S-nitrosothiol modifications of proteins are emerging as an important nitric oxide-mediated signaling pathway. Our laboratory has focused on S-nitrosation of the metal binding protein metallothionein and the resulting effects on zinc homeostasis, gene and protein expression and nitric oxide (NO) mediated signaling in the pulmonary endothelium. Statement of public health significance: The pulmonary endothelium is responsible for filtering the blood before it enters systemic circulation, and as such it is extremely vulnerable to injury by inhaled toxicants in the environment as well as those that circulate in the bloodstream. As the endothelium constitutively produces NO, we are interested in studying NO-mediated signaling in order to lay a foundation that will allow us to better understand diseases such as asthma, pulmonary hypertension and sepsis in which dysregulation of NO-mediated signaling is thought to be a contributing factor to the disease pathology. To this end we have used both recombinant DNA and biochemical techniques to examine the relationship between metallothionein, zinc homeostasis and the metal responsive transcription factor MTF-1. We demonstrated that exposure to NO results in zinc release from metallothionein, which in turn activates MTF-1, resulting in nuclear translocation of the protein and NO-dependent increases in metallothionein protein expression. We hypothesized that S-nitrosation of the sulphydryl groups in metallothionein were the cause of NO-mediated zinc release and downstream protein expression effects. We used a fluorescent modification of the biotin switch assay in combination with two-dimensional electrophoresis and mass spectroscopy to extend our study of NO-mediated signaling through S-nitrosation of protein thiols to identify S-nitrosated metallothionein in endothelial cells exposed to NO donor, and used the technique in further studies to illuminate the proteome of pulmonary endothelial cells. We were able to identify several potential targets of S-nitrosation in endothelial cells including cytoskeletal, cytoprotective, glycolytic and chaperone proteins. The proteomic assay that we developed is a useful screening tool, and may lead to new insights in post-translational S-nitrosothiol modifications of endothelial proteins, and eventually to new perspectives regarding diseases exacerbated by dysregulation of this NO-mediated signaling pathway.

Background: Breast cancer is a worldwide public health concern. Breast cancer now ranks first not only in the industrialized world but also in the developing world. In the United States, breast cancer is the most common non-skin cancer and the second leading cause of cancer-related death in women. In the past fifty years, a woman's lifetime risk of breast cancer more than tripled in the United States, to one in seven today. This trend parallels a staggering increase of chemicals in the environment. Given the increasing number of women in the workforce, it is possible that increases in breast cancer incidence may be caused by occupational exposure. Methods: Application of literature review results of breast cancer risk factors and screening efforts at workplaces to determine the cost-benefit analyses for applications in an occupational medicine practice. Results: Review of epidemiologic studies on suspected environmental risk factors for breast cancer shows that at risk populations can readily be found in the workplace. Effective screening efforts by occupational medicine physicians can reduce mortality in the workforce. Although, conclusions drawn here are limited, it is advisable to develop national policies to reduce chemical exposures that may be associated with breast cancer. Conclusions: Occupational physicians may be an important and appropriate healthcare provider with the opportunity to screen on at risk population, (workforce- female from 18- 65) and influence a wide range of well established and suspected environmental risk factors for breast cancer by incorporating prevention into occupational medicine clinic visits. Mammography and the clinical breast exam have a potential to detect suspicious lesions and may be implemented in occupational medicine clinics. Integrating screening into pre-employment or periodic examinations would expend minimal time and reasonable expenses while potentially preventing worker mortality. The integration of breast cancer screening into occupational medicine may simultaneously improve worker health and increase the value of the occupational medicine physician.

