Shuk-Mei Ho - US grants

The rate of production of toxic, partially reduced species of oxygen is thought to increase in hyperoxic lungs, eventually overwhelming endogenous cellular defenses and leading to lung cell damage. However, development of oxygen tolerance in adult rats was often observed after exposure to 85% O2 for 5-7 days and which is correlated with an increased activity of both the copper/zinc and manganese superoxide dismutases in lung tissue. The overall goal of this research is to understand the molecular basis for regulation of gene expression of these antioxidant enzymes in rat lungs in response to oxidant stress. In order to perform these studies, a cDNA clone coding for the CuZn SOD and an additional cDNA clone coding for the Mn SOD have been isolated. These cDNA probes will be employed to quantitate the level of CuZn and Mn SOD mRNAs in in vitro pulmonary endothelial cells and alveolar type II cells with or without hyperoxic exposure to define whether expression of individual SOD gene in response to hyperoxia is different in various types of lung cells. In situ hybridization at electron microscope level will be performed to determine if the observation of changing mRNA level in in vitro lung cells correlates closely with the results obtained in in vivo lung cells. Elevation of SOD mRNA level in lung cells after hyperoxic insult should direct to investigating the mechanisms that are involved in mRNA accumulation. We would further measure the effect of hyperoxia on the copy number of the SOD gene, rate of RNA transcription and mRNA transportation and stability of mRNAs. The role of cis and trans acting elements in regulation of SOD gene expression will also be defined by DNA binding assays and gene transfer experiments if we observed transcriptional activation of these genes in response to hyperoxia. Finally, translation efficiency of these mRNAs and stability of these enzymes will also be determined by pulse labelling and immunoprecipitation experiments to define if hyperoxia affects these enzyme activities on translational and/or post-translational levels. Antioxidant enzymes are critical for cellular defense against oxidant stress. Unravelling of the effect of hyperoxia on the modulation of SOD gene expression is essential for us to uncover the signal(s) generated by hyperoxia, and should also provide valuable information to design strategies for augmenting antioxidant defense in the lungs.

This is a Research Opportunities for Women Career Advancement Award, to enable Dr. Ho to determine the feasibility of studying estrogen receptor expression in estrogen target tissues of the freshwater painted turtle, Crysemys picta.

The production of toxic, partially reduced species of oxygen is greatly enhanced in hyperoxic lungs and may eventually overwhelm the endogenous cellular capacity of antioxidant defenses resulting in lung cell damage. This kind of pulmonary injury is observed not only in experimental animals, but also in humans receiving oxygen therapy for respiratory insufficiency. Previously studies have demonstrated that augmentation of intracellular antioxidant enzyme activities [superoxide dismutase (SOD) and/or catalase] in cells or animals by treating them with liposome-encapsulated antioxidant enzymes, or exposing animals to a sublethal concentration of oxygen would provide dramatic protection to the recipient cells or animals against hyperoxic insults. In this application, a novel approach will be undertaken to further evaluate the effectiveness of each of the antioxidant enzymes in protecting lung cells from oxygen-mediated injury. Initially, five lines of transgenic mice each carrying a specific antioxidant enzyme transgene will be generated. These transgenes are being designed to be under the control of a human beta-actin promoter to insure high levels of transcription in all types of cells in animals. Additionally, mice containing a combination of two transgenes will also be obtained by mating two transgenic founder mice each bearing a different transgene. Expression of these transgenes in different tissues including the lung will be examined at both mRNA and protein levels. Immunocytochemistry will be used to identify the cellular sites, distribution and concentrations of the antioxidant enzymes in both control and exposure will be used to determine the degree of protection provided by the augmented antioxidant enzyme activities. Antioxidant enzymes are critical for cellular defense against oxidant stress. Unravelling the effectiveness of each antioxidant enzyme should help design more efficient antioxidant therapies to block or reduce oxygen- mediated lung injury. the transgenic mice generated in this study also can be used for elucidating the role of antioxidant enzymes in other diseases associated with the over-production of partially reduced oxygen species, such as some types of lung fibrosis and reperfusion injury of ischemic heart tissue.

health science research support; university;

biomedical equipment purchase;

Activation of Manganese-containing superoxide dismutase (MnSOD) gene expression in adult rat lungs during exposure to a sublethal concentration of oxygen (85%) may play a critical role in allowing animals to survive a subsequent exposure to lethal concentrations of oxygen. The overall goal of this research is to define the molecular mechanisms for regulation of MnSOD gene in rat lungs in response to oxidant stress. Previous studies have shown that activation of transcription of MnSOD gene occurs only after three days of exposure to 85 % oxygen and at that time inflammation of the lungs is also evident. These observations suggest that induction of MnSOD gene transcription during hyperoxia may result from the response of lung cells to various cytokines generated during lung inflammation including tumor necrosis factor (TNF), interleukin-1 and interferon-gamma which are known to be capable of inducing MnSOD gene expression, rather than from a direct effect of oxygen tension. Expression/induction studies on transgenic mice carrying various rat MnSOD-I promoter deletion/chloramphenicol acetyltransferase (CAT) fusion genes have allowed us to map one of the hyperoxic injury responsive elements to between positions -507 and -405 and one of the TNF-alpha responsive elements to between -405 and -289 of the rat MnSOD-I promoter. In this application, the cis-acting regulatory DNA elements responsible for hyperoxia and cytokines will be mapped to the nucleotide level by initially constructing various CAT reporter genes under the transcriptional control of (1) promoter internal deletion mutants, (2) promoter base substitution mutants and (3) fusion promoters consisting of herpes thymidine kinase promoter and various regions of MnSOD-I promoter. Transgenic mice and tissue culture cells harboring these fusion genes will be generated, and the inducibility of expression of the reporter genes by hyperoxia and cytokines will be assessed. The similarity between the hyperoxic injury responsive element(s) and cytokine responsive elements will then be determined. The sites of interactions between promoter sequences and nuclear protein factors in lungs of rats and transgenic mice with or without exposure to hyperoxia or cytokine treatments will also be studied by in vivo footprinting. Finally, comparison will be made between the sites of DNA-protein interactions and sequences of hyperoxia and cytokines responsive elements. Accomplishment of the proposed research should provide valuable basic information toward understanding the nature of the regulatory elements which control the expression of this gene, as well as uncovering the molecular signal driven in vivo in the lungs by hyperoxia and cytokines. This knowledge should then direct development of strategies for augmenting MnSOD expression as a part of antioxidant defense against oxidant-induced lung diseases.

Traditionally sex hormones are recognized as non-genotoxic carcinogens that induce neoplastic transformation epigenetically via enhancement of cell proliferation in target tissues. We have obtained recent evidence that estrogens, per se or via metabolic conversion, can cause genetic damage in vivo and hence may act as tumor initiators in the prostate. A rat model with features of the human prostatic cancer is used as an experimental model of study. Treatment of intact Noble (NBL) rats with testosterone (T) and estradiol- 17beta (E2beta) consistently induced a 100% dysplasia and adenocarcinoma in the dorsolateral lobe (DLP) of the rat gland. Dysplasia was however, not induced when rats were exposed to T, DHT or E2beta alone. We observed that the induction of dysplasia was preceded by the drastic increase in DLP mitotic activity and type II estrogen receptor level. More recently, we found a significant increase in DNA-strand breaks and lipid peroxidation exclusively in DLPs harboring the dysplasia. We therefore conclude that, in addition to mitogenic action, the sex steroids may have direct genotoxicity in this prostatic lobe. We hypothesize that the synergism between genetic and epigenetic action of the sex hormones provides the mechanistic basis of tumor initiation and promotion which eventually leads to neoplastic transformation and tumor formation in rat DLP. In this application we propose the following specific aims to evaluate the relateive importance of the genotoxic and mitogenic action of sex hormones in prostatic carcinogenesis in NBL rats: 1) to ascertain if E2beta is the component in the E2beta treatment that actually induces DNA damage in the rat DLP; 20 to evaluate whether the T + E2beta-induced DNA damage in the rat DLP is a primary event induced by E2beta genotoxicity and not a secondary phenomenon caused by cell proliferation; 3) to determine whether the occurrence of increase in DNA damage precedes the appearance of cell proliferation and dysplasia and thereby provides evidence that genetic-damage is an event likely involved in tumor initiation; 40 to examine if catechol estrogens (CEs) formation is the metabolic step responsible for the conversion of E2beta to DNA- reactive products in the DLP, but not in the VP, of the rat gland and to determine whether prostatic CE formation activity in NBL rats is higher than that found in Sprague-Dawley (SD) rats. This difference may then explain the difference in cancer susceptibility of the two rat strains; 5) to assess the effectiveness of T + diethylstilbestrol (DES, a synthetic estrogen with known genotoxicity) treatment in inducing DNA damage, cell proliferation and dysplasia in the two major prostatic lobes, VP and DLP, of the rat gland. Data from this study are expected to identify the principal roles of sex- hormones in prostatic carcinogenesis and help shape our views on the mechanisms of hormonal carcinogenesis in, not just the prostate, but other hormone-sensitive tissues as well. This study also offers the prospect of using DNA damage and enhancement of cell proliferation as short-term and points from assessing the caner risk of a sex hormone.

