In Vitro-In Vivo Extrapolation of Hepatic Biotransformation Data for Fish. III. An In-depth Case Study with Pyrene.

Computational models that predict chemical bioaccumulation in fish generally account for biotransformation using an apparent first-order whole-body rate constant (k ; d ). The use of such models requires, therefore, that methods exist for estimating k , ideally without the need to expose live animals. One promising approach for estimating k involves the extrapolation of measured in vitro intrinsic clearance (CL ) to the whole animal (in vitro-in vivo extrapolation, [IVIVE]).

Biotransformation Potential of Cationic Surfactants in Fish Assessed with Rainbow Trout Liver S9 Fractions.

Biotransformation may substantially reduce the extent to which organic environmental contaminants accumulate in fish. Presently, however, relatively little is known regarding the biotransformation of ionized chemicals, including cationic surfactants, in aquatic organisms. To address this deficiency, a rainbow trout liver S9 substrate depletion assay (RT-S9) was used to measure in vitro intrinsic clearance rates (CL ; ml min g liver ) for 22 cationic surfactants that differ with respect to alkyl chain length and degree of methylation on the charged nitrogen atom.

Addition of Phenylmethylsulfonyl Fluoride Increases the Working Lifetime of the Trout Liver S9 Substrate Depletion Assay, Resulting in Improved Detection of Low Intrinsic Clearance Rates.

The activity of a trout liver S9 substrate depletion assay has been shown to decline over time, presumably due to proteolytic degradation of biotransformation enzymes. To address this problem, assay performance was evaluated following the addition of phenylmethylsulfonyl fluoride (PMSF) or a general-purpose protease inhibitor cocktail to liver homogenization buffers and/or S9 reaction mixtures. Addition of PMSF to liver homogenization buffers and/or S9 reaction mixtures had little or no effect on clearance of phenanthrene, a model cytochrome P450 substrate, in short-term (25 or 30 min) depletion experiments but resulted in significant improvements in retention of this initial activity over time.

In vitro-in vivo extrapolation of hepatic and gastrointestinal biotransformation rates of hydrophobic chemicals in rainbow trout.

Hepatic in vitro biotransformation assays, in combination with in vitro-in vivo extrapolation (IVIVE) and bioaccumulation modeling, can be used to support regulatory bioaccumulation assessments. In most applications, however, these methods ignore the possibility of extrahepatic metabolism. Here we evaluated intestinal biotransformation in rainbow trout using S9 fractions prepared from the upper intestinal (GIT) epithelium.

Biotransformation of Polycyclic Aromatic Hydrocarbons by Trout Liver S9 Fractions: Evaluation of Competitive Inhibition Using a Substrate Depletion Approach.

Environmental contaminants frequently occur as part of a chemical mixture, potentially resulting in competitive inhibition among multiple substrates metabolized by the same enzyme. Trout liver S9 fractions were used to evaluate the biotransformation of 3 polycyclic aromatic hydrocarbons (PAHs): phenanthrene, pyrene, and benzo[a]pyrene, tested as binary mixtures. Initial rates of biotransformation were determined using a substrate-depletion approach.

Allometric scaling of hepatic biotransformation in rainbow trout.

Biotransformation may substantially impact the toxicity and accumulation of xenobiotic chemicals in fish. However, this activity can vary substantially within and among species. In this study, liver S9 fractions from rainbow trout (4-400 g) were used to evaluate relationships between fish body mass and the activities of phase I and phase II metabolic enzymes.

Toxicokinetics of the neonicotinoid insecticide imidacloprid in rainbow trout (Oncorhynchus mykiss).

Studies were conducted to determine the distribution and elimination of imidacloprid (IMI) in rainbow trout. Animals were injected with a low (47.6 μg/kg), medium (117.

Measurement of kinetic parameters for biotransformation of polycyclic aromatic hydrocarbons by trout liver S9 fractions: Implications for bioaccumulation assessment.

substrate depletion methods developed by the pharmaceutical industry are being used with increasing frequency to support chemical bioaccumulation assessments for fish. However, the application of these methods to high log chemicals poses special challenges. Biotransformation of three polycyclic aromatic hydrocarbons (PAHs) was measured using trout liver S9 fractions.

