The sediments of Bayou Trepagnier, at the western border of the La Branche wetlands, were contaminated with heavy metals in industrial waste water discharged before the Clean Water Act. Our study of the Pb, Cr, Cu, and Zn content of sediments and associated pore waters at 27 sites along the bayou began in June of 1994. At the beginning of this study, cooling water and storm water runoff from the Shell Norco Manufacturing Complex entered the bayou at its headwaters. This effluent was diverted to the Mississippi River in late February of 1995 as required by the EPA. The effluent, which is warm and slightly brackish, counted for only a small fraction of the water feeding the bayou with the major portions being rainwater runoff from the neighboring swamp along with tidal and wind-driven inputs from Lake Pontchartrain. A profile of the conditions of the bayou in 1994, along with updated monthly analyses for metals dissolved in bottom water samples before and since the diversion, will be presented. It appears that the change in water chemistry due to the removal or the plant discharge has not caused a significant release of contaminates currently sequestered in the sediments / acase@tulane.edu

<p>The liver is a primary target for toxicants and/or their metabolites. Selected fish species now serve as model organisms for laboratory investigations of toxic responses in the liver. One such model is the Japanese medaka (<italic>Oryzias latipes</italic>), a small freshwater teleost with a robust history of usage in liver and biliary toxicity studies. The structural components of the medaka hepatobiliary system have been well-described by recent studies in two- and three-dimensional contexts, but efforts to characterize the molecular mechanisms underlying critical medaka liver functions during normalcy remain sparse. This dearth of information makes it difficult to definitively characterize toxic responses in this model organism. A crucial transcription factor underlying proper hepatobiliary function in both mammalian and non-mammalian species is the farnesoid X receptor alpha (FXR&alpha;), a member of the nuclear receptor superfamily that plays a key role in bile acid homeostasis. This dissertation describes the function of medaka fxr&alpha; during both normalcy and toxicity.</p><p>To achieve this overall objective, <italic>in vitro</italic> techniques were first employed to study the function of medaka <italic>fxr&alpha;</italic>. Two isoforms of <italic>fxr&alpha;</italic> that differ in the AF1 domain, Fxr&alpha;1 and Fxr&alpha;2, were isolated from liver cDNA and are the result of alternative splicing of one gene locus. Fxr&alpha;2 responded significantly to C24 bile acids and the synthetic FXR&alpha; agonist GW4064. On the other hand, Fxr&alpha;1, despite having an identical ligand-binding domain to that of Fxr&alpha;2, showed no response to any agonists tested by transient transactivation assays. Furthermore, Fxr&alpha;2 interacted with nuclear receptor coactivators PGC-1&alpha; and SRC-1 in mammalian two-hybrid assays while Fxr&alpha;1 did not. These findings point to a significant importance of the AF1 domain to overall receptor structure and function. </p><p>Following <italic>in vitro</italic> functional characterization, <italic>in vivo</italic> experiments using medaka larvae were performed to determine <italic>fxr&alpha;'s</italic> function during normalcy. Quantitative, real-time PCR data demonstrated that Fxr&alpha;1 is highly expressed in adult liver, while Fxr&alpha;2 is expressed predominantly in gut. Fxr&alpha;1's expression was higher than Fxr&alpha;2 in embryos and larvae at all developmental timepoints tested. In vivo exposures of medaka hatchlings to GW4064 at various doses significantly altered expression of defined FXR&alpha; targets, including: bile salt export protein (BSEP), small heterodimer partner (SHP), and cytochrome P450 7A1 (CYP7A1). Surprisingly, numerous sublethal hepatic alterations to hepatocytes and bile preductular epithelial cells (BPDECs) were observed following exposure to GW4064; alterations included: lipid accumulation, glycogen depletion, mitochondrial swelling and rupture of mitochondrial membranes, disruption of endoplasmic reticulum, and apoptosis. Significant lipid accumulation, as revealed by oil red O whole mount staining of larvae, was also noted at lower doses of GW4064. These findings were the first observations of sublethal hepatotoxicity of GW4064; to date, no studies in the mammalian literature reported alterations following its administration.