The primary objective and public health relevance of this investigation was to develop a chemical-animal model with a toxicological and therapeutic approach. The results outlined here are developed from the latest techniques being employed in the chemical and biomedical fields. This research outlines a model building approach that progressed from a preliminary agent screening technique (quantitative structure-activity relationship/structure-activity relationship, QSAR/SAR) and in vivo testing using the Chernoff-Kavlock (CK) assay through to in vitro testing in transgenic adenocarcinoma of the mouse prostate (TRAMP) cell lines. The preliminary investigation involved development of a QSAR/SAR model to predict the teratogenicity of a series of related chemical agents (dopamine mimetics). This QSAR/SAR model was then validated using a complete leave one out cross-validation. The predictivity of a more general QSAR/SAR model of developmental toxicity was then tested experimentally in vivo using the chemical agent retinoic acid. The second model was based on in vivo animal screening using the CK assay. The CK assay involves the dosing of pregnant animals, either mice or rats, during the organogenesis period of fetal development. This assay quantitatively measures effects on fetal viability and growth, and allows for a more qualitative assessment of teratogenicity by recording obvious malformations. The third segment of this study was an in vitro evaluation of the effects of a series of microtubule perturbing agents on cell viability, cell death and gene expression of the TRAMP cell lines. This research could contribute to the development of drug treatments that would be more effective against human prostate cancer. In the first section of my thesis, a mathematical model was generated with experimental data from the literature on a congeneric series of twelve dopamine mimetics. Based on a single physicochemical parameter, the final model is 100% effective at predicting biological activity (teratogenicity) of dopamine mimetics. We also found inconsistencies in the original biological data that might influence the choice of final model. The second section of my thesis involves the experimental validation of a general QSAR/SAR model that predicted retinoic acid would be positive for developmental toxicity. Retinoic acid was therefore tested in a standard mouse CK assay (the same assay used to generate the data used to generate the model) to test the SAR model prediction. Significant increases in the incidence of both fetal death and intrauterine growth retardation were observed in the offspring of the treated mice. Statistical analysis revealed these effects were dose-dependent. These results demonstrated, in a quantitative manner, the developmental toxic effects of retinoic acid in the mouse, as were predicted by the SAR model and as expected from developmental literature. The final segment of my thesis dealt with the preliminary in vitro screening of four promising anticancer agents, Analog II, 4-methoxy Analog II, JR oxime I and TDH 169 on the clonal TRAMP cell lines C1A, C2H and C2N. 4-Methoxy Analog II displayed the most promising antiproliferative effects and apoptosis inducing effects. A microarray analysis of mRNA expression in response to 4-methoxy Analog II was conducted to determine agent-induced expression alterations in the C1A cell line. Upregulation of the apoptosis activating genes Bok and Siva-pending was observed, while the apoptosis inhibiting genes Birc 4, Dad1 and Atf5 were significantly downregulated.

The respiratory tract is the target of multiple infectious agents. Because the lungs are continually exposed to infectious pathogens in inspired air, natural defense mechanisms have devolved to prevent infection. These defense mechanisms coordinate with each other to provide efficient protection against infection. As a result, pulmonary infections can be viewed not just as a consequence of exposure to a virulent pathogen but as a result of a breakdown of natural host defenses. SPLUNC1 or SPURT (secretory protein in upper respiratory tracts) is small, secreted protein that is expressed in epithelial areas of the nose, mouth, pharynx and lungs. It may be involved in host defense because it is highly homologous to bactericidal/permeability-increasing protein (BPI); a protein that mediates LPS related bacteria killing. In this study, we established a constitutively overexpress CCSP-spurt transgenic mouse model to examine the biological function of spurt. We compared mRNA expression of CCSP-spurt in transgenic mice and their transgenic negative litermates. We determined that mRNA expression of CCSP-spurt was elevated in transgenic mice. The tissue distribution of overexpressed spurt in CCSP-spurt mice was confirmed to be only at trachea and lung and exist in no other tissues. Bronchoalveolar lavage (BAL) fluid from unchallenged CCSP-spurt mice had higher spurt concentration as was determined by ELISA and western blot. BAL from unchallenged CCSP-spurt mice also exhibited antibacterial activity. Furthermore, CCSP-spurt mice display enhanced bacterial clearance than wild-type mice after both groups of mice were challenged with aerosolized gram negative microorganisms Pseudomonas aeruginosa (PAO1) infection. BAL of CCSP-spurt mice had lower levels of proinflammatory cytokines than BAL of their wild-type littermates after the challenge with aerosolized P. aeruginosa. Results from our studies suggest that spurt is a novel BPI-like antibacterial protein that may play a critical role in airway specific innate immunity. Further studies of this protein might have potential public health significance in providing better understanding of natural defense processes in respiratory tract.

