Freshwater mussels (Bivalvia: Unionidae) are among the most threatened aquatic species in the world. One of the major issues implicated in this decline is water pollution. Polycyclic aromatic hydrocarbons (PAHs) are a suite of hydrophobic environmental pollutants common in terrestrial and aquatic ecosystems. These compounds are largely derived from petroleum related sources (e.g., gasoline, oil) and are of major concern from transportation-related runoff to aquatic systems due to the acute and chronic (e.g., mutagenic and carcinogenic) toxic properties of many members of this class. The effects of exposure to PAHs have been investigated in many species of bivalves; however, to date no comprehensive study of the effects of exposure to these compounds on all life stages of native freshwater mussels have been completed. The goals of this study therefore were to investigate the effects of exposure to PAHs on all life stages of freshwater mussels and to develop diagnostic tests that are rapid, accurate, inexpensive, and of minimal impact to the mussels. This study examined the acute (48 h) toxicity of PAHs to the glochidial (larval) and juvenile stages of mussels and the sub-acute (7 d) toxic effects on adult mussels. Additionally, the study examined the use of genetic damage as a biomarker of exposure of mussels to PAHs by utilizing the Comet assay to determine levels of DNA strand breakage following aqueous exposure. Finally, mussels were collected from areas of high and low environmental levels of PAHs and were analyzed to validate laboratory findings and to examine relations to previously obtained field PAH mussel, water and sediment measurements. We found that there were no acute toxic effects of PAHs on glochidia or juveniles of the two species of freshwater mussels examined, up to concentrations approaching water solubility, and well exceeding those commonly measured in the streams of North Carolina. Experiments with adult Elliptio complanata, both in the laboratory and from the field, indicated that genetic damage due to PAH exposure was likely present, however the results were highly variable and the potential for biological, ecological, and toxicological consequences were uncertain. Further development and improvement of assay methods may reduce this variation. Generally, mussels from streams with higher average daily traffic counts (ADTC) exhibited greater levels of genetic damage compared to mussels from streams with lower ADTC values. Data obtained from the laboratory study generally showed increasing DNA damage relative to increasing PAH concentration. Based on the data generated, however, PAHs are not likely contributing to acute toxicity of mussels in North Carolina streams, but the chronic, long-term pervasive effect of PAHs on native freshwater mussels remains uncertain.

Innate immunity is the initial response of a host in defending against pathogen invasion (Beutler 2004, Janeway and Medzhitov 2002). The key features of an innate immune response are that it is: non-specific (broad-spectrum), rapid (often within minutes to hours) and conserved in its pattern of recognition. Antimicrobial polypeptides (AMPP) are a key component of the innate system and present in virtually all life forms (Zasloff 2002). One of the most widespread AMPP in fish are the piscidins. Piscidins have potent, broad-spectrum activity against viruses (Chinchar et al 2004), bacteria (Silphaduang and Noga 2001), fungi (Lauth et al 2002) and parasites (Colorni et al, Accepted). Another family of AMPP are histone-like proteins (HLPs), which are highly homologous to core nuclear histones (Robinette et al 1998, Noga et al 2002). Because disease is often the major cause of economic losses in fish farming, understanding how stress affects innate immunity (e.g., expression of AMPP) and thus disease susceptibility could be highly beneficial to the industry. One very important factor that can contribute to stress in aquaculture is inadequate nutrition. Feed is the single largest expenditure in semi-intensive and intensive farms (Lunger et al 2007, Twibell et al 2003, Mbahinzireki et al 2001). Thus, reducing feed costs while still maintaining optimal health is critical for successful production. As nutrient intake decreases, metabolic processes are impaired and susceptibility to disease increases. I anticipated that this would be reflected in the levels of piscidins and HLPs. The overall goal was to investigate the levels of these antibiotics that indicate fish are stressed prior to any pathological signs. For this first, an ELISA for piscidin 4 was developed and validated and its application to asses stress was confirmed. Using this ELISA and various other independent assays the presence of piscidin 4 related AMPP were detected in important aquacultured fish. Next, I found that nutritional deprivation has a deleterious effect on piscidin 4 expression and that susceptibility to an ich increases in hybrid striped bass (Morone saxatilis âx M. chrysops â). The nutritional deprivation also lead to dermatological condition in channel catfish.

