Cadmium is an ubiquitous, nonessential trace element that has demonstrated toxic effects in whole placental tissue. Among these, cadmium has been shown to alter placental perfusion parameters, zinc transfer to the fetus, trophoblast viability, and protein hormone production. Furthermore, in women who are exposed to cadmium either occupationally or via cigarette smoke, the incidence of delivering low birth weight infants is increased. One factor associated with decreased birthweight among neonates is prematurity. It is suspected that cadmium disrupts endocrine regulatory mechanisms in the placenta, resulting in premature onset of labor and, hence, prematurity. Based on this information, we have hypothesized that cadmium bioaccumulation in placental trophoblast cells occurs in a dose and time-dependent manner, subsequently resulting in the inhibition of progesterone release via reduction in cell viability, inhibition of trophoblast differentiation, and/or interference in the steroidogenic pathway Human trophoblast cells exposed to cadmium in culture accumulated the metal in both a time- and dose-dependent fashion up to the 10 muM concentration. This accumulation further resulted in a concomitant, dose-dependent decrease in progesterone release by trophoblast cells. The differentiation of cytotrophoblast progenitor cells into the endocrinologically-active syncytiotrophoblast was examined under identical culture conditions. Utilizing H & E stained whole mounts, we were able to observe the presence of syncytial elements in cadmium-treated preparations From subsequent studies, the mechanism by which cadmium diminished progesterone production was determined to be independent of trophoblastic death and differentiation. Employing the MTT assay, no demonstrable effect was observed on mitochondrial succinate dehydrogenase activity, a metabolic activity associated with cellular respiration and, hence, viability. Additionally, results from DNA isolated from control and cadmium-treated cells and analyzed via agarose gel electrophoresis confirmed that apoptosis and/or necrosis were not responsible for inhibiting progesterone release. Further, immunocytochemical localization of cellular boundaries and the nuclei contained within showed that syncytial maturation in cadmium-treated cells was not inhibited, thereby suggesting that the alteration in steroid hormone release occurs at a site within the progesterone pathway Cytotrophoblasts allowed to mature into syncytial elements prior to cadmium exposure demonstrated a dose-dependent inhibition in progesterone release into the media, confirming that the effect of cadmium on progesterone release is independent of the differentiation event. Beginning with the initial step in the progesterone biosynthetic pathway, mRNA transcripts for LDL-R were assayed utilizing a quantitative competitive RT-PCR technique. Results indicated that the abundance of LDL-R mRNA transcripts was reduced in a dose-dependent manner in response to cadmium exposure. Therefore, the conduit by which cholesterol substrate is supplied to syncytial mitochondria for progesterone synthesis is one potential mechanism by which cadmium interferes with the production/release of progesterone / acase@tulane.edu

In the last decade, AIDS has evolved from a deadly medical curiosity of unknown cause into a major epidemic of a new viral disease. AZT administration has been shown to decrease mortality and the frequency of opportunistic infections in AIDS patients. However, drug-induced bone marrow toxicity and the development of resistant viruses remain a major limitation in the usefulness of AZT as a long-term therapeutic agent In order to elucidate the mechanisms of AZT-induced toxicity to the bone marrow cells, the following hypotheses were formulated and tested: 1. HIV-Tat protein enhances AZT-induced toxicity to bone marrow progenitor cells. AZT was significantly more toxic to the cells obtained from the Tat mouse with an IC50 of 0.44 muM in comparison to 2.4 muM for the cells obtained from the normal mouse. In the experiments using recombinant Tat protein, a combination of Tat (10 ng/ml) with 5 muM AZT caused a significant inhibition of 81.9% of CFU-E derived colony formation 2. AZT causes a concentration dependent decline in mRNA expression of GM-CSF receptors. AZT causes a concentration-dependent inhibition of GM-CSF receptor mRNA levels. There is 50% inhibition at 5muM concentration and 97.42% inhibition at 10.0 muM concentration of AZT. The effect of AZT on the GM-CSF receptor mRNA levels was selective and not attributed to generalized inhibition of gene expression. In contrast, the degree of inhibition by AZT of the IL-3 receptor gene expression, another hematopoietic growth factor closely related to GM-CSF, was significantly less than GM-CSF receptor down regulation. Furthermore, the expression of c-myc, one of the proto-oncogenes involved in the erythroid cellular proliferation remains unaffected by AZT, even at 10 muM concentration, confirming the selective nature of the toxicity 3. IL-3 has a protective role in AZT-induced toxicity to the bone marrow progenitor cells at the level of GM-CSF receptor gene expression . IL-3 is an important cytokine required in erythroid lineage and has a synergistic role with erythropoietin, (Epo) which leads to proliferation of progenitor cells and their eventual commitment to lineage specific differentiation. We have shown that IL-3 reduces the AZT-induced toxicity to hematopoietic progenitor cells as evidenced by an increase in the mRNA levels for GM-CSF receptors, in the presence of AZT / acase@tulane.edu

