Determination of Xenobiotic Intrinsic Clearance in Freshly Isolated Hepatocytes from Rainbow Trout (Oncorhynchus mykiss) and Rat and Its Application in Bioaccumulation Assessment

Abstract

Bioaccumulation in fish depends on the dynamics of various processes that involve fish uptake, storage, and elimination of xenobiotics. Elimination via fish biotransformation is a primary process that can be evaluated in an in vitro system to improve the performance of the prediction of xenobiotic bioaccumulation potentials. In this study, values of intrinsic clearance (CLint) of seven reference compounds (atrazine, molinate, 4,4-bis(dimethylamino)-benzophenone, 4-nonylphenol, 2,4-di-tert-butylphenol, trifluralin, benzo(a)pyrene) in hepatocytes freshly isolated from rainbow trout and rat were determined using a substrate depletion approach. Atrazine was metabolized in rat hepatocytes with a CLint value of 3.81 +/- 1.96 mL/h/ 10(6) cells, whereas in trout hepatocytes, the clearance was not significant until very high cell concentration was used and the rate was estimated to be approximately 0.002 mL/h/10(6) cells. Intrinsic clearance values for all other compounds were 5.5-78.5-fold lower in trout hepatocytes than those in rat hepatocytes. Trout hepatic clearance (CL(H)) values were extrapolated from the CLint values using a "well-stirred" liver model. Biotransformation rate constants (kMET) of the compounds in trout were subsequently estimated and used as inputs to a kinetic model for the prediction of bioconcentration factors (BCF) in fish. Compared to the BCF values predicted without consideration of fish biotransformation, the inclusion of estimated kMET values significantly improved fish BCF predictions for the reference compounds. This study demonstrates a framework for future bioaccumulation assessment of xenobiotics using combined information of the physical-chemical properties of the compounds and the biotransformation potentials of the compounds in fish.

Toward improved models for predicting bioconcentration of well‐metabolized compounds by rainbow trout using measured rates of in vitro intrinsic clearance

“…Equation 3 differs from that given by Han et al [11] with respect to inclusion of a new term, C HEP /(2 Â 10 6 ), which corrects for differences in hepatocyte concentration. Binding values for trout used to develop the original relationship were measured in a solution containing 2 Â 10 6 hepatocytes/mL.…”

“…The binding term f U,HEP is calculated using a modification of the relationship given by Han et al [11] …”

“…One approach that may satisfy this need involves the use of in vitro metabolizing systems derived from liver tissue. Using procedures developed initially by the pharmaceutical industry, several groups have extrapolated in vitro biotransformation data for fish to the intact animal and used this information to predict impacts on chemical accumulation [11][12][13][14][15]. In each case, incorporating in vitro information into established models of chemical accumulation substantially improved model performance, resulting in predicted BCFs that were in better agreement with measured values than predictions obtained assuming no biotransformation.…”

Multimedia modeling of human exposure to chemical substances: The roles of food web biomagnification and biotransformation

“…Biotransformation rate estimates for fish were obtained from a database [20], whereas rates in birds and mammals were assumed to be five times faster than those in fish on a per body weight basis unless otherwise noted. This extrapolation factor was assumed for illustrative purposes in the present study based on evidence that biotransformation rates in birds and mammals are generally faster than those in fish [21][22][23]. A further rationale for this assumed extrapolation is provided in the Supplemental Data.…”

Fish Toxicity Testing Framework

“…These assays borrow from mammalian ADME (Absorption, Distribution, Metabolism, and Excretion) techniques used for pharmaceutical development, and most focus on the liver as the main site of metabolism. Primary hepatocyte isolations from rainbow trout and carp have been used to predict in vivo metabolism to refine bioaccumulation estimates (Han et al 2007, Cowan-Ellsberry et al 2008, as have subcellular fractions such as fish S9 or microsomes. There are also several fish liver cell lines that demonstrate metabolic capacity (reviewed in Castaño et al 2003), though these rates tend to be lower than those seen in primary liver cells or subcellular fractions (Weisbrod et al 2009).…”