The vascular effects of arsenic in drinking water are a global public health concern that contribute to disease in millions of people worldwide. However, the cellular and molecular mechanisms for these pathogenic effects of arsenic are not well defined. This thesis examined the hypothesis that arsenic stimulates pathogenic signals through surface receptors on liver sinusoidal endothelial cells (LSECs) to stimulate NADPH oxidase (NOX) activity that is required for arsenic-stimulated LSEC capillarization. In mice and isolated LSECs, we demonstrated that exposure to arsenic promoted capillarization and increased expression of platelet endothelial cell adhesion molecule (PECAM-1) through a time and dose dependent mechanism. Superoxide generating NOX enzyme complexes participate in vascular remodeling and angiogenesis and are central to arsenic stimulated cell signaling. LSEC arsenic exposure increased NOX dependent superoxide generation that was inhibited using gp91ds-tat protein, NSC23766, a Rac1-GTPase inhibitor, or quenched by the intracellular superoxide scavenger, Tempol. These inhibitors also blocked arsenic-stimulated LSEC PECAM-1 expression and defenestration. In vivo arsenic exposures failed to promote LSEC capillarization in p47phox knockout mice. These data demonstrated that arsenic stimulates capillarization through a NOX dependent mechanism. Given that arsenic rapidly activates NOX in vascular cells, we hypothesized that signaling for these responses was receptor mediated. Since arsenic-stimulated LSEC defenestration and capillarization is Rac1 and NOX dependent, we examined whether a g-protein coupled receptor (GPCR) upstream of Rac1 initiated these effects. Pre-treatment LSECs with Pertussis toxin (PTX), an inhibitor of Gi/o, prevented arsenic-stimulated defenestration. Since capillarization is a gain in barrier function, LSEC expression of the sphingosine-1-phosphate type 1 (S1P1) receptor, a major Gi/o linked regulator of endothelial barrier function, and its role in arsenic-stimulated defenestration were investigated. S1P1 was highly expressed in LSECs relative to large vessels. In ex vivo studies, inhibiting LSEC S1P1 with a selective antagonist, VPC23109, blocked arsenic-stimulated superoxide generation, defenestration, and PECAM-1 expression. These data demonstrated that arsenic targets a specific LSEC GPCR to promote vascular remodeling, and the first demonstrating that S1P1 regulates oxidant-dependent LSEC capillarization. Taken together, these data demonstrate that S1P1 activated NOX stimulates LSEC capillarization, which aids in our understanding of mechanisms underlying arsenic-induced liver disease.

Normal cardiac rhythms are generated by an organized propagation of depolarization and repolarization. The voltage-gated transient potassium current (Ito) is a major determinant of cardiac action potential. The Ito is expressed in a gradient across the left ventricular wall of the hearts, which is essential for the proper repolarization sequence in the left ventricle. Altered expression of Ito is seen in hypertrophied and failing hearts and may contribute to the increased incidence of cardiac sudden death. Thus, elucidating mechanisms underlying the expression of Ito channels will provide basic knowledge essential for the prevention and treatment of cardiac diseases with high public health significance. In the mammalian heart, Ito is produced by assembly of pore-forming Kv4 and accessory KChIP2 subunits. Differential expression of Kv4.2 gene underlies transmural gradient of Ito in the left ventricle in small rodents, whereas the size of Ito is correlated with different levels of KChIP2 in large animals. Recent studies have shown that atypical homeodomain Iroquois proteins are distributed in a gradient in the left ventricle and influence the expression of Kv4.2 and Ito. Therefore, this thesis examines the hypothesis that Irx proteins control Kv4.2 gene transcription in a cell-type specific manner and analyzes the underlying molecular mechanism. Irx3 and Irx5 are differentially expressed in a steep gradient in the left ventricle of rat hearts in an inverse pattern to Kv4.2 expression, whereas Irx4 is equally abundant in the ventricle. Irx5 activates Kv4.2 promoter in several non-myocyte cell lines, whereas the transcription factorinhibits the promoter activity in neonatal ventricular myocytes. Moreover, Irx4 prevents Irx5 to activate the channel promoter. Structure-function studies establish that the C-terminus of Irx5 is required for its regulation of channel promoter, whereas the N-terminus of Irx4 mediates its action. Addition of histone deacetylase inhibitor relieves the inhibitory effect of Irx4. Deletion and mutation analyses demonstrate the presence of a previously unidentified Irx5-responsive element in the Kv4.2 distant promoter region. Collectively, these results indicate that the interplay between Irx4 and Irx5 contributes to the heterogeneous expression of Kv4.2 gene, and hence Ito density, in the left ventricle of rat hearts.

