Program/Grant Name: Center for Ecogenetics and Environmental Health
Funding Agency: NIH/NIEHS (P30 ES07033)
Many diseases of public health importance, such as most cancers, Alzheimer's Disease, Parkinson's disease, asthma, and even birth defects, arise through a complex interaction of genetics and environment. Investigators in the Center for Ecogenetics and Environmental Health are conducting research to understand how specific genetic traits increase or decrease an individual's likelihood of contracting a chronic disease or illness. For example, researchers are studying how genetic differences in the way people break down (metabolize) drugs and other chemicals might increase their chances of getting cancer when exposed to potentially cancer-causing chemicals in their workplace, diet or lifestyle activities (e.g., smoking). Other researchers in the Center are studying genetic traits that, when combined with exposure to substances in the diet or general environment, make individuals more likely to develop chronic neurological diseases such as Parkinson's disease. Other researchers are investigating the link between air pollution and cardiovascular disease in genetically susceptible populations, and environmental factors that might cause birth defects in genetically susceptible individuals. In addition to basic research, the Center includes community engagement activities that include consideration of the ethical, legal and social issues related to the use, and potential misuse, of genetic susceptibility information. The Community Outreach and Ethics Core within the CEEH develops workshops and educational materials, and promotes community education, in the environmental health sciences. Although based in the School of Public Health and Community Medicine, CEEH Investigators come from a variety of schools and departments across the University. Research findings from CEEH investigators will help unravel how genetics and environment interact to produce disease, and are an important extension of the exciting new information coming from the Human Genome Project.
Microphysiological systems as novel in vitro approaches for toxicity assessment: Microphysiological systems (MPS) represent a new frontier in biology, where 3-dimensional cultures of human primary tissue-derived cells can be assembled into flow-through devices that allow in vitro experimentation, but are much more representative of in vivo human response than traditional 2-D static monocultures of immortalized cell lines. Such MPS, once validated, may provide a paradigm shift in how potentially toxic new chemical entities are screened for potential human health risks. My lab is working with Nortis, Inc. on several projects, two of which are described below:
Hazard-based values for regulatory toxicology are traditionally estimated from laboratory animal studies, which are expensive, low throughput, and at times not predictive of human toxicity. The increased presence of chemical contaminants in the environment is an undeniable concern to human health and ecosystems. The need to develop more efficient toxicity assessment systems in order to satisfy the overburdening demand to adjudicate toxicity of this vast library of synthetic chemicals is critical. There are two major avenues being pursued to overcome this bottleneck in animal testing, in silico prediction of compound toxicity, which requires a comparison with similar compounds that are associated with known toxicological data, and in vitro models that faithfully recapitulate the in vivo outcomes at the molecular, cellular, organ, and organ systems level.
#1) We propose to develop and commercialize a human liver 3-dimensional (3D) flow through “microphysiological system” (MPS) based on liver tissue derived cell clusters that, like tissue slices, retain heterotypic cell communication native to the liver. In Phase 1, we will use rat livers to optimize the system due mainly to the ease of tissue procurement. In Phase 2, we will adapt these methods to human derived tissue obtained from core biopsies. We anticipate minor modification to protocols during this transition between species because most of our proposed experimental methods are independent of tissue source. In Phase 1, we will optimize the MPS architecture, cell cluster production, and cell seeding strategies by monitoring the viability and basic liver functions of MPS seeded clusters. We will use real-time continuous assessment of lactate dehydrogenase, albumin, and urea release in the downstream outflow and compare them with periodic assessment of live and dead cell populations by vital dyes. We also intend to develop a unique assay to determine if the cell clusters are perfused through the sinusoid by exploiting the impressive phagocytic activity of Kupffer cells toward quantum dots (Qdots). This assay will also prove valuable in reporting the 3D cellular location and phagocytic activity of this important cell type under various conditions. Once the system is optimized in terms of viability we will look at the higher order liver functions in response to chemicals known modulate activity in vivo, including P450 mRNA levels by qPCR, P450 enzymatic activity by EROD and BROD assays, and nuclear localization of HNFa by immunohistochemistry. These qPCR and enzymatic assays require that after treatment with drugs we remove the cell clusters from the MPS. For these assays to be useful as we move to human tissue in Phase 2 it is necessary the reproducibility of these assays in our system be understood when measuring a functional outcome. We intend to build enough statistical power to evaluate the quality of our assay and thus qualify its use for other researchers.
Our ultimate aim is to develop a biologically high content and relevant, inexpensive, marketable 3D liver bioassay system for improved xenobiotic toxicity analysis and prediction in humans. The perfusion platform we envision is adaptable to various throughput needs, from a single assay to hundreds of assays at a time. This system should enhance relatively long-term metabolic functions, allow for precise drug toxicity testing in vitro, and be suitable for real-time analysis of metabolic functions and long-term drug toxicity profiles.
#2) Here we propose to utilize a novel MPS developed by Nortis, Inc. to create an in vitro test system for human liver carcinogens. Using a novel approach that allows us to identify and monitor, in real time (by confocal fluorescence microscopy), the initiation of single preneoplastic cells and their clonal expansion to glutathione S-transferase-positive (GSTP+) preneoplastic foci, we will for the first time establish an in vitro carcinogenesis bioassay using human cells. Initial approaches will utilize ’initiated’ liver cells obtained from rats exposed in vivo to carcinogens using the well established ‘Solt-Farber’ medium term bioassay protocol. We will then establish conditions that allow for in vitro initiation within Nortis MPS devices. We will then expand this to human primary liver cells in 3D. This MPS approach has the potential to develop a screening process that will identify genotoxic initiators of hepatocarcinogenesis in less than a month, using primary human liver cells in a fully in vitro system.