Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "August 2000." Typescript. Vita. Includes bibliographical references (leaves 113-122). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 170-186).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 122-136).

Dioxins are environmental contaminants that raise concern because they are potent and persistent. The most potent dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes a wide variety of biochemical and toxic effects in laboratory animals and in humans. Major toxicities of TCDD are initiated by their binding to the AH receptor (AHR), a ligand-activated transcription factor that regulates expression of numerous genes. However, the specific genes whose dysregulation leads to major toxicities such as wasting, hepatotoxicity, and lethality are unknown. The objective of this thesis research was to identify the molecular mechanisms by which dioxins cause lethality. To this end, a powerful genetic rat model was utilized – the Han/Wistar (Kuopio) rat which is highly resistant to dioxin toxicity due to a major deletion in the AHR’s transactivation domain (TAD) leading to 3 potential AHR variant transcripts. We found that insertion-variant transcripts (IVs) are the dominant forms of AHR expressed in H/W rats, constitutively and after TCDD treatment. Gene expression array analysis revealed that the total number of TCDD-responsive genes in liver was significantly lower in H/W rats (that carry the TAD deletion) than in dioxin-sensitive rats (that carry wildtype AHR). Genes that are well-known to be AHR-regulated and dioxin-inducible  such as CYP1 transcripts  remained responsive to TCDD in H/W rats; thus the TAD deletion selectively interferes with expression of a subset of hepatic genes rather than abolishing global AHR-mediated responses. Genes that differed in response to TCDD between dioxin-sensitive rats and dioxin-resistant rats are integral parts of pathways known to be disrupted by dioxin treatment such as protein synthesis/degradation, fatty acid transport/metabolism, and apoptosis. These genes are worthy candidates for further mechanistic studies to test their role in major dioxin toxicities. Numerous differentially-regulated genes were downregulated; however, microRNAs, which downregulate mRNA levels in other systems, likely play no role in downregulation of mRNAs by dioxins in adult liver and are unlikely to be involved in hepatotoxicity. Findings in this research support the hypothesis that H/W rats are resistant to TCDD lethality because the TAD deletion prevents the AHR from dysregulating specific mRNA transcripts but not hepatic miRNAs.

Many physiological responses to dioxin-like compounds (DLCs), including polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are mediated by the aryl-hydrocarbon receptor (AhR). In birds, activation of the AhR stimulates the transcription of cytochrome P4501A (CYP1A) genes, including CYP1A4 and CYP1A5, and ultimately leads to expression of biotransformation enzymes, including ethoxyresorufin-O-deethylase (EROD). It is well established that potencies of different DLCs range over several orders of magnitude. There is also a wide variation among birds in their responsiveness to DLCs both in efficacy and threshold for effects. A molecular basis for this differential sensitivity has been suggested. Specifically, a comparison of the AhR ligand-binding domain (LBD) indicated that key amino acid residues are predictive of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) sensitivity. Based on sequencing of the AhR LBD from numerous avian species a sensitive classification scheme has been proposed (in order of decreasing sensitivity, chicken (type I; sensitive) > Common pheasant (type II; moderately sensitive) > Japanese quail (type III; insensitive)). A series of egg injection studies with White-leghorn chicken (<i>Gallus gallus domesticus</i>), Common pheasant (<i>Phasianus colchicus</i>) and Japanese quail (<i>Coturnix japonica</i>) were performed to determine whether molecular and biochemical markers of exposure to DLCs are predictive of the proposed classification scheme. In addition, I was interested in determining whether this classification scheme applies to other DLCs, specifically dibenzofurans. Determining which species are "chicken- like", "pheasant-like" and "quail-like" in their responses to DLCs should allow more refined risk assessments to be conducted as there would be less uncertainty about the potential effects of DLCs in those species for which population-level studies do not exist.