Kim Boekelheide - US grants

The hexacarbons, acrylamides and carbon disulfide are all industrial compounds produced in large quantity which pose a serious health risk due to their induction of a similar toxic syndrome consisting of nervous system dysfunction and testicular atrophy following subchronic workplace or environmental exposure. This laboratory has identified abnormalities in microtubule assembly resulting from intoxication of rats with the biologically active n-hexane metabolite 2,5-hexanedione and has associated these microtubule abnormalities with the induction of testicular atrophy. Based on these preliminary observations, we propose the following hypothesis for the pathogenesis of testicular injury induced by this class of toxicants: after metabolic activation, the hexacarbons, acrylamides and carbon disulfide produce crosslinks between lysyl Epilson-amine groups of tubulin; crosslinked tubulin subunits nucleate microtubule formation; the presence of additional toxicant induced nucleating centers within cells alters the number and length of cytoplasmic microtubules; testicular atrophy results because of the unique dependence of the seminiferous tubule upon Sertoli cell cytoskeletal integrity for the maintenance of spermatogenesis and testicular homeostasis. This hypothesis will be tested with a Gamma-diketone treatment protocol which controls for systemic and nervous system toxicity allowing identification of biochemical and morphological events specific for testicular atrophy. The reversibility of the testicular injury will be determined. The nature of the underlying tubulin modification which results in abnormal microtubule assembly will be elucidated. Tissue culture and immunologic techniques will be developed to allow early assessment of additional compounds which may induce testicular atrophy by Sertoli cell cytoskeletal disruption.

The hexacarbons, acrylamides and carbon disulfide are all industrial compounds produced in large quantity which pose a serious health risk due to their induction of a similar toxic syndrome consisting of nervous system dysfunction and testicular atrophy following subchronic workplace or environmental exposure. This laboratory has identified abnormalities in microtubule assembly resulting from intoxication of rats with the biologically active n-hexane metabolite 2,5-hexanedione and has associated these microtubule abnormalities with the induction of testicular atrophy. Based on these preliminary observations, we propose the following hypothesis for the pathogenesis of testicular injury induced by this class of toxicants: after metabolic activation, the hexacarbons, acrylamides and carbon disulfide produce crosslinks between lysyl Epilson-amine groups of tubulin; crosslinked tubulin subunits nucleate microtubule formation; the presence of additional toxicant induced nucleating centers within cells alters the number and length of cytoplasmic microtubules; testicular atrophy results because of the unique dependence of the seminiferous tubule upon Sertoli cell cytoskeletal integrity for the maintenance of spermatogenesis and testicular homeostasis. This hypothesis will be tested with a Gamma-diketone treatment protocol which controls for systemic and nervous system toxicity allowing identification of biochemical and morphological events specific for testicular atrophy. The reversibility of the testicular injury will be determined. The nature of the underlying tubulin modification which results in abnormal microtubule assembly will be elucidated. Tissue culture and immunologic techniques will be developed to allow early assessment of additional compounds which may induce testicular atrophy by Sertoli cell cytoskeletal disruption.

A group of large volume, commercially important environmental and industrial toxicants - including the acrylamides, organophosphorus esters and the hexacarbons - selectively injure the nervous system and male reproductive system. Research in this laboratory, supported by ROl ES05033 entitled "Environmental/Industrial Toxicants and Testicular Injury" and funded through 08/31/93, has focused on the testicular Sertoli cell cytoskeleton as a target for these toxicants. A Research Career Development Award provides an opportunity to expand the scope of research by emphasizing a multifaceted approach to they investigation of these combined neuronal/testicular toxicants. The candidate, Kim Boekelheide, M.D., Ph.D., is Assistant Professor (tenure track} the Department of Pathology and Laboratory Medicine at Brown University. Receipt of a Research Career Development Award will relieve him of non-research-related administrative and teaching burdens which currently occupy 50% of his time. The expectation is that Dr. Boekelheide will continue to take advantage of the fine basic science research available and advance in his research career at Brown University. A manuscript recently accepted for publication in The Journal of Cell biology (M.D. Neely and K. Boekelheide, "Sertoli cell processes have axoplasmic features: an ordered microcubule distribution and an abundance high molecular weight microtubule associated protein (cycoplasmic dynein)"), describes the comparative approach taken to elucidate shared features of neuronal and testicular cytoskeletons. This proposal will continue the basic description of common cytoskeletal features and examine unique neuronal/ testicular cytoskeletal components as targets for toxicant-induced injury. The following specific hypothesis will be tested: 1) intoxication with some toxicants inhibits the function of the cytoplasmic dynein found in Sertoli cells and axons, 2) inhibition of the function of this microtubule motor blocks retrograde transport in the axon and vesicle transport in the Sertoli cell, and 3) failure of retrograde axonal transport results in a localized axonopathy with associated distal degeneration while failure of Sertoli cell vesicle transport leads to germ cell necrosis and sloughing.