The primary goal of this thesis was to identify new biomarkers of exposure for developing neural tissues in aquatic species. It was found that the cytokine, interleukin-1$\beta$ (IL-1$\beta$) and its type 1 receptor are expressed in the very earliest functional neural circuits that regulate early locomotor behavior in the frog embryo. Even though IL-1$\beta$ is cleaved by ICE (interleukin-I$\beta$ converting enzyme), an enzyme that initiates apoptosis, IL-1$\beta$ expression is not associated with the expression of poly (ADP-ribose) polymerase (parp), a marker of apoptosis, indicating that IL-1$\beta$ expression is not a marker of programmed cell death in the developing frog embryo. Thus, IL-1$\beta$, like other neurotrophins, may play a role in regulating cell growth or survival, or in regulating synapse formation and/or validation. Exposure of developing tadpoles to methylmercury chloride, a potent aquatic environmental contaminant, dramatically reduced IL-1$\beta$ levels within specific neural cell types. Thus, IL-1$\beta$ serves as a potential molecular biomarker of methylmercury exposure in the developing nervous system / acase@tulane.edu

acase@tulane.edu

Occupational exposure to polyisocyanates is common during the manufacture and application of polyurethane spray paints and is associated with a variety of pulmonary diseases. However, the toxicology and molecular mechanisms by which inhaled polyisocyanates cause lung disease are not known. A novel exposure system was developed to allow large scale exposure of animals to HDI polyisocyanates at a stable concentration and aerosol size distribution. This system was used to characterize lung pathology induced by inhalation of HDI polyisocyanates in mice, which included impairment of lung function, edema, neutrophilic inflammation, cellular proliferation and histologic lesions in terminal bronchioles and alveolar ducts. Analysis of 512 transcripts by gene array and 24 transcripts by RNase protection assay identified cytokine, growth factor, and apoptosis-related genes whose message levels were altered by inhalation of HDI polyisocyanates in mice To examine the role of tachykinin-containing sensory nerves in polyisocyanate-induced lung injury, genetically manipulated mice with altered innervation of the airways were exposed to HDI polyisocyanates to determine whether manipulation of airway innervation can alter the magnitude of polyisocyanate-induced lung injury. Transgenic mice overexpressing nerve growth factor (NGF) from the lung-specific Clara cell secretory protein (CCSP) promoter were used. CCSP-NGF mice exhibit hyperinnervation of the airways by sympathetic and sensory nerve fibers. Mice with a targeted mutation in the gene encoding the low affinity NGF receptor, which are deficient in sensory nerve fibers, were also studied. Pulmonary function impairment, inflammation, and airway cell proliferation induced by HDI polyisocyanates inhalation were attenuated in mice with enhanced airway innervation compared with those with reduced innervation. The role of a receptor for tachykinins, the neurokinin 1 (NK1) receptor, in modulating HDI polyisocyanate-induced lung injury was also assessed using transgenic mice overexpressing NK1 receptors in ciliated airway epithelial cells and alveolar type II epithelial cells. HDI polyisocyanate-induced inflammation was attenuated in NK1 receptor transgenic mice compared with wild-type mice. These findings suggest that airway sensory nerves may protect the lung from lung injury and inflammation induced by HDI polyisocyanates and the inhibition of HDI polyisocyanate-induced inflammation may be mediated by through NK1 receptor signaling / acase@tulane.edu