The production of toxic, partially reduced species of oxygen is greatly enhanced in hyperoxic lungs and may eventually overwhelm the endogenous cellular capacity of antioxidant defenses resulting in lung cell damage. This kind of pulmonary injury is observed not only in experimental animals, but also in humans receiving oxygen therapy for respiratory insufficiency. Previously studies have demonstrated that augmentation of intracellular antioxidant enzyme activities [superoxide dismutase (SOD) and/or catalase] in cells or animals by treating them with liposome-encapsulated antioxidant enzymes, or exposing animals to a sublethal concentration of oxygen would provide dramatic protection to the recipient cells or animals against hyperoxic insults. In this application, a novel approach will be undertaken to further evaluate the effectiveness of each of the antioxidant enzymes in protecting lung cells from oxygen-mediated injury. Initially, five lines of transgenic mice each carrying a specific antioxidant enzyme transgene will be generated. These transgenes are being designed to be under the control of a human beta-actin promoter to insure high levels of transcription in all types of cells in animals. Additionally, mice containing a combination of two transgenes will also be obtained by mating two transgenic founder mice each bearing a different transgene. Expression of these transgenes in different tissues including the lung will be examined at both mRNA and protein levels. Immunocytochemistry will be used to identify the cellular sites, distribution and concentrations of the antioxidant enzymes in both control and exposure will be used to determine the degree of protection provided by the augmented antioxidant enzyme activities. Antioxidant enzymes are critical for cellular defense against oxidant stress. Unravelling the effectiveness of each antioxidant enzyme should help design more efficient antioxidant therapies to block or reduce oxygen- mediated lung injury. the transgenic mice generated in this study also can be used for elucidating the role of antioxidant enzymes in other diseases associated with the over-production of partially reduced oxygen species, such as some types of lung fibrosis and reperfusion injury of ischemic heart tissue.

Activation of Manganese-containing superoxide dismutase (MnSOD) gene expression in adult rat lungs during exposure to a sublethal concentration of oxygen (85%) may play a critical role in allowing animals to survive a subsequent exposure to lethal concentrations of oxygen. The overall goal of this research is to define the molecular mechanisms for regulation of MnSOD gene in rat lungs in response to oxidant stress. Previous studies have shown that activation of transcription of MnSOD gene occurs only after three days of exposure to 85 % oxygen and at that time inflammation of the lungs is also evident. These observations suggest that induction of MnSOD gene transcription during hyperoxia may result from the response of lung cells to various cytokines generated during lung inflammation including tumor necrosis factor (TNF), interleukin-1 and interferon-gamma which are known to be capable of inducing MnSOD gene expression, rather than from a direct effect of oxygen tension. Expression/induction studies on transgenic mice carrying various rat MnSOD-I promoter deletion/chloramphenicol acetyltransferase (CAT) fusion genes have allowed us to map one of the hyperoxic injury responsive elements to between positions -507 and -405 and one of the TNF-alpha responsive elements to between -405 and -289 of the rat MnSOD-I promoter. In this application, the cis-acting regulatory DNA elements responsible for hyperoxia and cytokines will be mapped to the nucleotide level by initially constructing various CAT reporter genes under the transcriptional control of (1) promoter internal deletion mutants, (2) promoter base substitution mutants and (3) fusion promoters consisting of herpes thymidine kinase promoter and various regions of MnSOD-I promoter. Transgenic mice and tissue culture cells harboring these fusion genes will be generated, and the inducibility of expression of the reporter genes by hyperoxia and cytokines will be assessed. The similarity between the hyperoxic injury responsive element(s) and cytokine responsive elements will then be determined. The sites of interactions between promoter sequences and nuclear protein factors in lungs of rats and transgenic mice with or without exposure to hyperoxia or cytokine treatments will also be studied by in vivo footprinting. Finally, comparison will be made between the sites of DNA-protein interactions and sequences of hyperoxia and cytokines responsive elements. Accomplishment of the proposed research should provide valuable basic information toward understanding the nature of the regulatory elements which control the expression of this gene, as well as uncovering the molecular signal driven in vivo in the lungs by hyperoxia and cytokines. This knowledge should then direct development of strategies for augmenting MnSOD expression as a part of antioxidant defense against oxidant-induced lung diseases.

Although cadmium (Cd) is a proven carcinogen in rodents and may be a "sufficient" carcinogen in men, the mechanism of Cd-induced carcinogenesis remains unknown. Additionally, while Cd is cytotoxic to a wide spectrum of tissues in the body, its carcinogenicity is limited to the prostate, testes and lungs. It has been postulated that the expression of the metal- binding protein, metallothionein (MT), plays a role in determining tissue susceptibility to Cd toxicity and carcinogenicity. We, and others, have shown that rat ventral prostate (VP) and testes, both target organs of Cd carcinogenesis, lack MT expression. The aim of this proposal is to determine whether MT plays a protective role in Cd carcinogenesis. We hypothesize that "induced" expression of MT in rat VP would render the gland resistant to Cd carcinogenicity. Using a transgenic animal approach, we shall attempt to induce MT expression in rat VP. A chimeric gene will be synthesized by fusing the rat probasin (PB) promotor and 5'-flanking region to a mouse MT coding region cDNA (PB-MT construct). The PB gene 5'- flanking region contains two androgen responsive elements and a sequence that targets a transgene expression specifically to the prostate of a transgenic animal. The expression of PB-MT will he induced in an androgen- receptor positive PC-3-cloned prostatic cancer cell line (AR+ PC-3) transfected with the chimeric gene and, later, in transgenic animals carrying the construct by androgen activation. Alterations in cellular or tissue susceptibility to Cd carcinogenicity after induction of MT expression will he assessed by monitoring a set of "early or intermediate" biomarkers. Specifically, these biomarkers are: 1) cytotoxicity, as measured by the expression of a cell death-associated gene (TRPM-2) and "internucleosomal DNA fragmentation", 2) proliferation, as quantitated in situ by levels of histone-3 transcripts and proliferation cell nuclear antigen (PCNA), 3) genotoxicity as assessed by the degree of nuclear DNA strand breaks and DNA-protein formation, and 4) epithelial atypical hyperplasia, as a morphological marker of neoplastic development. To measure tumorigenicity per se, tumor incidence will he enumerated histologically and malignancy of the primary tumors will be assessed by anchorage-independent growth, cellular ploidy and in vivo tumor formation efficiency in isogeneic hosts. Should our theory be proven, we expect to observe either abolition or delay in development of the early or intermediate biomarkers of carcinogenesis, reduction of tumor incidence, and/or reduced malignancy of the primary tumors formed in the VPs of transgenic rats expressing MT, as compared to those found in normal VPs. Data from these experiments are expected to yield important information concerning the role of MT in Cd tumorigenesis. They should illuminate the mechanisms of prostatic carcinogenesis and decipher the causes of heavy metal toxicity.