Comparison of trout hepatocytes and liver S9 fractions as in vitro models for predicting hepatic clearance in fish.

Isolated hepatocytes and liver S9 fractions have been used to collect in vitro biotransformation data for fish as a means of improving modeled estimates of chemical bioaccumulation. To date, however, there have been few direct comparisons of these 2 methods. In the present study, cryopreserved trout hepatocytes were used to measure in vitro intrinsic clearance rates for 6 polycyclic aromatic hydrocarbons (PAHs).

Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: activity enhancement by alamethicin, a pore-forming peptide.

1. An existing assay for UDP-glucuronosyltransferase (UGT) activity in trout liver microsomes was optimized using trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5'-diphosphoglucuronic acid (UDPGA), substrate (p-nitrophenol) and alamethicin, a pore-forming agent added to eliminate latency.

Toxicokinetics of perfluorooctane sulfonate in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) confined to respirometer-metabolism chambers were dosed with perfluorooctane sulfonate (PFOS) by intra-arterial injection and sampled to obtain concentration time-course data for plasma and either urine or expired water. The data were then analyzed using a 2-compartment clearance-volume model. Renal and branchial clearance rates (mL/d/kg) determined for all experiments averaged 19% and 81% of total clearance, respectively.

Toxicokinetics of perfluorooctanoate (PFOA) in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) confined to respirometer-metabolism chambers were dosed with perfluorooctanoate (PFOA) by intra-arterial (i.a.) injection and sampled to obtain concentration time-course data for plasma, urine, and expired water.

Optimizing the use of rainbow trout hepatocytes for bioaccumulation assessments with fish.

Biotransformation rates measured using cryopreserved trout hepatocytes can be extrapolated to the whole animal to predict metabolism impacts on chemical bioaccumulation. Future use of these methods within a regulatory context requires, however, that they be optimized and standardized. Specifically, questions exist concerning gender differences in metabolism, cryopreservability of cells, and the accuracy of in vitro-in vivo scaling factors.

Hepatic clearance of 6 polycyclic aromatic hydrocarbons by isolated perfused trout livers: prediction from in vitro clearance by liver S9 fractions.

Isolated perfused trout livers were used to evaluate in vitro-in vivo metabolism extrapolation procedures for fish. In vitro depletion rates for 6 polycyclic aromatic hydrocarbons (PAHs) were measured using liver S9 fractions and extrapolated to the intact tissue. Predicted hepatic clearance (CLH) values were then compared with values exhibited by intact livers.

Enantiomer-specific in vitro biotransformation of select pharmaceuticals in rainbow trout (Oncorhynchus mykiss).

The occurrence of pharmaceuticals in the environment represents a challenge of emerging concern. Many pharmaceuticals are chiral compounds; however, few studies have examined the relative toxicity of pharmaceutical enantiomers to wildlife. Further, our understanding of stereospecific pharmacokinetics remains largely informed by research on humans and a few well-studied laboratory test animals, and not by studies conducted with environmentally relevant species, including fish.

Comparative pharmaceutical metabolism by rainbow trout (Oncorhynchus mykiss) liver S9 fractions.

The occurrence of pharmaceuticals in the environment presents a challenge of growing concern. In contrast to many industrial compounds, pharmaceuticals undergo extensive testing prior to their introduction to the environment. In principle, therefore, it may be possible to employ existing pharmacological safety data using biological "read-across" methods to support screening-level bioaccumulation environmental risk assessment.

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

Models were developed to predict the bioconcentration of well-metabolized chemicals by rainbow trout. The models employ intrinsic clearance data from in vitro studies with liver S9 fractions or isolated hepatocytes to estimate a liver clearance rate, which is extrapolated to a whole-body biotransformation rate constant (kMET ). Estimated kMET values are then used as inputs to a mass-balance bioconcentration prediction model.

Optimization of an isolated perfused rainbow trout liver model: Clearance studies with 7-ethoxycoumarin.