Cytochrome c (cyt c) binds with high affinity with anionic phospholipids such as phosphatidylserine (PS) and cardiolipin (CL) to form a complex with peroxidase activity capable of oxidizing polyunsaturated phospholipids, including CL. Release of cyt c from mitochondria plays a pivotal role in cytosolic triggering of apoptotic caspase cascades whereby CL oxidation is involved in mitochondrial membrane permeabilization. Thus control of CL oxidation is critical to regulation of early stages of apoptosis. Given the lipid antioxidant potency of etoposide as well as its ability to induce apoptosis, we hypothesized that cyt c catalyzed CL oxidation during apoptosis can be sensitive to etoposide, hence affect execution of the apoptotic program. We analyzed how the apparent inability of etoposide to prevent apoptosis is related to the mechanism of mitochondrial cardiolipin oxidation by hydrogen peroxide catalyzed by cyt c molecules which has public health relevance since etoposide is a commonly used anti-tumor drug. In a model biochemical system, we showed that 160 pmol lipid hydroperoxides / nmol CL were generated when 100µM liposomes containing a mixture tetralinoleoyl-CL (TLCL) with dioleoyl phosphatidylcholine (DOPC) [1:1] were incubated with 4µM cyt c and 100µM H2O2 or 250µM AMVN. Etoposide inhibited CL hydroperoxide production in a concentration dependent manner but with different sensitivity to the different oxidizing systems. Etoposide was more effective in AMVN system (I50=3µM) compared to cyt c/H2O2 system (I50=15µM), suggesting CL is not oxidized randomly but via a definite pathway. Next we tried to outline the pathway of cardiolipin oxidation using EPR techniques and PAGE studies. Characterizing the protein derived (tyrosyl) radical and etoposide-phenoxyl radical we noted that cardiolipin oxidation occurs via the heme of the cyt c peroxidase and also partly by the protein derived (tyrosyl) radical generated as result of cyt c peroxidase reaction. Etoposide, in the model system inhibits cardiolipin oxidation by preventing formation of protein derived (tyrosyl) radical. However, in HL-60 cells, etoposide enhanced CL oxidation while suppressing AMVN-induced oxidation of other phospholipids. Thus etoposide-dependent inhibition of CL oxidation is not likely to interfere with the execution of apoptotic program via prevention of mitochondrial membrane permeabilization.

The purpose of this study was to investigate the prevalence of smoking among workers at a major academic medical center including hospitals and other medical and health care facilities. It also was designed to investigate the prevalence of smoking among employees while at the workplace and the amount of time spent doing so. Finally, this study investigates the possible connection between the level of addiction to nicotine and the amount of time spent smoking while at work. The findings in this study may help employers target employees who smoke and assist them in efforts to quit. This study has public health significance because it may improve the health of the employees and those around them, as well as decrease costs and increase productivity. An internet-based survey questionnaire addressing the issues of smoking, demographics, the prevalence of smoking at the workplace, second-hand smoke, and level of dependence to nicotine was distributed via e-mail to employees of a major academic medical center and its affiliated university. The results of this study revealed that 12% of those that responded to the survey are current smokers and half of these individuals smoke at work. Those individuals who have been smoking for a longer period of time seem to be more likely to smoke at work and take more breaks to smoke. Those individuals who have a higher level of dependency to nicotine, according to their score on the Fagerstrom score of nicotine dependency, appear to be more likely to smoke at work, take more breaks to smoke, and have more difficulty refraining from smoking at work for one day than those with lower levels of dependency.