</p><p>Because of studies in the mammalian literature that demonstrated alleviation of cholestatic injury induced via the classic hepatotoxicant &alpha;-naphthylisothiocyanate (ANIT) by GW4064, it was originally hypothesized that a similar finding would be observed in medaka coexposed to these compounds. However, because of GW4064's ability to induce sublethal hepatic alterations in medaka, it was anticipated that its coadministration with ANIT would result in enhanced toxicity rather than alleviation as described in rodent models. However, despite the sublethal alterations induced by 1 uM GW4064, alleviation of toxicity following exposure to 15 uM ANIT was observed. Surprisingly, reduction of GW4064's toxicity was also observed in larvae exposed to both compounds. These investigations of <italic>fxr&alpha;</italic> function are an important and essential component in furthering our understanding of hepatobiliary toxicity in small aquarium fish models of human liver disease. These collective findings have created molecular underpinnings necessary for understanding medaka hepatobiliary function during normalcy and toxicity.</p> / Dissertation

<p>Polycyclic aromatic hydrocarbons (PAHs) are important contaminants that are found in increasing amounts in aquatic ecosystems. One of the sites that that is contaminated by extremely high levels of PAHs is the Atlantic Wood Industries Superfund Site on the Elizabeth River, VA. The Atlantic killifish (<italic>Fundulus heteroclitus</italic>) from this site exhibit increased levels of antioxidants, increased sensitivity to hypoxia, and increased expression of enzymes involved in glycolytic metabolism, suggesting that exposure to PAHs in the environment may induce changes in mitochondrial function and energy metabolism. Normal mitochondrial activity is crucial to an organism's survival. Therefore, gaining a better understanding of how mitochondria are affected by environmental contaminants such as PAHs is a pressing research objective. As a first step in understanding changes in cellular bioenergetics of aquatic organisms in response to PAHs, this research focused on the effect of benzo[a]pyrene (BaP), a representative PAH, on mitochondria the killifish model and on comparison of the mitochondria of the PAH-adapted killifish from the Elizabeth River Superfund Site to reference site fish. In order to assess the extent of mitochondrial DNA damage in the killifish, a PCR-based assay (LA-QPCR) for nuclear and mitochondrial DNA (nDNA, mtDNA) damage was adapted to this model and validated in with UV exposure and BaP exposure studies, as well as with <italic>ex situ</italic> study examining DNA damage in killifish inhabiting the Elizabeth River Superfund site. With the newly adapted LA-QPCR, mtDNA and nDNA damage in the killifish from the Elizabeth River Superfund site and from a reference site (King's Creek, VA) that were treated with BaP were examined. Similar increases in mitochondrial and nuclear DNA damage were observed in King's Creek fish treated with BaP. Killifish from the Elizabeth River showed high levels of basal nDNA and mtDNA damage compared to fish from the reference site, but the level of damage induced due to BaP treatment was much lower in Elizabeth River killifish. Laboratory-reared offspring from both populations showed increased BaP-induced damage in mtDNA, relative to nDNA. Similar to the adult experiment, the Elizabeth River larvae had higher levels of basal DNA damage than those from the reference site, but were less impacted by BaP exposure. Results suggest that BaP exposure can have important energetic consequences and that multi-generational exposure in the wild may lead to adaptation that dampens DNA damage arising from BaP exposure. Since the toxic effects of many PAHs are the result of bioactivation by cytochrome P4501A (CYP1A), the existence of enzymes that can potentially metabolize PAHs in mitochondria was verified. Using Western blot, protein similar in size to microsomal CYP1A was identified with monoclonal antibody against scup CYP1A in the mitochondrial fraction from adult male killifish livers. The size of the protein in the mitochondria was the similar to that of microsomal CYP1A. Fish dosed with BaP had increased EROD activity in the liver mitochondrial fraction compared to controls. In killifish larvae dosed with BaP and benzo[k]fluoranthene (BkF), CYP1A protein levels as well as enzyme activity were elevated. However, fish from the Elizabeth River Superfund site showed recalcitrant mitochondrial CYP1A protein levels and enzyme activity in a