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In Vitro Hepatobiliary Disposition Assessment of 15 Non-Therapeutic Chemicals in Sandwich-Culture Rat Hepatocytes using B-CLEAR® Technology
Citation:
Wambaugh, J., J. Jackson, K. Brouwer, A. Herman, G. Honda, R. Pearce, AND R. Thomas. In Vitro Hepatobiliary Disposition Assessment of 15 Non-Therapeutic Chemicals in Sandwich-Culture Rat Hepatocytes using B-CLEAR® Technology. Presented at Society of Toxicology Annual Meeting, San Antonio, Texas, March 11 - 15, 2018. https://doi.org/10.23645/epacomptox.6987284
Impact/Purpose:
This is a late breaking abstract for the Society of Toxicology annual meeting. This presentation describes billiary clearance contract work that was conducted to reduce uncertainty in the application of the CSS RED HTTK tool.
Description:
Toxicokinetic (TK) data are important but often unavailable for chemicals associated with environmental exposure. Relatively high throughput, in vitro TK (HTTK) methods have been developed to predict TK using intrinsic hepatic clearance and plasma protein binding. However total clearance is often underestimated by current HTTK methods, suggesting that some major clearance mechanisms may be poorly defined within the HTTK approach. Biliary clearance is an important hepatic clearance mechanism describing the removal of a molecule from the blood into the hepatocyte (e.g. hepatic uptake) followed by biliary excretion of the molecule into the bile. To determine the potential impact of this clearance mechanism on HTTK model predictions, the biliary clearance and hepatocyte partitioning (Kp, the ratio of cellular concentration to nominal concentration) were evaluated in sandwich-cultured rat hepatocytes (SCRH) using B-CLEAR® technology. Hepatocytes cultured in a sandwich-configuration form functional bile canalicular networks and maintain the expression and function of key uptake and efflux transporters relative to hepatocytes in vivo. In vitro predictions of biliary clearance utilizing B-CLEAR® technology has been demonstrated to strongly correlate with in vivo biliary clearance in both humans and rats. Fifteen chemicals from the ToxCast library were selected for analysis. HTTK data underestimates overall clearance (renal and hepatic metabolism) by more than a factor of ten for 12 of the 15 chemicals. SCRH with tight junctions intact or tight junctions modulated (e.g. open) were exposed to each compound at two concentrations (10 and 30 µM) for 10 and 30 minutes. Post incubation supernatant and cell lysates were analyzed using LC-MS/MS. Kp values for 7 out of the 15 compounds ranged from 3 to 30, indicating significant hepatic accumulation. In addition, 3 out of 15 compounds had Kp values less than 1, and three compounds had Kp values approximately equal to 1. These differences were evident at both time points and both concentrations, and indicated the importance of taking hepatic accumulation into account. Observed cellular accumulation was compared with predictions from a mathematical model for predicting in vitro partitioning (Armitage et al., 2014), finding reasonable correspondence. Accumulation of three chemicals (Diclosulam, Quinclorac, and Monobutyl phthalate) was significantly over-predicted by the partitioning model, indicating a possible role for efflux transporters (basolateral/biliary) for these chemicals although biliary excretion was not directly observed. Overall, biliary clearance does not appear to explain the underestimation of clearance, pointing to a potential role for extra-hepatic metabolism. This abstract does not necessarily reflect U.S. EPA policy. Armitage, et al. Environmental science & technology 48.16 (2014): 9770-9779.
URLs/Downloads:
DOI: In Vitro Hepatobiliary Disposition Assessment of 15 Non-Therapeutic Chemicals in Sandwich-Culture Rat Hepatocytes using B-CLEAR® Technology
SOT-POSTER-B-CLEAR_FINAL032018_M.PDF (PDF, NA pp, 610.682 KB, about PDF)