acase@tulane.edu

The drug 3,4-methylenedioxymethamphetamine (MDMA), more commonly known as 'Ecstasy', is a synthetic amphetamine analogue that possesses neurotoxic properties in the rat. The neurotoxicity produced by MDMA is characterized by a chronic depletion of serotonin and 5-HIAA, inhibition of tryptophan hydroxylase and the destruction of serotonergic nerve axon terminals in the brain. The mechanism by which MDMA produces this neurotoxicity is unknown. However, a popular theory is that this neurotoxicity is mediated by a metabolite of MDMA and not by the parent compound. Thus, the purpose of this present study was to investigate further the role of metabolism in the mediation of the neurotoxicity produced by MDMA. Since MDMA is metabolized by a family of enzymes known as cytochrome P450, a specific isozyme may be responsible for the formation of a neurotoxic metabolite, or metabolites, from MDMA The present study suggests that cytochrome P4502E1 (CYP2E1), a specific isozyme of cytochrome P450, may be involved in the formation of a neurotoxic metabolite from MDMA. This study found that the completion of serotonin produced by MDMA can be attenuated or blocked by the inhibition of CYP2E1 activity. Three distinct inhibitors of CYP2E1 were used in this study including, disulfiram, acetone and the physiologic condition of fasting. All three attenuated the depletion of serotonin in cerebral cortex produced by the subcutaneous administration of MDMA Additionally, the intracerebroventricular administration of an ultrafiltrate extract of a microsomal preparation incubated in the presence of MDMA caused the depletion of serotonin in cerebral cortex at seven days. The degree of depletion of serotonin was linear with respect to the incubation time of the microsomal preparation containing MDMA. Furthermore, the ability of the ultrafiltrate extract to deplete serotonin was blocked by the incubation of the microsomal preparation with MDMA in the presence of acetone, the CYP2E1 inhibitor. Taken together, these observations suggest that CYP2E1 is involved in the formation of a neurotoxic metabolite from MDMA / acase@tulane.edu

acase@tulane.edu

Interstitial pulmonary fibrosis (IPF) affects millions of individuals worldwide. Common features of this pathology are varying degrees of acute and chronic inflammation, stiffening of the lung over time and shortness of breath due to impairment of gas exchange resulting from excess deposition of scar tissue. We and others have identified a group of potent polypeptide intracellular messengers, known as growth factors, which are likely to be mediators of the fibroproliferative process in the lung consequent to asbestos inhalation. Thus, we focus on the hypothesis that peptide growth factors such as platelet-derived growth factor, transforming growth factor-beta and tumor necrosis factor-alpha secreted by responding macrophages and epithelial cells are key mediators of the fibroproliferative response. We use a mouse model of IPF in order to investigate the early stages of this disease because most of the pathognomonic histopathologic changes occur within 48 hours of even a brief asbestos exposure. I demonstrate here that compared to C57BL/6 mice, the 129 mouse strain exhibits (1) reduced cell proliferation and growth factor expression at sites of fiber deposition in vivo and (2) failure to develop fibrogenic lesions at these same sites in the lung. I further demonstrate that (3) primary mouse lung fibroblasts (MLFs) isolated from 129 mice exhibit reduced proliferative capacity in vitro in both serum and in serum free defined media containing PDGF when compared to primary MLFs from C57BL/6 mice and (4) that the MLFs isolated from the 129 inbred strain exhibit a reduced responsiveness to factors which upregulate expression of mRNA for procollagen. These findings support the postulate that these peptide growth factors contribute to fibroproliferative lung disease consequent to injury / acase@tulane.edu

Cadmium is a heavy metal which is naturally found in soil and water. It has similar properties to copper, zinc and calcium, and it may disrupt essential metal homeostasis. The toxicity of cadmium is magnified by its extremely long biological half-life in the body, between 10 to 30 years. Although the primary body burden is found in the liver and kidneys, the testis is extremely sensitive to cadmium, and acute exposure results intesticular edema and hemorrhagic necrosis. While most animals with scrotal testes are susceptible to the testicular toxicity of cadmium, strain-related differences to this effect occur in mice. For this dissertation, the mechanism(s) of murine strain differences to cadmium-induced testicular toxicity was studied using sensitive (129/J) and resistant (A/J) mice. We found significantly decreased cadmium concentrations in the testis, epididymis and seminal vesicle 24 hr after subcutaneous cadmium exposure in the resistant A/J mice, as compared to the sensitive 129/J mice. The transport of cadmium across vascular barriers was also markedly different in the two strains. Influx rates of $\sp{109}$Cd into the brain, testis and epididymis were found to be significantly lower in A/J mice. To help identify the natural ligand for the transport system in these tissues, $\sp{65}$Zn influx rates were studied. Zinc is an essential element, and a transport system should exist to facilitate passage into tissues. Since cadmium is a nonessential and toxic metal, specific mechanisms would not be expected to be present to maintain homeostatic concentrations of this cation. Cadmium and zinc share many properties, and the two elements have been shown to compete for transport. Thus, cadmium may use the zinc transporter to enter tissues. The resistant A/J mice demonstrated a significantly reduced zinc entry into the testis and brain when compared to $\sp{129}$/J mice. Cadmium and zinc transport were significantly lower in A/J mice, suggesting that the resistant mice may have a decreased number of zinc transporters, or a mutation in the transporters that makes them less active. Therefore, the nature of murine strain resistance to the testicular effects of cadmium may be related to a relative deficiency or a mutation in the zinc transporter / acase@tulane.edu