similar manner to microsomal CYP1A. Finally, the hypothesis that energy metabolism of BaP-treated fish may be different from the control group and that killifish from the Elizabeth River Superfund site may also have altered energy metabolism compared to reference site fish was tested. Respiration of killifish embryos treated with BaP from both populations was measured. Compared to the King's Creek control fish, all other treatment groups showed decrease in oxygen consumption, indicating lower respiration rate. However, when activities of key enzymes involved in glycolysis (PK) and anaerobic metabolism (LDH) in adult killifish liver and muscle were measured, no differences in the enzyme activities were observed in BaP-treated group compared to the control group. Moreover, metabolomic analysis on BaP treated King's Creek and Elizabeth River killifish showed no difference in the profile in all four treatment groups. The findings in this thesis contribute to the understanding of how BaP, a common environmental pollutant in the aquatic ecosystem, targets the mitochondria in fish model. Nevertheless, deeper examination of how BaP may impact mitochondrial function in killifish and potentially influence adaptation of killifish at a highly contaminated site is necessary. Further studies will elucidate whether such impacts can potentially affect the energy budget and organism level fitness in populations in the wild.</p> / Dissertation

<p>Chronic exposure to toxicant mixtures is a serious threat to environmental and human health. It is especially important to understand the effects of these exposures for contaminants, such as polycyclic aromatic hydrocarbons (PAHs), which are toxic, ubiquitous, and increasingly prevalent. Furthermore, estuarine systems are of particular concern, as they are highly impacted by a wide variety of pollutants; fish there are often exposed to some of the highest levels of contaminants of any vertebrate populations, along with other stressors such as fluctuations in water level, dissolved oxygen, and temperature. A population of <italic>Fundulus heteroclitus</italic> (the Atlantic killifish or mummichog, hereafter referred to as killifish) inhabits a Superfund site heavily contaminated with a mixture of PAHs from former creosote operations; they have developed resistance to the acute toxicity and teratogenic effects caused by the mixture of PAHs in sediment from the site. The primary goal of this dissertation was to better understand the mechanism(s) by which Elizabeth River killifish resist the developmental toxicity of a complex mixture of PAHs and to investigate the tradeoffs associated with this resistance. Because the aryl hydrocarbon receptor (AHR) pathway plays an important role in mediating the effects of PAHs, one major hypothesis of my work was that suppression of the AHR response plays an important role in the resistance of Elizabeth River killifish. For this reason, investigation of the activation of the AHR pathway, as measured by CYP induction, is a unifying thread throughout the work. Another major hypothesis of this work is that adaptation to PAHs has secondary consequences for Elizabeth River killifish, such as altering their response to other xenobiotics. To investigate these hypotheses, a series of experiments were carried out in PAH-adapted killifish from the Elizabeth River and in reference fish. The morpholino gene knockdown technique was modified for use in killifish; we demonstrated that CYP1A knockdown exacerbates PAH-driven cardiac teratogenesis and AHR2 (but not AHR1) knockdown rescues PAH-driven cardiac teratogenesis. Using acute toxicity tests of larval killifish, we showed that Elizabeth River killifish are less sensitive than reference larvae to chlorpyrifos, permethrin, and carbaryl. These results demonstrated that the adaptation was able to protect from multiple xenobiotics, not just PAHs. Using the in ovo ethoxyresorufin-o-deethylase (EROD) assay and a subjective cardiac deformity screen, we showed that the adaptation was spread throughout the killifish subpopulations of the Elizabeth River estuary. However, the adaptive response varied greatly among the subpopulations, which showed that AHR pathway suppression was not required for some level of protection from PAH toxicity. Finally, using the quantitative real-time PCR, the EROD assay, and cardiac deformity screening, we demonstrated that the adaptation was heritable for two generations of fish reared in clean laboratory conditions. The findings in this dissertation will help to reveal how mixtures of PAHs exert their toxic action in un-adapted organisms. Furthermore, these studies will hopefully demonstrate how chronic exposure to PAH mixtures can affect organisms at the population and even evolutionary level. Perhaps most importantly, they will help us to better predict the consequences and tradeoffs for organisms and populations persisting in PAH-contaminated environments.</p> / Dissertation

