Antonio Planchart

Per- and polyfluoroalkyl substances (PFAS) are emerging as a major public health problem in North Carolina and across the United States. PFAS comprise a class of over 5,000 compounds. Their unique chemical properties have been harnessed to make consumer and industrial products more water, stain, and grease resistant; they are found in products as diverse as cosmetics and flame-retardants. PFAS are resistant to degradation, move easily through the environment, and accumulate in living organisms. Exposure to PFAS has been associated with health effects including cancer and toxicity to the liver, reproductive development, and thyroid and immune systems. Despite widespread detection in the environment and evidence of increasing human exposure, understanding about PFAS toxicity, its bioaccumulative potential in dietary sources such as aquatic organisms, and effective remediation remain notably understudied. The recent discovery by this proposed Center’s Deputy Director, Dr. Detlef Knappe, of widespread PFAS contamination in the Cape Fear River watershed in NC underscores that these compounds are in need of immediate investigation.. The goal of our Center is to advance understanding about the environmental and health impacts of PFAS. To meet this goal we are employing a highly trans-disciplinary approach that will integrate leaders in diverse fields (epidemiology, environmental science and engineering, biology, toxicology, immunology, data science, and advanced analytics); all levels of biological organization (biomolecule, pathway, cell, tissue, organ, model organism, human, and human population); state-of-the-art analytical technologies; cutting-edge data science approaches; a recognized track record in interdisciplinary, environmental health science (EHS) training; and well-established partnerships with government and community stakeholders.

Primary liver cancer, the vast majority of which is hepatocellular carcinoma (HCC) is one of the few cancers with increasing incidence in the US. Incidence of HCC has tripled since 1980, which is particularly worrisome given that HCC confers a median survival of less than two years. The steepest increases in incidence are in Southern rural states and among ethnic minorities. While the prevalence of HCC had paralleled high rates of viral hepatitis in the last several decades, recent increases in the prevalence of nonalcoholic fatty liver disease (NAFLD) and its progression to nonalcoholic steatohepatitis (NASH) with fibrosis and cirrhosis, has fueled HCC in recent years. Yet, these factors alone do not explain the substantial regional and ethnic variation in HCC progression. One understudied but potentially potent HCC risk factor with increasing prevalence that disproportionately affects ethnic minorities, is exposure to environmental contaminants. These contaminants degrade slowly and therefore persist in the environment, providing a stable exogenous source for human exposure. Toxic metal(oid)s such as cadmium and arsenic are classified as probable carcinogens, and emerging data from murine models suggest that exposure is associated with hepatic steatosis, cirrhosis and liver cancer. Per- and poly-fluoroalkyl substances (PFAS) exposure in humans is associated with obesity and NASH. Further, emerging evidence indicates that these environmental exposures can induce epigenetic alterations that may promote adverse effects on the liver, but we lack longitudinal human data. These data underscore the need for longitudinal human data to assess whether and how these contaminants impact HCC risk. To address these knowledge gaps, and in response to RFA-CA-20-049, we propose the Southeastern Liver Health Study, a longitudinal cohort study of two sub-cohorts comprising 16,000 males and females aged 40 years and older in two Southeastern states, North Carolina and Georgia. We will test the overarching hypothesis that cadmium alone or in a mixture with other toxic metals and PFAS increases the risk of progression from NAFLD to liver fibrosis and HCC. The cohort will be recruited from community clinics including Federally Qualified Health Centers and University Health Systems’ Primary Care Centers and Hepatology programs at Duke, UNC Chapel Hill and Emory. Sub-cohort I will comprise 10,000 otherwise healthy adults who will be followed for 1–5 years, anticipating that ~1,100 fibrosis cases, including cirrhosis, will develop, and sub-cohort II will comprise 6,000 advanced fibrosis cases, anticipating ~750 HCC cases will develop. We will nest case-control studies within the cohorts, evaluate associations between environmental exposures and HCC incidence, and identify epigenetic marks responsive to contaminants that predict progression to HCC. Impact: This will be the first large-scale effort to longitudinally determine the link between environmental contaminants, liver disease and cancer in a residentially and ethnically diverse population. Additionally, we will create a data and specimen repository that will provide the research community with an invaluable resource to study HCC and other cancers.