DESCRIPTION (provided by applicant): Age-dependent alterations in sex hormone milieu and tissue oxidant status are believed to be endogenous risk factors for the development of prostate cancer (PCa) in humans. We previously demonstrated that simultaneous treatment of Noble (NBL) rats with testosterone (T) and estradiol-17beta (E2) for 16 weeks induced epithelial dysplasia, a proliferative lesion that closely resembles human prostatic intraepithelial neoplasia (PIN), exclusively in the dorsolateral prostate (DP/LP), but not in the ventral prostate (VP), of all treated rats. In the LP of the treated rats, an inflammatory response similar to one that associates with human proliferative inflammatory atrophy (PIA), a purported precursor to PIN and PCa, also develops. Longer treatment of NBL rats with the combined hormone regimen causes a high incidence of prostatic adenocarcinoma in the DLPs of all treated animals. We now showed that, 1) in addition to induction of dysplasia/neoplasia, T+E2 induced hyperprolactinemia and attended inflammation in the LPs, 2) it caused oxidative stress (OS)-related damages directly in the epithelia of DPs/LPs of the treated rats, 3) the T+E2 dysplasia-inducing action in rat DP/LP was partially blocked by co-treatment with bromocriptin (Br), an inhibitor of pituitary prolactin (PRL) release, or completely inhibited by co-treatment: with 1C1182,780 (ICI), an antiestrogen that inhibits both local estrogen action and systemic hyperprolactinemia. The two co-treatments also eliminated the: hyperprolactinemia-induced inflammation in the LPs of T+E2-treated rats, 4) using a low density microarray for gene profiling we identified and validated' unique molecular changes in the LP that are associated with dysplasia induction, and 5) we have developed a prostate organ culture system that preserves epithelial-stromal architecture under serum-free condition to define tissue responsiveness to individual or combined hormone supplements. T, 5alpha-dihydrotestosterone (DHT), PRL and E2 were shown to exert different proliferation- and dysplasia-promoting actions on explants established from the VP, LP and DP. Taken together, our data suggest that androgen, estrogen as well as PRL are responsible for the genesis of dysplasia which likely contribute to PCa development in this model and raise the possibility that OS and/or inflammatory may corroborate this process. Based on these findings we hypothesize that 1, DHT, E2 and PRL, singularly or in combination, contribute to early neoplastic transformation of the rat prostate, via: distinct and synergistic molecular pathways, which are definable by a comprehensive gene profiling study, coupled with parallel validations in the animal model and in the organ culture system, as well as functional studies in human cell lines and human specimens. The Aims of this: project are: 1) To determine the unique transcriptomes that are associated with evolution of dysplasia in the DP (free of inflammation but associate with OS) and that in the LP (associated with inflammation and OS), 2) To identify the transcriptomes associated with the purported dysplasia-inducing action: of T+E2 and of T+ PRL in order to delineate if a difference exists between the two, 3) to identify sets of genes associated with each hormonal stimulation. lobe-specificity, OS and inflammation among the various prostatic lobes, 4) to identify the genes associated with specific stages of premalignant and malignant transformation, and 5) to test the functionality of selected genes or pathways in human cell lines and validate their expression in human PCa specimens. Data from these studies are expected to uncover the underlying mechanisms of early prostate carcinogenesis.

The long term objective of our research is to understand the basis of diethylstilbestrol (DES) and cadmium(Cd) toxicities in the ventral prostate (VP) of young and old testosterone (T)-supported rats. We and others have shown that the VP exhibits unique susceptibilities to these toxicants. Our preliminary data revealed that both T+DES- and Cd- treatment of rats induced severe oxidative stress (OS) as well as aberrant proliferative and apoptotic activities in this prostatic lobe. T+DES and Cd separately induced VP tumors in life-time studies, and synergistically early dysplastic changes. We found that the VP expresses minimal levels of mRNA of metallothioneins (MTs) which are heavy metal binding proteins believed to function as intracellular metal-ion chelators and/or antioxidants. We recently reported marked increases in lipid peroxidation status and drastic decline in the activities of reactive oxygen species (ROS) detoxification enzymes in the VPs of aged rats. Taken together, we hypothesize that T+DES and Cd are potent carcinogens for rat VP because, inter alia, they exacerbate oxidative stress (OS) in the aging VP. We thus postulate that 1) induction of OS and associated events, such as DNA single-strand breaks and apoptosis- cell proliferation imbalance, are early events leading to T+DES- and/or Cd-induced dysplasia and cancer development in rat VP, and 2) the VP of an aged rat is more susceptible to tumor induction by these toxicants than its younger littermates. Experiments have been designed to determine whether l) T+DES and/or Cd induce OS, DNA damage, and imbalance in cell proliferation-apoptosis in the VP and these events precede the development of dysplasia 2) these OS-inducers activate MT gene expression 3) the aged VPs have reduced ROS detoxification capability and thus are more sensitive to T+DES and/or Cd action 4) T+DES and Cd, given simultaneously to rats, have compounded effects on OS, dysplasia, and tumor induction in the VP and 5) T+DES with and without Cd can induce OS parameters in organ cultures of VP of young and aged rats and, if so, whether antioxidants added in vitro can reverse these damages. Results form this work will enhance our understanding of aging and OS in toxicant- induced carcinogenesis and the potential application of antioxidants as anti-cancer chemopreventive measures.

DESCRIPTION (Provided by the applicant) It is currently believed that estrogens may have profound effects on prostatic cell growth, differentiation, and neoplastic transformation. Recently our laboratory has obtained exciting new data indicating estrogen receptor-beta (ER-beta) is a key mediator of prostate carcinogenesis and prostate cancer cell growth. Based on our published and unpublished data (summarized in our application) our long term goals are to determine if: 1) ER-beta promotes cell differentiation and/or inhibits growth in normal prostatic epithelium and whether progressive loss of receptor expression is causally linked to malignant transformation, and 2) if re-expression of ER-beta in metastatic prostate cancer cells is essential for cancer cell survival/or maintenance of malignant phenotypes, and activation of the beta receptor, via known antiestrogens or newly devised ligands (the Hanson compounds), leads to cell cycle arrest or cell death which is associated with downregulation of survivin, TERT (the catalytic subunit of the telomerase), and other genes identified by cDNA microarray. The following specific aims are proposed to address these long-term goals. Aim 1: To identify novel (the Hanson compounds) or known (tamoxifene, ICI-182,780, and Raloxifene) estrogen-like compounds that show pronounced subtype-selective differences in ligand binding, prostatic cell growth inhibition and/or transcriptional potency or efficacy for ER-beta. Aim 2: To determine whether the antiestrogen-induced growth inhibition in human prostate cancer cell lines, that express only ER-beta, involves altered expression of survivin, TERT, thymidylate synthase (TS) and metallothionein-II (MT-II). Additionally, cDNA microarray and ER-beta antisense technology will be employed to identify additional ER-beta/antiestrogen regulated genes in this process. Aim 3: A) To further identify genes that are upregulated during the T+E2-induced dysplasia and their reversal induced by co-treatment with ICI in the NBL rats. B) To determine the efficacy of selected antiestrogens and/or Hanson compounds in preventing dysplasia induction by T+E2 in the Noble rats and ascertain whether the chemoprevention is mediated via altered expression of Fra-2 and GADD45 as well as genes identified in Sub-aim 3a. Data from these studies are expected to identify new ER-beta SERMs and provide sights into the structural requirements of ER-beta selectivity. They will also identify new ER-beta/antiestrogen regulating genes and illuminate potential roles of ER-beta in prostate carcinogenesis and utility of ER-beta SERMs in prostate cancer treatment.