Humans are exposed daily to both pharmaceutical estrogens and xenoestrogens (PEXE) due to their presence in many household products, food products, soil, air, water and estrogen based medications. These PEXEs have been implicated in various human health outcomes, such as breast cancer in women and testicular dysgenesis syndrome including testicular cancer. They also can have adverse reproductive effects on aquatic wildlife through sex reversals, production of intersex individuals, alterations in mating, and prevention of gonadal maturation. There are many sources and types of PEXEs in air, water, soil, household products and food products, but the focus for this research is on the transport and fate of PEXEs from all media into surface water, especially through municipal waste water treatment plant (WWTP) sources. This dissertation consists of three related research papers. The first examines the sources and types of PEXEs in municipal WWTPs. The second documents and compares aquatic exposure concentrations of PEXEs to their Predicted No effect concentration (PNECs) to determine aquatic species protectiveness or risk. The third paper conducts an aquatic hazard assessment of the xenoestrogen, Bisphenol A (BPA). The findings of the research suggest that PEXEs; contain compounds that can mimic estrogens, are mostly introduced into the environment through municipal WWTP effluent sources, and are discharged directly into rivers and lakes at environmentally relevant concentrations. Specifically, BPA, a compound widely used in plastics may be present in surface waters at hazardous concentrations that may present a risk for aquatic receptors. The public health significance of this research is that approximately sixty percent of Americans obtain their drinking water from surface water sources. Thus, understanding PEXEs and their concentrations present of WWTP effluents is imperative for environmental public health tracking of associated disease states, and in the regulation of fish or wildlife consumption from rivers and lakes. Further, to examine adverse health effects in the biotic aquatic system is to indirectly explore possible exposure and health effects on humans since species in the wild are sentinels for human exposure (the canary in the mine). Sentinel animals may provide early warning of potential risks before disease develops in human populations.

Numerous epidemiological studies associate chronic inhalation of metal mixtures with increased risk of pulmonary diseases. Although exposure to metal mixtures is of great public health relevance, the integration of cellular responses to metals within these mixtures that promote disease is poorly understood. This dissertation investigated the hypothesis that chromium (VI) (Cr(VI)) stimulates signaling that alters transcriptional complexes to silence protective gene induction by nickel (Ni). In airway epithelial (BEAS-2B) cells, Cr(VI) activated signal transducer and activator of transcription 1 (STAT1)-dependent signaling within 1 h of exposure. This activation was dependent on Src family kinases (SFKs) since inhibiting SFKs prevented Cr(VI)-stimulated STAT1 signaling. Moreover, Cr(VI) activated STAT1 in wild-type mouse embryonic fibroblast (MEF) cells, but no response was observed in MEF cells null for the SFKs, Src, Yes, and Fyn. However, reconstituting human Fyn in the deficient MEF cells restored the Cr(VI) response. These data indicate that Cr(VI)-activated STAT1 is mediated by Fyn. This signaling may be detrimental as STAT1 has been implicated as an inflammatory mediator in asthma patients that is specifically activated in bronchial epithelial cells (1). Metallothionein (MT) and vascular endothelial growth factor A (VEGFA) are involved in protecting the lung from injury by sequestering metals and promoting wound repair, respectively. Ni-induced MT2A, the most abundant human isoform, required zinc (Zn) redistribution which directly activated metal transcription factor-1 (MTF-1). A prolonged induction was mediated by secondary signaling pathways. Cr(VI) negatively regulated the secondary pathway and had no effect on Zn mobilization. For VEGFA induction, Ni activated a complex signaling cascade involving ERK. Ni-stimulated ERK was upstream of hypoxia-inducible factor-1á (HIF-1á) and Src-mediated Sp1 transactivation. Cr(VI) inhibited Ni-activated ERK, HIF-1á stabilization, Src phosphorylation, and VEGFA induction. The current study demonstrated that Cr(VI)-activated STAT1 is responsible for the silencing of inducible genes. In BEAS-2B cells stably expressing STAT1 shRNA, Cr(VI) no longer had an inhibitory effect on Ni-induced MT or VEGFA mRNA expression and positively interacted with Ni to induce both genes. These data indicate that Cr(VI)-activated STAT1 may play a role in the pathogenesis of Cr(VI)-induced pulmonary diseases by silencing the protective gene transcription in the airway epithelium.