DESCRIPTION (Adapted from the Investigator's Abstract): Infertility affects one out of every eight couples, with male dysfunction contributing to half of this incidence. The causes of male reproductive failure are usually unknown, though recent attention has focused on environmental toxicants and their effects. Microtubule disrupters - including model compounds (colchicine, taxol) and environmental contaminants (the benomyl metabolite, carbendazim, and the n-hexane metabolite, 2,5-hexanedione) - are male reproductive toxicants which target Sertoli cell microtubules and produce irreversible testicular injury and infertility. Past work using predominantly biochemical and cell biological in vitro approaches has suggested that these toxicants initiate a cascade of events: inhibition of Sertoli cell microtubule-dependent transport leads to decreased seminiferous tubule fluid formation and germ cell death. This working hypothesis is tested in this project with new methods developed to assess Sertoli cell functions in vivo. In preliminary studies, selective infection of rat Sertoli cells was accomplished by in vivo injection of replication-deficient adenovirus into the rete testis. Initial experiments will characterize this new technique, evaluating onset and persistence of adenovirus-directed gene expression. Next, two properties of this adenovirus injection system will be exploited to study the in vivo consequences of Sertoli cell microtubule disruption. First, since adenovirus is known to use microtubule tracks for translocation from the cell periphery to the nucleus, the time required for adenovirus to appear in the Sertoli cell nucleus after rete injection will be used to measure microtubule-dependent transport rates in this target cell. Second, the gene transfer capability of adenovirus will be used to over-express gamma-tubulin or to knock-out kinesin in the Sertoli cell, permitting germ cell viability to be evaluated in the setting of a selective failure of Sertoli cell microtubule-dependent transport. Together, these complimentary approaches will critically examine key steps in the pathogenetic sequence by which microtubule disrupters alter the supportive capacity of Sertoli cells resulting in loss of germ cells and infertility.

Germ cell apoptosis is the final common pathway of injury following exposure to toxicants that target the interacting cell types necessary for successful spermatogenesis. Model toxicants have been developed as functional probes of each of these interacting cell types: 2,5-hexanedione, carbendazim, and mono-(2-ethylhexyl)phthalate target Sertoli cells, ethane-1,2-dimethane sulfonate targets Leydig cells, and x-irradiation targets germ cells. Over the past quarter century, many labs have contributed to a rich database describing how each of these model toxicants acts one at a time to produce testicular injury. However, real world exposures, like those occurring at Superfund sites and Brownfields in Rhode Island, involve complex mixtures of hazardous chemicals. This project takes the next step toward mechanistic understanding of complex exposures by combining these model testicular toxicants in a novel co-exposure paradigm. 2,5-Hexanedione exposure is characterized by a 3-week prodromal phase followed by the rapid onset of Sertoli cell dysfunction and germ cell loss. In preliminary experiments, we show that early during 2,5-hexanedione exposure, the seminiferous epithelium is highly susceptible to co-exposure toxicity by carbendazim, a Sertoli cell toxicant with the same subcellular target as 2,5-hexanedione, even though each toxicant alone produces only a modest injury response. This 2,5-hexandeione-induced sensitization of the seminiferous epithelium is exploited by model toxicant co-exposure to test the following working hypothesis: the extent of co-exposure synergy following 2,5-hexanedione priming of the seminiferous epithelium depends upon targeting and shared molecular perturbations. We address this hypothesis by pursuing these Specific Aims: 1) Characterize the dose response and time dependence of 2,5-hexanedione-induced sensitization to carbendazim co-exposure 2) Determine the dependence of the co-exposure response to model toxicants in the 2,5-hexanedione-primed testis on cellular and subcellular targeting 3) Use gene chips to obtain molecular fingerprints of testicular sensitization and the model toxicant injury responses to predict co-exposure toxicity.