Ovarian cancer (OC) is the highest-ranking cause of death from gynecological cancers among American women. Over 90 percent of OC are epithelial carcinoma derived from the human ovarian surface epithelium or HOSE. Epidemiological studies suggest that exposure of HOSE to pelvic inflammatory agents or to toxicants from the reproductive tract may lead to OC development. In addition, systemic exposure to environmental or dietary factors may contribute to the etiology since smoking and caffeine are linked to higher risks. Cytochrome P450-1 enzymes (CYP1 enzymes) are key enzymes responsible for activating procarcinogens to carcinogens. They are highly inducible by endogenous and exogenous factors such as inflammation, hypoxia, sex hormones, caffeine, polycyclic aromatic hydrocarbons, polychlorinated biphenols, and dioxins. Furthermore, their activities regulate estrogen inactivation and activation to genotoxic metabolites. Although overexpression of CYP1s has been linked to the development of several neoplasms an analogous knowledge base for OC is presently unavailable. Recently, we have demonstrated a) overexpression of CYP1A1 enzyme, but not CYP1B1 enzyme, in epithelial OC specimens, b) increased transcription of CYP1A1 in epithelial OC cells when compared to normal HOSE cells, c) existence of a novel CYP1A1 mRNA variant and its marked overexpression in epithelial OC cells, and d) differential growth response of normal and malignant HOSE cells to estrogen. Based on these preliminary data we hypothesize that overexpression of CYP1A1 enzyme is involved in the pathogenesis of epithelial OC, in part via modulation of estrogen metabolism, and that differential expression of wildtype and variant CYP1A1 transcript in vivo is a reflection of dissimilar roles played by these molecules during ovarian carcinogenesis. The goal of our proposed research is to elucidate 1) the role played by CYP1A1 in ovarian carcinogenesis, 2) the relationship between CYP1A1 activity and estrogen metabolism, and 3) the biological relevance of the newly identified CYP1A1 mRNA variant. Four aims have been put forth to test the hypothesis: Aim 1- Stable transfectants, with capabilities of conditional expression of wildtype- and/or variant CYP1A1 enzyme, will be used to determine whether their overexpression in immortalized non-tumorigenic HOSE cells leads to acquisition of malignant phenotypes. Aim 2- Epithelial OC cell lines will be inhibited from CYP1A1 enzyme expression, via a CYP1A1-specific antisense oligonucleotide (ODN), to determine if malignant phenotypes are diminished/abolished. Aim 3- Reverse phase HPLC will be used to determine whether HOSE cells and epithelial OC cells exhibit differential rates/patterns of estrogen metabolism, and if these differences could be explained by differential levels of CYP1A1 enzyme activity as measured by a functional assay (ethoxyresorufin O-deethylase activity). Aim 4- Using discarded human epithelial OC specimens, we will determine by real-time RT-PCR whether expression of CYP1A1 wildtype and/or variant transcript in laser-capture microscopy (LCM)-assisted microdissected carcinoma foci varies among different histopathological types and/or tumor grades of OC, and whether immuno-reactivity of the enzyme correlates with the relative abundance of one or both transcripts in the carcinoma foci.

[unreadable] DESCRIPTION (provided by applicant): Cadmium (Cd) is a significant and growing environmental contaminant. Cd exposure has been linked to prostate cancer (PCa) in some, but not all epidemiological studies. In rats, Cd is a proven carcinogen of the prostate. Although little is known about the mechanisms of Cd-carcinogenicity, several lines of evidence point to an involvement of metallothioneins (MTs), a family of Cd-copper-zinc binding proteins in the process. During our last funding period, we have made several major contributions to the field of Cd and metallothionein research: a) We have developed highly sensitive, sequence-specific quantitative RT-PCR protocols for rodent MT-I and MT-Il quantification and demonstrated that, despite of their low expression, MT-I and -Il genes are inducible in rat ventral prostate, b) We have generated the first ribozymes (Rzs) which can induce sequence-specific degradation of rat and mouse MT-I and MT-lI mRNA, respectively. Significantly, sequence analyzes predict that these Rzs Will degrade many human MTs. Cell transfection studies demonstrated that MT Rzs work effectively in cellulo and degradation of MT mRNA increases cellular susceptibility to Cd cytotoxicity, c) Importantly, transgenic expression of MT Rzs in human prostatic cancer cell lines induces a dramatic apoptotic response, suggesting that MTs are cell survival factors and MT Rzs potent anticancer agents, and d) We have generated a prostate-specific, MT-I overexpresser using a third generation probasin (PB) promoter, ARR2, to target MT-1 expression. This animal, referred as PBMT-1, exhibits intra-epithelial atypia beginning at 26-week of age and widespread prostatic dysplasia by 56-week of age. Based on these findings we now hypothesize that overexpression of MT in prostatic epithelial cells increases cell survival and favors accumulation of premalignant and early malignant cells. Hence, we predict that the PBMT-1 transgenic animal will be more susceptible to Cd-induced neoplastic tansformation. Our recent preliminary findings support this premise since we found widespread dysplasia developed in the VPs of PBMT-1 within 96 hours after a single dose of CdCl2 injection. Furthermore, we predict Rz-mediated degradation of MT-I mRNA will reverse dysplasia/neoplasia development in PBMT-1. These hypotheses will be tested in vivo. Aim 1: To determine whether PBMT-1 mice are more/less susceptible to Cd-induced cytotoxicity/carcinogenicity. Aim 2: Part-1 Human adenovirus vectors (AdV) will be used to deliver MT Rz to PBMT-I, via an orthotopic route, to determine if down-regulation of MT-1 mRNA levels will reverse the development of dysplasia/neoplasia in its prostates Part-2 By cross-breeding with a mouse, named CAR, which expresses membrane Coxsackie/human Adenovirus Receptors (hCAR) on all body cells, with PBMT-1 we will generated the CAR/PBMT-1 mouse. We will then test whether expression of hCAR on murine cells will enhance hAdV-mediated delivery of MT Rz to murine prostatic cells and increase Rz efficacy. These studies are expected to advance our understanding of the relationship between MTs and Cd-cytotoxicity/carcinogencity, generate new preclinical models for future development of gene therapy protocols based on the MT Rz technology.

DESCRIPTION (provided by applicant): The developing fetus is uniquely sensitive to long-term imprinting effects of estrogenic chemicals that are known to have profound effects on adult disease susceptibility. Genistein, a naturally occurring isoflavone found in soy, is recognized as a dietary estrogen as it interacts with both estrogen receptor (ER)-alpha and ER-beta. Human exposure to genistein is predominantly through dietary consumption of soy products, however, its use as nutritional supplements and pharmaceutical agents has dramatically increased due to the presumed beneficial effects of phytoestrogens in preventing cardiovascular diseases and cancer. Ironically, the potential deleterious effects of dietary estrogens, especially if exposure occurs during critical stages of development, remain poorly understood. Of concern are the well-documented carcinogenic effects of prenatal exposure to diethylstilbestrol (DES) and the wide exposure of infants and children to soy-based formulas. We have developed a mouse model to investigate the potential adverse effects of exposing the developing routine neonate to genistein. Outbred female CD-1 mice were treated on days 1-5 with equivalent estrogenic doses of genistein (50 mg/kg/day) or DES (0.001 mg/kg/day) dissolved in corn oil, or corn oil alone (control), and sacrificed at varying ages. A 35% and 31% incidence of uterine adenocarcinoma were found in aged mice neonatally treated with genistein (50 mg/kgf day) and DES (0.001mg/kg/day), respectively. A higher dose of DES (lmg/kg/day) caused a 95% incidence of the cancer. In this proposal, we will test the hypothesis that neonatal exposure to estrogenic compounds induces 1) aberrant methylation of 5'CpG islands of specific genes, and 2) that these changes persist in adult uterine tissues leading to "permanent" changes in gene expression profiles in the adult uterus, resulting in a higher propensity for uterine tumorigenesis in this mouse model. In Aim 1,we will use a highly innovative technology (methylation-sensitive DNA fingerprinting or MS-AP-PCR) to identify genes that are hyper- or hypomethylated in neonatal and adult uteri from mice neonatally exposed to DES or genistein as compared to control (unexposed) animals. In Aim 2, we will perform global gene profiling analyses to identify gene sets that are up - or down - regulated in adult uteri from animals exposed neonatally to estrogens when compared to those without exposure. By comparing the results obtained from Aim 1 and Aim 2 we expect to establish a link between hyper- (silencing) or hypo- (release from silencing) of specific genes induced by neonatal estrogen exposure that are of relevancy to cancer susceptibility and development in the adult uteri, Results from these studies are expected to enhance our understanding of the mechanistic basis of adult diseases caused by developmental exposure to dietary estrogens. Future studies along this line of investigation will address impacts of other environmental estrogens.