The purpose of this research is to provide a preliminary description of the morphological and genetic variation of a cosmopolitan freshwater mussel E. complanata from one North Carolina river system, Cape Fear River (CFR). Individuals from CFR were collected and compared with known specimens of E. complanata (topotype). Multivariate analyses, such as factor and discriminant analyses were utilized to differentiate the individuals based on thirty morphological shell landmarks. Genetic analyses involved the use of diversity estimates and cluster analyses based on cytochrome oxidase I (COI) sequence and Amplified Fragment Length Polymorphism (AFLP) fingerprint data. Factor analysis suggest that E. complanata from CFR maybe differentiated based on the thickness of posterior and anterior shell angles, and obesity of the shells. Significant differences between the CFR samples and topotypes were demonstrated by discriminant analysis of morphological data and by COI gene diversity estimates. This difference corroborated earlier work suggesting geographic delineation of E. complanata shell form. Genomic fingerprinting suggests further variation even within the topotypes. Phenotype of the topotypic materials seems to support this genomic variability. Heirarchical cluster analyses of morphometry and genetic data further showed different groups supporting earlier research suggesting high form variation within the E. complanata species.

Knuckles, Travis Lee. In Vitro Cardiotoxicity of Residual Oil Fly Ash. (Under the direction of Kenneth B. Adler and Kevin L. Dreher). Epidemiological studies have shown an association between air pollution particulate matter (PM) and adverse cardiovascular effects. Although numerous mechanisms have been proposed, the actual mechanism(s) as well as emission sources and associated causal properties by which PM affects the cardiovascular system remain elusive. At least some adverse PM health effects can be attributed to bioavailable constituents, most notably, the transition metal content of the particles. Toxicological studies in rats using residual oil fly ash (ROFA) combustion source particles show extrapulmonary effects ranging from thermo-regulatory alterations, myocardial necrotic lesions, to fatal cardiac arrhythmias. Exposure of rats to ROFA via intratracheal instillation shows a rapid and transient increase in plasma metal content as early as 15mins post-exposure, suggesting that PM constituents are bioavailable to both the systemic circulation and perfused organs. However, the impact of this systemic exposure on extrapulmonary organs at the cellular and molecular levels is unknown. In this study, cardiomyocytes derived from 1-day-old rat pups were exposed to determine the direct effects of a particle free residual oil fly ash leachate (ROFA-L). Using concentration of leachate relevant to amount that were found in the plasma of rats following pulmonary deposition, I have shown that ROFA bioavailable constituents cytotoxicity in cardiomyocyte cultures and alter cardiomyocyte gene expression and transcription factor activation profiles consistent with alteration in cardiomyocyte growth and function.

Perfluorooctane sulfonate (PFOS) is a perfluoroalkyl acid that has unique surfactant properties and is used widely in industrial and household products. Furthermore, PFOS is an environmentally persistent compound, and its presence has been detected in both humans and wildlife. The maternal and developmental toxicities of PFOS were evaluated in Sprague-Dawley rats given 1, 2, 3, 5, or 10 mg/kg PFOS daily from gestational day (GD) 2 to GD 20. Controls received 0.5% Tween 20 vehicle (1 ml/kg). Serum thyroxine (T4) and triiodothyronine (T3) in the PFOS-treated dams were significantly reduced as early as one week after chemical exposure, although no feedback response of thyroid stimulating hormone (TSH) was observed. The effects of sub-chronic exposure to PFOS (3 mg/kg/day for eight days) on thyroid hormone economy are further characterized in adult male rats, and the thyrotoxic effects of PFOS are compared to propylthiouracil (PTU), a classic goitrogen. This work substantiates the endocrine disrupting effects of PFOS, but fails to provide evidence for its physiological consequences in comparison to PTU based on two biomarkers of hypothyroidism: myocardial beta-adrenergic receptor density and hepatic malic enzyme activity. Furthermore, in vitro static incubation of the pituitary is used to investigate the observed lack of response of the hypothalamic-pituitary-thyroid axis. PFOS does not significantly alter thyrotropin releasing hormone (TRH) induced secretion of TSH from the pituitary. Therefore, it is not likely that PFOS-induced changes in circulating thyroid hormones are due to a disruption at the level of the pituitary.