DESCRIPTION (provided by applicant) This Superfund Basic Research and Training Program-Reuse in RI: A State-Based Approach to Complex Exposures-tackles through research and outreach three inter-related thematic challenges: 1) land reuse, 2) a state-based approach, and 3) complex exposures. Rhode Island is small, densely populated, and burdened by a long history of industrial activity. There are few barriers to academics, government leaders, and community members working together to address basic and translational research issues, management decisions, and communication complexities inherent to re-using hazardous waste sites. The research clusters 5 Biomedical and 3 Engineering Projects into Interdisciplinary Focus Areas: Nanotechnology &Metals, Molecular Epidemiology &Reproduction, and Semi-Volatile Organics &Vapor Intrusion, that investigate toxicant-induced disease mechanisms and develop potential biomarkers associated with co-exposures, and identify, characterize, separate, and remediate complex mixtures. These projects are supported by an Analytical Core-Chemistry and Biostatistics, a Molecular Pathology Core, and a Training Core. The Research Translation and Community Outreach Cores facilitate the flow of knowledge and information among the participating academics, government leaders, and community members in a true university-state-community partnership. The Administrative Core provides the infrastructure to promote communication, critical self-evaluation, and resource management. This program provides Rhode Islanders with a responsive center of technical excellence that takes a research-oriented approach to resolving the complex scientific, engineering, and societal issues that arise when considering the reuse of hazardous waste sites, by focusing on the following specific objectives: * Provide Leadership - achieve excellence in basic and applied research, teaching, and service in environmental health and technology. * Catalyze Research - increase understanding of health risks and solve technological challenges associated with remediation. * Enhance Connectivity - build bridges across fields and create new interdisciplinary training and educational opportunities. * Research Translation and Community Outreach - communicate health hazards, guide plans for reuse of contaminated sites, and increase the capacity of state agencies and community groups. ADMINISTRATIVE CORE (Boekelheide) Description (provided by applicant) The primary objective of the Administrative Core (Core A) is to promote, organize and facilitate the cross disciplinary research activities of the SBRP around environmental health, land re-use and co-exposure issues in Rhode Island. Brown University's SBRP, "Reuse in Rhode Island: A State-Based Approach to Complex Exposures," began funding in April 2005. The elements that guide the investigators focus on Mixed Exposures, take a State-Based approach to environmental health research, technology development, and contaminated land re-use with Rhode Island as their laboratory. During their initial three years of funding, the investigators have established themselves as an innovative program of intellectual activity and of basic research and applied science at Brown and throughout Rhode Island. The newly established Center for Environmental Health and Technology (CEHT), officially recognized by the Brown University Corporation in spring, 2007, has strengthened the identity, status and capabilities of our SBRP. CEHT provides a structure for expanding the investigators research and core activities, and facilitates collaboration with other programs within Brown University, and with community and government agencies within the State of Rhode Island. The Specific Aims of the SBRP include: Specific Aim 1: Develop processes that support and accommodate the growth of the interdisciplinary basic and applied research and training. Specific Aim 2: Maintain a communication structure that strengthens team building and facilitates the dissemination of information. Specific Aim 3: Implement mechanisms that will optimally promote the program's objectives and ensure the best and responsible use of the available resources.

