3R-Project 108-07

In-vitro fish hepatocytes as a source of metabolic clearance data in alternative approaches to reduce in-vivo bioaccumulation testing of fish

Catharina Lany and Helmut Segner
Centre for Fish and Wildlife Health, Vetsuisse Faculty, University of Berne, 3001 Berne, Switzerland
helmut.segner@vetsuisse.unibe.ch

Keywords: fish; liver; ecotoxicology; pharmacology; toxicology; cell cultures: organ-specific; replacement; toxicity testing: xenobiotics

Duration: 2 years Project Completion: 2011

Background and Aim
Bioaccumulation is generally referred to as a process in which the chemical concentration in an organism achieves a level exceeding the one in the respiratory medium (in the case of fish: water), in the diet, or both (Arnot and Gobas 2006). The bioaccumulation process encompasses absorption, distribution, metabolism and excretion (ADME) of the chemical. While bioaccumulation occurs as a result of chemical uptake through all routes of chemical exposure, bioconcentration is a result of chemical exposure within the water but not of dietary uptake (Arnot and Gobas 2003). The extent to which chemicals bioaccumulate is expressed by quantities such as the Bioconcentration Factor (BCF), i.e. the ratio of steady-state chemical concentration in the organism versus chemical concentration in the respiratory media; the Bioaccumulation Factor (BAF) also considers chemical concentration in the food. In a regulatory context, bioaccumulative substances in fish are usually identified using BCF.
The standard test procedure used for determining BCF values of chemicals is the in-vivo fish test according to OECD (Organisation for Economic Co-operation and Development) Test Guideline 305 (ECETOC 2005), which is associated with high animal use (n = 108 fish per test). The worldwide implementation of new chemical regulations will drastically increase the extent of bioconcentration testing for regulatory purposes. In Europe, for instance, REACH requires testing of the bioconcentration potential in fish for chemicals with log K_ow ≥ 2.7 that are produced at > 100 tons per year, meaning that some 3,000-5,000 existing substances will have to be tested for BCF. This may entail a drastic increase of animals used for BCF testing. Thus, there is an urgent need to develop alternative methods to reduce the numbers of in-vivo bioaccumulation testing of fish.
In recent years, various non-animal strategies for bioaccumulation assessment have been discussed. A critical issue in the implementation of non-animal based predictions of bioaccumulation is the incorporation of information on chemical biotransformation (de Wolf et al. 2007, Nichols et al. 2007, Weisbrod et al. 2009). This data in principle can be procured by in-silico models, in-chemico assays or metabolically competent in-vitro systems such as hepatic S9 preparations, microsomes or isolated hepatocytes.
The aim of this project is to evaluate the potential of an in-vivo assay with fish hepatocytes to provide data on chemical biotransformation rates for alternative non-animal testing.

Method and Results
Hepatocytes are isolated from rainbow trout livers by a two-step-perfusion method. Fresh suspensions of the isolated cells are then dosed with the test chemical and incubated at physiological temperature (12-16°C) over a 2-hour-period (Figure 1).

Figure 1: Principle of the In-vitro hepatocyte assay to measure intrinsic metabolic clearance rates of xenobiotics

Samples are taken from the incubation medium at regular intervals and processed for chemical analysis. The disappearance rate of the test compound from the incubation medium over time provides the intrinsic clearance rate (ml/h/10⁶ cells). Physiology-based prediction models allow translation of in-vitro measured intrinsic clearance rates into the metabolic rates of intact fish (Cowan-Ellsberry et al. 2007), hence permit extrapolation from intrinsic clearance rates measured in isolated trout hepatocytes to the metabolic rate km of the test chemical in intact fish. This value can then be used to calculate BCF or BAF.
The project succeeded in establishing a protocol of good intra-laboratory repeatability for measuring intrinsic hepatic clearance of lipophilic xenobiotics using freshly isolated liver cells of rainbow trout, Oncorhynchus mykiss. Seasonal fluctuation of trout metabolic capabilities has been identified as a major source of variability of the in-vitro assay (Figure 2).