<p> Several concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or 2,3,7,8-tetrachlorodibenzofuran (TCDF) (triolein vehicle) were injected into the air cells of Japanese quail, Common pheasant and chicken eggs. Liver from 14 d post-hatch chicks was harvested for analysis of CYP1A4 and CYP1A5 mRNA abundance by quantitative real-time PCR (Q-PCR), and EROD activity. Lowest observed effective concentration (LOEC) and relative potency (ReP) values for CYP1A mRNA abundance and EROD activity were determined and used to make comparisons of sensitivity between each species and DLC potency within each species.<p> The TCDD is widely considered to be the most potent DLC and this is supported by the rank order of LOEC values for CYP1A5 mRNA abundance in White-leghorn chicken (TCDD > PeCDF > TCDF). CYP1A4 mRNA abundance and EROD activity in White-leghorn chicken were significantly increased in the lowest dose exposure groups of each of the three DLCs, so the potency of these compounds based on these endpoints was not established. Interestingly, TCDD was not the most potent DLC in Common pheasant and Japanese quail. In Common pheasant, PeCDF is the most potent as a CYP1A4 mRNA inducer, followed by TCDD and TCDF. However, TCDF was the most potent EROD activity inducer for Common pheasant, followed by PeCDF, and then TCDD. No significant increases were found in CYP1A5 mRNA abundance in pheasant within the tested dose ranges for all the three DLCs. No significant increases in either CYP1A5 mRNA abundance or EROD activity were found in Japanese quail. In addition, PeCDF and TCDF, but not TCDD, significantly increased CYP1A4 mRNA abundance.<p> According to the predicted relative sensitivity by comparing the AhR LBD amino acid sequences, the White-leghorn chicken is more responsive to DLCs than the Common pheasant which is more responsive than the Japanese quail. By comparing the relative sensitivity calculated based on the LOEC values from my study, the sensitivity order to TCDD and TCDF support the proposed molecular based species sensitivity classification scheme (chicken > pheasant > quail), while pheasant is almost as sensitive as chicken to PeCDF ( pheasant ¡Ý chicken > quail).<p> Taken together, the data suggest that TCDD is the most potent DLC in White-leghorn chicken, but not in Common pheasant, or or Japanese quail. The data suggest that in type II avian species PeCDF may be more potent than TCDD. In addition, I found in my study that different biomarkers have different responses, which depends on species and chemicals as well. These data provide further insight into avian sensitivities to DLCs.</p>

Dioxins are environmental contaminants that raise concern because they are potent and persistent. The most potent dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes a wide variety of biochemical and toxic effects in laboratory animals and in humans. Major toxicities of TCDD are initiated by their binding to the AH receptor (AHR), a ligand-activated transcription factor that regulates expression of numerous genes. However, the specific genes whose dysregulation leads to major toxicities such as wasting, hepatotoxicity, and lethality are unknown. The objective of this thesis research was to identify the molecular mechanisms by which dioxins cause lethality. To this end, a powerful genetic rat model was utilized – the Han/Wistar (Kuopio) rat which is highly resistant to dioxin toxicity due to a major deletion in the AHR’s transactivation domain (TAD) leading to 3 potential AHR variant transcripts. We found that insertion-variant transcripts (IVs) are the dominant forms of AHR expressed in H/W rats, constitutively and after TCDD treatment. Gene expression array analysis revealed that the total number of TCDD-responsive genes in liver was significantly lower in H/W rats (that carry the TAD deletion) than in dioxin-sensitive rats (that carry wildtype AHR). Genes that are well-known to be AHR-regulated and dioxin-inducible  such as CYP1 transcripts  remained responsive to TCDD in H/W rats; thus the TAD deletion selectively interferes with expression of a subset of hepatic genes rather than abolishing global AHR-mediated responses. Genes that differed in response to TCDD between dioxin-sensitive rats and dioxin-resistant rats are integral parts of pathways known to be disrupted by dioxin treatment such as protein synthesis/degradation, fatty acid transport/metabolism, and apoptosis. These genes are worthy candidates for further mechanistic studies to test their role in major dioxin toxicities. Numerous differentially-regulated genes were downregulated; however, microRNAs, which downregulate mRNA levels in other systems, likely play no role in downregulation of mRNAs by dioxins in adult liver and are unlikely to be involved in hepatotoxicity. Findings in this research support the hypothesis that H/W rats are resistant to TCDD lethality because the TAD deletion prevents the AHR from dysregulating specific mRNA transcripts but not hepatic miRNAs.

Persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs) are widespread in aquatic systems. These toxicants bioaccumulate in the tissues of aquatic organisms, especially fish as they occupy a position near the top of the aquatic food web. Teleost fish respond to stressors, including toxicants, by activating a co-ordinated network of adaptive responses, collectively termed the integrated stress response, which allows animals to regain homeostasis. Depending on the nature of the stressor, this stress response may be a generalised endocrine response that occurs at the organismal level and/or a cellular response involving protein synthesis. The cellular response to PCB insult involves aryl hydrocarbon receptor (AhR) activation and the induction of biotransformation enzymes, including cytochrome P4501A (Cyp1A). However, little is known about the mode of action of PCBs in affecting the adaptive stress response in animals. The objective of this thesis was to investigate the role played by AhR in mediating PCB impact on the highly conserved physiological responses to secondary stressors in fish. The experimental approach involved whole animal exposure studies with PCBs both in a laboratory setting as well as using feral fish. Also, in vitro mechanistic studies with pharmacological agents [AhR agonist (β-naphthoflavone) and antagonist (resveratrol), Hsp90 inhibitor (geldanamycin), proteasomal inhibitor (MG-132) and transcription (Actinomycin D) and translational inhibitors (cycloheximide D)] were carried out to understand AhR regulation in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. Also, a targeted trout cDNA microarray was developed as a tool to identify stress-responsive genes and signaling networks in fish. Short-term (3 day) exposure to PCBs, while inducing liver AhR and Cyp1A expression, did not modify the adaptive plasma cortisol response to an acute handling disturbance in rainbow trout. However, PCBs exposure did modify the metabolic response that is critical for recovery from an acute stressor in rainbow trout. To assess the impact of chronic PCB exposure on cellular stress response, two feral populations of Arctic char (Salvelinus alpinus) from Bjørnøya Island, Norway, were utilized. This is because the average PCB load in char liver from Lake Ellasjøen was approximately 25-fold higher than in individuals from Lake Øyangen, providing a natural setting to compare long-term toxicant impact on stress proteins. Liver Cyp1A expression was elevated in the high PCB fish suggesting AhR activation. Changes in mRNA abundance and/or protein expression of glucocorticoid receptor (GR), heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) in fish from the high PCB lake leads to the proposal that chronic exposures to PCBs is proteotoxic to the fish. In vitro mechanistic studies with trout hepatocytes revealed for the first time that AhR is autoregulated in response to ligand activation in rainbow trout. Furthermore this AhR regulation as well as AhR signaling involves both the molecular chaperone Hsp90 and the proteasome in hepatocytes. AhR signaling appears to play a role in the cellular response to heat shock in trout hepatocytes. Specifically, AhR signaling appears to be involved in the heat shock-induced Hsp70 and Hsp90 protein expression in trout hepatocytes. This modulation of Hsps by AhR may involve the proteasome. Overall, the results point to a cross-talk between the AhR and Hsps signaling pathways, while the precise mechanism(s) remains to be elucidated. A targeted rainbow trout cDNA microarray was constructed as a tool to identify stress-responsive genes in trout. This custom cDNA array consisted of 147 rainbow trout genes designed from conserved regions of fish sequences available in GenBank. The targeted genes had established roles in physiological processes, including stress and immune function, growth and metabolism, ion and osmoregulation and reproduction. This targeted array revealed changes in gene expression suggesting a rapid liver molecular reprogramming as critical for the metabolic adjustments to an acute stressor in fish. Also, transcripts not previously implicated in the stress response process in fish, including genes involved in immune function and protein degradative pathways, were found to be stress-responsive. Many of these transiently elevated stress-responsive transcripts were also shown previously to be glucocorticoid-responsive in fish implicating a key role for genomic cortisol signaling in stress adaptation. Overall, this thesis demonstrates that PCBs impact the organismal and cellular stress response in fish. AhR autoregulation may be a key aspect of PCBs impact on the cellular stress response pathways. Hsp90 and the proteasome may be involved in AhR regulation and PCB-mediated signaling in fish. The results suggest a cross-talk between AhR and Hsp signaling pathways in fish. Finally, the targeted cDNA microarray will be a useful tool to further expand our knowledge on PCBs impact on the cellular stress signaling pathways in fish.

Persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs) are widespread in aquatic systems. These toxicants bioaccumulate in the tissues of aquatic organisms, especially fish as they occupy a position near the top of the aquatic food web. Teleost fish respond to stressors, including toxicants, by activating a co-ordinated network of adaptive responses, collectively termed the integrated stress response, which allows animals to regain homeostasis. Depending on the nature of the stressor, this stress response may be a generalised endocrine response that occurs at the organismal level and/or a cellular response involving protein synthesis. The cellular response to PCB insult involves aryl hydrocarbon receptor (AhR) activation and the induction of biotransformation enzymes, including cytochrome P4501A (Cyp1A). However, little is known about the mode of action of PCBs in affecting the adaptive stress response in animals. The objective of this thesis was to investigate the role played by AhR in mediating PCB impact on the highly conserved physiological responses to secondary stressors in fish. The experimental approach involved whole animal exposure studies with PCBs both in a laboratory setting as well as using feral fish. Also, in vitro mechanistic studies with pharmacological agents [AhR agonist (β-naphthoflavone) and antagonist (resveratrol), Hsp90 inhibitor (geldanamycin), proteasomal inhibitor (MG-132) and transcription (Actinomycin D) and translational inhibitors (cycloheximide D)] were carried out to understand AhR regulation in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. Also, a targeted trout cDNA microarray was developed as a tool to identify stress-responsive genes and signaling networks in fish. Short-term (3 day) exposure to PCBs, while inducing liver AhR and Cyp1A expression, did not modify the adaptive plasma cortisol response to an acute handling disturbance in rainbow trout. However, PCBs exposure did modify the metabolic response that is critical for recovery from an acute stressor in rainbow trout. To assess the impact of chronic PCB exposure on cellular stress response, two feral populations of Arctic char (Salvelinus alpinus) from Bjørnøya Island, Norway, were utilized. This is because the average PCB load in char liver from Lake Ellasjøen was approximately 25-fold higher than in individuals from Lake Øyangen, providing a natural setting to compare long-term toxicant impact on stress proteins. Liver Cyp1A expression was elevated in the high PCB fish suggesting AhR activation. Changes in mRNA abundance and/or protein expression of glucocorticoid receptor (GR), heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) in fish from the high PCB lake leads to the proposal that chronic exposures to PCBs is proteotoxic to the fish. In vitro mechanistic studies with trout hepatocytes revealed for the first time that AhR is autoregulated in response to ligand activation in rainbow trout. Furthermore this AhR regulation as well as AhR signaling involves both the molecular chaperone Hsp90 and the proteasome in hepatocytes. AhR signaling appears to play a role in the cellular response to heat shock in trout hepatocytes. Specifically, AhR signaling appears to be involved in the heat shock-induced Hsp70 and Hsp90 protein expression in trout hepatocytes. This modulation of Hsps by AhR may involve the proteasome. Overall, the results point to a cross-talk between the AhR and Hsps signaling pathways, while the precise mechanism(s) remains to be elucidated. A targeted rainbow trout cDNA microarray was constructed as a tool to identify stress-responsive genes in trout. This custom cDNA array consisted of 147 rainbow trout genes designed from conserved regions of fish sequences available in GenBank. The targeted genes had established roles in physiological processes, including stress and immune function, growth and metabolism, ion and osmoregulation and reproduction. This targeted array revealed changes in gene expression suggesting a rapid liver molecular reprogramming as critical for the metabolic adjustments to an acute stressor in fish. Also, transcripts not previously implicated in the stress response process in fish, including genes involved in immune function and protein degradative pathways, were found to be stress-responsive. Many of these transiently elevated stress-responsive transcripts were also shown previously to be glucocorticoid-responsive in fish implicating a key role for genomic cortisol signaling in stress adaptation. Overall, this thesis demonstrates that PCBs impact the organismal and cellular stress response in fish. AhR autoregulation may be a key aspect of PCBs impact on the cellular stress response pathways. Hsp90 and the proteasome may be involved in AhR regulation and PCB-mediated signaling in fish. The results suggest a cross-talk between AhR and Hsp signaling pathways in fish. Finally, the targeted cDNA microarray will be a useful tool to further expand our knowledge on PCBs impact on the cellular stress signaling pathways in fish.