[unreadable] DESCRIPTION (provided by applicant): An apparent increase in the prevalence of human male reproductive tract abnormalities during the past half century have led Skakkebaek to articulate "testicular dysgenesis syndrome" as an explanation for this phenomenon. According to Skakkebaek, the observed abnormalities-including malformed external genitalia (hypospadias), undesecended testis (cryptorchidism), spermatogenic defects, and testis germ cell cancer- result from the alterations in the in utero and perinatal hormonal milieu causing disruption of sensitive male reproductive tract development events. Attention is focused on environmental endocrine disrupting chemicals as possible causes of this developmental disruption. [unreadable] [unreadable] Phthalates are ubiquitous environmental contaminants and endocrine disrupting chemicals. Rats exposed to di-(n-butyl)phthalate) (DBP) during critical in utero window of male reproductive tract development have altered gene expression and a number of reproductive tract abnormalities associated with testicular dysgenesis syndrome, including underdeveloped or absent reproductive organs, hypospadias, cryptorchidism and decreased sperm production. Although testis germ cell cancer has not been observed in this rat model, the fetal testes of DBP-exposed male rats contain dysplastic, multinucleated gonocytes. [unreadable] [unreadable] In this project, a new mouse model of gestational DBP-induced testicular dysgenesis is established. After characterizing alterations in gene expression in this genetically tractable species, p53-deficient mice will be used to allow DBP-induced dysplastic gonocytes to persist in the postnatal testis rather than degenerate and die. We expect these persistent dysplastic gonocytes to become transformed in the p53-deficient environment, resulting in tumorigenesis and the development of testis germ cell cancer. These goals will be pursued with the following working hypothesis as a guide: Disruption of mammalian in utero hormonal environment by phthalates produces a common spectrum of abnormalities across species, including a predilection to testicular carcinogenesis unmasked in p53-deficient mice. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant) [unreadable] [unreadable] The purpose of this series of workshops is to provide members of academia, community groups, and government agency staff a better understanding of hazardous waste site investigation and [unreadable] remediation practices. This workshop series has the potential to impact several stakeholders, [unreadable] and would be an important adjunct to the wide-range of topics currently being addressed by the [unreadable] Brown University SBRP Research Translation and Outreach Cores. We anticipate that each of the targeted audiences (members of academia, community groups and government agency staff) will benefit. This series of workshops will consist as four separate one-half day sessions, and will be conducted over the period of four to eight weeks. Each workshop will relate to and complement subsequent workshops, but will contain adequate background to stand-alone. The format of four separate workshops was selected to encourage attendance by reducing the required time commitment (per workshop) for the targeted audiences. Each of the workshops is designed to provide adequate background, such that attendance of all four workshops is not mandatory. A single two-day conference including all of the four workshops could be considered as the conference is further developed. [unreadable] [unreadable] [unreadable] [unreadable]

The primary objective of the Administrative Core (Core A) is to promote, organize and facilitate the crossdisciplinary research activities of the SBRP around environmental health, land re-use and co-exposure issues in Rhode Island. Brown University's SBRP, REUSE IN RHODE ISLAND: A STATE-BASED APPROACH TO COMPLEX EXPOSURES, began funding in April 2005. The elements that guide us focus on MIXED EXPOSURES, take a STATE-BASED approach to environmental health research, technology development, and contaminated land re-use with RHODE ISLAND as our laboratory. During our initial three years of funding, we have established ourselves as an innovative program of intellectual activity and of basic research and applied science at Brown and throughout Rhode Island. Our newly established Center for Environmental Health and Technology (CEHT), officially recognized by the Brown University Corporation in Spring, 2007, has strengthened the identity, status and capabilities of our SBRP. CEHT provides a structure for expanding our research and core activities, and facilitates collaboration with other programs within Brown University, and with community and government agencies within the State of Rhode Island. The Specific Aims of our SBRP include: Specific Aim 1: Develop processes that support and accommodate the growth of the interdisciplinary basic and applied research and training. Specific Aim 2: Maintain a communication structure that strengthens team building and facilitates the dissemination of information. Specific Aim 3: Implement mechanisms that will optimally promote the program's objectives and ensure the best and responsible use of the available resources.

Environmental exposures, like those occurring at Superfund sites and Brownfields in Rhode Island, nvolve complex mixtures of hazardous chemicals. We have established a novel co-exposure paradigm to expand our mechanistic understanding of testicular injury resulting from such complex exposures. Model esticular toxicants are used as functional probes of the interacting cell types within the seminiferous epithelium: 2,5-hexanedione, carbendazim, and mono-(2-ethylhexyl) phthalate target Sertoli cells, and xrradiation and 1,2-dibromo-3-chloropropane target germ cells. Using the adult rat as the animal model, the co-exposure paradigm combines subacute exposure to 2,5-hexanedione with acute exposure to another Sertoli cell or germ cell toxicant. In the initial funding period, dose-response behavior and phenotypic alterations were determined for the testicular toxicants. Compared with acute toxicant exposure alone, the co-exposure paradigm attenuated or enhanced the germ cell apoptotic response, the final common pathway of testicular injury, depending on dose and cellular target. Strikingly, the gene array analysis supports a positive correlation between a muted or exaggerated gene expression response and the extent of co-exposure attenuation or enhancement of germ cell apoptosis. These exciting results provide a phenotypic anchor for further molecular analyses, and underscore the ability of the co-exposure paradigm to provide new insight into the testicular response to complex exposures. In the next funding period, laser capture microdissection will be used for cell-type and stage-specific enrichment of mRNA and protein from the seminiferous epithelium, extending the co-exposure paradigm to low doses. In addition, novel sperm biomarkers of effect will be identified for chronic cell-type specific testicular injury. The work will be guided by the following working hypotheses: 1) co-exposure attenuation or enhancement of toxicity depends upon dose, targeting, and the extent of molecular perturbation, and 2) the testicular response to cell type specific toxicant exposure can be identified through molecular analysis of sperm. This project contributes to the Molecular Epidemiology &Reproduction Interdisciplinary Focus Area, and identifies principles of paracrine-dependent co-exposure-induced toxicity that are broadly applicable to many organ systems.