Figure 2: Relation between benzo(y)pyrene intrinsic clearance rates (ml//(10⁶ cells) of individual liver cell preparations, and their cytochrome P450 1A (CYP1A) expression levels at different seasons of the year. CYP1A data are expressed as MNE (mean normalized expression versus 18 S mRNA). Data derives from three individual hepatocyte isolations done in September, October and November 2011. Incubations took place at 10°C using a cell density of 2 million cells/ml. The results indicate correlation of enzyme expression levels (capacity for metabolism) with actual metabolic rates, and a decrease in both of them towards the winter season.

To overcome this limitation, methods for cryopreservation of trout hepatocytes have been developed. The project tested two extrapolation models for prediction of in-vivo metabolic rates of xenobiotics from in-vitro intrinsic clearance rates as measured in the hepatocyte assay (Han et al. 2007, Cowan-Ellsberry et al. 2008). The models work well but would gain from improved knowledge on physiological scaling factors (cf. Tables 1, 2).

Figure 3: Assumptions on fish scaling values as used in the prediction models of Han et al. (2007) and Cowan-Ellsberry et al. (2007).

Figure 4: Consequences of variation in fish physiological parameters on model predictions

Metabolic rate values and BCF predictions for three chemicals were generated and compared to in-vivo values. The in-vitro hepatocyte assay well predicted the in-vivo BCF values, and, importantly, correctly classified the test compounds as “non-bioaccumulative compounds”. These findings show the principal utility of the hepatocyte assay, however, the currently existing database is too small to support general conclusions.

Conclusions and Relevance for 3R
This project provides proof of principle that the fish hepatocytes assay can contribute the required biotransformation rate data to a non-animal testing strategy of bioaccumulation assessment. Such a strategy has major implications for the reduction of animal testing as new chemical regulations such as REACH will require BCF information on thousands of chemicals. The next steps required to implement the test in regulatory praxis are (i) testing a broader range of chemicals in order to learn more about the applicability of the assay, and (ii) performing inter-laboratory comparisons to validate the assay for regulatory use. Both activities are underway.

(see also 3R-INFO-BULLETIN Nr. 47)

References
Arnot JA, Gobas F (2006) A review of bioconcentration factor (BCF) and bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms. Environ Rev 14:257-330.

Cowan-Ellsberry CS, Dyer S, Erhardt S, Bernhard MJ, Roe A, Dowty M, Weisbrod A (2008) Approach for extrapolating itro metabolism data to refine bioconcentration factor estimates. Chemosphere 70: 1804-17.

de Wolf W, Comber M, Douben P, Gimeno S, Holt M, Léonard M, Lillicrap A, Sijm D, van Egmond R, Weisbrod A, Whale G (2007) Animal use replacement, reduction, and refinement: development of an integrated testing strategy for bioconcentration of chemicals in fish. Integr Environ Assessm Mngmt 3: 3-17.

Han X, Nabb D, Mingoia R, Yang C (2007) Determination of xenobiotic intrinsic clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and rat and its application in bioaccumulation assessment. Environ Sci Technol 41: 3269-76.

Nichols J, Erhardt S, Dyer S, James M, Moore M, Plotzke K, Segner H, Schultz I, Thomas K, Vasiluk L, Weisbrod A (2007). Workshop Report: Use of in vitro absorption, distribution, metabolism, and excretion (ADME) data in bioaccumulation assessments for fish. Human Ecol Risk Assess 13:1164-1191.

Weisbrod AV, Sahi J, Segner H, James MO, Nichols J, Schultz I, Erhardt S, Cowan-Ellsberry C, Bonnell M, Hoeger B (2009). The state of in vitro science for use in bioaccumulation assessments for fish. Environmental Toxicology and Chemistry 28:86-06.

Mayer P, Wernsing J, Tolls J, de Maagf PGJ, Sijm DTHM (1999). establishing and controlling concentrations of hydrophobic organics by partitioning from a solid phase. Environ Sci Technol 33:2284-2290.

Nichols J, Erhardt S, Dyer S, James M, Moore M, Plotzke K, Segner H, Schultz I, Thomas K, Vasiluk L, Weisbrod A (2007). Workshop Report: Use of in vitro absorption, distribution, metabolism, and excretion (ADME) data in bioaccumulation assessments for fish. Human Ecol Risk Assess 13:1164-1191.