Hepatic Clearance of 6 Polycyclic Aromatic Hydrocarbons by Isolated Perfused Trout Livers: Prediction From In Vitro Clearance by Liver S9 Fractions

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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). These rates were extrapolated to estimates of in vivo intrinsic clearance and used as inputs to a well stirred liver model to predict hepatic clearance. Predicted rates of hepatic clearance were then evaluated by comparison with measured rates determined previously using isolated perfused livers. Hepatic clearance rates predicted using hepatocytes were in good agreement with measured values (<2.1-fold difference for 5 of 6 compounds) under 2 competing binding assumptions. These findings, which may be attributed in part to high rates of PAH metabolism, are similar to those obtained previously using data from liver S9 fractions. For 1 compound (benzo[a]pyrene), the in vivo intrinsic clearance rate calculated using S9 data was 10-fold higher than that determined using hepatocytes, possibly due to a diffusion limitation on cellular uptake. Generally, however, there was good agreement between calculated in vivo intrinsic clearance rates obtained using either in vitro test system. These results suggest that both systems can be used to improve bioaccumulation assessments for fish, particularly when vitro rates of activity are relatively high, although additional work is needed to determine if the chemical domain of applicability for each system differs. Environ Toxicol Chem 2017;36:463-471. Published 2016 SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 88%

{true[normalP normalA normalH true]}_{f r e e} = {true[normalP normalA normalH true]}_{f i b e r} / {normalK}_{P D M S}

The concentration of total PAH in the suspension was then determined, and the fraction of unbound chemical was calculated as

f_{U, H E P} = {[P A H]}_{f r e e} / {[P A H]}_{s u s p e n s i o n}

…”

Section: Methodsmentioning

confidence: 99%

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

Fay

Fitzsimmons

Hoffman

et al. 2016

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“…The initial protein content of the pooled sample was 25.6 ± 0.6 mg mL –1 , whereas the CYP content was 68.9 ± 1.6 pmol P450 mg protein –1 . All values are within the ranges of values determined previously for S9 fractions isolated from trout of the same age and strain (Nichols et al , ).…”

Section: Resultssupporting

confidence: 85%

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

Nichols

Ladd

Hoffman

et al. 2019

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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. The resulting data were then fitted by simultaneous nonlinear regression to a competitive inhibition model. In each case, the PAH possessing the lower Michaelis‐Menten affinity constant (KM) competitively inhibited biotransformation of the other compound. Inhibition constants determined for the lower‐KM compound were generally close to previously determined KM values, consistent with the suggestion that phase I biotransformation of PAHs is largely catalyzed by one or a small number of cytochrome P450 enzymes. The use of a substrate‐depletion approach to perform enzyme‐inhibition studies imposes practical limitations on experimental design and complicates the interpretation of derived kinetic constants. Nevertheless, the resulting information may have utility for chemical hazard assessments as well as the design and interpretation of controlled laboratory studies. Depletion experiments informed by measured chemical concentrations in tissues may also provide a means of determining whether enzyme inhibition occurs under relevant environmental conditions. Environ Toxicol Chem 2019;38:2729–2739. Published 2019 Wiley Periodicals, Inc. on behalf of SETAC. This article is a US government work, and as such, is in the public domain in the United States of America

“…Chemical binding in trout liver S9 fractions as a function of log K OW. Measured binding values, which are plotted as the log of the bound/free ratio (log (1–ϕ S9 /ϕ S9 )), are from Nichols et al (; gray circles), Nichols et al (; solid circles), Escher et al (2011; solid squares), Lo et al (; open triangles), and Lee et al (2014; gray diamonds). The lines represent binding algorithms given by Nichols et al (; dotted line), Lee et al (2017; dashed line), and Han et al (2009; solid line).…”

Section: Resultsmentioning

confidence: 99%

Concentration dependence of in vitro biotransformation rates of hydrophobic organic sunscreen agents in rainbow trout S9 fractions: Implications for bioaccumulation assessment

Saunders¹,

Fontanay²,

Nichols

et al. 2019

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In vitro biotransformation studies were performed to support the bioaccumulation assessment of 3 hydrophobic organic ultraviolet filters (UVFs), 4‐methylbenzylidene camphor (4‐MBC), 2‐ethylhexyl‐4‐methoxycinnamate (EHMC), and octocrylene. In vitro depletion rate constants (kdep) were determined for each UVF using rainbow trout liver S9 fractions. Incubations performed with and without added cofactors showed complete (4‐MBC) or partial (EHMC and octocrylene) dependence of kdep on addition of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), suggesting that hydrolysis of EHMC and octocrylene by NADPH‐independent enzymes (e.g., carboxylesterases) is an important metabolic route. The concentration dependence of kdep was then evaluated to estimate Michaelis–Menten parameters (KM and Vmax) for each UVF. Measured kdep values were then extrapolated to apparent whole‐body biotransformation rate constants using an in vitro–in vivo extrapolation (IVIVE) model. Bioconcentration factors (BCFs) calculated from kdep values measured at concentrations well below KM were closer to empirical BCFs than those calculated from kdep measured at higher test concentrations. Modeled BCFs were sensitive to in vitro binding assumptions employed in the IVIVE model, highlighting the need for further characterization of chemical binding effects on hepatic clearance. The results suggest that the tested UVFs are unlikely to accumulate to levels exceeding the European Union Registration, Evaluation, Authorisation, and Restriction regulation criterion for bioaccumulative substances (BCF > 2000 L kg−1). However, consideration of appropriate in vitro test concentrations and binding correction factors are important when IVIVE methods are used to refine modeled BCFs. Environ Toxicol Chem 2019;38:548–560. © 2018 SETAC