A computer simulation model of blood cholinesterase inhibition in rats resulting from a single oral or dermal dose of parathion was developed and tested. The model consists of a set of non-linear differential equations describing the absorption, distribution and metabolism of parathion and the kinetics of inhibition and reactivation of erythrocyte and plasma cholinesterase. The equations are numerically integrated to produce the time-course of each of the cholinesterase activities of the blood. The model was tested for validity by a comparison of the activities predicted by the model to values determined experimentally in male rats. The model successfully simulated plasma cholinesterase activity after a four milligram per kilogram oral dose, and total blood cholinesterase activity after a one milligram per kilogram oral dose. Erythrocyte cholinesterase activity was closely predicted for the first three hours after a four milligram per kilogram oral dose, but inhibition was overestimated during the next two hours. Agreement between model output and observed erythrocyte cholinesterase activity was good for the first twenty four hours after initiation of a fifteen milligram per kilogram dermal dose, but the rate of activity regeneration was overestimated. Es-timated erythrocyte cholinesterase inhibition is sensitive to changes in the values of parameters controlling liver paraoxon metabolism and the phosphorylation reaction between paraoxon and the enzyme, but in general, the model is not sensitive to small perturbations of parameter values. The model has potential for interpreting human exposures to parathion and for investigating the relationships between various parameters and blood cholinesterase inhibition.

Oxidation of the guanosine (G) moiety, yielding the oxidized lesion 8-hydroxy-2'-deoxyguanosine (oxo82dG), in DNA has become a hallmark biomarker in assessing cellular outcomes induced by oxidative stress. It is well established that the guanosine nucleotide triphosphate pool is also susceptible to oxidative stress and suggested to be more available for oxidation than DNA due to the lack of protective histones and robust repair mechanisms for reducing the levels of all the products of oxidation. The oxidation of guanosine in the nucleotide triphosphate pool, resulting in oxidized guanosine 5'-triphoshate (oxo8GTP), has been overlooked due to the lack of a reliable method. Oxo8GTP has been shown to precede oxidation to G incorporated into DNA and modulate cell processes such as G-protein signaling and RNA synthesis. Evidence is presented in this study of a reliable method to quantify oxo8GTP, a proposed mechanism for the oxidative modification of GTP in the presence of copper and L-ascorbic acid, and evidence of oxo8GTP as an inhibitor of soluble guanylyl cyclase (sGC). A significant induction of oxo8GTP in cell-free preparations as well as in PC12 and HEK 293T cells exposed to physiologically relevant oxidative conditions generated with 10 ?M copper sulphate and 1mM L-Ascorbic Acid (Cu/Asc) is also reported. Exposure to oxidative conditions by Cu/As leads to elevations in oxo8GTP significant enough to result in a reduction of the sGC product, cyclic guanosine monophosphate (cGMP), by as much as half in pure sGC and PC12 cells. GTP is protected from oxidation in the presence of reduced glutathione and this subsequently rescues sGC activity. This suggests that oxo8GTP is produced by free radicals in vivo and can significantly impact neuronal cell functions regulated by sGC activity in the central nervous system such as synaptic plasticity. Alterations in copper homeostasis and oxidative stress have been implicated in several neurodegenerative disorders including Alzheimer's and Parkinson's diseases as well as Amyotrophic Lateral Sclerosis. Based upon evaluation of the data presented herein, we hypothesize that neuronal deficiencies in such disorders might be due to oxidation of the GTP pool and the ensuing effects on neuronal function.

