The liver is the site of action for many toxicants due to the interaction with liver-specific phase I, II, and III clearance pathways. In vitro models of hepatocellular response are desirable to study mechanisms of action, reduce animal-to-animal variability from in vivo experiments, and allow examination of various patterns of toxicant exposure (dose-dependence, acute or chronic, single agents or mixtures). Current in vitro models of liver tissue are limited by the instability of the hepatocyte phenotype in vitro and the lack of compartmentalized gene expression that underlies the regional toxicity of hepatotoxins such as carbon tetrachloride. The investigators have previously developed a stable model of liver tissue in vitro (approximately 2 months) by co-cultivating primary rat hepatocytes with non-parenchymal cells using microfabrication techniques. In addition, they have demonstrated that exposure of co-cultures to physiologic gradients of dissolved oxygen in a parallel plate bioreactor induced compartmentalized ('zonal') functions and susceptibility to toxicants through a pathway that may be dependent on hypoxia inducible factor-la (HIFla, an oxygen-sensitive transcription factor). They hypothesize that the adverse effects of toxic environmental agents that chronically perturb hepatic clearance or produce toxic metabolites can be studied in a microscale platform of stable, zonated liver tissue. To test this idea, the Specific Aims of this proposal are, 1) to develop a miniaturized, stable murine liver tissue model based on micropatterning and co-cultivation with non-parenchymal cells to study the response to known liver toxicants (chlorinated hydrocarbons); 2) to study the oxygen-dependent zonal responses and dependence on HIFla using fluorescent reporter hepatocytes derived from transgenic animals (CYP2B2-GFP) and Cre-LoxP technology to excise HIFla in vitro; and 3) to develop a perfused microscale array of zonated engineered liver tissues to study the response to mixtures of toxicants. Development of a microscale platform of stable liver tissue is anticipated that can be easily interrogated with small quantities of toxicants, their mixtures, and water samples will provide a unique tool to study mechanisms of action and establish biomarkers to predict liver toxicity due to environmental exposure.
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