Sex-, species-, and chain length-dependent renal elimination is the hallmark of mammalian elimination of perfluorocarboxylates (PFCAs) and has been extensively studied for almost 30 years. In this review, toxicokinetic data of PFCAs (chain lengths ranging from 4 to 10) in different species are compared with an emphasis on their relevance to renal elimination. PFCAs vary in their affinities to bind to serum albumins in plasma, which is an important factor in determining the renal clearance of PFCAs. PFCA-albumin binding has been well characterized and is summarized in this review. The mechanism of the sex-, species-, and chain length-dependent renal PFCA elimination is a research area that has gained continuous interest since the beginning of toxicological studies of PFCAs. It is now recognized that organic anion transport proteins play a key role in PFCA renal tubular reabsorption, a process that is sex-, species-, and chain length-dependent. Recent studies on the identification of PFCA renal transport proteins and characterization of their transport kinetics have greatly improved our understanding of the PFCA renal transport mechanism at the molecular level. A mathematical representation of this renal tubular reabsorption mechanism has been incorporated in physiologically based pharmacokinetic (PBPK) modeling of perfluorooctanoate (PFOA). Improvement of PBPK models in the future will require more accurate and quantitative characterization of renal transport pathways of PFCAs. To that end, a basolateral membrane efflux pathway for the reabsorption of PFCAs in the kidney is discussed in this review, which could provide a future research direction toward a better understanding of the mechanisms of PFCA renal elimination.

The detection of perfluorinated organic compounds in the environment has generated interest in their biological fate. 8-2 Fluorotelomer alcohol (8-2 FTOH, C(7)F(15)CF(2)CH(2)CH(2)OH), a raw material used in the manufacture of fluorotelomer-based products, has been identified in the environment and has been implicated as a potential source for perfluorooctanoic acid (PFOA) in the environment. In this study, the in vitro metabolism of [3-(14)C] 8-2 FTOH and selected acid metabolites by rat, mouse, trout, and human hepatocytes and by rat, mouse, and human liver microsomes and cytosol were investigated. Clearance rates of 8-2 FTOH in hepatocytes indicated rat > mouse > human >/= trout. A number of metabolites not previously reported were identified, adding further understanding to the pathway for 8-2 FTOH metabolism. Neither perfluorooctanoate nor perfluorononanoate was detected from incubations with human microsomes. To further elucidate the steps in the metabolic pathway, hepatocytes were incubated with 8-2 fluorotelomer acid, 8-2 fluorotelomer unsaturated acid, 7-3 acid, 7-3 unsaturated acid, and 7-2 secondary fluorotelomer alcohol. Shorter chain perfluorinated acids were only observed in hepatocyte and microsome incubations of the 8-2 acids but not from the 7-3 acids. Overall, the results indicate that 8-2 FTOH is extensively metabolized in rats and mice and to a lesser extent in humans and trout. Metabolism of 8-2 FTOH to perfluorinated acids was extremely small and likely mediated by enzymes in the microsomal fraction. These results suggest that human exposure to 8-2 FTOH is not expected to be a significant source of PFOA or any other perfluorocarboxylic acids.

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.

Metabolism plays an important role in bioaccumulation of xenobiotics in fish. The applicability of trout liver microsomes and S9 fraction in bioaccumulation assessment of xenobiotics in fish was investigated in the present study. Basal-level activities of 7-ethoxyresorufin-O-dealkylase, testosterone 6beta-hydroxylase, glutathione-S-transferase, and uridine 5'-diphospho-glucuronosyltransferase in trout liver microsomes and S9 were significantly lower than those in rat liver microsomes and S9. The in vitro-to- in vivo scaling factors, which are the values of liver microsomal and S9 protein contents per unit weight of trout liver, were determined to be 38.4 +/- 5.1 (mean +/- standard deviation throughout) and 95.9 +/- 11.9 mg/g, respectively. Intrinsic clearance (CL(int)) values for a number of reference compounds obtained from trout liver S9 were lower than those from trout liver microsomes. After correction with the scaling factors, trout liver microsomes and S9 provided equivalent prediction of trout hepatic clearance (CL(H)) using the well-stirred liver model, but their CL(H) values were significantly lower than those obtained from freshly isolated trout hepatocytes. Consequently, trout liver microsomes and S9 showed poorer prediction of the bioconcentration factors of the reference compounds compared with trout hepatocytes. Unit conversion revealed that CL(int) values obtained from trout liver microsomes and S9 were 6.3 to 22.4% of those from trout hepatocytes, which explained, to a large extent, the differences in their CL(H) and bioconcentration factor prediction.

In vitro assays are widely employed to obtain intrinsic clearance estimates used in toxicokinetic modeling efforts. However, the reliability of these methods is seldom reported. Here we describe the results of an international ring trial designed to evaluate two in vitro assays used to measure intrinsic clearance in rainbow trout. An important application of these assays is to predict the effect of biotransformation on chemical bioaccumulation. Six laboratories performed substrate depletion experiments with cyclohexyl salicylate, fenthion, 4-n-nonylphenol, deltamethrin, methoxychlor, and pyrene using cryopreserved hepatocytes and liver S9 fractions from trout. Variability within and among laboratories was characterized as the percent coefficient of variation (CV) in measured in vitro intrinsic clearance rates (CLIN VITRO, INT; ml/h/mg protein or 106 cells) for each chemical and test system. Mean intralaboratory CVs for each test chemical averaged 18.9% for hepatocytes and 14.1% for S9 fractions, whereas interlaboratory CVs (all chemicals and all tests) averaged 30.1% for hepatocytes and 22.4% for S9 fractions. When CLIN VITRO, INT values were extrapolated to in vivo intrinsic clearance estimates (CLIN VIVO, INT; l/d/kg fish), both assays yielded similar levels of activity (<4-fold difference for all chemicals). Hepatic clearance rates (CLH; l/d/kg fish) calculated using data from both assays exhibited even better agreement. These findings show that both assays are highly reliable and suggest that either may be used to inform chemical bioaccumulation assessments for fish. This study highlights several issues related to the demonstration of assay reliability and may provide a template for evaluating other in vitro biotransformation assays.

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Measured rates of intrinsic clearance determined using cryopreserved trout hepatocytes can be extrapolated to the whole animal as a means of improving modeled bioaccumulation predictions for fish. To date, however, the intra- and interlaboratory reliability of this procedure has not been determined. In the present study, three laboratories determined in vitro intrinsic clearance of six reference compounds (benzo[a]pyrene, 4-nonylphenol, di-tert-butyl phenol, fenthion, methoxychlor and o-terphenyl) by conducting substrate depletion experiments with cryopreserved trout hepatocytes from a single source. O-terphenyl was excluded from the final analysis due to nonfirst-order depletion kinetics and significant loss from denatured controls. For the other five compounds, intralaboratory variability (% CV) in measured in vitro intrinsic clearance values ranged from 4.1 to 30%, while interlaboratory variability ranged from 27 to 61%. Predicted bioconcentration factors based on in vitro clearance values exhibited a reduced level of interlaboratory variability (5.3-38% CV). The results of this study demonstrate that cryopreserved trout hepatocytes can be used to reliably obtain in vitro intrinsic clearance of xenobiotics, which provides support for the application of this in vitro method in a weight-of-evidence approach to chemical bioaccumulation assessment.

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