title: Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse, rat, and human primary hepatocyte proliferation.
abstract: Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). The assay was multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. An optimized defined medium cocktail was developed to sensitize hepatocytes to cell cycle progression. The baseline EdU response to defined medium was greatest for mouse, followed by rat, and then human. Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on increased gene expression for Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of EdU labeling in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The observed species differences are consistent with CAR activators inducing a proliferative response in rodents, a key event in the liver tumor mode of action that is not observed in humans.
authors.affiliation: The Hamner Institutes for Health Sciences, Institute for Chemical Safety Sciences, 6 Davis Drive, PO Box 12137, Research Triangle Park, NC 27709, USA.
authors.forename: Valerie
authors.initials: V
authors.lastname: Soldatow
authors.affiliation: Syngenta Crop Protection, LLC, P.O. Box 18300, 410 Swing Road, Greensboro, NC 27419-8300, USA.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Peffer
authors.affiliation: The Hamner Institutes for Health Sciences, Institute for Chemical Safety Sciences, 6 Davis Drive, PO Box 12137, Research Triangle Park, NC 27709, USA.
authors.forename: O Joseph
authors.initials: OJ
authors.lastname: Trask
authors.affiliation: Syngenta Crop Protection, LLC, P.O. Box 18300, 410 Swing Road, Greensboro, NC 27419-8300, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Cowie
authors.affiliation: The Hamner Institutes for Health Sciences, Institute for Chemical Safety Sciences, 6 Davis Drive, PO Box 12137, Research Triangle Park, NC 27709, USA; ScitoVation, LLC, 6 Davis Drive, PO Box 110566, Research Triangle Park, NC 27709, USA.
authors.forename: Melvin E
authors.initials: ME
authors.lastname: Andersen
authors.affiliation: The Hamner Institutes for Health Sciences, Institute for Chemical Safety Sciences, 6 Davis Drive, PO Box 12137, Research Triangle Park, NC 27709, USA.
authors.forename: Edward
authors.initials: E
authors.lastname: LeCluyse
authors.affiliation: The Hamner Institutes for Health Sciences, Institute for Chemical Safety Sciences, 6 Davis Drive, PO Box 12137, Research Triangle Park, NC 27709, USA; ScitoVation, LLC, 6 Davis Drive, PO Box 110566, Research Triangle Park, NC 27709, USA. Electronic address: deisenroth.chad@epa.gov.
authors.forename: Chad
authors.initials: C
authors.lastname: Deisenroth
mesh_terms: D000082:Acetaminophen / Q000633:toxicity; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics; D001681:Biological Assay; D049109:Cell Proliferation / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D000090702:Constitutive Androstane Receptor; D051544:Cytochrome P-450 CYP3A / Q000235:genetics; D000072467:Cytochrome P450 Family 2 / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D010091:Oximes / Q000494:pharmacology; D010634:Phenobarbital / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D013250:Steroid Hydroxylases / Q000235:genetics; D013844:Thiazoles / Q000494:pharmacology
chemical_list: D000090702:Constitutive Androstane Receptor; D008565:Membrane Proteins; D010091:Oximes; D011725:Pyridines; D018160:Receptors, Cytoplasmic and Nuclear; D013844:Thiazoles; D000082:Acetaminophen; D004815:Epidermal Growth Factor; C028474:1,4-bis(2-(3,5-dichloropyridyloxy))benzene; D004247:DNA; C475093:6-(4-chlorophenyl)imidazo(2,1-b)(1,3)thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C101679:Cyp2b10 protein, mouse; C496508:Cyp3a11 protein, mouse; D051544:Cytochrome P-450 CYP3A; D000072467:Cytochrome P450 Family 2; D010634:Phenobarbital
keywords: Car; Cell proliferation; High content imaging; Primary hepatocytes