Determination of Metabolic Stability Using Cryopreserved Hepatocytes from Rainbow Trout (<i>Oncorhynchus mykiss</i>)

Fay, Kellie A.; Nabb, Diane L.; Mingoia, Robert T.; Bischof, Ina; Nichols, John W.; Segner, Helmut; Johanning, Karla; Han, Xing

doi:10.1002/0471140856.tx0442s65

Cited by 11 publications

(12 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This apparent difference in assay performance was probably due to increased variability inherent to the use of cryopreserved cells. For the RT-HEP assay, the user must reconstitute frozen samples through a series of steps, count live cells, and dilute the sample accordingly ( Fay et al , 2015 ). The diluted sample is then counted a second time to determine the number of cells that will be used to normalize in vitro rate determinations.…”

Section: Discussionmentioning

confidence: 99%

“…All donor fish were drawn from the same mixed sex population of animals. RT-HEP were obtained using methods given by Fay et al (2015) . Each sample lot contained cells from 7 animals.…”

Section: Methodsmentioning

confidence: 99%

“…Thawed hepatocytes were evaluated in the presence of 0.04% trypan blue to determine cell yield and viability ( Fay et al , 2015 ). These suspensions were then diluted to the desired concentration of viable cells (1 or 2 × 10 6 cells/ml) in L-15 and recounted for accuracy.…”

Section: Methodsmentioning

confidence: 99%

“…In vitro intrinsic clearance rates were measured using a substrate depletion approach ( Fay et al , 2015 ; Johanning et al , 2012a ). Brief descriptions of each assay are provided as Supplementary Data ( In vitro methods).…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Reliability of In Vitro Methods Used to Measure Intrinsic Clearance of Hydrophobic Organic Chemicals by Rainbow Trout: Results of an International Ring Trial

Nichols

Fay

Bernhard

et al. 2018

Toxicological Sciences

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

“…All donor fish were drawn from the same mixed sex population of animals. RT-HEP were obtained using methods given by Fay et al (2015) . Each sample lot contained cells from 7 animals.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Reliability of In Vitro Methods Used to Measure Intrinsic Clearance of Hydrophobic Organic Chemicals by Rainbow Trout: Results of an International Ring Trial

Nichols

Fay

Bernhard

et al. 2018

Toxicological Sciences

Self Cite

View full text Add to dashboard Cite

show abstract

“…In a first step, existing in vitro methods were adapted to be used as metabolic stability assays, and standard protocols were developed (Segner 1998;Braunbeck and Segner 2000;Segner and Cravedi 2001;Mommsen and Moon 2005). Focus was given to freshly isolated hepatocytes (Han et al 2007(Han et al , 2009Fay et al 2015) and liver S9 fractions from rainbow trout, Oncorhynchus mykiss (Johanning et al 2012). In parallel, cryopreservation techniques were developed to allow the reliable provision of the biological materials (Mingoia et al 2010).…”

Section: Introductionmentioning

confidence: 99%

In Vitro Biotransformation Assays Using Liver S9 Fractions and Hepatocytes from Rainbow Trout (Oncorhynchus mykiss): Overcoming Challenges with Difficult to Test Fragrance Chemicals

Kropf

Begnaud

Gimeno³

et al. 2020

Enviro Toxic and Chemistry

Self Cite

View full text Add to dashboard Cite

In vitro metabolic stability assays using rainbow trout (Oncorhynchus mykiss) isolated hepatocytes (RT-HEP) or hepatic S9 fractions (RT-S9) were introduced to provide biotransformation rate data for the assessment of chemical bioaccumulation in fish. The present study explored the suitability of the RT-HEP and RT-S9 assays for difficult test chemicals, and the in vitro-based predictions were compared to in silico-based predictions and in vivo-measured bioconcentration factors (BCFs). The results show that volatile or reactive chemicals can be tested with minor modifications of the in vitro protocols. For hydrophobic chemicals, a passive dosing technique was developed. Finally, a design-of-experiment approach was used to identify optimal in vitro assay conditions. The modified assay protocols were applied to 10 fragrances with diverse physicochemical properties. The in vitro intrinsic clearance rates were higher in the S9 than in the hepatocyte assay, but the in vitro-in vivo (IVIV) predictions were comparable between the 2 assays. The IVIV predictions classified the test chemicals as nonbioaccumulative (BCF < 2000), which was in agreement with the in vivo data but in contrast to the in silico-based predictions. The findings from the present study provide strong evidence that the RT-HEP and RT-S9 assays can provide reliable estimates of in vivo biotransformation rates for test chemicals with difficult physicochemical properties.

show abstract

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

Fay

Fitzsimmons

Hoffman

et al. 2016

Enviro Toxic and Chemistry

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Determination of Metabolic Stability Using Cryopreserved Hepatocytes from Rainbow Trout (Oncorhynchus mykiss)

Cited by 11 publications

References 54 publications

Reliability of In Vitro Methods Used to Measure Intrinsic Clearance of Hydrophobic Organic Chemicals by Rainbow Trout: Results of an International Ring Trial

Reliability of In Vitro Methods Used to Measure Intrinsic Clearance of Hydrophobic Organic Chemicals by Rainbow Trout: Results of an International Ring Trial

In Vitro Biotransformation Assays Using Liver S9 Fractions and Hepatocytes from Rainbow Trout (Oncorhynchus mykiss): Overcoming Challenges with Difficult to Test Fragrance Chemicals

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

Contact Info

Product

Resources

About