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TOXICITY OF SELECTED BIOACTIVATED COMPOUNDS IN PRIMARY RAT HEPATOCYTES CULTURED IN MICROPATTERNED CO-CULTURES
EP22543
Poster Title: TOXICITY OF SELECTED BIOACTIVATED COMPOUNDS IN PRIMARY RAT HEPATOCYTES CULTURED IN MICROPATTERNED CO-CULTURES
Submitted on 05 Dec 2014
Author(s): Okechukwu Ukairo, Julianne Shi, Justin Jackson, Kelly Rose, Melvin E. Andersen, and Edward L. LeCluyse
Affiliations: The Hamner Institutes, Hepregen Corporation
This poster was presented at 2014 Eurotox
Poster Views: 1,731
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Poster Information
Abstract: Drug-induced liver injury is often caused by cytochrome P450-dependent activation of drugs into reactive metabolites. In vitro models, which can mimic in vivo responses and allow the evaluation of initial and adaptive responses to bioactivated compounds over prolonged periods, offer potentially valuable tools for toxicological assessment. We have previously developed a model in which primary hepatocytes (rat, human) are seeded onto ECM-coated domains of optimized dimensions and subsequently co-cultivated with murine embryonic fibroblasts [i.e. micropatterned cocultures (MPCC)]. This model retains key biochemical functions of in vivo liver with long term stability. Here, we assess the bioactivation and cytotoxicity of acetaminophen (APAP) and other compounds in the 96-well rat MPCC. APAP is a well-known hepatotoxin and exerts its toxic effects through bioactivation associated, in part, with cytochrome P450 3A (CYP3A). Rat MPCCs were exposed to increasing concentrations of APAP (over 5 days) and assessed for changes in hepatic ATP content, glutathione (GSH) levels and urea synthesis. Similar concentration-dependent cytotoxicity profiles (AC50=8.4 ± 2.4mM for GSH depletion and 14.17 ± 3.5mM for urea synthesis inhibition) were obtained over the course of the 4-week study. Addition of 200μM L- buthionine (S, R)-sulfoximine (BSO), an inhibitor of GSH synthesis, or 10μM dexamethasone (DEX), an inducer of rat CYP3A1/2, to rat MPCCs potentiated APAP-induced hepatotoxicity in these cultures irrespective of culture age (over 4 weeks). These findings are consistent with the known in vivo mechanisms of APAP toxicity in rats. In conclusion, rat MPCCs provided reproducible APAP-induced cell cytotoxicity profiles over a 4 week period and can be used to assess the effects of chronic exposure to bioactivated compounds. The toxicity profiles of selected bioactivated compounds are also reported here.Summary: Drug Induced Liver Injury (DILI) modeled in a novel co-culture liver model. Here, we assess the bioactivation and cytotoxicity of acetaminophen (APAP) and other compounds in the 96-well rat MPCC.References: 1.Gómez-Lechón MJ, Lahoz A, Gombau L, Castell JV, Donato MT. In vitro evaluation of potential hepatotoxicity induced by drugs. Current pharmaceutical design 2010;16(17):1963-1977
2.Khetani, S.R. & Bhatia, S.N. Microscale culture of human liver cells for drug development. Nat Biotechnol. 26(1), p120-126 (2007).
3.LeCluyse EL, Witek RP, Andersen ME, Powers MJ. Organotypic liver culture models: Meeting current challenges in toxicity testing. Crit Rev Toxicol. 2012 Jul;42(6):501-48.
4.Ukairo O, Kanchagar C, Moore A, Shi J, Gaffney J, Aoyama S, et al. Long-term stability of primary rat hepatocytes in micropatterned cocultures. J Biochem Mol Toxicol. 2013 ; 27 (3): 204-12
5.Ukairo O, McVay M, Krzyzewski S, Aoyama S, et al. Bioactivation and Toxicity of Acetaminophen in Rat Hepatocyte Micropatterned Co-culture System. J Biochem Mol Toxicol. 2013; 27 (10): 471-8.
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