DESCRIPTION (provided by applicant): Ovarian carcinoma (OCa) continues to be the leading cause of death due to gynecologic malignancies. Epidemiological data suggest that progesterone (P4) offers protection against OCa while estrogens promote OCa development. We recently demonstrated in normal and malignant human ovarian surface epithelium (HOSE) cells a growth inhibitory action of P4 and a growth promotional effect of estrogens. P4 was shown to activate an extrinsic apoptosis-initiation pathway that involves caspase-8, but not the intrinsic, mitochondrion-related caspase-9' pathway. Importantly, our results have revealed involvement of the Fas/FasL signaling as an important mechanism of P4-induced apoptosis in normal and malignant OSE cells. This finding has incited us to examine the relationship between this steroid and the TNF-related apoptosis-inducing ligand (TRAIL)TRAIL receptor signaling pathway, which holds greater promises for targetted therapies for a variety of cancers.; TRAIL has several advantages over the other TNF-family ligands;1) it is a "pure" apoptosis-inducer, 2) it has preferential cell kill activity against cancer cells, 3) it has a very favorable in vivo toxicity profile, 4) its apoptotic action is independent of p53 status, and 5) its action is often enhanced by combination with other therapeutic agents. In addition, we have discovered that, contrary to the existing dogma, estrogens down regulate progesterone receptors (PRs) in normal and malignant HOSE cells and therefore can negate the protective effects of P4. We therefore hypothesize that P4 and TRAIL independently and/or synergistically induce apoptosis in HOSE and OCa cells through activation of an extrinsic pathway of apoptosis and offer protection against ovarian cancer while E2 via downregulation of PR can negate the effects of P4. Five broad specific aims have been proposed to test these hypotheses: 1. To characterize and compare the apoptotic responses induced by TRAIL and P4, individually or conjointly, in HOSE and OVCA cell lines in order to discern any synergistic effects. In parallel, the effects of P4 on TRAIL and TRAIL receptor, expression will be studied to gain a better understanding of the mechanistic link between the two. Special attention will be placed on detecting whether the apoptotic response belong to type I (extrinsic alone) or type II (extrinsic plus intrinsic amplification). 2: To compare the efficacies of natural progesterone and synthetic progestins such as MPA and NETA in inducing apoptosis in HOSE and OVCA cell lines and to determine whether they have comparable synergistic actions on TRAIL-induced apoptosis. Their action on activating iNOS via the MAPkinase signaling pathway will also be investigated. 3. To determine the anti-tumor activity of P4, MPA, NETA, and recombinant human TRAIL in vivo. 4. To determine whether estrogens antagonize the P4-induced apoptosis via induction of PR downregulation and to determine which estrogen is most effective. 5. To determine if estrogens and SERMs negate the effects of P4 by blocking the influences of P4 on the expression of TRAIL and TRAIL receptors. Results from this proposal have the potential to generate new strategies for the prevention and treatment of OCa.

DESCRIPTION (provided by applicant): Age-dependent alterations in sex hormone milieu, tissue oxidant status, and inflammation are purported endogenous risk factors of human prostate cancer (PCa). Treatment of Noble rats, a human PCa-relevant model, with testosterone (T) and estradiol-17beta (E2) for 16 weeks induced epithelial dysplasia, a proliferative lesion that resembles human prostatic intraepithelial neoplasia (PIN), in the lateral prostates (LPs), but not in the ventral prostate (VPs) of treated rats. The LP dyslasia is frequently attended by inflammation and resembles the human proliferative inflammatory atrophy (PIA), a putative precursor to PIN and PCa. Longer treatment of rats with T causes PCa exclusively in the LPs of 100 percent of the treated rats. T treatment also causes hyperprolactinemia, which induces inflammation in the LP. Marked oxidative stress (OS)- and nitrative stress (NS)-related damages, aberrant expression of cyclooxygenase-2 (COX-2), NAD(P)H oxidases (NOXs), and NO synthases (NOSs), as well as disruption of anti-oxidant defenses were noted in the dysplastic LPs. We here hypothesize that T-supported E2 action contributes to early neoplastic development directly in rat LP by induction of chronic OS/NS that ultimately leads to tumorigenesis. Moreover, the, E2-induced hyperprolactinemia activates tissue inflammatory responses that inflict additional OS/NS damages, thus exacerbating the process. Three aims are proposed. Aim I- To determine if T+ E2 induced LP dysplasia is accompanied by induction of OS/NS directly in the prostatic epithelial compartment and that this process is exacerbated by the presence of inflammation. Laser-capture-microscopy will be used to sample normal and dysplastic epithelia, and their adjacent stroma, in areas with and without evidence of inflammation, to assess the hormone- induced versus inflammation-mediated OS/NS-associated changes. Five types of biomarkers will be evaluated: 1) disruption of OS/NS generation/defense pathways, 2) OS/NS-induced lipid, DNA and protein damages, 3) altered expression of T+E2 induced oncogenes, 4) imbalances in cell proliferation and apoptosis, 5) production of inflammation mediators via COX and/or lypoxygenase (LOX) pathways. Aim 2 - To determine if combined celecoxib (a specific COX-2 inhibitor) and zileuton (a 5-LOX inhibitor) treatment negates or diminishes inflammation and/or dysplasia in the LPs of T+E2 treated rats and if this effect is attended by diminution of OS/NS-associated biomarkers. Aim 3 - Using a LP organ culture system, we will further elucidate the direct cancer-promoting actions of E2, T, DHT, and PRL, in the absence of inflammation or systemic confounding factors. Aim 4-To determine whether advancement of age renders the rat prostate more susceptible to hormone-induced inflammation and/or carcinogenesis. Results from our studies should help explicate the relation between inflammation and carcinogenesis in the human prostate, a topic of intense debate.

A 5-year renewal of the Center for Environmental Genetics (CEG) is proposed. The unifying research focus of the Center is to study the impact of genetic diversity on individual susceptibility to toxic environmental agents. The CEG has a long history (10 years) and broad range of accomplishments in environmental genetics, and this research focus has become increasingly important in environmental health science research. The Specific Aims are: Aim 1. Facilitate the identification of susceptibility genes that may interact with environmental agents to induce complex diseases such as type II diabetes, asthma, intracranial strokes, hypertension, and lung cancer. Aim 2. Facilitate studies to analyze the impact of genetic variants of known genes on individual response to specific environmental agents such as airborne particulates, environmental estrogens, metals, and ionizing radiation. Aim 3. Facilitate studies to examine signal transduction pathways induced by response to environmental agents whose exposure is associated with a disease or tissue/cellular damage. Aim 4. Encourage investigators to direct their research toward a focus in environmental genetics. Aim 5. Develop a partnership with the surrounding community to provide education, awareness of environmental health issues, and public policies through the Community Outreach and Education Program (COEP) Core. We have four highly successful Research Cores that have been in existence for at least 5 year, Ecogenetics, Genetic Toxicology, Respiratory Toxicology, and Signal Transduction. The Human Populations Research Core was initiated to reflect our emphasis to perform human studies related to our research focus. In this renewal application, we will respond to new advances in genomics, proteomics and bioinformatics, and genetic epidemiology by modifying the Facilities and Services Cores to offer the following services to CEG members: 1) Bioinformatic data analysis procedures, 2) Microarray expression and protein analysis methodology, and 3) Procedures for conducting human populations studies. The goal of the Center is to understand and thus, either prevent or intervene in complex human disease by determining the role of gene- environment interaction in the development of diseases.

[unreadable] DESCRIPTION (provided by applicant): This proposal seeks funds to provide 25 travel awards of $1000 each to trainee investigators (graduate students, residents, clinical and postdoctoral fellows and faculty below the rank of Assistant Professor). The travel awards are for participation in the Fall 2008 Society for Basic Urologic Research (SBUR) meeting to be held in Phoenix, AZ on November 20th-23rd. The SBUR is a society of scientists specializing in research on benign and malignant urologic diseases. The Society has held its independent Fall Annual Symposia for 18 consecutive years with great success. Most timely subject of the year is addressed in each meeting, with special emphasis on cross-fertilization between urology and other relevant fields of research. The 2008 meeting will focus on the theme "Epigenetics and Genitourinary Disorders". This year's meeting is structured as a forum for the attendees to learn about the latest advancements in the field of epigenetics (e.g. micro-RNA, DNA methylation, histonemodification) and their applications to investigative research in GU diseases. Scientific exchange at this meeting will open up new avenues for the discovery of diagnostic/prognostic epigenetic markers and for devising novel therapeutics for GU diseases. Key advancements in benign and malignant diseases of the bladder, prostate, and kidneys will be emphasized through a mini-symposium on novel therapeutics and management strategies. The trainees will have opportunities to network with peers and senior members of the Society. They will also have an opportunity to present their data at poster sessions and, in selected cases, as podium presentations. The invited speakers are leaders in their respective fields, offering extensive knowledge and experience. The atmosphere of the meetings is friendly and informal, providing encouragement and unique opportunities for trainees to participate and establish collaborations. In order for such interactions to occur, the trainees need support for their travel expenses. If funded, this grant will contribute significantly to the educational objectives of this meeting. [unreadable] [unreadable] [unreadable]

Significance and Specific Aims Significance: The Administrative Core (AC) is structured to improve and maintain the strong center we have built, and enable us to reach new heights through attainment of our new vision in understanding how gene and environmental interaction, through epigenetics, influences human diseases susceptibility and outcome. Based on the research strengths of our members four broad focus areas will be pursued in the coming funding cycle: endocrine disruption/cancer, cardiovascular/lipid disorders, immune/allergic diseases and neurology/neurobehavior research. The AC is the hub around which all of the participating disciplines and components of the CEG rotate. It intends to ensure maximum efficiency while conserving resources, expediting progress while seizing new opportunities, and enhance operational effectiveness among individual projects and cores while maintaining overall cohesiveness. t he Core promotes pro-active intellectual leadership, effective communication, and exceptional administrative support to integrate all components of the Center, activities into a highly effective program that aims at generating the highest caliber of environmental health sciences (EHS) research, training, and communications with NIEHS and the community. The Core actively seeks advice and input from the Internal (lAB) and External (EAB) Advisory Boards, strives to fulfill fiscal responsibility to the highest standards, and vigorously seeks new avenues of translation.