Amyotrophic lateral sclerosis (ALS) is a devastating disease resulting from loss of motor neurons, leading to paralysis and eventually death. There are no effective therapies for ALS. The major disease hallmark found in the vast majority (>90%) of all ALS patients is called TDP-43, a protein that accumulates in the brain and spinal cord and is implicated in the death of neurons. Unfortunately, targeting or removing TDP-43 with drugs is not a logical step forward, as reducing overall TDP-43 levels can be quite toxic to cells, posing unique challenges in devising new therapies for the majority of sporadic ALS patients. Our recent work suggests that activation of HSF-1, a critical factor that controls the cell’s ability to refold toxic aggregated proteins, is capable of preventing TDP-43 from accumulation and therefore may restore normal cellular function to diseased neurons affected by ALS. Thus, we hypothesize that HSF-1 activator drugs represent a new avenue for ALS therapies that could prolong the survival of ALS patients. The implications are clear; preclinical testing of new drugs that slow disease progression by preventing TDP-43 accumulation could lead to new and effective ALS drugs. We made significant progress towards this goal already by showing that a particular drug called HSF1A, which turns on HSF-1, is able to prevent TDP-43 from aggregating in cells. Now, we can move this idea forward and test the extent to which HSF1 activation produces a therapeutic response in an animal model of ALS. Our study could dramatically accelerate the development of new drugs that act to “disaggregate” TDP-43; these drugs represent potential therapies for the majority of those currently afflicted by ALS.

Cadmium (Cd) is an extremely toxic industrial and environmental pollutant classified as a human carcinogen. It is a metal found ubiquitously in the earth’s crust and extracted in the production of other metals such as copper, lead, and zinc. Human exposure occurs through consumption of contaminated food, cigarette smoke, and fossil fuel combustion. Cd is associated with nephro-, neuro-, and osteotoxicicity, and carcinogenesis. Animal and epidemiological studies have linked exposure to learning disabilities, autism spectrum disorders (ASD), and hyperactivity. Unlike other toxic heavy metals, Cd is not thought to directly damage DNA. Instead toxicity may occur through epigenetic changes, such as altered DNA methylation or chromatin modification, although the mechanisms remain unclear. Given the ubiquitous presence of cadmium and the increasing prevalence of ASD and associated conditions such as attention deficit hyperactivity disorder, improved understanding of Cd’s mechanism of action and potential role in the etiology of these disorders is critical. Our laboratory uses the zebrafish as a powerful comparative model to explore conserved mechanisms underlying environmentally influenced phenotypes. Our preliminary data indicate that developmental exposure to cadmium in zebrafish results in hyperactivity and significant (>five-fold) downregulation of the ring finger protein 2 (Rnf2; previously Ring1b), which is an E3 ubiquitin ligase and a major regulator of chromatin structure via ubiquitination of histone H2A, as it is a subunit of the polycomb repressive complex 1. In addition, hyperactivity was recapitulated in Rnf2 heterozygous knockout fish as well as in wildtype fish exposed to an Rnf2 specific inhibitor, indicating that levels of Rnf2 protein are a critical factor in the observed hyperactivity. Based on these findings, we hypothesize that downregulation of Rnf2 coupled with altered chromatin structure may play an important role in Cd-mediated hyperactivity. To probe this hypothesis, we will explore two fundamental questions: 1) does restoration of Cd-mediated downregulation of Rnf2 rescue exposure-related hyperactivity? and 2) does downregulation of Rnf2 alter levels of ubiquitinated H2A and chromatin structure in regions of neurodevelopmentally critical genes? This project will leverage cutting-edge epigenetic techniques to provide novel insights into the mechanisms underlying Cd-mediated behavioral abnormalities.

Amyotrophic Lateral Sclerosis (ALS) is a progressive fatal disease with a median survival period of three years from symptom onset. There are no effective disease modifying therapeutics and the etiology of the disease remains largely outstanding. While some cases (<10%) are due to known genetic mutations (i.e., familial), the vast majority of cases (>90%) are referred to as sporadic – occurring at random. Thus, it has been suggested that one’s local environment plays a major role in the risk for ALS. In addition, recent research emphasizes the link between early-life exposures and increased risk of adult disease onset. Herein, we hypothesize that there is overlap amongst the processes that are perturbed in both genetic and sporadic cases of ALS. To probe this hypothesis, we will leverage a transgenic zebrafish line with an ALS-associated mutation in the superoxide dismutase 1 (SOD1) gene in the presence and absence of a suspected neurotoxicant, beta-methyl-amino alanine (BMAA). Deep protein sequencing and phenotypic assays will be evaluated to provide insight into the molecular pathways that contribute to the genetic and environmental mechanisms of ALS and motor neuron degeneration.

