Abstract
In vitro hepatocyte culture systems have inherent limitations in capturing known human drug toxicities that arise from complex immune responses. Therefore, we established and characterized a liver immuno-competent co-culture model and evaluated diclofenac (DCF) metabolic profiles, in vitro-in vivo clearance correlations, toxicological responses, and acute phase responses using liquid chromatography tandem mass spectrometry. DCF biotransformation was assessed after 48 h of culture, and the major phase I and II metabolites were similar to the in vivo DCF metabolism profile in humans. Further characterization of secreted bile acids in the medium revealed that a glycine-conjugated bile acid was a sensitive marker of dose-dependent toxicity in this 3D liver microphysiological system. Protein markers were significantly elevated in the culture medium at high μM doses of DCF, which were also observed previously for acute drug induced toxicity in humans. In this immuno-competent model, lipopolysaccharide treatment evoked an inflammatory response that resulted in a marked increase in the overall number of acute phase proteins (APPs). Kupffer cell-mediated cytokine release recapitulated an in vivo pro-inflammatory response exemplified by a cohort of 11 cytokines differentially regulated following LPS-induction, e.g., IL-1β, IL-1Ra, IL-6, IL-8, IP-10, TNF-α, RANTES, G-CSF, M-CSF, MIP-1β, and IL-5. In summary, our findings indicate that 3D liver microphysiological systems may serve as a preclinical investigational platforms from the perspectives of the discovery of a set of clinically relevant biomarkers including potential reactive metabolites, endogenous bile acids, excreted proteins and cytokines to predict early drug-induced liver toxicity in humans.
- anti-inflammatory drugs
- cytokines/chemokines
- hepatocytes
- liver injury/toxicity (DILI)
- mass spectrometry/MS
- organotypic models
- pharmacokinetics
- protein binding
- proteomics
- toxicology
- The American Society for Pharmacology and Experimental Therapeutics