The innate immune response is the first barrier against external stimuli arising from disease-causing pathogens. The primary membrane associated Toll-Like receptors (TLRs) play an important role in the innate immune response by recognizing pathogen-associated molecular patterns (PAMPs). Recently nucleotide-binding oligomerization domain (NOD) proteins have been shown to serve as intracellular receptors that are also involved in the innate immune response. Caveolae are small plasma membrane invaginations that exist in a wide range of cell types. The overall goals of this proposal are to examine the molecular determinants of the interaction of the intracellular pathway (NOD2) with plasma membrane (TLR2) mediated events in the response of epithelial cells to bacterial pathogens and to identify the role of caveolae in the signaling events that may underlie this association. Revealing the mechanisms of interaction between NOD2 and pathogens has significant importance to control or prevent Crohn's disease and benefits the public health issues. Accordingly, the specific aims are: Aim 1: To reveal the molecular mechanism for the interaction between NOD2 and MDP. We hypothesize that a cognitive sequence in the LRR of NOD2 recognizes MDP and the cognitive sequence accounts for the interaction of PGN from Gram-positive microorganism in polarized epithelial cells. Aim 2: to determine the requisite role of NOD2/MDP pathway in response to Gram-positive infection. We hypothesize that constitutive levels of NOD2 are low in polarized epithelial cells. Gram-positive infection results in synthesis of TNFa that increases NOD2 expression and TNFa in an autocrine fashion and enables the cells to participate in host defense via synthesis of IL-8. Aim 3: to identify an interaction between NOD2 and TLR2 pathways in mediating Gram-positive bacterium Staphylococcus aureus activation (IL-8 synthesis) in polarized MDCK cells. IL-8 synthesis in response to gram-positive infection will be contrasted in wildtype MDCK cells vs. cells in which NOD2, TLR2 or both have been silenced by siRNA. We also hypothesize that cross talk between NOD2 and TLR2 pathways occurs at a MAP kinase step and accordingly, experiments will be repeated in the presence of pharmacological (or genetic, e.g. dominant negative) inihibitors of p38, JNK and MAPK.

The adverse health consequences of air pollution are well recognized and range from minor upper respiratory system irritation to severe chronic lung disease. The identity and mechanisms of these pollutants, as well as how toxicity is influenced by additional risk factors are unclear. This study elucidates the relationship between air pollution and microbial agents and explores the mechanisms by which the two stimuli interact to cause adverse health effects with important public health relevance. Mycoplasma fermentans is a species of atypical bacteria with immune-regulatory properties and potential to establish chronic latent infections. Particulate matter (PM) is a complex and diverse component of air pollution associated with adverse health effects. The hypothesis of this study is that M. fermentans infection modulates the cellular responses induced by exposure to residual oil fly ash (ROFA), a type of PM particularly rich in metals. Using human lung fibroblasts (HLF) as an in vitro model I measured the release of the immune-modulating cytokine interleukin-6 (IL-6) as a biomarker of stress-induced cell activation after exposure to various chemical and microbial challenges alone or together. The synergistic interaction between live M. fermentans and ROFA to stimulate IL-6 release and gene expression in HLF was demonstrated. This effect was specific for PM that contains high amounts of water-soluble metal and was recapitulated when NiSO4 was substituted for ROFA. The potentiating effect of live infection was mimicked by exposure to M. fermentans-derived macrophage-activating lipopeptide-2 (MALP-2), a Toll-like receptor-2 agonist. Experiments with consecutive singular exposures to MALP-2 and NiSO4 revealed that pre-treatment of cells with NiSO4 facilitated MALP-2-induced IL-6 production, while pre-exposure to MALP-2 failed to influence the response to Ni. Facilitation of MALP-2 response by NiSO4 depended, in part, upon Ni-induced activation of the ERK1/2 MAP kinase. These interactive effects were studied at the level of gene transcription using a series of IL-6 promoter-luciferase reporter constructs and mutants.