Bioturbation, the biological process through which many species of infaunal benthic invertebrates suspend bottom sediments into the water column through their burrowing, feeding, respiratory, and locomotor activities, may be a sub-lethal endpoint that can be exploited to assess the toxicity of aquatic sediments. Therefore, we developed a novel test method that used bioturbation (BioTurbTox test) generated by the activities of second in-star Chironomus tentans larvae as the toxicity endpoint (Chapter 2). To validate this method, copper (Cu) and fluoranthene were individually spiked into relatively uncontaminated aquatic sediment to assess changes in bioturbation and mobilization of the chemicals into the overlying water. Turbidity production responded to the chemicals in the sediment in a concentration-dependent manner and was an excellent indicator of sediment toxicity. Moreover, substantial concentrations of Cu were released into the overlying water from the Cu-spiked sediment, whereas little fluoranthene was mobilized into the overlying water from the fluoranthene-spiked sediment. Sediment samples were then collected from the field and used to evaluate the similarity of response of the BioTurbTox test to other more standardized toxicity tests. In the summer of 2003, sediment samples were collected at six sites in the Neuse River of North Carolina tested for toxicity, and analyzed for chemical contaminants (Chapter 3). Atrazine was the most frequently detected current-use pesticide and pyrene and fluoranthene were measured at relatively high concentrations from the Neuse River sites. Concentrations of fluoranthene were correlated with results from the Ceriodaphnia dubia porewater and BioTurbTox tests. We concluded that the new BioTurbTox test was useful as a rapid screening method for sediment toxicity information, but required normalization to the clay content or to the total organic carbon content of field collected sediments. In Chapter 4, the toxicity of environmental pharmaceuticals and personal care products (PPCPs) were evaluated with the BioTurbTox and C. dubia reproductive tests. Fluoxetine and bisphenol A significantly affected bioturbation caused by C. tentans, especially at high concentrations (1-2 mg/L), and the turbidity change induced by caffeine, fluoxetine, and bisphenol A showed a concentration-response relation. Triclosan affected reproduction of C. dubia at relatively low concentrations (IC50: 85.4 ?Ýg/L). However, most of the tested PPCPs were not acutely toxic at environmentally relevant concentrations, but were relatively toxic at high concentrations. In Chapter 5, two sediment-spiking methods (extract mixing vs. whole sediment dilution methods) were compared with the BioTurbTox test and a gradient response was observed from both methods. Based on the similarity of the toxic response, we determined that either of the spiking methods was appropriate for estimating the toxicity of aquatic sediments in screening level assessments. The overall conclusion from this research was that the newly developed BioTurbTox test shows promise as a tool to assess the toxicity and mobilization of contaminants from aquatic sediments.

Dibromoacetic acid (DBA) and bromochloroacetic acid (BCA) are prevalent disinfection by-products of drinking water known to produce defects in spermatogenesis and fertility in adult rats. Previous work in our laboratory demonstrated that BCA compromises both the fertility of cauda epididymal sperm and SP22, a sperm membrane protein highly correlated with the fertility of these sperm. Herein, we administered DBA and BCA, individually and in combination, to determine whether levels of SP22 on sperm and fertility were diminished in an additive fashion. Moreover, we wished to validate an immunoassay for quantitation of SP22 to replace the tedious two-dimensional (2D) gels quantitation. The initial study consisted of 8 treatment groups and a water vehicle control group, on which animals were exposed by oral gavage daily for 14 days. BCA was administered alone at 1.6, 4, and 8 mg/kg, and DBA was given in equimolar fashion at 2, 5, and 10 mg/kg. BCA and DBA were also given as two binary mixtures: 1.6 mg/kg BCA + 2 mg/kg DBA and 4 mg/kg BCA + 5 mg/kg DBA. Proximal cauda epididymal sperm membrane proteins (30 ìg) were resolved following concentration/desalting in 2D gels under denaturing conditions (2D SDS-PAGE) and the SP22 protein was quantified. In addition, SP22 was quantified by enzyme-linked immunosorbant assay (ELISA). Full length rat recombinant SP22 (rSP22) was used to generate a standard curve and affinity purified sheep anti-rSP22 was used as primary antibody. The ED50 for the decrease in SP22 quantified by 2D SDS-PAGE for DBA and BCA was 7.15 and 4.61 mg/kg. The ED50 for the decrease in SP22 quantified by ELISA for DBA and BCA was 8.10 and 5.93 mg/kg. The second study consisted of 2 and 4 mg/kg DBA, 1.6 and 3.2 mg/kg BCA, and a 2 mg/kg DBA + 1.6 mg/kg BCA mixture. In this study proximal cauda sperm were also used for in utero insemination to assess fertility. The ED50 for the decrease in fertility for DBA and BCA was 3.5 and 2.7 mg/kg. Immunostaining for SP22 in the testis revealed staining of both round and elongating spermatids and decreased staining in testes exposed to the DBA + BCA mixture. An evaluation of SP22 in testicular parenchyma was also performed by ELISA and Western blotting. Both evaluations revealed a treatment-related decrease in SP22. For either the 2D SDS-PAGE or ELISA quantitation of SP22 on sperm or the fertility of sperm, additivity or synergy is indicated. Finally, the correlation between SP22 levels measured by ELISA versus fertility was r2=0.74, compared to 0.82 for SP22 levels measured by 2D SDS-PAGE versus fertility, suggesting the ELISA could be used to supplant the time-intensive SDS-PAGE.