The purpose of this research work was to assess the dermal permeation of biocides in metalworking fluids (MWFs) to develop predictive QSAR models and to develop an appropriate training set of chemicals to enhance the predictive ability of QSAR models for dermal permeation. Estimation of the amount of chemicals absorbed through skin plays a vital role in dermal risk assessment. Approximately 1.2 million US workers are occupationally exposed to MWFs annually. Different components of MWFs especially biocides, contribute to adverse health effects including irritant and allergic contact dermatitis as well as carcinogenesis. These adverse effects may be positively correlated to their dermal absorption and may cause systemic toxicity if absorbed in significant amount in workers involved in metalworking operations. A lack of scientific data exists regarding the dermal permeation of MWF components, particularly biocides. Therefore, the first two studies were conducted to (1) determine the dermal permeation of biocides and other chemicals (used as training set to develop Linear Solvation Energy Relationship (LSER) models) in commercial and generic MWFs; and (2) develop a LSER model for predicting dermal permeation of other biocides, not used in these studies. Dermal permeation was evaluated in dermatomed porcine skin by utilizing a flow through diffusion cell system. Chemical analysis was performed by employing gas chromatography with a solid phase micro-extraction technique and ultra performance liquid chromatography with a solid phase extraction technique. LSER models, which are a subset of quantitative structure activity relationship models, were constructed by multiple linear regression analysis with permeability coefficient as the response variable and solvatochromic descriptors as the predictor variables. The LSER model is useful to quantitatively measure the difference in interaction between the two phases (skin and vehicle) as well as a predictive tool. Since the training set used to develop a LSER model was not optimally diverse in terms of structure and chemical space, the third study focused on developing a training set of chemicals representing a wider chemical space (in terms of descriptor values) using a best possible chemical selection method. The results from the first two studies demonstrated that (1) the dermal permeation of biocides as well as other chemicals was highest in aqueous solution followed by synthetic, semi-synthetic and soluble oil type of MWFs; (2) addition of water to MWFs for dilution increased dermal permeation; (3) the LSER model adequately predicted the dermal permeability of biocides in MWFs and also shed light on the chemical interactions resulting in reduced permeability. An optimal and less subjective method (uniform coverage design) to select chemicals representing a wider chemical space was identified in the third study. The LSER model based on the new selected training set of chemicals performed statistically better over the LSER model based on the training set used in the previous study. In summary, the aforementioned results demonstrated that there is a difference in the absorption profile of chemicals among the type of MWFs and dilution of MWFs with water increases the dermal permeation of chemicals; the LSER model can be useful to explain the change in vehicle solvatochromic properties upon addition of water as well as can be an effective prediction model for dermal permeation of chemicals in mixtures; finally, a structurally diverse training set of chemicals representing a wider chemical space is required to improve the predictive capability of a model. All of these results will augment the dermal risk assessment of the chemicals in mixtures and contribute to the improvement of computational predictive models.

The purpose of this research was to understand the mechanisms in which blood clots form and are degraded. The research studies examined the role of an endogenous protease, plasmin, which is found in all vertebrates, and the optimal doses of plasmin required to dissolve blood clots. When we investigated clot lysis with plasmin we examined blood clots from several species to which varying concentrations of plasmin were added, as well as varying methods of plasmin administration. The results of these studies not only highlighted important dose-response relationships of plasmin, but also demonstrated differences in the effect of human plasmin to dissolve blood clots compared to blood clot from the species tested. Porcine clots, in particular, were more resistant to lysis compared with human clots, while ovine clots had similar lysis compared with human clots. In addition, this research demonstrated that plasmin?s effectiveness to lyse thrombi increases with an increase in clot surface area, e.g, by fragmentation, or when plasmin is administered as an intrathrombic administration. In separate studies, to investigate clot formation, we uesd mice that lack the FVIII protein. For these experiments we investigated the formation of blood clot formation using rotational thromboelastography (ROTEG) that measures the formation of fibrin in whole blood. This method was shown to be extremely sensitive to low levels of factor VIII protein and may have applications to classify particular phenotypes of hemophilia patients, or as a research tool to evaluate novel FVIII molecules.