The Integrative Health Sciences Facility Core (IHSFC) will facilitate clinical investigations that enhance both forward and back translational research of environmental factors in disease causation. The overall objectives of the IHSFC will be accomplished through expert consultation services in medicine, epidemiology, statistics, exposure assessment, informatics tools, and Forth and Back clinical research conferences that will highlight the opportunities for translational research in environmental health. Through providing these services and access to technologies, this Core will foster research that integrates basic science with clinical research and public health. The goal of the Core is to accelerate the transfer of basic biomedical knowledge from the laboratory to clinical applications in prevention, early detection, and new treatments and, ultimately, to prolong and enhance life. The IHSFC programs and activities we propose also will foster greater ease in taking clinical findings or public health observations back to the basic science laboratory. We will capitalize on the potential for synergy between the activities of the IHSFC and other CEG activities such as the Career Development Program (CDP). As an example, the IHSFC can provide a stream of expert knowledge to both the Next-generation Biomedical Investigators (NGBI) and the Masters of Science Clinical Researcher (MSCR). The IHSFC will act as liaison to all other CEG service Cores (Bioinformatics Core and Integrative Technology Support Core) and services available within the University of Cincinnati Medical Center (UCMC) (General Clinical Research Center, Environmental Laboratory, Tissue Procurement, Metallomics Center) to help provide a coordinated and systematic approach to questions concerning translational biomedical research.

DESCRIPTION (provided by applicant): Developmental exposure to natural or environmental estrogens predisposes to prostate carcinogenesis with aging;however, the molecular underpinnings of this phenomenon are unclear. We present evidence that developmental reprogramming of the prostate by estrogens may be mediated, in part, through epigenetic alterations. Using methylation-sensitive fingerprinting (MSRP) as an initial screen for genome-wide methylation changes, we identified multiple prostatic genes whose methylation status was permanently altered in rats as a result of neonatal estradiol and bisphenol A (BPA) exposures at environmentally relevant doses. Detailed characterization of phosphodiesterase 4D4 (PDE4D4) and HPCAL, enzymes involved in cAMP breakdown and formation, respectively, revealed aberrant promoter CpG island methylation patterns with resultant changes in gene transcription as the animals aged. Importantly, these epigenetic alterations were associated with increased susceptibility to hormonal carcinogenesis of the rat prostate gland. Thus we hypothesize that early estrogenic imprinting of the prostate gland with resultant predisposition to carcinogenesis with aging is mediated through epigenetic modifications which permanently affect gene expression in the gland. The objectives of the present proposal are to further characterize our model of developmental reprogramming by low dose estradiol or BPA, to characterize in detail the prostatic gene methylation and transcriptional alterations which result from early life estrogenic exposures and to identify the methylation candidate genes contribute to increased carcinogenic potential in the developmentally estrogenized prostate glands. In Aim 1, we will determine the dose-response relationship for prostatic- BPA effects and establish the developmental windows of susceptibility. We will also use a novel tissue recombination model to test whether BPA modifies carcinogenic susceptibility and methylation patterns in human prostate-like structures. In Aim 2, we will characterize in detail the altered rat prostate methylome with resultant alterations in gene expression as a result of developmental exposures to environmentally relevant doses of BPA or estradiol. MSRP and methylation arrays will be used to expand our prostatic screen to identify a full panel of candidate genes and a stringent algorithm will be followed to identify candidates with regulatory CpG islands. Site-specific methylation and resultant transcriptional regulation will be confirmed. In Aim 3, we will directly test whether the genes epigenetically modified by estrogenic exposures play an active role in prostate gland carcinogenesis using a variety of in vitro and in vivo studies.

DESCRIPTION (provided by applicant): Developmental reprogramming is an important mechanism by which early life exposures to environmental agents increase susceptibility to adult disease. For xenoestrogens such as BPA, additional research is required to understand how specific estrogen receptor (ER) subtypes and genomic vs. nongenomic signaling mediate this developmental reprogramming. Xenoestrogens can act as agonists or antagonists in different tissues, also pointing to the need for a better understanding of how specific ER coregulators (CoR) influence their activity. Finally, additional research is needed to identify the epigenetic effects of reprogramming that persist into adulthood and underlie adult disease, including prostate cancer (PCa). This Grand Opportunity (GO) grant addresses these research needs with an integrated, mechanistic assessment of how BPA induces developmental reprogramming to increase susceptibility to prostate carcinogenesis. The Specific Aims are 1) Global analysis of the BPA epigenome to define changes associated with developmental reprogramming and increased PCa risk and 2) Define the role of specific ER subtypes and CoRs as determinants of BPA activity. This project is appropriate for a GO grant: it is large;multi-investigator, multi-institutional, multi-disciplinary;will address a specific research need and make a significant research impact in 2 years. The research team is uniquely poised to accomplish the objectives, the project is ready to implement now, utilizes innovative technologies, and the results will advance the NIH mission to improve health and prevent disease. Neonates exposed to BPA during prostate development will be followed into adulthood to quantitate BPA-associated prostate lesions and susceptibility to develop lesions will be correlated with BPA induced changes in the prostate epigenome. These studies will fill specific data gaps in the BPA literature with oral exposures, internal measurements of dose and dose-response data for relevant disease endpoints (PIN and carcinoma). The in vivo studies will be informed by in vitro, multi-parametric mechanistic studies to elucidate how specific ERs (?, or ? homo- and ?/? hetero-dimers), nongenomic signaling and the p160 class CoRs (SRC-1, -2, -3) determine the effects of BPA in prostate epithelial cells. The prevalence of PCa is increasing world-wide with 1 in 6 men in the US currently at risk for developing this disease. There is a tremendous need to understand the early origins of PCa to determine if additional public health measures are necessary to prevent exposure to BPA, or other xenoestrogens, during critical windows of development. This highly novel, scientifically vigorous, multidisciplinary approach to model the developmental effects of BPA on the prostate will set new standards for weight-of-evidence assessment of the toxicology of BPA and other endocrine disruptors, and will significantly advance research in the fields of gene-environment interaction, non-Darwinian disease susceptibility and hormone receptor action. PUBLIC HEALTH RELEVANCE: The broad question we wish to address in this proposal is how environmentally relevant low doses of BPA induce a long-lasting chromatin state via epigenetic reprogramming to increase the susceptibility to prostate carcinogenesis in adult life. The identification of a developmentally reprogrammable BPA-epigenome associated with prostate carcinogenesis and its dynamic changes throughout life will open opportunities to identify exposed and at-risk populations, which is the first step toward prostate cancer prevention. More importantly, the studies proposed in this application will provide the new and critical mechanistic data on the mechanism of action of BPA to assist agencies in setting regulatory guidelines for the safe use of BPA.