DESCRIPTION (provided by applicant): Testicular Dysgenesis Syndrome has been proposed to explain the reported increase in the prevalence of human male reproductive tract abnormalities during the past half century. According to this hypothesis, the observed abnormalities-including malformed external genitalia (hypospadias), undescended testis (cryptorchidism), spermatogenic defects, and testis germ cell cancer-result from alterations in the in utero and perinatal hormonal milieu causing disruption of sensitive male reproductive tract developmental events. Attention has focused on environmental endocrine disrupting chemicals as possible causes of this developmental disruption. Phthalates are ubiquitous environmental contaminants and endocrine disrupting chemicals. Rats exposed to di-(n-butyl)phthalate) (DBP) during a critical in utero window of male reproductive tract development have decreased fetal testicular testosterone secretion, altered gene expression (including decreased steroidogenic gene expression), and many of the reproductive tract abnormalities associated with testicular dysgenesis syndrome. Surprisingly, in recent work conducted in our laboratories, we have discovered that, unlike the rat, fetal mouse testes are resistant to the testosterone and steroidogenic enzyme inhibitory effects of DBP. This observation of species specificity in the testicular dysgenesis response raises two important questions addressed by this application: What are the molecular pathway determinants of an effect of endocrine active toxicants on fetal testicular steroidogenesis? And what is the human fetal testicular response to endocrine disrupting chemicals, including DBP? In this project, a xenograft bioassay of fetal testes transplanted into a rodent host is optimized, considering host hormonal status, exposure parameters, and intrinsic testicular versus host susceptibility. The dose response of rat, mouse, and human fetal testis xenografts to model endocrine active toxicants (diethylstilbestrol [DES] and DBP) will then be determined. Using molecular analyses, gene profiling, and modeling, the comparative biology of the fetal testicular response to these endocrine disruptors will be explored, identifying biomarkers of susceptibility. These goals will be pursued with the following working hypothesis as a guide: Exposure of a xenotransplant bioassay predicts human fetal testicular susceptibility to endocrine active toxicants. PUBLIC HEALTH RELEVANCE: Increases in human male reproductive tract abnormalities (falling sperm counts, hypospadias, cryptorchidism, and testis germ cell cancer) have been blamed on environmental exposures that alter the in utero and perinatal hormonal milieu. This project will develop a xenograft bioassay consisting of fetal testes implanted in a rodent host to directly assess human susceptibility to endocrine disrupting chemicals, including the plasticizer di-n-butyl phthalate.

With an increasing incidence worldwide. prostate adenocarcinoma is the most common non-skin cancer diagnosis and the second leading cause of death in men in the U.S. Estrogenicity during in utero development has been associated with later life risk of prostate adenocarcinoma, raising concern that early life exposures to endocrine disrupting chemicals may be contributing to this disease burden. This pilot project uses xenotransplantation of human fetal prostate (gestational age 12-22 weeks) as a model to examine the developmental effects of endocrine disrupting chemicals. Following characterization of the maturation sequence in the xenografts, dysregulation of this developmental sequence by estradiol exposure, with and without a second later exposure, will be determined. Histopathological and biomarker assessment will identify estradiol-induced pre-cancerous alterations in ductal morphogenesis, and the long-term effects of estradiol exposure, including the occurrence of prostatic intraepithelial neoplasia, will be identified. Building on this basic characterization of the model, early developmental exposures to the environmental endocrine disruptors bisphenol A and genistein will be compared with estradiol for epigenetic modifications. The working hypothesis is: Human fetal prostate xenotransplants respond to estrogenic endocrine disrupting chemical exposure with aberrant differentiation due to altered DNA methylation. Rodents have been useful models for the study of prostate carcinogenesis; however, spontaneous prostate neoplasia in rodents is rare while humans are uniquely susceptible. Therefore, this project offers the very distinct advantages of human relevance and enhanced susceptibility in the evaluation of environmental impacts on fetal prostate development. The goal of this pilot project is to build a human-based platform to evaluate the developmental origins of later life prostate disease associated with endocrine disrupting chemical exposure, focusing on the underlying epigenetic mechanisms that control disease induction and progression.