Maternal cigarette smoke exposure during pregnancy has been identified as a risk factor for adverse reproductive outcomes, a major public health concern. However, little is known about genetic susceptibility and possible interactions with environmental factors to increase risk of these events. This study was designed to investigate relative contributions of genetic and maternal environmental risk factor interactions to adverse reproductive outcomes. Maternal peripheral and umbilical cord blood samples from 1148 healthy mother/newborn pairs were genotyped for a panel of polymorphisms associated with the metabolic enzymes CYP1A1, CYP2E1, GSTM1, GSTT1 and NAT2* for several subgroups; low birthweight (<2500g, n=86), preterm delivery (<37th gestational week, n=93), premature birth (<2500g & <37th gestational week, n=53) and small for gestational age (SGA) at term (d37th gestational week, n=948) in comparison to the average for gestational age (AGA) group (n=948). Maternal cigarette smoking during the last trimester was significantly associated with birthweight reduction (Ý=101.4g, SE=32, p=0.002). Maternal GSTT1 null genotype was significantly associated with low birthweight (OR=1.97, 95% CI: 1.24-3.12, p=0.004), preterm delivery (OR=1.91, 95% CI: 1.22-2.98, p=0.004) and premature birth (OR=2.42, 95% CI: 1.38-4.26, p=0.002). The mean reduction of birthweight observed among the maternal GSTT1 null genotype group was 89.6g (SE=37, p=0.018) and the mean reduction in gestational age was 0.25 weeks (SE=0.1, p=0.049). In addition, African American women were more likely to have a smaller baby; the mean reduction of birthweight was 230g (SE=34.5, p<0.001) compared with Caucasians. An additive interaction between smoking, African American ethnicity and GSTT1 null genotype was observed (OR=7.81, 95% CI: 2.49-24.43, p<0.001). The mean birthweight reduction observed in this group was 570.0g (SE=117, p<0.001) and the mean gestational age reduction was 1.10 weeks (SE=0.4, p=0.007). A similar risk was observed for newborn GSTT1 null genotype in the presence of maternal smoking (426.7g,SE=111, p<0.001) and (1.0 weeks, SE=0.4, p=0.012). These results demonstrated a clear overrepresentation of maternal and newborn GSTT1 null genotype among adverse reproductive outcome cases. Furthermore, a gene-gene-environment interaction was observed where the combination of maternal and newborn GSTT1 null genotype in the presence of maternal cigarette smoke during pregnancy significantly increased risk of adverse reproductive outcomes.

A panel of metabolic enzyme genetic polymorphisms, which are involved in cigarette carcinogen metabolism, also a DNA repair gene involved in the nucleotide excision repair pathway were evaluated for associations with lung cancer risk in 203 lung cancer cases and 205 controls and in a case-only analysis of 177 lung cancer patients. Significant relationships between predicted high CYP1A2 activity, CYP1B1 (*1/*3 or *3/*3), GSTM3*A/*A, and XPD (Lys/Gln or Gln/Gln) genotypes and lung cancer risk were observed (adjusted ORs, 2.05; 95%CI, 1.13-3.73, 2.6; 95%CI, 1.19-5.69, 1.84; 95%CI, 1.03-3.31, 2.56; 95%CI, 1.45-4.51, respectively). The predicted mEPHX intermediate or high activity genotype also increased risk approximately 3-fold among females. The combined effects of carrying multiple genetic polymorphisms (gene-gene interaction) or of gene-environment interactions, for example, between the CYP1B1 (*1/*3 or *3/*3) genotype and packyear also resulted in significant levels of increased risk and early age onset of lung cancer. CYP mRNA expression levels were measured in 20 pairs of lung tumor and histologically normal tissues to evaluate the potential for local metabolic activation and as a smoking exposure biomarker. The detection of CYP1B1 and CYP2E1 mRNA expression in lung tissue suggests that local bioactivation of procarcinogens may occur. A relationship between lung CYP2E1 mRNA expression levels and cigarette smoke and/or other environmental carcinogens exposure was observed. The significant increase in the levels of CYP2E1 mRNA expression in lung tumors compared to their corresponding histological normal adjacent tissues among current and former smokers and nonsmokers who were exposed to petroleum and/or other environmental exposures further suggest a mechanistic link between environmental carcinogens exposures and lung cancer development. The results indicate that individual susceptibility to lung cancer determined by endogenous host factors such as genetic polymorphisms in metabolic and DNA repair genes, family history of lung and other cancers, early age onset, and interindividual differences in capacity of local procarcinogens bioactivation could interact with each other and/or interact with individual exposures or other exogenous factors such as cigarette smoke, environmental carcinogens and occupational exposures in modifying lung cancer risk.