The hypothalamic-pituitary-gonadal (HPG) axis was characterized in female mice lacking one or both forms of estrogen receptor (ERa, ERb) with the aim of elucidating the contribution of each receptor form to gonadotropin homeostasis and ovarian function. These studies consisted of a thorough evaluation of gene expression for the gonadotropin subunits in the pituitary and the components necessary for steroidogenesis in the ovary. These data were corroborated with evaluations of the plasma levels for each of the relevant pituitary and gonadal hormones. Females lacking ERb (bERKO) exhibit minimal disruption in HPG axis function but do exhibit deficits in gonadotropin responsiveness in the ovary. Females lacking ERa (aERKO) exhibit dramatic ovarian phenotypes of hemorrhagic and cystic follicles and exaggerated steroid synthesis in the ovaries. The phenotypes in the aERKO ovary are attributable to chronically elevated LH due to the loss of ERa function in the hypothalamus. Pharmacologic reduction of plasma LH levels in aERKO females abates the ovarian phenotypes. These studies indicate that the hypothalamic functions of ERa are most critical to ovarian function. To better understand the contribution of ERb to the manifestations of LH-hyperstimulation in the ovary, females lacking functional ERb but possessing elevated LH via a transgene (bERKOLHCTP) were generated. Characterization of bERKOLHCTP animals indicates the intraovarian functions of ERb are necessary for the induction of LH-associated cystic follicles but not amplified steroidogenesis. An additional novel finding in aERKO ovaries was ectopic expression of the Leydig cell specific enzyme, 17b-HSD III and correlating male-like testosterone synthesis. This phenotype is dependent on LH-hyperstimulation of ovaries lacking ERa function to manifest. In summary, the predominant contribution of ERa to ovarian function occurs in the hypothalamus, whereas ERb is more important within the ovary itself. Presence of Leydig cell specific gene expression in aERKO ovaries indicates a potential role for estradiol and ERa in gonadal differentiation.

Human epidermis is routinely subjected to DNA damage induced by solar radiation and keratinocytes have developed intricate mechanisms to respond to UVB-induced DNA damage. Despite these mechanisms, nonmelanoma skin cancer is the most common cancer in the US. Previous analysis of immortalized mouse keratinocytes has revealed that the bZIP transcription factor, CCAAT/enhancer binding protein alpha (C/EBP alpha), is induced by DNA damage and has a role in the G1 checkpoint. Here we demonstrate C/EBP alpha is induced in the epidermis of the human subjects exposed to UVB. To begin to determine the in vivo physiological significance of the up-regulation of C/EBP alpha by UVB, we generated an epidermal specific C/EBP alpha knockout (K5Cre;C/EBP alpha fl/fl) mouse on a SKH1 hairless background. Following UVB treatment, these mice displayed an impaired keratinocyte cycle arrest and abnormal entry of keratinocytes into S-phase. This impaired cell cycle checkpoint in UVB-treated C/EBP alphaï deficient skin was associated with greatly diminished p21 levels which occurred through a p53-independent mechanism. To further investigate whether C/EBP alpha could function as a tumor suppressor gene in UVB induced skin tumorigenesis, we exposed K5Cre;C/EBP alpha fl/fl and K5Cre control SKH1 mice to 20mJ/cm2 UVB three times weekly. The K5Cre;C/EBP alpha fl/fl mice displayed both increased tumor incidence and multiplicity, suggesting that loss of C/EBP alpha in the epidermis confers increased susceptibility to UVB-induced skin tumorigenesis. In addition, we also observed that human skin SCC and BCC display greatly reduced or absent C/EBP alpha levels, implicating that loss of C/EBP alpha contributes to the development of human nonmelanoma skin cancers. Collectively, our results demonstrate that C/EBP alpha is induced by UVB in human skin, inhibits cell cycle progression in response to UVB in vivo and is a tumor suppressor gene in UVB induced skin tumorigenesis.