DESCRIPTION (provided by applicant): Previous studies in rats have provided evidence that brief, early-life exposures to bisphenol A (BPA) at environmentally relevant doses results in developmental reprogramming of the prostate gland via epigenetic modifications that enhance carcinogenic susceptibility later in life. While dose-response profiles, BPA pharmacokinetics and route of exposure studies in rodent models are underway to validate these findings, there is a current and compelling need for research on BPA effects in the developing human prostate gland. The National Toxicology Program 2008 report summarily stated that "studies in laboratory animals provide only limited evidence for adverse effects on development and more research is needed to better understand their implications for human health". In response to this need, novel models have been developed with human prostate progenitor cells that permit a direct examination of the impact of low-dose BPA exposures as they form prostate-like structures in vitro and in vivo. The goals of the proposed studies are to determine if exposure to environmentally relevant levels of BPA during the early stages of human prostate development increases susceptibility to prostate carcinogenesis later in life and to identify the underlying mechanism of this reprogramming event. It is hypothesized that prostate epithelial progenitor cells are the direct targets of BPA action during early gland formation. Further, it is predicted that BPA-induced reprogramming is mediated through a combination of altered DNA methylation and histone modifications that are heritable as progenitor cells self renew, transmitting altered epigenomic information throughout the lifespan of the individual. The proposed research thus represents a new paradigm that human prostate carcinogenesis may begin early in life in response to adverse environmental influences that epigenetically alter progenitor cells. An innovative approach will be exploited to test this hypothesis and directly examine BPA effects on human prostate progenitor cells using two model systems: 1) a 3-D co-culture system with human primary prostate epithelial/stromal cells to form prostaspheres in vitro, and 2) as recombinants with rat urogenital sinus mesenchyme grafted to murine kidney capsules for 1-3 months to form chimeric prostate-like tissues in vivo. These novel approaches will permit an examination of differentiation defects (Aim 1) and carcinogenesis (Aim 2) in the human prostate epithelial cells as a function of developmental BPA exposures. These studies will be informed by genome-wide studies of DNA methylation patterns and heritable chromatin modifications using human gene promoter arrays and Solexa ChIP-seq analysis (Aim 3). Using integrative bioinformatics, it is expected to identify BPA reprogrammed gene candidates that may serve as biomarkers for early-life BPA exposures in human epidemiology studies. To determine the potential relevance of BPA-reprogrammed candidate genes to human prostate cancer, tissue microarrays (TMAs) of human prostate cancer will be utilized to screen for misexpression of gene candidates as a function of disease stage, progression and ethnicity. The information gained from the proposed studies will be of high impact on the scientific, medical and regulatory communities in terms of 1) providing strong and compelling evidence for negative effects of BPA in humans, 2) establishing a mechanistic framework for developmental reprogramming, 3) identifying BPA-reprogrammed candidate genes for use as biomarkers, 4) ascertaining relevance of BPA-genes to human prostate cancer, 5) validating a useful model system for screening other endocrine disrupting chemicals, and 6) establishing a basis for studies on BPA in other organ systems and diseases. PUBLIC HEALTH RELEVANCE: There is increasing evidence in rodent models that brief, early-life exposures to bisphenol A (BPA) at dose levels typically found in humans results in developmental reprogramming of the prostate gland and increases susceptibility to prostate cancer later in life. The present proposal will test for this possibility in human prostate tissue using newly developed model systems with human prostate progenitor cells. Identification of epigenetic marks and BPA-reprogrammed genes may serve as biomarkers for developmental BPA exposures and provide molecular insight into the epigenomic plasticity that predisposes to prostate cancer with aging. The findings will be of high value to the medical and regulatory communities and serve as a model for human exposures to prevalent environmental endocrine disruptors with suspected carcinogenic potential.

DESCRIPTION (provided by applicant): The Department of Environmental Health at the University of Cincinnati is a world leader in environmental and occupational health, with over $22M in external research in 2008. The building that houses this department is showing its age, with one wing already beyond repair. This project will renovate the 3rd floor Kehoe wing and Atrium space of Kettering Lab, to improve research facilities, and meet the following specific aims: 1. Contribute to the American Recovery and Reinvestment Act of 2009 with a "shovel ready" renovation project, which will establish the equivalent of full-time employment for 30 new construction-related positions each year of the project duration-estimated at 24 months. Completion of this work will expand our research capacity to accommodate up to twelve (12) new faculty positions and 60 - 80 support personnel, beyond the construction phase. 2. Renovate the of the 3rd floor Kehoe wing of Kettering Lab (8,910 gross square feet), and transform the under-utilized atrium into useful interdisciplinary research space (4,355 gross square feet), which will meet the physical needs of modern, trans-disciplinary research, with optimization of energy performance, improve energy efficiency, maintenance, and operations to extend the life of Kettering Lab for several decades. 3. Provide a research and training platform as a basis for Comparative Effective Research based on our successful mentoring and training programs, with a focus on epigenetic contributions to human disease. 4. Increase our ability to recruit exemplary scientists to the Ohio-Kentucky region by increasing our allure-to new faculty, staff, postdocs and graduate students. 5. Develop a physical home for transdisciplinary research in a yet-to-be-established Center for Epigenetic Epidemiology, which will house expertise in basic toxicology, computational modeling, bioinformatics, epidemiology, biostatistics, and community based participatory research. This team will focus on understanding how epigenetics links the environment to disease susceptibility in populations, translate research findings to the community, and increase scientific capital. PUBLIC HEALTH RELEVANCE (provided by applicant): The UC Department of Environmental Health is requesting funds to renovate and transform our research space from outdated facilities to modern areas that will bring together scientists from different disciplines. This will increase cooperation among our scientists, help us recruit high-caliber people to the Ohio- Kentucky region, and create an area where we can connect scientists with the community. These improvements will also reduce energy consumption and our carbon footprint.

DESCRIPTION (provided by applicant): Breast cancer (BCa) is the leading cause of death in women;incidence rates continue to rise globally. Genetic predisposition accounts for less than 15% of BCa risk while main etiological factors are those related to environmental exposure and lifestyle choices. Immigrants to the US from low-risk countries develop a higher BCa risk within one generation. Moreover, risk in the second generation is dependent on whether one is born to mother from homeland or US. The latter finding suggests BCa is of fetal origin. A diet high in fat is likely a major risk factor of BCa in the U.S. Rodent studies showed that the type of fat and the particular critical window of exposure are key determinants of this linkage. The environmental estrogen, Bisphenol A (BPA), found ubiquitously in US populations, is also a suspect. Rats exposed to BPA pre-/peri-natally are more susceptible to DMBA-induced BCa than their unexposed counterparts. Although both BPA and high-fat diets have been separately studied, whether the two, given together, have synergistic action has not been investigated. More importantly, epigenetics, a known mechanism underlying gene by environment interaction, has not been studied in the context of developmental origin of BCa. The investigators recently showed that rats prenatally exposed to high-fat diets (39% of kcal) exhibited a higher DMBA-induced BCa susceptibility than controls exposed to a reference diet (16% of kcal). The high-fat diets also induced marked increases in epithelial cell proliferation along with a unique proliferation gene signature identified by global transcriptome profiling. Using an unbiased methylome profiling technique, the investigators also generated preliminary data in support of epigenetic reprogramming of gene expression in adult mammary glands after prenatal exposure to high-fat diets. Here, they propose the gestational period is a critical developmental window for dietary fatty acids-BPA interaction, that reprograms the mammary methylome, resulting in aberrant gene expression and increased BCa risk in adulthood. Three specific alms are proposed to test this hypothesis and translate findings Into public health action through community outreach: Aim 1: To characterize the dose-response of dietary BPA on the developmental effects of high-fat diets on BCa risk in later-life; Aim 2: To delineate (a) if the type of fat matters in synergizing with an effective developmental dose of BPA in elevating adult BCa risk, and (b) if a methylome, along with an aberrant mammary epithelial cell proliferative gene signature, is reflective of the combined exposure;and Aim 3: To translate research findings into public awareness and action to reduce the burden of adverse lifestyle choices and environmental pollutants on BCa risk through an outreach program that partners with the Pink Ribbon Girls. This study will be the first to examine epigenetics as a mechanism underlying lifestyle choices by environmental toxicant Interaction.