Description (provided by applicant): The primary objective of the Administrative Core is to promote, organize and facilitate the interdisciplinary research and core activities of the Formative Center. The Program Director/Principal Investigator will provide leadership, supervision and organizational control in a manner that best utilizes the team's talents and skills towards four programmatic objectives: 1) provide leadership and innovation in basic and clinical research that furthers our understanding of environmental stressors and their impact on fetal development, 2) expand interdisciplinary research that broadens our understanding of the health risks associated with developmental exposures and the underlying molecular mechanisms associated with disease, 3) build bridges across fields and create new interdisciplinary research, training, and mentoring opportunities, and 4) effectively communicate the potential health hazards and translate the basic research findings for the benefit of public health. The Administrative Core will function as the hub of the program by developing the organizational structure and creating mechanisms for communication among the members of the Formative Center, with clinicians, with researchers, with the public, with NIEHS staff, and with other Children's Environmental Health Centers and Formative Centers. Responsible fiscal and resource management will be the primary responsibility of the Principal Investigator, aided by the Program Manager. The Administrative Core will pursue these Specific Aims: Specific Aim 1: Provide, support and develop processes that sustain and accommodate the growth of interdisciplinary basic and applied research and training. Specific Aim 2: Maintain a communication structure that strengthens team building and facilitates the dissemination of information. Specific Aim 3: Implement mechanisms that will optimally promote the program's objectives and ensure the best and most responsible use of the available resources. Public Health Relevance: The over-arching goal of this Formative Center is to develop novel biomarkers for the adverse effects of environmental exposures that impact fetal development and produce childhood and adult disease. Using an interdisciplinary approach, research, educational, and training interventions will be designed to address the concerns that pregnant women, families, and communities have about environmental chemicals and the health of their developing children.

Bisphenol A (BPA) is an endocrine disrupting chemical causing ubiquitous exposures in the developed world. Both animal studies and recent epidemiological evidence indicate that BPA targets the male reproductive tracl<, producing testicular injury and alterations in sperm parameters. Standard GLP-based approaches in rodent models have been relatively insensitive in detecting BPAinduced abnormalities in the male reproductive tract. We propose the use of well-established morphometric assessments ofthe testis, including quantitation of retained spermatid heads and the incidence of TUNELpositive germ cells, to enhance the sensitivity of detecting testicular effects of BPA exposure. In addition, we will identify molecular biomarkers of effect in the testis and sperm, by examining global mRNA and miRNA levels and genome-wide DNA methylation. Measuring these molecular biomarkers in sperm is expected to be particularly sensitive and specific, since sperm are an homogeneous population of cells that developed throughout their life history within the seminiferous epithelium. The GLP design provides multiple doses, and a positive estradiol control, allowing robust testing for the presence of a BPA-induced effect. In addition, we will assess both the paternal FO rats and their progeny, addressing differences across generations. The goal of this project is to test the following working hypothesis: addition ofthe enhanced morphological and molecular endpoints with the GLP design will provide more sensitive and specific biomarkers of BPA-induced male reproductive toxicity. The GLP study will be enhanced by morphometric assessments and molecular biomarkers of effects through the following Specific Aims: Specific Aim #1: will enhance the assessment of BPA-induced testicular injury using advanced histological and morphometric endpoints. Specific Aim #2: characterize the caudal sperm and testis mRNA and miRNA transcript content. Specific Aim #3: develop the caudal sperm and testis methylome.