Heart disease is the leading cause of death nationwide, killing one of every four Americans in all communities. The health care costs associated with heart disease have been estimated to surpass 300 billion dollars in 2010. Various preventative risk factors have been identified like obesity, diabetes, cigarette smoking, and air pollution. Pulmonary hypertension (PH) is a common secondary pathogenesis associated with cardiovascular disease. Understanding its exact physiological mechanisms will allow for better treatment options and better ways of preventing the disease. The importance of nitric oxide (NO) in biology was recognized in 1998 when the Nobel Prize in Physiology or Medicine was awarded for its discovery as a physiologic signaling molecule and was validated again in 1992 by naming it "the molecule of the year". NO, produced in the endothelium by the enzyme nitric oxide synthase (eNOS), is critical in maintaining blood pressure by functioning as vasodilator. Its lack in the vascular system leads to problems, like heart disease and PH. As a critical signaling molecule, NO is likely to be important in transducing the effects of a broad variety of environmental stimuli on vasomotor regulation and studying the regulation of NO's biosynthetic capacity is of great public health importance. Recent studies demonstrate that the glycoprotein thrombospondin-1 (TSP1) influences vascular responses by interfering with the NO-mediated vasodilatory pathway at downstream targets. We hypothesized that TSP1 directly modulates eNOS activity and, thus, endothelium-dependent relaxation of blood vessels. Further, we were interested in how the inhibitory effects of TSP1 on eNOS would translate in a disease setting, specifically PH. This study demonstrates TSP1's negative role in the protective NO-mediated vasodilatory pathway and establishes a clear role for TSP1 in the development of PH. Despite the advent of several new drugs for the treatment of PH mortality remains high. Importantly, these drugs aim at increasing NO's bioavailability. The studies described herein suggest that increased TSP1 expression may limit the efficacy of these drugs. A deeper understanding of the role of TSP1 in physiology and disease will open new avenues in our fight to prevent heart disease.

Group B Streptococcus is the most common cause of bacterial infection in the newborns. Infection by Group B Streptococcus often results in pulmonary hypertension. The bacterial components causing pulmonary hypertension had been identified as cardiolipin and phosphatidylglycerol. Cardiolipin not only induces pulmonary hypertension in human, but also in lambs. Due to very little research on cardiolipin and other phospholipids in lung injury, it is important to understand how cardiolipin and other phospholipids play a role in lung pathophysiology. In our study, we are the first to demonstrate that no significant difference in cell viability is shown in the treatment with phosphatidylcholine alone (300 uM) nor with a combination of cardiolipin (30uM) and phosphatidylcholine (70uM) in bovine pulmonary artery endothelial cells at 4 hr and 24 hr. However, cells treated with 30uM cardiolipin and 70 uM phosphatidylcholine for 24 hr have a significant increase in caspase-3 and caspase-7 activity. Furthermore, caspase-3 and caspase-7 activity was elevated by treatment with CL alone at 10uM and 30uM. Conclusions: Cardiolipin and co-treatment with phosphatidylcholine induces apoptosis pathways in caspase-3 and caspase-7 cascades in bovine pulmonary artery endothelial cells. Public health significance: This study provides insight into the potential role of cardiolipin in the pathophysiology of lung injury. This may further open new approaches for the development of therapeutic intervention for pulmonary diseases.