Environmental tobacco smoke (ETS) is a complex mixture of chemicals generated during the burning of tobacco products. The principle contributor to ETS is side-stream cigarette smoke (SSCS), the material emitted from the smoldering tobacco product between puffs Our hypothesis is that reactive oxygen species from SSCS are playing an essential role in disease promotion and antioxidant supplementation (a single form of alpha-tocopherol or a mixture of multiple antioxidants) will potentially prevent SSCS associated tissue damage, pulmonary dysfunction. The specific aims of the present study are to determine if: (1) SSCS would induce tissue lipid peroxidation and proinflammatory responses; (2) SSCS would provoke pulmonary and cardiac function changes; (3) SSCS would cause oxidative stress, reduce nutrient concentrations and suppress immune function in murine retrovirus infections; (4) dietary alpha-tocopherol, specifically, can enhance resistance to oxidative damage by SSCS and improve lung function; (5) multiple antioxidant supplementation can modulate proinflammatory cytokine secretion and tissue lipid peroxidation induced by SSCS exposure in old healthy mice. SSCS exposure methodology in a murine model was developed to facilitate these goals. For the first time we established a SSCS model in murine retrovirus infection. Also we conduced a SSCS dose-response model for a cardiac function study. We found that SSCS exposure in mice consistently increased oxidation, depleted tissue vitamin E levels, and promoted inflammatory cytokines production. SSCS exposure at 120-min/day, 5 days/week for 12 weeks decreased heart contractile function and increased vascular resistance. SSCS induced increased oxidative stress, reduced nutrient concentrations and suppressed immune function, which could make mice with murine retrovirus more susceptible to opportunistic infections. Dietary alpha-tocopherol enhanced resistance against SSCS-induced oxidation and improved lung function, primarily through the antioxidant property of alpha-tocopherol and its modulation of local cytokine production. The multiple antioxidant with beta-carotene, bioflavanoids, Coenzyme Q10, d-alpha-tocopherol, L-ascorbic acid, L-carnitine, magnesium, N-acetylcysteine, retinol, selenium and zinc given as a dietary supplementation prevented oxidation and IL-6 production in healthy old mice during SSCS exposure. (Abstract shortened by UMI.)

The consensus view is that cardiac hypertrophy is an adaptive response to increased work caused by a variety of stimuli. While hypertrophy can be defined as an increase in cell mass without an increase in cell number, not all increases are equivalent in type and amount of protein accumulated. Our goal in this study was to identify the common steps in the process of cardiac hypertrophy. Our working hypothesis was that in all forms of cardiac hypertrophy glucose utilization increases and that the percentage of energy derived from fatty-acid oxidation decreases. The first part of this study entailed the development and characterization of a neonatal rat heart cell model. The model had to provide uniform culture conditions for rapid development of hypertrophy by agents acting at different sites in the cardiomyocytes. The second part of this study was composed of an assessment of hypertrophy caused by four pharmacologically distinct agents: norepinephrine, angiotensin-II, endothelin-I and tetradecylglycidic acid. In this part we compared the quantity of protein accumulation and quality of hypertrophy cause by each agent. This task was accomplished by examining the effect of each agent on selected mRNA messages and alteration in DNA content of cardiomyocytes. Here we also examined the effect of protein kinase-C, endothelin-I and angiotensin-II inhibitors on hypertrophy caused by each agent. In the final part of this study, metabolic alteration in hypertrophy caused by each agent was assessed for a potential common pathway to hypertrophy. As part of this analysis, we examined changes in glucose and palmitate oxidation, glucose uptake and role of pentose pathway in hypertrophy resulting from treatment of cardiomyocyte by each agent.

