pourrait affecter le métabolisme glucidique hépatique et contribuer, en partie, au développement du syndrome métabolique. / Epidemiological studies have shown that exposure to certain xenobiotics is associated with an increased prevalence of metabolic diseases. Humans are exposed to mixtures of xenobiotics in a chronic and inevitable way. We studied the effects of the interaction of two xenobiotics on metabolism in the liver, the major organ for detoxification in the body. We chose two endocrine disruptors and persistent organic pollutants which activate different signaling pathways: 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), which uses the AhR (Aryl hydrocarbon receptor) pathway, and α-endosulfan, an organochlorine pesticide, which acts via the PXR (pregnane X receptor) and/or the ER (estrogen receptor) pathway. Our aim was to determine the effects of this pollutant mixture, as compared to each pollutant alone, on the regulation in vitro of some hepatic metabolism pathways in the human hepatic cell line, HepaRG. In the first publication, a transcriptomic study of differentiated HepaRG cells was performed. The cells were exposed for 30h to 25nM TCDD, to 10 µM α-endosulfan or to the mixture. We observed that the mixture strongly inhibited the expression of some genes involved in the metabolism of glucose and alcohol. In the second study, we studied the mechanism of action of the mixture of pollutants on the metabolism of glucose. The expression of two genes involved in hepatic gluconeogenesis, glucose transporter 2 (GLUT2) and glucose-6-phosphatase (G6Pc), were reduced 80% by the mixture. The expression of other glucose metabolism genes (pyruvate kinase, glycogen synthase, glycogen phosphorylase, pyruvate dehydrogenase 2) also was decreased suggesting that the mixture might impact markedly carbohydrate metabolism. Furthermore, glucose production decreased 40% with the mixture under gluconeogenic conditions. Under glycolytic conditions, the oxidation of glucose into CO2 decreased 30% after 72h of exposure of the cells to the mixture. Long-term treatment (8 days) with lower doses (0.2 to 5 nM TCDD, 3 µM α-endosulfan) similarly decreased G6Pc and GLUT2 expression. We showed that TCDD activated the AhR pathway, and that ER was partly involved in the α-endosulfan effect. In the third part of this thesis, we studied the regulation of several enzymes involved in the metabolism of alcohol (alcohol dehydrogenase, ADH, cytochrome P450 2E1, CYP2E1) after activation of AhR. AhR agonists led to a decrease in the amounts of mRNAs for ADH1, 4, 6 and CYP2E1 and the corresponding proteins. We showed that this regulation uses the AhR genomic pathway. Furthermore, this effect was also observed after 8 days of treatment with lower doses of TCDD. Chronic exposure of individuals to low doses of xenobiotics in mixtures might significantly affect hepatic carbohydrate metabolism and be a contributing factor for the development of the metabolic syndrome.

The main goal of this thesis was to develop new methods and knowledge that will explain and predict species differences in sensitivity to dioxin-like compounds (DLCs) in birds. The important achievements and results obtained from the four experimental chapters of this thesis are summarized as follow: (1) an efficient luciferase reporter gene (LRG) assay was developed for use with 96-well cell culture plates; (2) the results obtained from LRG assay were shown to be highly correlated to available in ovo toxicity data; (3) amino acids at positions 324 and 380 within the aryl hydrocarbon receptor 1 ligand binding domain (AHR1 LBD) were shown to be responsible for reduced Japanese quail (Coturnix japonica) AHR1 activity to induce a dioxin-responsive reporter gene in comparison to chicken (Gallus gallus domesticus), and ring-necked pheasant (Phasianus colchicus) AHR1 in response to different DLCs; (4) AHR1 LBD sequences of 86 avian species were studied and differences at amino acid sites 256, 257, 297, 324, 337 and 380 were identified. It was discovered that only positions 324 and 380 play a role in AHR1 activity to induce a dioxin-responsive gene; (5) in COS-7 cells expressing chicken AHR1, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) are equipotent inducers of the reporter gene and bind with similar affinity to chicken AHR1, however, in the cells expressing pheasant, Japanese quail and common tern (Sterna hirundo) AHR1, PeCDF is a stronger inducer than TCDD. PeCDF also binds with higher affinity to pheasant and quail AHR1 than TCDD. The results of this thesis show that embryo lethal effect of DLCs in avian species can be predicted by use of two new non-lethal methods: (1) the LRG assay and (2) determination of the identity of the amino acids at positions 324 and 380. The findings and methods described in this thesis will be of use for environmental risk assessments of DLCs.

No description available.