title: High content analysis of an in vitro model for metabolic toxicity: results with the model toxicants 4-aminophenol and cyclophosphamide.
abstract: In vitro models that accurately and rapidly assess hepatotoxicity and the effects of hepatic metabolism on nonliver cell types are needed by the U.S. Department of Defense and the pharmaceutical industry to screen compound libraries. Here, we report the first use of high content analysis on the Integrated Discrete Multiple Organ Co-Culture (IdMOC) system, a high-throughput method for such studies. We cultured 3T3-L1 cells in the presence and absence of primary human hepatocytes, and exposed the cultures to 4-aminophenol and cyclophosphamide, model toxicants that are respectively detoxified and activated by the liver. Following staining with calcein-AM, ethidium homodimer-1, and Hoechst 33342, high content analysis of the cultures revealed four cytotoxic endpoints: fluorescence intensities of calcein-AM and ethidium homodimer-1, nuclear area, and cell density. Using these endpoints, we observed that the cytotoxicity of 4-aminophenol in 3T3-L1 cells in co-culture was less than that observed for 3T3-L1 monocultures, consistent with the known detoxification of 4-aminophenol by hepatocytes. Conversely, cyclophosphamide cytotoxicity for 3T3-L1 cells was enhanced by co-culturing with hepatocytes, consistent with the known metabolic activation of this toxicant. The use of IdMOC plates combined with high content analysis is therefore a multi-endpoint, high-throughput capability for measuring the effects of metabolism on toxicity.
authors.affiliation: National Research Council, Research Associate Program, Washington, DC, USA stephanie.d.cole9.ctr@mail.mil.
authors.forename: Stephanie D
authors.initials: SD
authors.lastname: Cole
authors.affiliation: US Army, Research, Development, and Engineering Command, Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD, USA.
authors.forename: Janna S
authors.initials: JS
authors.lastname: Madren-Whalley
authors.affiliation: In Vitro ADMET Laboratories, Inc., Columbia, MD, USA.
authors.forename: Albert P
authors.initials: AP
authors.lastname: Li
authors.affiliation: US Army, Research, Development, and Engineering Command, Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD, USA.
authors.forename: Russell
authors.initials: R
authors.lastname: Dorsey
authors.affiliation: US Army, Research, Development, and Engineering Command, Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD, USA Department of Homeland Security, Chemical Security Assessment Center, Aberdeen Proving Ground, MD, USA.
authors.forename: Harry
authors.initials: H
authors.lastname: Salem
mesh_terms: D041721:3T3-L1 Cells / Q000187:drug effects / Q000378:metabolism; D000627:Aminophenols / Q000633:toxicity*; D000818:Animals; D018920:Coculture Techniques; D003520:Cyclophosphamide / Q000633:toxicity*; D005453:Fluorescence; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008658:Inactivation, Metabolic; D051379:Mice; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D000627:Aminophenols; D003520:Cyclophosphamide; C026729:4-aminophenol
keywords: 3T3-L1 cells; co-culture; high content analysis; human hepatocytes; in vitro toxicology; metabolism