2016 Progress Report: Organotypic Models of Mammalian Liver as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials

EPA Grant Number: R835738C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R835738
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Predictive Toxicology Center for Organotypic Cultures and Assessment of AOPs for Engineered Nanomaterials
Center Director: Faustman, Elaine
Title: Organotypic Models of Mammalian Liver as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
Investigators: Kavanagh, Terrance J , Eaton, David
Institution: University of Washington
EPA Project Officer: Klieforth, Barbara I
Project Period: December 1, 2014 through November 30, 2018 (Extended to November 30, 2019)
Project Period Covered by this Report: December 1, 2015 through November 30,2016
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text |  Recipients Lists
Research Category: Safer Chemicals , Health , Human Health

Objective:

The objectives are to develop an organotypic 3D model of human and rodent liver using a microphysiological device, and evaluate its suitability for assessing the adverse effects of engineered nanomaterials and heavy metals.

Progress Summary:

Primary human and rat hepatocytes were used to populate a dual-chamber Nortis microphysiological system. These devices were then evaluated for cell morphology, viability, functionality (cytochrome P450 activity; HNF4α expression; albumin production). Human and rat hepatocytes were also evaluated for their ability to metabolize vitamin D and to metabolize and activate the nephrotoxin aristolochic acid (AA) into metabolites that were toxic toward proximal tubule epithelial cells (PTEC) cultured in tandem in another Nortis MPS device. Human hepatocytes were also evaluated for their ability to metabolize the liver hepatotoxicant and carcinogen aflatoxin B1.

Results indicate that hepatocytes cultured in the 3D MPS maintain greater viability and hepatic functions for at least 14 days whereas 2D cultures only exhibited these for 5-6 days. Compared to 2D monolayers, hepatocytes in 3D organotypic cultures also exhibit enhanced albumin production, HNF4α expression, cytochrome p450 activity and inducibility (Cyp1A1; Cyp3A4), presence of bile canaliculi, multi-drug resistance protein-2 (MRP2) transporter expression and function, vitamin D hydroxylation, and AA bioactivation (as indicated by a 4 to 10-fold increase in toxicity of AA toward PTEC when AA is passed through the liver prior to PTEC, vs. direct exposure of PTECs to AA). The hepatocytes were also able to metabolize the aflatoxin as indicated by specific adducts on DNA (evaluated with immunofluorescence).

We also evaluated the performance of newer generation Nortis microphysiological chambers. One of these chambers is a wide-bore system that better accommodates primary hepatocyte cultures. Cells were viable in these chambers for up to 28 days.

We also evaluated the survival, morphology and differentiated phenotype of human primary hepatocytes seeded into Nortis triple well chamber devices. These newer devices are designed to accommodate hepatocytes such that they can form cord-like structures reminiscent of hepatic sinusoids present in intact liver. The morphology of these structures is cord-like and staining for the hepatocyte differentiation marker HNF4α. These new chambers have provided a convenient model of hepatocyte differentiation and structure that is largely representative of human hepatoctyes in vivo.

We also demonstrated that the Nortis devices can be connected in series to identify important 'organ-organ interactions in toxicology.' We connected a human liver chip with human kidney (proximal tubule cells, PTECs) and demonstrated that the highly specific kidney toxin, aristolochic acid, is 5 times more toxic to the kidney when first passed through the liver.

Future Activities:

In the future, we will continue to characterize hepatocyte function in the 3D MPS system and assess their polarized phenotype (presence of bile canaliculi; xenobiotic transporter expression and localization).  We will evaluate the effects of silver nanoparticles (AgNPs), quantum dot nanoparticles, silver and cadmium ions and other heavy metals in hepatocytes cultured in 2D monolayers vs. 3D MPS. These will include quantitative measures of viability, function, the induction of glutathione pathway genes and proteins, metallothionein (MT) expression, and oxidative stress biomarker expression.  In a manner similar to that we have described for tandem organ on a chip evaluation of AA toxicity, we will examine the ability of hepatocyte expression of MT to deliver Cd and Ag to PTECS and determine if such delivery influences kidney cell viability and function.  We also have developed lentiviral vectors that report on cellular redox status, and these will be applied to this system as well as other systems in the UW PTC.


Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other subproject views: All 10 publications 4 publications in selected types All 4 journal articles
Other center views: All 134 publications 39 publications in selected types All 38 journal articles
Type Citation Sub Project Document Sources
Journal Article Chang S-Y, Weber EJ, Van Ness KP, Eaton DL, Kelly EJ. Liver and kidney on chips: microphysiological models to understand transporter function. Clinical Pharmacology & Therapeutics 2016;100(5):464-478. R835738 (2016)
R835738 (2017)
R835738 (2018)
R835738C002 (2016)
R835738C003 (2016)
R835738C003 (2017)
R835738C005 (2017)
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  • Journal Article Chang S-Y, Voellinger JL, Van Ness KP, Chapron B, Shaffer RM, Neumann T, White CC, Kavanagh TJ, Kelly EJ, Eaton DL. Characterization of rat or human hepatocytes cultured in microphysiological systems (MPS) to identify hepatotoxicity. Toxicology In Vitro 2017;40:170-183. R835738 (2016)
    R835738 (2017)
    R835738 (2018)
    R835738C002 (2016)
    R835738C002 (2017)
    R835738C003 (2016)
    R835738C003 (2017)
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  • Supplemental Keywords:

    3D organotypic cultures, microphysiological systems, hepatocytes, mouse, human, rat, nanoparticles, quantum dots, aristolochic acid, cadmium, silver, cytotoxicity, redox status, cellular stress response

    Relevant Websites:

    The Predictive Toxicology Center (PTC) for Organotypic Cultures Exit

    Progress and Final Reports:

    Original Abstract
  • 2015 Progress Report
  • 2017 Progress Report
  • 2018

  • Main Center Abstract and Reports:

    R835738    Predictive Toxicology Center for Organotypic Cultures and Assessment of AOPs for Engineered Nanomaterials

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R835738C001 Airway Epithelium Organotypic Culture as a Platform for AdverseOutcomesPathway Assessment of Engineered Nanomaterials
    R835738C002 Organotypic Model of Human Kidney as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
    R835738C003 Organotypic Models of Mammalian Liver as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
    R835738C004 Organotypic Model of Testis as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
    R835738C005 Integrating Liver, Kidney and Testis Nanomaterial Toxicity using the Adverse Outcome Pathway Approach