Although the incidence of new cases of asthma has decreased in recent years, the prevalence of asthma morbidity continues to be a significant clinical and public health issue. The measures of morbidity include the need for urgent medical care and high-dose asthma medications due to uncontrolled asthma symptoms. However, the risk factors for uncontrolled asthma symptoms are poorly defined, especially for the adult asthmatic. Much interest in the host-environment interaction has evolved in response to the greater morbidity observed in adult asthmatics. Thus, the need to identify risk factors is greater than ever. An underlying problem is that surveillance for asthma does not exist at the local or state level. Here we address the concept of environmental health surveillance by demonstrating the utility of local asthma hospitalization data to estimate the burden of asthma morbidity in hopes of identifying environmental risk factors. We examine this burden within 2 geographic settings: 1. a selected urban-rural setting in Pennsylvania and 2. within the 89 zip codes in Allegheny County, Pennsylvania. We also demonstrate that such hospitalization records are a rich source for data needed to generate hypotheses with respect to the prevalence of environmental risk factors and chronic disease morbidity. Lastly, we demonstrate the use of a non-invasive biomarker (i.e. antibodies specific for atypical respiratory pathogens) to assess the risk of exposure to a biological environmental agent to adult asthma morbidity. Given the poor understanding of risk factors for adult asthma prevalence and morbidity, this research is both relevant and important in addressing environmental public health disparities.

This study recruited local anglers to catch catfish from 3 locations within the Pittsburgh Pool and an upstream location on Allegheny River at Kittanning Dam to compare the As, Hg and Se levels in catfish fillet. The objectives were: to find if there exist locational differences of As, Hg and Se levels in catfish flesh; use catfish as sentinels to identify the sources of pollution; determine if any catfish had mercury levels above the EPA criterion; and assess the consumption risk for semi-subsistence anglers and their families. Local store-bought catfish are also compared with river-caught samples. Fish tissue was prepared following EPA method 3052. As and Se were analyzed by collision cell ICP-MS with calibration by standard addition methods. Mercury was analyzed by isotope dilution cold vapor ICP-MS. Data were log-transformed and analyzed by ANOVA with Tukey Post-Hoc Comparisons. There were no significant differences in As, Hg and Se concentrations among the Pittsburgh Pool catch, so we combined these data. Significantly higher levels of Hg and Se were found in Kittanning-caught fish even given significantly smaller fish sizes compared to those caught in the Pittsburgh Pool. The store-bought fish were significantly lower in As, Hg and Se than those caught in the Pittsburgh Pool. In addition, 23% of samples caught in Kittanning had higher mercury levels than the EPA criterion. Hg and Se levels in samples are significantly positively correlated. Using upper 95% CI of mean mercury level in Kittanning-caught catfish flesh, the maximum monthly allowable fish consumption limit for adult anglers is 4 meals, for children below 16 years old is 2 meals, and for women of childbearing age is 3 meals. Conclusions: The Hg and Se levels in catfish in Pittsburgh Rivers vary significantly by location. Fishers are exposed to higher Hg and Se levels when they eat the fish caught near Kittanning and Pittsburgh pool than bought from the fish market. Public health implications: River areas upstream from Pittsburgh may have higher mercury levels than those nearer Pittsburgh because of deposition of emissions from coal-fired power plants. Location specific fish consumption advisories are needed for local fishers.