CORE SUMMARY/ABSTRACT The Brown Superfund Research Program (SRP) addresses the difficult scientific challenges inherent to Superfund and Brownfield sites by engaging academic-government-community partnerships to inform a broad- based state-of-the-art research enterprise focused on providing Integrated Biomedical & Engineering Solutions to Regulatory Uncertainty. With four projects organized into Biomedical-Engineering Project Collaborations (Vapor Intrusion Modeling and Health Monitoring, Nanotechnology Applications and Safety) and within- discipline integrating work (Biomarkers & Toxicity Testing, Nanomaterial Vapor Barriers), the Center investigates vapor intrusion, develops biomarkers of reproductive health, and designs safe nanomaterials for site remediation. These research efforts are supported by the Cores and State Agencies Liaisons that provide dynamic, interdisciplinary, and bi-directional interactions with government and community partners. The Administrative Core is the hub of the Center and develops the organizational structure and mechanisms to facilitate communication among its members and with the Rhode Island Departments of Environmental Management and Health, the community, and numerous stakeholders, including the NIEHS, USEPA, ATSDR, and other SRPs. Kim Boekelheide, M.D., Ph.D., Center Director, Eric Suuberg, Ph.D., Associate Director, and Robert Hurt, Ph.D., Associate Director, comprise an experienced leadership team that provides vision, research insight, and management expertise to the Center. A Biostatistics and Bioinformatics Specialist, Susan Huse, Ph.D., is supported by the Administrative Core and provides an integrative resource for data analysis. Linda Covington, Center Manager, is responsible for the day-to-day management tasks of the Program. Brown University continues its strong support of the Brown SRP with a commitment to invest $200,000 per year over the next five year cycle, and $500,000 bridging support in the no cost extension year, enhancing the scope of environmental health and remediation research, and augmenting research integration, translation, and community engagement. Responsible fiscal and resource management are the primary responsibility of the Center Director and Associate Directors, aided by the Center Manager. The Administrative Core brings together all efforts of the Center members through these Specific Aims: Specific Aim 1: Provide strong leadership and develop processes that support and accommodate the growth of interdisciplinary basic and applied research, translational activities, community engagement, and training. Specific Aim 2: Maintain administrative structures that strengthen team building and ensure effective communication within the program and to all stakeholders. Specific Aim 3: Implement mechanisms that optimally promote the program's objectives and ensure best practices for responsible use of available resources.

PROJECT SUMMARY/ABSTRACT This project develops sperm molecular biomarkers to improve detection and monitoring of toxicant- induced testicular injury. Monitoring now relies upon semen parameters and serum hormone levels, which are insensitive and variable indicators of testicular toxicity. Sperm mRNA transcripts and DNA methylation marks are acquired during spermatogenesis and reflect the integrity of that process; therefore, measuring these sperm biomarkers provides insight into the testicular response to environmental and occupational exposures. In the past grant period, we characterized rat models of toxicant-induced testicular injury and identified rat sperm mRNA transcript alterations that are sensitive and specific indicators of testicular toxicity. We have also shown that human sperm mRNA transcripts and DNA methylation marks can be measured and are indicative of sperm dysfunction. In this grant period, additional rat models of toxicant exposure, including mixed exposures, will be evaluated (Specific Aim 1) to identify rat sperm mRNA transcripts and DNA methylation marks that reflect testicular toxicity (Specific Aim 2a). Building on these rat studies, we will collaborate with Project 3 to study trichloroethylene exposure, a key vapor intrusion indoor air contaminant (Specific Aim 2b). An optimized human-relevant sperm molecular biomarker panel will be developed by characterizing sperm mRNA and DNA methylation alterations in men receiving chronic methotrexate treatment in comparison to a control group of normal men (Specific Aim 3). This basic-to-clinical translational effort will be guided by the following working hypothesis: Sperm mRNA transcripts and DNA methylation marks are integrated molecular biomarkers of testicular injury that can be used to monitor low level mixed environmental exposures. The goal of this project is to apply novel sperm molecular biomarkers to well-defined animal models of testicular toxicity, enabling the translation of this technology for monitoring environmental and occupational exposures in humans. This goal will be achieved by fulfilling the following Specific Aims: Specific Aim 1: Evaluate the phenotypic and functional consequences of low level exposures to testicular toxicants that produce molecular alterations in rat sperm, including changes in testis histopathology, fertilizing capacity, and early embryogenesis. Specific Aim 2: a) Identify rat sperm mRNA transcripts and DNA methylation marks that are molecular biomarkers of testicular dysfunction following low-level exposure to cell type specific toxicants and mixtures of toxicants. b) In conjunction with Project 3, evaluate the male reproductive tract effects of trichloroethylene exposure in the rat with phenotypic and functional assays, and sperm biomarkers. Specific Aim 3: Translate these findings by examining sperm molecular biomarkers in an epidemiology study of normal men compared to men treated with methotrexate for autoimmune arthritis.

