Xenobiotic intrinsic clearance in freshly isolated hepatocytes from rainbow trout ( Oncorhynchus mykiss): Determination of trout hepatocellularity, optimization of cell concentrations and comparison of serum and serum-free incubations

Abstract

Metabolism plays an important role in bioaccumulation of xenobiotics in fish. In vitro determination of xenobiotic intrinsic clearance (CL int) in trout hepatocytes and subsequent extrapolation to in vivo hepatic clearance (CL H) using the “well-stirred” liver model greatly improved our current practice of bioaccumulation assessment [Han, X., Nabb, D.L., Mingoia, R.T., Yang, C.H., 2007. Determination of xenobiotic intrinsic clearance in freshly isolated hepatocytes from rainbow trout ( Oncorhynchus mykiss) and rat and its application in bioaccumulation assessment. Environ. Sci. Technol. 41, 3269–3276]. In an effort to further optimize this approach, we experimentally obtained the value of trout hepatocellularity ( H T), a key scaling factor in the “well-stirred” liver model. H T was determined to be (540 ± 12) × 10 6 cells/g liver for male trout. We also investigated the potential effect of different cell concentrations on the determination of CL int values of molinate, 4,4-bis(dimethylamino)benzophenone, 4-nonylphenol, 2,4-di- tert-butylphenol, and benzo(a)pyrene. Linear relationships were established between clearance rates and cell concentrations at 1 × 10 6, 2 × 10 6, 5 × 10 6, and 10 × 10 6 cells/mL. This suggests that under our experimental conditions, CL int determination was independent of hepatocyte concentrations. In order to better understand the “in vitro binding” effect in in vitro-to-in vivo scaling, we obtained CL int values for the above-mentioned compounds in trout hepatocytes that were suspended in trout serum. Incubations in serum, in general, resulted relatively larger prediction of CL H values. Our findings suggest that in bioaccumulation assessment, the traditional medium incubation method offers a conservative estimate on fish metabolism of xenobiotics and the serum incubation approach could be used for certain classes of compounds that are of challenge for in silico prediction of their plasma and in vitro binding properties.