The objective of these studies was to functionally characterize canine cytochromes P450 from subfamilies 2B and 3A. Studies of the canine hepatic 2B form PBD-2 had previously revealed that this enzyme exhibits a species specific ability to metabolize 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) and catalyze progesterone 21-hydroxylation. Expression of the putative PBD-2 cDNA, 2B11, in three different heterologous systems revealed that the recombinant enzymes' apparent substrate specificities varied based upon the expression system. 2B11 expressed in COS cells and yeast did not metabolize progesterone in a manner consistent with that of the purified hepatic enzyme, suggesting that 2B11 did not encode PBD-2. However, subsequent expression of 2B11 in E. coli confirmed that this recombinant enzyme did metabolize progesterone into 21- and 16α-hydroxyprogesterone. The structural determinants of 2B11-mediated progesterone 21-hydroxylation were then examined via site-directed mutagenesis of amino acid residues 114, 290, 363, and 365. Consistent with the importance of these residues in androstenedione and 245-HCB metabolism, amino acid alterations Val 114 $\to$ Ile, Asp-290 → Ile, and Ile-363 → Val each decreased 2B11 progesterone 21-hydroxylation. 2B11 mutants at position 365 differed in their regioselective metabolism of progesterone, whereas these mutations had little affect on the stereoselective metabolism of androstenedione. Cytochrome P450 3A forms are of intense interest due to the wide range of therapeutic compounds metabolized by these enzymes. To determine if canine liver contained multiple 3A forms which exhibit substrate specificities similar to those of other species, the canine 3A form PBD-1 (3A12) was expressed in E. coli. 3A12, rabbit 3A6 and human 3A4 metabolized steroids and macrolide antibiotics into the same products at comparable rates. A 3-fold difference in the rate of troleandomycin demethylation between liver microsomes and 3A12, the identification of a novel putative 3A hepatic protein via NH₂-terminal sequencing, and isolation of a distinct 3A cDNA provided evidence for 3A multiplicity in canine liver microsomes. The identification of functionally distinct canine 3A forms could provide the framework for structure-function analysis of these clinically important enzymes. Furthermore, 3A multiplicity and the conservation of substrate specificity between canine and human forms suggests that the canine may be an appropriate model of human 3A metabolism.

<p> Nonalcoholic fatty liver disease (NAFLD) has become a worldwide, chronic liver disease of increasing clinical significance. It is closely associated with the rising epidemics of obesity and insulin resistance. Up to 17% of the United States population may progress from the disease stage characterized as simple, benign steatosis to the more severe, inflammatory stage of nonalcoholic steatohepatitis (NASH). This progression occurs through 2^nd 'hits' of increased oxidative stress and inflammation to a liver that has been sensitized by lipotoxic stress. NASH is also characterized by increased collagen deposition resulting in fibrosis and architectural rearrangement of the liver. Progressive NAFLD is currently recognized as an important contributor to the development of cryptogenic cirrhosis and subsequent liver-related mortalities (estimated at 30-40% in these patients). </p><p> The pathological progression of NAFLD, as described by the 'two hit' hypothesis, characterizes the different stages of liver injury. However, the mechanism(s) responsible for the progression to NASH are unknown. Profiling global gene expression and metabolite patterns in human liver samples representing the full spectrum of progressive human NAFLD may reveal potential mechanisms of progressive disease. Human liver samples representing each stage of NAFLD progression were analyzed by methodologies such as high-throughput microarrays, high resolution mass spectrometry, and protein immunoblot techniques. Bioinformatics tools and gene expression/regulation database software were utilized in several studies to characterize the altered hepatic profiles of these patients. </p><p> Hepatic transcriptomic profiles of ADME (absorption, distribution, metabolism and elimination) and ER (endoplasmic reticulum) stress response genes exhibited initiated hepatoprotective responses in patients with NASH. The endogenous pathways of BA (bile acid) synthesis and BCAA (branched chain amino acid) metabolism also showed evidence of coordinately regulated alterations in response to disease-induced stress in NASH. The transcriptional regulation of the investigated pathways was confirmed by transcription factor binding sites enrichment analysis. The collective response to hepatic stress in human NAFLD, demonstrates a coordinated, hepatoprotective intent that may be utilized for future therapeutics in the battle against progressive liver disease.</p>