Endometrial cancer (ECa) is the most common cancer diagnosed in women. Genetics can only account for 5-10% of ECa risk and the rest lies in hormonally and environmentally influences. Observational studies strongly support unopposed estrogen exposure including endocrine-active substance and its associated metabolic complications are linked to a higher risk of ECa in human. In rat studies, neonatal exposure to BPA affects the adult uterine response to hormone and induced uterotrophy, uterine hyperplasia and cancer. However, data on a more human relevant exposure regimen that involves a low-dose lifespan oral exposure is non-existent. Additionally, the molecular mechanisms underlying it pathogenesis remains incompletely understood. This project aims to address these data gap gaps by assessing the impact of chronic low dose exposure to bisphenol A (BPA) on uterine hyperplasia and carcinogenesis by using a well controlled GLP platform. Our goal is to identify BPA-driven early cancer risk markers to promote translation into public health policy. Our specific aims will be as follows. In aim 1, we will establish a dose-response curve between chronic BPA exposure and the development of uterine atypical hyperplasia or adenocarcinoma and to determine an effective dose of BPA that will induce uterine tumor and/or hyperplasia/dysplasia in 75% of the 2-year-old rat. In aim 2, we will identify BPA-associated eariy ECa biomarkers using an Exploration Approach, which combined genome-wide methylation promoter array analysis and global transcriptome profiling, and a Knowledge-based Approach, which we select a set of genes whose methylation status was discovered and confirmed in another ongoing study. In aim 3, we will seek to determine the time course of changes of the candidate genes confirmed in Aim 2 to identify the BPA-driven early uterine cancer marker genes.

DESCRIPTION (provided by applicant): The University of Cincinnati Center for Environmental Genetics (CEG) is applying for supplemental funds to re-establish a Community Outreach and Education Core (COEC) that will link CEG scientists to local communities through effective outreach and education activities. The investigators intend to revise P30 ES006096 to incorporate a COEC that will establish bidirectional communication between CEG investigators and target audiences, including community members and key opinion leaders, health care professionals, and public health policy makers. The mission of the proposed COEC is to effectively translate environmental health science research findings into practical health promotion, disease prevention information, tools and resources for the target audiences. The investigators are particularly interested in translating environmental health information that concerns susceptible populations, including minority and underserved com munities that are disproportionately affected by environmental contaminants and exposures. The COEC will develop and evaluate effective communication strategies that will reach target audiences through a variety of mechanisms, including online learning, puppet shows, websites, workshops, and targeted education for physicians using innovative outreach and education mechanisms. The outreach and education materials and information developed will be distributed for national implementation. The new COEC will formalize bi-directional community networks and collaborations and provide tools and resources for communicating science to specific audiences. The new COEC will: 1). Work with community stakeholders and opinion leaders to identify local environmental health problems that require research; 2). Work with CEG researchers to ensure that the research is conducted in a sound, ethical manner that will provide meaningful results to the community; 3). Ensure direct community benefit from research through public health policy and communication; 4). Determine the appropriate mechanisms to translate research findings to target audiences.

DESCRIPTION (provided by applicant): The overall goal of the proposed work is to determine whether developmental or adult exposures to inorganic arsenic (iAs) increase prostate cancer (PCa) risk by reprogramming human prostate stem cells. Prior studies have revealed that environmental iAs exposure from natural sources (e.g. drinking water) increases PCa incidence. Recent findings also indicate that iAs directly alters stem cells and augments transformation and studies with PCa cell lines support that this may occur in the prostate gland. To further delineate iAs action(s) in normal human prostate stem cells, determine windows of sensitivity for human exposures and link iAs stem cell exposures to PCa development, novel human prostate stem cell models have been established using primary epithelial cells from disease-free human prostates and human embryonic stem cells (hESC). Importantly, in vivo models using either hESC or adult prostate stem cells mixed with rat inductive mesenchyme have been produced to generate chimeric prostate tissues with normal human prostate epithelium. These original approaches are in place to directly address several unresolved issues regarding iAs exposures and stem cell reprogramming events and to potentially link iAs to growth of human PCa. To accomplish this three specific aims are proposed. Specific Aim 1: Delineate the effects of iAs in modulating normal human prostate and embryonic stem cell self-renewal and differentiation to epithelial cell lineages. To accomplish this, FACS and prostasphere assay of adult human prostate stem cells and directed differentiation of hESC in vitro will be utilized to interrogate te actions of a range of iAs doses in perturbing normal prostate development and growth. Specific Aim 2: Elucidate the molecular underpinnings of As reprogramming of prostate stem cells by identifying DNA methylation modifications and resultant changes in gene expression. In parallel with Specific Aim 1, analysis of prostasphere DNA methylome, DNA hydroxymethylome and transcriptome will be undertaken to identify genome-wide marks in stem cells with resultant As gene expression signatures. Specific Aim 3: Determine whether As exposure initiates carcinogenesis, acts as a co-carcinogen or promotes PCa progression in human prostate epithelium using novel in vivo chimeric prostate models. Adult prostate stem cells from normal men, PCa patients or hESC will be used to generate chimeric tissues grown as renal grafts in nude mice. Carcinogenesis in normal epithelium will be monitored after iAs exposures alone or with steroids as co-carcinogens. PCa progression will be examined in PCa stem cell-derived grafts exposed to iAs. Finally, aberrant expression of iAs-reprogrammed genes will be evaluated in tissue microarrays constructed from PCa patients for translational relevance. The present approaches using fresh human specimens with in vivo models combined with state-of-the-art epigenomic technologies are a marked advance over current approaches that will undoubtedly provide significant new and useful information pertaining to human prostate health. Together, these studies will generate novel information on how iAs increases PCa risk and provide a rationale framework for iAs exposure assessment.

Water pollution is a widespread problem. According to a recent Gallup Poll, ground and drinking water pollution are Americans' top environmental concerns. Some of the most serious pollutants include pesticides and heavy metals, such as lead and arsenic. These substances not only get into our drinking water, but their accumulation in plants and animals can make food unsafe in many communities, adversely affecting human health. Yet, bodies of water are often tested only once a year due to manual collection procedures and large, costly equipment to measure pollutants. This project will create small, printable sensors to simultaneously measure toxic heavy metals and pesticides on-site to enable widespread environmental surveillance in bodies of water, and to measure levels of heavy metals in human populations. The project will recruit and train a diverse workforce to design, test, and produce these new types of sensors. Through collaborations at four universities, the project will leverage the unique skills and facilities at Boise State University, Louisiana Tech University, the University of Alabama at Birmingham, and the University of Arkansas for Medical Sciences to produce and test the sensors. In addition to traditional K-12 outreach activities and recruitment to increase diversity in science, technology, engineering, and mathematics (STEM), this project has developed a novel, hybrid pre-mentoring research experience (PRE Program) to recruit and train underrepresented minority students that will enhance training and increase retention. The PRE Program will be run by the University of Arkansas at Pine Bluff, an HBCU institution. Students will engage in learning and professional development activities for several months prior to working in one of the other four universities for a summer research experience, gaining essential knowledge and experience to ensure success in a STEM career. Furthermore, the project leadership will work with economic development teams to establish manufacturing capabilities to commercialize the sensors for large global markets and to employ project trainees, which will amplify investment in this project. The sensors have the potential to enable a future convergence with the Internet of Things, artificial intelligence, and consumer cell phone apps to provide widespread surveillance and analysis of environmental toxins in water and in human populations. This convergence will create new research and commercialization opportunities contributing to the sustainability of the project beyond the life of the award.<br/><br/>The project will advance chemical and materials engineering, sensor design, environmental research, and human safety. Our research will produce databases of eco-friendly, printable sensor inks for microelectronic devices and functionalized photonic carbon dots for detection of toxic chemicals, as well as electronic and photonic sensor detection methods for multianalyte measurements. Using this fundamental research, the project team will design and optimize economical, multianalyte, eco-friendly sensors that avoid or use only a minute amount of precious metals. The project will develop non-invasive, human HM sensors for on-site use in the home and in community screening clinics. Sensors will be deployed to areas with toxic spills or persistent leakage to evaluate initial exposure and monitor exposure over time, and to gage the progress of remediation procedures. This research project will (1) provide a fundamental understanding of the emergent electrochemical properties of nanocomposite network coatings (NNCs) that avoid or use only minute amounts of precious metals versus Si-based sensors; (2) discover catalytic effects of NNC inks and their sensing mechanisms; (3) develop optimal jet printing parameters for NNC inks; (4) explore novel dopant and functionalization methods for luminescent carbon dot (CD) sensors, including upcycling of papermill and plastics industry waste; (5) learn how the electronic energy gap shifts between functionalized CDs and specific heavy metals; and (6) optimize sensing parameters for multianalyte detection for printed ink and CD sensors. These economical sensors will enable wider use and more frequent monitoring of toxic chemicals which will facilitate a greater understanding of the impact of human activity in the environment and how to minimize the spread of toxic chemicals to humans.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.