Functional xenobiotic metabolism and efflux transporters in trout hepatocyte spheroid cultures

Uchea, Chibuzor; Owen, Stewart F.; Chipman, J.K.

doi:10.1039/c4tx00160e

Cited by 27 publications

(23 citation statements)

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“…There was no effect of any of the pharmaceuticals on spheroid viability (≤ 320 μg L -1 ), measured using a tetrazolium salt reduction assay, in any of the repeated exposures. Our choice of single fish replicates in this experiment was to assess the importance of the differences between individual animals and possible differences within each culture batch over time that might potentially affect the rate of xenobiotic metabolism [18, 31]. We suggest this is likely to reflect differences among fish in vivo and could contribute to the five-fold measured variability in circulating propranolol concentrations already reported in trout [32].…”

Section: Resultsmentioning

confidence: 99%

“…In recent years, the use of in vitro techniques in the bioaccumulation assessment of PPCPs has received more attention, with a particular focus on the measurement of metabolism and the extrapolation of this data to an in vivo scale, to aid improved predictions of the bioconcentration potential of these chemicals [15]. In particular, the use of three-dimensional (3-D) hepatic fish cultures or ‘spheroids’ has been proposed as an alternative model with which to assess metabolism, efflux and bioaccumulation potential of PPCPs in aquatic environments due to their in vivo -like physiology [10, 16–18]. …”

Section: Introductionmentioning

confidence: 99%

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Pharmaceutical Metabolism in Fish: Using a 3-D Hepatic In Vitro Model to Assess Clearance

et al. 2017

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At high internal doses, pharmaceuticals have the potential for inducing biological/pharmacological effects in fish. One particular concern for the environment is their potential to bioaccumulate and reach pharmacological levels; the study of these implications for environmental risk assessment has therefore gained increasing attention. To avoid unnecessary testing on animals, in vitro methods for assessment of xenobiotic metabolism could aid in the ecotoxicological evaluation. Here we report the use of a 3-D in vitro liver organoid culture system (spheroids) derived from rainbow trout to measure the metabolism of seven pharmaceuticals using a substrate depletion assay. Of the pharmaceuticals tested, propranolol, diclofenac and phenylbutazone were metabolised by trout liver spheroids; atenolol, metoprolol, diazepam and carbamazepine were not. Substrate depletion kinetics data was used to estimate intrinsic hepatic clearance by this spheroid model, which was similar for diclofenac and approximately 5 fold higher for propranolol when compared to trout liver microsomal fraction (S9) data. These results suggest that liver spheroids could be used as a relevant and metabolically competent in vitro model with which to measure the biotransformation of pharmaceuticals in fish; and propranolol acts as a reproducible positive control.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Pharmaceutical Metabolism in Fish: Using a 3-D Hepatic In Vitro Model to Assess Clearance

et al. 2017

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“…Gene expression studies indicate that fish possess a diverse array of membrane transport proteins [42][43][44][45][46], many of which have been shown to transport xenobiotic compounds [47][48][49][50][51][52][53][54][55]. Functional characterization studies have been performed on several adenosine 5 0 -triphosphate binding cassette (ABC) transporters from fish.…”

Section: Toxicokinetics Of Pfos In Troutmentioning

confidence: 99%

Toxicokinetics of perfluorooctane sulfonate in rainbow trout (Oncorhynchus mykiss)

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Hoffman

Fitzsimmons

et al. 2016

Enviro Toxic and Chemistry

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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. Expressed as mean values for all experiments, the steady-state volume of distribution was 277 mL/kg and the terminal half-life was 86.8 d. Additional animals were exposed to PFOS in water, resulting in an average calculated branchial uptake efficiency of 0.36%. The renal clearance rate determined in the present study is approximately 75 times lower than that determined in earlier studies with perfluorooctanoate (PFOA). Previously, it was suggested that PFOA is a substrate for membrane transporters in the trout kidney. The present study suggests that glomerular filtration may be sufficient to explain the observed renal clearance rate for PFOS, although a role for membrane transporters cannot be ruled out. These findings demonstrate that models developed to predict the bioaccumulation of perfluoroalkyl acids by fish must account for differences in renal clearance of individual compounds.