Brevetoxins (PbTx) are potent lipid soluble polyether neurotoxins produced by the marine dinoflagellate Karenia brevis, an organism linked to periodic red tide blooms. Brevetoxins have been linked to deaths in marine mammals, which are exposed through ingestion of organisms harboring high brevetoxin concentrations and through the inhalation of aerosolized brevetoxins. Humans are also at risk through the same routes of exposure. Evidence suggests that respiratory exposure to brevetoxins can lead to a severe inflammatory response. Brevetoxins exert their toxicity by interacting with neurotoxin receptor site five associated with domain IV of the alpha subunit of the voltage gated sodium channel. Brevetoxin binding to tissues that contain voltage gated sodium channels on excitable cells results in membrane depolarization, repetitive firing, and increase in sodium currents. The goal of the current research project is to determine how these marine toxins evoke an immune response, specifically by examining the effects of brevetoxin and brevetoxin antagonists on T cells. The hypothesis is that brevetoxins alter cell proliferation, cause DNA damage, disrupt normal signal transduction, and ultimately cause cell death, possibly through an apoptotic mechanism, and the natural and synthetic brevetoxin antagonists are able to prevent and reverse these deleterious effects, making them a useful pharmaceutical agent not only for brevetoxin exposure treatment, but also for human diseases exhibiting similar inflammatory congestion, like Asthma and Cystic Fibrosis. The effects of brevetoxins and antagonists were determined by cell proliferation assays, comet assays and examination of caspase activation, Poly ADP- ribose polymerase (PARP) cleavage and gene expression. Exposure of Jurkat E6-1 Cells to Brevetoxin 2, 3, 6, and 9 at doses of 10-5M severely inhibited proliferation. Further analysis revealed positive staining for apoptosis, PARP cleavage, caspase 3/7 and caspase 8 activation, decreased intracellular sodium and increased intracellular calcium concentrations. PCR array gene expression analysis revealed significant change in gene expression. The apoptosis PCR array yielded 30 up-regulated and 4 down-regulated genes divided into the following families: TNF ligand, TNF receptor, Bcl-2, caspase, IAP, CARD, death domain, CIDE, p53 and DNA damage response, and anti-apoptosis. The DNA Damage Signaling Pathway Array had 20 up-regulated genes and 5 down-regulated genes from the following families: apoptosis, cell cycle arrest, cell cycle checkpoint, DNA repair-damaged DNA binding, DNA repair-double strand break repair, DNA repair-mismatch repair, and other genes related to DNA repair. The Common Cytokine PCR array had 50 up-regulated genes from the following gene families: interferons, interleukins, bone morphogenic protein and TGF-ï¢, PDGF/VEGF, TNF Superfamily, and other growth factors/cytokines. Exposure of Jurkat E6-1 cells to brevetoxin in combination with the antagonists brevenal, alpha naphthoyl, and beta naphthoyl did not improve cell proliferation, which is in stark contrast to experiments conducted with CHO-K1 BH4 cells. This disparity may be due to the difference in voltage gated sodium channel subtypes that these cells possess. The results demonstrate that brevetoxins can induce apoptosis and DNA damage and that the antagonists may show selectivity based on VGSC subtype.

The first phase of this study involved examination of lead (Pb) concentrations in adult Eastern elliptio (Elliptio complanata) mussel tissue and sediment at 40 sites throughout North Carolina, ranging in average daily traffic count, land-use patterns and watershed characteristics. Data from these sites revealed an increased Pb concentration of about 2 μg/g in sediment downstream of bridge crossings compared to upstream for high traffic areas of >20,000 vehicle crossings/day (vc/d), as well as a significantly greater average Pb concentration in mussels from sites with traffic count >500 vc/d compared to sites with <500 vc/d. Two 28-d toxicity tests with Pb and adult Eastern elliptio mussels were conducted in the second phase of the study to compare the field-derived Pb bioaccumulation data and to examine sublethal biomarkers of Pb exposure and toxicity. Endpoints for the tests included mussel survival, metal accumulation, shell length and weight. For the first test, δ-aminolevulinic acid dehydratase (ALAD) inhibition was assessed as a biomarker of exposure and effect in mussel hemolymph. In the second test, Na+,K+-ATPase activity in mussel gill tissue was examined as a biomarker of exposure and effect, along with measurements of hemolymph ion concentrations. Pb was measured in mussel hemolymph in both tests at near-exposure levels, and at the greatest exposure concentration in the first test (396 μg/L), the hemolymph Pb concentration was 4.5 times the amount present in test water. In contrast, the greatest exposure concentration in the second test (458 μg/L), had hemolymph concentrations only 0.12 times the amount present in test water. This finding suggested a threshold effect concentration for Pb exposure and hemolymph concentrations. The average Pb concentration in mussel tissue was strongly correlated with Pb exposure concentration (R²= 0.99) at day 28 for both tests. The enzyme ALAD was not present in measurable concentrations in freshwater mussel hemolymph, gill or mantle tissue. The Na+,K+-ATPase activity was found to be a strong biomarker of Pb exposure in freshwater mussel gill tissue (~50% of total ATPase) using a K-free salt solution for detection, and was found to be negatively correlated with average tissue Pb concentration (R2 = 0.85) on day 28. Pb accumulated rapidly in mussel tissue during the first two weeks of the second exposure, with lesser accumulation during the final two weeks, with only the 121 µg/L treatment group changing significantly. Because Pb was being removed from the test water throughout the duration of exposure, I conclude that the mussels were approaching equilibrium with the aqueous environment and were eliminating the Pb in lysosomes and granulocytes through pseudo-feces. Although the major contribution of Pb to the environment from now-banned leaded gasoline is still evident in North Carolina, the concentrations measured in mussel tissue and sediment were not at levels which would cause immediate or short-term reductions in Eastern elliptio populations. Moreover, the sub-lethal effects of Pb on Na+,K+-ATPase activity, although a strong biomarker of exposure, appear not to be significantly effected at environmentally relevant concentrations of Pb. However, other species of freshwater mussels may be more sensitive to Pb contamination and further assessment is needed to verify the role Pb may be having in the overall declines in mussel species diversity being observed worldwide.