The goal of this proposed project is to establish a state-­‐of-­‐the-­‐art zebrafish research core facility at North Carolina State University (NCSU) to 1) advance basic, environmental and biomedical research; 2) increase the competitiveness of our faculty to secure extramural funding; 3) strengthen present and cultivate future collaborations; 4) provide opportunities to grow research programs and recruit new faculty; and 5) enable the potential for collaborative development of new technologies using the zebrafish model. The zebrafish (Danio rerio) has emerged as a critically important model for human biomedical research. Initially established as a model for genetics and developmental biology, its applications have extended to many cutting edge areas such as cancer biology, drug discovery, regeneration, epigenetics and understanding the underlying mechanisms of diverse human diseases from cardiovascular to neurodegenerative conditions. Zebrafish are highly fecund with short generation times of 3-­‐5 months. Eggs can be obtained in abundance (hundreds per female in a single day). Zebrafish eggs are fertilized externally, are relatively large (0.6 mm), transparent and readily manipulated by microinjection techniques. Rapid development from a zygote to the hatching period (~48 hours) provides many advantages over mammalian models for observation throughout organogenesis and for dissecting the molecular events that regulate diverse developmental processes. Zebrafish are significantly more economical to maintain than mammalian models (<1%), enabling many more experimental opportunities. Gene transfer procedures, in situ hybridization protocols, and morpholino-­‐based knockdown technology have been refined and are well-­‐accepted experimental approaches for zebrafish. Despite the evolutionary distance (~400 million years), its genetics and signaling pathways are highly conserved with humans. The combination of these features makes the zebrafish uniquely suited for high impact discovery-­‐based and technologically innovative biomedical research. NCSU recognizes the importance of this model for high impact research and technology development. In recent years, the University has been building a critical mass of faculty using zebrafish that currently focus on craniofacial development, neurodevelopment, immune system function and toxicology. Currently, these faculty and their resources are distributed across three colleges despite significant overlap in interests and resources. In order to coordinate research and training activities, this group self-­‐organized in 2012 and have been conducting cross-­‐ laboratory meetings on a monthly basis to develop plans to improve communication and resource sharing. This proposal stems from these activities and reflects the collaborative potential and commitment by each faculty member (including a recent recruit that will begin at NCSU in January 2014) to the importance of building a centralized resource at NCSU. Successful funding of this proposal and establishment of a centralized zebrafish core facility will have an immediate positive impact on the productivity and research potential of a critical mass of NCSU faculty. It will directly improve cost-­‐ and resource sharing and facilitate new collaborations that will better leverage the collective investments and expertise of our faculty.

Toxicity from exposure to naturally occurring inorganic arsenic in drinking water is a significant global health threat. Chronic arsenic exposure is associated with teratogenicity and increased levels of morbidity and mortality manifested by heightened risks to certain cancers, innate immune deficiencies, hypertension and stroke, diabetes and respiratory diseases. Despite the immune risks associated with arsenic toxicity, there have been very few advances in our understanding of immunity-related cellular and molecular mechanisms affected by arsenic exposure. The objective of the proposed multidisciplinary research is to investigate effects of chronic exposure to low concentrations of inorganic arsenic (10 and 100 parts per billion) on the cellular and molecular functions of macrophages and neutrophils, which form essential components of the innate immune system. These experiments will be performed primarily in an in vivo setting by leveraging specific immune properties of embryonic- and larval-stage zebrafish (Danio rerio). Early-stage zebrafish rely exclusively on innate immunity to defend from external challenges. This property, along with rapid development from egg to larvae, developmental transparency, and availability of transgenic lines expressing fluorescent macrophages and neutrophils, make zebrafish a superior model for studies of immune function. Successful completion of the aims in this proposal will result in a substantial increase in our knowledge of the role of arsenic toxicity in immune deficiencies, will pave the way for more comprehensive explorations of how arsenic alters molecular pathways important in immune function, and will establish the utility of innovative in vivo assays for research of a broader chemical space.