Pharmaceutical Metabolism in Fish: Using a 3-D Hepatic In Vitro Model to Assess Clearance

Abstract

At high internal doses, pharmaceuticals have the potential for inducing biological/pharmacological effects in fish. One particular concern for the environment is their potential to bioaccumulate and reach pharmacological levels; the study of these implications for environmental risk assessment has therefore gained increasing attention. To avoid unnecessary testing on animals, in vitro methods for assessment of xenobiotic metabolism could aid in the ecotoxicological evaluation. Here we report the use of a 3-D in vitro liver organoid culture system (spheroids) derived from rainbow trout to measure the metabolism of seven pharmaceuticals using a substrate depletion assay. Of the pharmaceuticals tested, propranolol, diclofenac and phenylbutazone were metabolised by trout liver spheroids; atenolol, metoprolol, diazepam and carbamazepine were not. Substrate depletion kinetics data was used to estimate intrinsic hepatic clearance by this spheroid model, which was similar for diclofenac and approximately 5 fold higher for propranolol when compared to trout liver microsomal fraction (S9) data. These results suggest that liver spheroids could be used as a relevant and metabolically competent in vitro model with which to measure the biotransformation of pharmaceuticals in fish; and propranolol acts as a reproducible positive control.

Comparison of Alternative Methods for Bioaccumulation Assessment: Scope and Limitations of In Vitro Depletion Assays with Rainbow Trout and Bioconcentration Tests in the Freshwater Amphipod Hyalella azteca

“…Converting our CL INT, IN VITRO rate for diclofenac to be expressed on a milliliters per hour per gram liver basis and assuming a hepatocellularity number of 510 × 10 6 cells/g liver (Nichols et al 2013; Organisation for Economic Co-operation and Development 2018c), a depletion rate of 31.11 is derived. This is higher than the one obtained from trout S9 fractions of 9.5 (Connors et al 2013) but lower than the one obtained from trout hepatocyte spheroids of 49.8 (Baron et al 2017). This implies that the depletion rate calculated in the present study for diclofenac in rainbow trout hepatocytes is comparable to the rates for diclofenac determined in other hepatic trout in vitro systems (Connors et al 2013;Baron et al 2017), even though it would not have been considered valid in the guideline.…”

“…This is higher than the one obtained from trout S9 fractions of 9.5 (Connors et al 2013) but lower than the one obtained from trout hepatocyte spheroids of 49.8 (Baron et al 2017). This implies that the depletion rate calculated in the present study for diclofenac in rainbow trout hepatocytes is comparable to the rates for diclofenac determined in other hepatic trout in vitro systems (Connors et al 2013;Baron et al 2017), even though it would not have been considered valid in the guideline. To estimate IVIVE BCFs for ionic compounds such as diclofenac, updated extrapolation models which are not based on the hydrophobicity of the tested chemicals will be necessary.…”

“…Two of the substances tested in the present study, prochloraz and diclofenac, expressed an unusual depletion behavior; and the experiments would not be considered valid according to the standards set in the OECD guideline. However, because of its use as a pharmaceutical, diclofenac has been tested in different in vitro studies including assays using rainbow trout S9 fractions (Connors et al 2013) and hepatocyte spheroids (Baron et al 2017). Converting our CL INT, IN VITRO rate for diclofenac to be expressed on a milliliters per hour per gram liver basis and assuming a hepatocellularity number of 510 × 10 6 cells/g liver (Nichols et al 2013; Organisation for Economic Co-operation and Development 2018c), a depletion rate of 31.11 is derived.…”

<i>In vitro</i> metabolism of pesticides and industrial chemicals in fish

“…[15][16][17] This method has been successfully applied to pesticides, pharmaceuticals, polyaromatic hydrocarbons (PAHs), and ionizable chemicals, as described in OECD guidance document No. 280, 15) the international ring trial 18) and other references, 19,20) and it plays a great role in estimating their bioconcentration factor in fish at a satisfactory level. Furthermore, the risk assessment of relevant metabolites in fish has been recently required especially in the EU regulation of pesticides.…”

Metabolite profiling of carbamazepine and ibuprofen in Solea senegalensis bile using high-resolution mass spectrometry

“…Nonetheless, the rate of metabolic conversion in fish may vary largely between substrates as suggested by the quantitative results from in vitro studies obtained with trout liver S9 fractions [18] and trout liver spheroids [19]. Comparison of intrinsic clearance values and depletion rate constants, respectively, for sets of environmentally relevant drugs demonstrated extensive biotransformation in some cases while under the assay conditions, other drugs were resistant to metabolic conversion.…”