Introduction. Chlorination of surface water for human consumption results in a number of potentially toxic by-products. Recent investigation of the effects of these by-products on reproductive outcomes has demonstrated small increases in the risk of both intrauterine growth retardation and low birthweight with increasing exposure. / Objective. The primary objective of this study was to examine the relationship between one group of disinfection by-products, trihalomethanes, and intrauterine growth retardation. The secondary objective was to compare the relationship found with the commonly used estimate of exposure, THM concentration, to that found with aggregate exposure measures. / Methods. A case-control study was carried out with 200 cases and 200 matched controls. The risk of THM exposure was estimated using conditional logistic regression, controlling for the confounding effect of maternal smoking, prepregnancy weight and education. / Results. A small, but imprecise, increase in the risk of IUGR was found with increasing levels of the brominated THMs, particularly bromoform. A decrease in the odds ratio was observed with increasing exposure information in the aggregate measures. / Conclusions. The results of this study support previous studies demonstrating a relationship between THMs and intrauterine growth retardation, particularly the brominated THMs. However, the small sample size does not allow any firm conclusions to be drawn about this relationship.

<p> The Brownsberg Nature Park (BNP) in Suriname is home to eight monkey species: <i>Saguinus midas, Saimiri sciureus, Cebus apella, Alouatta seniculus. Pithecia pithecia, Cebus olivaceus, Chiropotes satanas (sagulatus), </i>and <i>Ateles paniscus.</i> Several studies have undertaken the task to better study the feeding and behavioral ecology of these species within the park. However, studies on the black spider monkey (<i>Ateles paniscus</i>) have been absent. As part of my thesis, I decided to conduct a baseline feeding and behavioral ecology study of this species during the period May 2008 &ndash; July 2008. In addition, I developed a field method for determining mercury levels (in parts per million = ppm) in fecal and urine samples of wild monkeys. Since the park is under enormous pressure from illegal gold mining activities, I decided to collect baseline data on potential exposure of wild monkeys to mercury in the environment. I also collected samples from monkeys at the zoo in Paramaribo and monkeys born in captivity at Hiram College in Ohio. I collected data on the frequency of feeding, resting, and traveling by black spider monkey subgroups every 10 minutes during all day follows. Feeding ecology data consisted of identifying fruits eaten by these subgroups. For the mercury analysis I used the OSUMEX LTD. home testing kit. Results from the behavioral data show the following frequencies of activities for the entire study period: 32% feeding, 43% resting, and 25% traveling. The feeding data further justifies spider monkeys as ripe fruit frugivores: 76% of food items consisted of ripe fruit, while 22% consisted of leaves, and 2% was comprised of flowers. The mercury testing results from the Brownsberg and zoo populations ranged between 0.025 ppm to 0.1 ppm (toxic level = 0.8 ppm). The Hiram College monkeys all displayed levels at 0.000 ppm. The results from the mercury analyses indicate that 1) wild monkeys in the vicinity of gold mining activities may not be under the same threat as humans, with regards to mercury exposure through food, and 2) that wild monkeys are still relative exposed to mercury in the environment whether it be natural or anthropogenic.</p>