Previous results have shown a strong correlation between vegetation and dissipation of polycyclic aromatic hydrocarbon (PAH) contamination over long time frames. Evidence of phytoremediation over shorter time frames is difficult to detect, particularly in sites where overall PAH concentrations have not begun to significantly decrease. This study assessed the impact of vegetation on a highly contaminated sediment over a short time frame in order to track the effects of plant derived organic carbon (PdOC) in a variety of sediment compartments. The Indiana Harbor Canal is an industrialized area with historic petroleum contamination of near-shore sediments. In 2002, near-shore sediments were planted with hybrid poplar and willow trees as part of a phytoremediation treatability study. Site disturbance allowed for colonization by cattails and Phragmites. In 2004, non-vegetated and vegetated oiled-shore sediments were collected and analyzed for 42 polycyclic aromatic hydrocarbons (PAHs), total organic carbon, and percent modern carbon (14C radiocarbon). Sediments were collected from several locations, field-composited, homogenized, sieved, and chemically fractionated. There is a significant input of modern carbon in both labile and recalcitrant fractions of vegetated sediments that is absent in the non-vegetated control. Phragmites sediments contained more modern carbon (plant carbon) and more weathered PAH ratios than willow, cattail, and non-vegetated sediments. Organic carbon-normalized PAH concentrations were greater in Phragmites humin than other sediments. Humin fractions of willow trees were more weathered than non-vegetated humin, but modern carbon values were not always different. Cattail humin contained more modern carbon than non-vegetated humin, but only de-ashed humin was more weathered than non-vegetated de-ashed humin. Results show that sediment composition and PAH weathering indices are not indicative of reduced PAH concentrations. Instead, PAH weathering appears related to increased modern carbon (plant carbon) content. Substantial increases in modern carbon content were observed after two years of plant growth. These results suggest that PdOC is diffusing into a variety of sediment compartments where it may be enhancing carbon cycling and beginning to mitigate PAH concentrations.

Genistein, the primary phytoestrogen in soy, was investigated for potential adverse effects on the developing female reproductive system with particular focus on the ovary. Mice were treated with genistein at doses that span the range of human exposure including vegetarian mothers during pregnancy and lactation to infants on soy based infant formulas. Neonatal genistein exposure caused the formation of multi-oocyte follicles (MOFs) in the ovary. This effect is mediated by ERâ as mice lacking this receptor do not develop MOFs while mice lacking ERá do. Further study of genistein?s effects on the ovary revealed inhibition of neonatal oocyte nest breakdown; oocytes were still attached by intercellular bridges and the normal progression of apoptosis was attenuated. Mechanistic studies of MOF formation revealed alterations in cell adhesion molecules. In addition, genistein is not unique in its ability to cause ovarian disruption; other environmental estrogens caused MOFs as well as altered cell adhesion molecule expression. Further, these effects appear to be exacerbated by preferential binding to ERâ. Assessment of reproductive function showed that mice treated with genistein (0.5 and 5 mg/kg) showed signs of early reproductive senescence while mice treated with genistein (50 mg/kg) exhibited infertility characterized by fewer, smaller, implantation sites as well as reabsorptions; ovaries from these mice had no corpora lutea. Stimulation with exogenous gonadotropins restored ovulation, suggesting problems with the hypothalamic-gonadal axis. These data taken together demonstrate that neonatal exposure to genistein at environmentally relevant doses causes adverse effects on the developing reproductive system and in particular on the ovary.