Aggressive penetration of nanomaterials in different spheres of our life from novel technologies to a plethora of consumer products, raises concerns about their possible adverse effects on public health. Several studies report that nanotubes cause lung toxicity. With increase in day-to-day applications of carbon nanotubes, particulate exposure either under occupational or environmental settings is inevitable. In the classic inflammatory response to nanotubes, emigration of neutrophils (PMNs) followed by macrophages into sites of particle deposition has been observed. The major role of the cells is to phagocytoze and promotes particulate clearance and the clearance might be essentially dependent on effective recognition. Carbon nanotubes are not effectively recognized by professional phagocytes and delayed clearance of particles within the lung parenchyma can thus be majorly attributed to impaired phagocytosis or deficiency in components involving their effective degradation. We in our research coated nanotubes with biomolecules to promote recognition, uptake and biodegradation by professional phagocytes. Coating nanotubes with eat-me-phospholipid signal, phosphatidylserine proved to be an effective strategy for targeting particles to professional phagocytes, specifically macrophages both in vitro and in vivo. However, opsonization of nanotubes made them competent for both macrophages and neutrophils. This targeting also enhanced the biodegradation in neutrophils and to a lesser extent in macrophages via action of myeloperoxidase and its potent oxidants whose critical role in biodegradation was delineated in cell free based in vitro studies. Further, in vivo experiments using wild type and myeloperoxidase null mice showed a significantly lower degree of biodegradation and particle elimination in latter animal type, underscoring the role of neutrophil peroxidase in biodegrading carbon nanotubes. Using contemporary techniques- confocal, transmission and scanning electron microscopy, Vis-NIR and Raman spectroscopy, we evaluated the hypothesis. Taken together, the results from the doctoral work suggest that targeting of nanotubes to professional phagocytes can be achieved by coating with certain biomolecules and this targeting can reduce the biopersistence and inflammation associated due to the presence of otherwise relatively biodurable nanotubes in biological ambience. The dissertation also foresees functionalization of nanotubes as a strategy to combat potential toxic effects of nanotubes which pose potential risk to the public health.

The Greater Pittsburgh Area is famous for its three rivers: the Allegheny, Monongahela, and Ohio Rivers. These rivers have a history of being polluted by decades of mine runoff and chemicals released by industrial sites. New problems, such as pollution from endocrine disrupting compounds and xenoestrogens, have recently been discovered in this well known aquatic environment and are suspected to be caused by the failing sewer system. Personal care products, pharmaceuticals and plasticizers all have the potential to enter the water supply though both treated and untreated sewage. Many of these compounds are known or suspected endocrine disruptors. Estrogenic potential of fish extracts from flesh/fat tissue captured from Freeport and Ford City was studied via the E-Screen Assay on MCF-7, T47D and BT-20 human breast cancer cell lines. Results showed weak estrogenic responses in both MCF-7 and T47D cell lines, with no significant differences for fish gender, weight, or sample location. Estrogenic potential of extracts from fish brain tissue was tested via Bromodeoxyuridine MCF-7 Analysis and paired with High Performance Liquid Chromatography-Mass Spectrometry to investigate the presence of specific xenoestrogens in the fish extracts. Fifty eight fish were sampled from rivers in the Greater Pittsburgh Area. All samples were non-detectable for methyl, ethyl, propyl and butyl parabens. Bisphenol A (BPA) was detected in 44 of the 58 samples, with a range from non-detectable to 120 pg/gram. The Harmarville sample location had higher results for all analyses when compared to all other sample sites. In summary, this dissertation supported all previous available literature leading to the conclusion that parabens are safe to remain on the market and are not a significant environmental concern. In particular, there does not seem to be any need for concern over paraben levels detected in the Greater Pittsburgh Area river system and water supply. The BPA portion of this research was in agreement with previous literature as to its bioconcentration tendencies; however, new implications regarding the public health significance of the effects from BPA in brain tissue may require some re-evaluation of concerns about BPA transport and fate in the environment around Pittsburgh and elsewhere.