The influence of distribution kinetics on estimates of the intrinsic clearance of PAHs by HepaRG

Renate M. Groot1*, Floris A. Groothuis1, Nynke I. Kramer1

1 Institute for Risk Assessment Sciences, Utrecht University, PO Box 80177, 3508 TD Utrecht, The Netherlands

* corresponding author, [email protected]

 

The distribution characteristics of chemicals tested in in vitro assays is often overlooked, yet essential to parameterizing quantitative in vitro-in vivo extrapolation (QIVIVE) models. Especially for hydrophobic chemicals, free concentrations in exposure medium may be significantly reduced by binding to assay components, like plate plastic and medium constituents. In our study of the distribution of polycyclic aromatic hydrocarbons (PAHs) in intrinsic clearance assays with the human hepatoma cell line HepaRG, we observed that the traditional, short (<2h) observation periods, combined with the substrate depletion method, is not a reliable procedure to estimate intrinsic clearance (CLint). We demonstrate the impact of chemical distribution on the CLint estimates of non-cytochrome P450 enzyme inducing PAHs in an intrinsic clearance assay with differentiated HepaRG cells on 12-wells plates. We analytically measured and modelled, using a three-compartmental model, the depletion of PAHs in the medium, as well as the amount in plastic and in cells over time, for at least 8h. Our results show two phases in PAH depletion from the medium: 1. fast depletion from medium and accumulation of the chemicals in cells and plastic during the first hours. 2. slower elimination over the next 6 hours where concentrations of PAH in cells decrease, though amounts in plastic and medium remain constant. The results indicate a clear two compartment system, which assumes distribution preceding the sought elimination through metabolism. To conclude, short observation periods may ignore the effects of distribution, resulting in an overestimation of metabolic rate and intrinsic clearance.

Keywords: QIVIVE, intrinsic clearance, PAH.