? DESCRIPTION (provided by applicant): The Brown Superfund Research Program (SRP)-Toxicant Exposures in Rhode Island: Past, Present, and Future-uses its highly successful paradigm of academic-government-community partnerships to tackle environmental health and remediation concerns in Rhode Island, an appropriate geopolitical context for interdisciplinary initiatives. This partnerships model resolves the longstanding problem of how to bring affected parties together to address issues of site contamination by complex mixtures. The scientific theme for this re-submission is Integrated Biomedical & Engineering Solutions to Regulatory Uncertainty, pursued by four projects (two biomedical and two engineering/environmental science) tackling complex issues of past, present and future environmental degradation resulting from legacy chemicals, current discharges, and emerging contaminants. This state-of-the-art research effort addresses key scientific uncertainties facing the Superfund program using the latest research tools, approaches, and technologies in Biomedical-Engineering Project Collaborations (Vapor Intrusion Modeling and Health Monitoring, Nanotechnology Applications and Safety), and within-discipline integration (Biomarkers & Toxicity Testing, Nanomaterial Vapor Barriers). State Agencies Liaisons in Engineering and Environmental Health are a unique feature of the Brown SRP. These positions have enabled the Research Translation Core and Community Engagement Core to develop academic-government-community partnerships with hazardous waste sites contaminated with mixtures of toxicants, both Superfund sites (Centredale Manor Restoration Project, Peterson/Puritan) and Brownfields sites (Fisherville Mill, Gorham/Textron/Mashapaug Pond, Industrial Lane Johnston, and Narragansett Tribal Lands/Waters). The Administrative Core, Training Core, and Molecular Pathology Core support interdisciplinary research and training involving these sites and their contaminants. The Brown SRP provides Rhode Islanders with a responsive center of technical excellence that takes a research-oriented approach to resolving the complex scientific, engineering, and societal issues that are associated with the remediation of hazardous waste sites, by focusing on the following Specific Aims: Specific Aim 1: Build on existing strong academic-government-community partnerships to address local environmental health and contaminant remediation issues as a national model of effective intervention at Superfund and Brownfields sites. Specific Aim 2: Discover innovative scientific solutions to regulatory uncertainty and environmental contamination by working at the biomedical-engineering interface. Specific Aim 3: Pursue interdisciplinary approaches to research, training, and communication to maximize the value and impact of our work locally and nationally.

PROJECT SUMMARY Historically, toxicity testing has relied on high dose exposures in animals with default methods for extrapolating to low level exposures in human populations, producing (at high cost) great uncertainty for human health risk assessments. This Brown University Bioengineering Research Partnership (BRP)?Human 3D Microtissues for Toxicity Testing via Integrated Imaging, Molecular and Functional Analyses? implements a transformative change by developing novel 3D human microtissues as a bridging technology based on integrating imaging, molecular and functional analyses that will facilitate more rapid, cost-effective toxicity testing of environmental chemicals and emerging toxicants. The project has two phases. Phase 1 establishes 3D microtissues of five tissue types (prostate, ovary, lung, brain, heart) to address the key challenges facing development of these predictive biology platforms: reproducibility, biological complexity, integrated endpoints, and human variability. Phase 2 selects two of these 3D microtissue models for computational systems biology analysis with sufficient dose- and time-response data to define adverse points of departure for an in vitro-to-in vivo extrapolation and safety assessment. Working with collaborators and commercial partners, the BRP team includes faculty from biology, engineering, mathematics, and medicine who have formed the Center to Advance Predictive Biology (https://www.brown.edu/research/projects/center-to-advance-predictive-biology/). The following working hypothesis guides the project: In vitro pathology assessment of human 3D microtissues within a computational systems biology framework identifies toxicant-induced adverse points of departure for safety assessment. The high content, high throughput platforms for these evaluations are 3D microtissue test systems that re-capitulate the differentiated features and characteristic cellular functions of humans tissues. Progress toward the goal of transforming toxicity testing will be made by addressing these Specific Aims: ? Specific Aim 1. Innovate the 3D microtissue platform with engineering solutions for improved well designs, confocal imaging, and high-throughput workflows ? Specific Aim 2. Optimize 3D microtissues as predictive biology platforms ? Specific Aim 3. Streamline image acquisition, reconstruction, and quantitative analysis for the in vitro pathology assessment of 3D microtissues ? Specific Aim 4. Integrate imaging, molecular, and functional endpoints within a computational systems biology framework for the purpose of human safety assessment This Brown University BRP will accelerate development and commercialization of human 3D microtissue platforms as alternatives to animal toxicity testing.