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“…While fish cell lines are increasing in availability, compared to mammalian cells few are available from validated depositories for in vitro toxicity testing (Hahn, 2011; Lee et al, 2009; Schirmer, 2006). New in vitro fish liver models have been developed including spheroids grown from primary rainbow trout hepatocytes in low adherence plates (Baron et al, 2012; Uchea et al, 2015) and an ex vivo model using cod liver slices (Eide et al, 2014). However, the rainbow trout model showed loss of cyp1a expression by day 5 when compared to the original primary cells, indicating the importance of establishing baseline responsiveness throughout the duration of cell culture (Uchea et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…New in vitro fish liver models have been developed including spheroids grown from primary rainbow trout hepatocytes in low adherence plates (Baron et al, 2012; Uchea et al, 2015) and an ex vivo model using cod liver slices (Eide et al, 2014). However, the rainbow trout model showed loss of cyp1a expression by day 5 when compared to the original primary cells, indicating the importance of establishing baseline responsiveness throughout the duration of cell culture (Uchea et al, 2015). The model presented here uses the fish liver cell line PLHC-1 to form a 3D spheroidal microtissue through self-assembly in a scaffold-free agarose hydro-gel (Napolitano et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

A 3D fish liver model for aquatic toxicology: Morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures

et al. 2017

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To identify the potential environmental impacts of aquatic pollutants, rapid and sensitive screening tools are needed to assess adaptive and toxic responses. This study characterizes a novel fish liver microtissue model, produced with the cell line PLHC-1, as an in vitro aquatic toxicity testing platform. These 3D micro-tissues remain viable and stable throughout the 8-day testing period and relative to 2D monolayers, show increased basal expression of the xenobiotic metabolizing enzyme cytochrome P450 1A (Cyp1a). To evaluate pulsed, low-dose exposures at environmentally relevant concentrations, microtissue responsiveness to the model toxicant benzo(a)pyrene was assessed after single and repeated exposures for determination of both immediate and persistent effects. Significant induction of Cyp1a gene and protein expression was detected after a single 24 h exposure to as little as 1 nM benzo(a)pyrene, and after a 24 h recovery period, Cyp1a expression declined in a dose-dependent manner. However, cell death continued to increase during the recovery period and alterations in microtissue architecture occurred at higher concentrations. To evaluate a pulsed or repeated exposure scenario, microtissues were exposed to benzo(a)pyrene, allowed to recover, then exposed a second time for 24 h. Following pre-exposure to benzo(a)pyrene, cyp1a expression remained equally inducible and the pattern and level of Cyp1a protein response to a second exposure were comparable. However, pre-exposure to 1 μM or 5 μM of benzo(a)pyrene resulted in increased cell death, greater disruption of microtissue architecture, and alterations in cell morphology. Together, this study demonstrates the capabilities of this PLHC-1 microtissue model for sensitive assessment of liver toxicants over time and following single and repeated exposures.

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Functional xenobiotic metabolism and efflux transporters in trout hepatocyte spheroid cultures

Abstract: Prediction of xenobiotic fate in fish is important for the regulatory assessment of chemicals under current legislation.

Cited by 27 publications

References 83 publications

Pharmaceutical Metabolism in Fish: Using a 3-D Hepatic In Vitro Model to Assess Clearance

Pharmaceutical Metabolism in Fish: Using a 3-D Hepatic In Vitro Model to Assess Clearance

Toxicokinetics of perfluorooctane sulfonate in rainbow trout (Oncorhynchus mykiss)

A 3D fish liver model for aquatic toxicology: Morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures

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