To date, research with isolated perfused fish livers has been limited by the relatively short time period during which stable performance can be achieved. In the present study, modifications to existing methods were employed with the goal of extending the usable life of an isolated perfused trout liver preparation. Liver performance was evaluated by measuring O(2) consumption (VO(2)), vascular resistance, K(+) leakage, glucose flux, lactate flux, and clearance of a model metabolic substrate, 7-ethoxycoumarin (CL(H,7-EC)).

Kinetics and effects of dichloroacetic acid in rainbow trout.

Halogenated acetic acids (HAAs) produced by chlorine disinfection of municipal drinking water represent a potentially important class of environmental contaminants. Little is known, however, about their potential to adversely impact fish and other aquatic life. In this study we examined the kinetics and effects of dichloroacetic acid (DCA) in rainbow trout.

Use of online microdialysis sampling to determine the in vivo rate of phenol glucuronidation in rainbow trout.

A quantitative microdialysis (MD) sampling method was used to study phenol (PH) glucuronidation in vivo in rainbow trout. The method employs internal calibrators to account for changes in MD probe performance (in vitro-to-in vivo and sample-to-sample) and yields data of high temporal resolution that are well suited for developing kinetic models. Initially, trout were dosed with phenyl glucuronide (PG) by intravascular infusion for 24 h and then depurated for 48 h.

Quantification of phenol, phenyl glucuronide, and phenyl sulfate in blood of unanesthetized rainbow trout by online microdialysis sampling.

ABSTRACT Free concentrations of phenol (PH), phenyl glucuronide (PG), and phenyl sulfate (PS) were measured in the bloodstream of unanesthetized rainbow trout by online microdialysis (MD) sampling. Preliminary studies were conducted to optimize the MD system and evaluate three retrodialysis calibration standards: p-nitrophenyl glucuronide (PNPG), p-nitrophenyl sulfate (PNPS), and [(14)C]-phenol ((14)C-PH). PG and PNPG exhibited nearly identical dialyzing properties in vitro (saline and plasma) and in vivo (muscle tissue and dorsal aorta).

Tissue distribution and metabolism of benzo[a]pyrene in embryonic and larval medaka (Oryzias latipes).

The need to understand chemical uptake, distribution, and metabolism in embryonic and larval fish derives from the fact that these early life stages often exhibit greater sensitivity to xenobiotic compounds than do adult animals. In this study, a 6-h acute waterborne exposure immediately after fertilization was used to quickly load the egg with benzo[a]pyrene (BaP). This exposure was used to mimic the initial egg concentration of a persistent bioaccumulative toxicant that could result from maternal transfer.

In vitro-in vivo extrapolation of quantitative hepatic biotransformation data for fish. II. Modeled effects on chemical bioaccumulation.

Hypothetical in vitro biotransformation rate and affinity values for fish were extrapolated to a set of in vivo whole-body metabolism rate constants. A one-compartment model was then used to investigate potential effects of metabolism on chemical bioaccumulation as a function of octanol/water partitioning (Kow). In a second model-based effort, in vitro data were incorporated into a physiologically based toxicokinetic (PBTK) model for fish.

A compilation of in vitro rate and affinity values for xenobiotic biotransformation in fish, measured under physiological conditions.

Scientific literature from the past 25 years was searched to obtain in vitro biotransformation rate and affinity data for fish. To maximize the environmental relevance of this dataset, we focused on studies conducted at multiple substrate concentrations, and established acceptance criteria with respect to assay temperature and pH. Altogether, enzyme rate and affinity parameters are provided for 43 species and 77 compounds.

In vitro-in vivo extrapolation of quantitative hepatic biotransformation data for fish. I. A review of methods, and strategies for incorporating intrinsic clearance estimates into chemical kinetic models.

Scientists studying mammals have developed a stepwise approach to predict in vivo hepatic clearance from measurements of in vitro hepatic biotransformation. The resulting clearance estimates have been used to screen drug candidates, investigate idiosyncratic drug responses, and support chemical risk assessments. In this report, we review these methods, discuss their potential application to studies with fish, and describe how extrapolated values could be incorporated into well-known compartmental kinetic models.