Etomoxir (ET) is a member of a family of substituted 2-oxirane-carboxylic acids that inhibit mitochondrial long-chain fatty acid beta-oxidation (FAO), ketogenesis and gluconeogenesis. Once converted to its CoA ester, ET irreversibly binds to the CPT-1 catalytic site and prevents long chain fatty acids from entering the mitochondrion. Along with this inhibition of FAO, ET causes a shift in energy substrate utilization from fatty acids to glucose, leading to systemic hypoglycemia, hypoketonemia, and hypotriglyceridemia. These effects make ET potentially useful in the treatment of non-insulin-dependent diabetes mellitus (NIDDM). The compound has been shown to induce cardiac and hepatic hypertrophy in animals and, therefore, has not been fully developed as an antidiabetic agent to date. It is well established that ET activates the peroxisome proliferator activated receptor-alpha (PPARá) which can cause both oxidative stress and dysregulation of the cell cycle control gene program. The goal of this research was to evaluate the ET-induced alterations in gene expression profiles in hepatocytes to elucidate the possible role of cell growth dysregulation and/or oxidative stress in ET-induced hepatic toxicity. In HepG2 cells treated with a high dose of ET, gene expression strongly suggestive of oxidative stress was observed and this was supported by decreased levels of reduced glutathione, reduced/oxidized glutathione ratio (GSH/GSSG), concurrent increase in oxidized glutathione (GSSG) and superoxide generation. A significant decrease in mitochondrial membrane potential and ATP levels implicated impairment of mitochondrial energy metabolism. Other gene expression findings suggested activation of p53, DNA repair and cell cycle arrest. In rats, ET induced a strong mitogenic response in the livers of rats 24 h after administration of one 25 mg/kg dose, that was consistent with the cell proliferation caused by peroxisome proliferators. This finding was coincident with a predominance of cell proliferation/growth-related gene expression alterations. Oxidative stress genes were down regulated; suggesting that this is not a viable etiologic mechanism for induction of ET-induced hepatic hypertrophy. PPARá appears to play a role in ET-induced hepatic hypertrophy, as shown by the early cell proliferation followed by increased level of PPARA mRNA, peroxisome proliferation and increase of PPARá-related genes.

Pittman, Kristianna Marie. Exploring the regulation of iNOS and the effects of NO production in endotoxin-induced uveitis (EIU). (Under the direction of Janice B. Allen and Kenneth Adler). In this study, we hypothesized that during anterior uveitis NF-kappaB activity and subsequent iNOS gene expression increase as a result of the decrease in TGFbeta2 activity. Increased iNOS expression leads to increased NO activity and production of peroxynitrite. We further proposed that exogenous administration of TGFbeta2, i.e. increasing TGFbeta2 activity or scavenging peroxynitrite may protect against EIU. Scavenging of peroxynitrite was examined by using the peroxynitrite scavenger, ebselen. All studies were undertaken using either the in vivo model of anterior uveitis, endotoxin-induced uveitis (EIU) or the in vitro system of iris ciliary body cells stimulated with IL-1beta. Results demonstrated that TGFbeta2 decreases iNOS expression during EIU, however, the mechanism is NF-kappaB-independent. Exogenous administration of TGFbeta2 did not protect against EIU. These studies identified peroxynitrite as a component of EIU, through the identification of nitrated MnSOD, a peroxynitrite target. Scavenging of peroxynitrite by the compound ebselen did protect against EIU. Ebselen mediated inhibition of EIU by scavenging peroxynitrite, inhibiting the recruitment of polymorphonuclear (PMN) leukocytes and suppressing NF-kappaB activation. This study demonstrated that TGFbeta2 negatively regulates iNOS expression during EIU and that iNOS, NO, and peroxynitrite are key mediators in the pathogenesis of EIU. Attentuation of uveitis occurs when suppression of these mediators is coupled to PMN suppression and inhibition of NF-kappaB activation.

The objective of this research was to characterize the percutaneous absorption of the organophosphorous pesticide, malathion, across the skin of bullfrogs (Rana catesbeiana) and marine toads (Bufo marinus) using in vitro models. An established mammalian model for percutaneous absorption, the two-compartment Teflon flow-through diffusion cell assay, was adapted to anuran skin to examine species and anatomical site differences in absorption and partitioning of C¹⁴-radiolabeled malathion. Malathion absorption was greater across the ventral skin compared to dorsal skin in both bullfrogs and marine toads but did not differ significantly between species. The issue of short-term storage and viability of anuran skin for diffusion cells was examined using glycerol preservation and cryopreservation techniques. Bullfrog skin viability was retained for 28 days, while marine toad skin viability significantly decreased after 7-10 days. A novel in vitro model, the harvested perfused anuran limb (HPAPL) preparation, which maintained an intact microvasculature to the skin, was developed. The HPAPL represented an improvement over diffusion cells by retaining the anatomic and physiologic integrity of the skin. Doppler ultrasound was used to determine the perfusion rate for the HPAPL by measuring the physiologic blood flow of the pelvic limb in vivo. In addition to the characterization of the percutaneous absorption of malation in anurans, effects of sublethal doses on brain acetyl cholinesterase activity in bullfrogs and marine toads, were examined using a modified Ellman spectrophotometric technique. Sensitivity to environmental toxins make anurans potentially important animal models for studying the impacts of organophosphorous insecticide contamination of the environment.