Many physiological responses to dioxin-like compounds (DLCs), including polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are mediated by the aryl-hydrocarbon receptor (AhR). In birds, activation of the AhR stimulates the transcription of cytochrome P4501A (CYP1A) genes, including CYP1A4 and CYP1A5, and ultimately leads to expression of biotransformation enzymes, including ethoxyresorufin-O-deethylase (EROD). It is well established that potencies of different DLCs range over several orders of magnitude. There is also a wide variation among birds in their responsiveness to DLCs both in efficacy and threshold for effects. A molecular basis for this differential sensitivity has been suggested. Specifically, a comparison of the AhR ligand-binding domain (LBD) indicated that key amino acid residues are predictive of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) sensitivity. Based on sequencing of the AhR LBD from numerous avian species a sensitive classification scheme has been proposed (in order of decreasing sensitivity, chicken (type I; sensitive) > Common pheasant (type II; moderately sensitive) > Japanese quail (type III; insensitive)). A series of egg injection studies with White-leghorn chicken (<i>Gallus gallus domesticus</i>), Common pheasant (<i>Phasianus colchicus</i>) and Japanese quail (<i>Coturnix japonica</i>) were performed to determine whether molecular and biochemical markers of exposure to DLCs are predictive of the proposed classification scheme. In addition, I was interested in determining whether this classification scheme applies to other DLCs, specifically dibenzofurans. Determining which species are "chicken- like", "pheasant-like" and "quail-like" in their responses to DLCs should allow more refined risk assessments to be conducted as there would be less uncertainty about the potential effects of DLCs in those species for which population-level studies do not exist.<p> Several concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or 2,3,7,8-tetrachlorodibenzofuran (TCDF) (triolein vehicle) were injected into the air cells of Japanese quail, Common pheasant and chicken eggs. Liver from 14 d post-hatch chicks was harvested for analysis of CYP1A4 and CYP1A5 mRNA abundance by quantitative real-time PCR (Q-PCR), and EROD activity. Lowest observed effective concentration (LOEC) and relative potency (ReP) values for CYP1A mRNA abundance and EROD activity were determined and used to make comparisons of sensitivity between each species and DLC potency within each species.<p> The TCDD is widely considered to be the most potent DLC and this is supported by the rank order of LOEC values for CYP1A5 mRNA abundance in White-leghorn chicken (TCDD > PeCDF > TCDF). CYP1A4 mRNA abundance and EROD activity in White-leghorn chicken were significantly increased in the lowest dose exposure groups of each of the three DLCs, so the potency of these compounds based on these endpoints was not established. Interestingly, TCDD was not the most potent DLC in Common pheasant and Japanese quail. In Common pheasant, PeCDF is the most potent as a CYP1A4 mRNA inducer, followed by TCDD and TCDF. However, TCDF was the most potent EROD activity inducer for Common pheasant, followed by PeCDF, and then TCDD. No significant increases were found in CYP1A5 mRNA abundance in pheasant within the tested dose ranges for all the three DLCs. No significant increases in either CYP1A5 mRNA abundance or EROD activity were found in Japanese quail. In addition, PeCDF and TCDF, but not TCDD, significantly increased CYP1A4 mRNA abundance.<p> According to the predicted relative sensitivity by comparing the AhR LBD amino acid sequences, the White-leghorn chicken is more responsive to DLCs than the Common pheasant which is more responsive than the Japanese quail. By comparing the relative sensitivity calculated based on the LOEC values from my study, the sensitivity order to TCDD and TCDF support the proposed molecular based species sensitivity classification scheme (chicken > pheasant > quail), while pheasant is almost as sensitive as chicken to PeCDF ( pheasant ¡Ý chicken > quail).<p> Taken together, the data suggest that TCDD is the most potent DLC in White-leghorn chicken, but not in Common pheasant, or or Japanese quail. The data suggest that in type II avian species PeCDF may be more potent than TCDD. In addition, I found in my study that different biomarkers have different responses, which depends on species and chemicals as well. These data provide further insight into avian sensitivities to DLCs.</p>

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 113-122). Also available on the Internet.