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3R-Project 108-07
In vitro fish hepatocytes as source of metabolic clearance data in alternative approaches for the reduction or replacement of in vivo bioaccumulation testing with fish.
Catharina Lany and Helmut Segner
Centre for Fish and Wildlife Health, Vetsuisse Faculty, University of Berne, CH – 3001 Berne, Switzerland
helmut.segner@itpa.unibe.ch
Keywords: fish; liver; ecotoxicology; pharmacology; toxicology; cell cultures: organ-specific; replacement; toxicity testing: xenobiotics
Duration: 2 years End of the Project: 2010
Background and Aim
Bioaccumulation is the uptake of chemicals from the environment into the fish, be it via water (i.e. bioconcentration) or via food (i.e. biomagnification). The bioaccumulation process encompasses absorption, distribution, metabolism and excretion (ADME) of the chemical. In ecotoxicologicall risk assessment, fish tests are used to determine the bioconcentration factor (BCF) of chemicals. With the worldwide implementation of new chemical regulations such as, e.g., REACH in Europe, the extent of bioconcentration testing for regulatory purposes will drastically increase. Thus, there is a need to develop alternative methods enabling either the reduction or the replacement of the in vivo bioaccumulation test with fishes. During recent years, various alternative strategies for BCF assessment have been discussed, however, a major obstacle to the implementation of such alternatives is the lack of validated in vitro methods for estimating the rate of metabolic clearance of xenobiotics (Nichols et al. 2007).
A promising in vitro approach to provide metabolic rate values for xenobiotics is the fish hepatocyte assay. While the principal suitability of isolated fish hepatocytes to determine biotransformation rates is undisputed, the utility of this assay in a regulatory context remains to be demonstrated. In particular, there exist questions on the standardization and reproducibility of the fish hepatocyte assay, as well as on its ability to correctly predict in vivo bioaccumulation.
The aims of the present project are to provide answers to these questions, i.e. to investigate the extent of variability in the rainbow trout hepatocyte assay, and to evaluate the predition of in vivo BCF values on the basis of in vitro metabolic clearance data determined in the fish hepatocyte assay.
Method and Results
in progress (present status)
Freshly isolated trout hepatocytes are used as suspension in serum-free media. To characterize between-isolation differences in biotransformation capabilities, expression levels of biotransformation enzymes such as cytochrome P4501A in the isolated cells are measured. To monitor the metabolism of the test chemicals, the substrate depletion approach which measured disappearnce of the parent compound, is used.
The findings from the first project year show that the substrate depletion approach works well with rainbow trout hepatocytes and enables accurate determination of metabolic rates. The change of substarte concentration per time is used to calculate the in vitro intrinsic clearance which is normalized to cell number (CLint, mL/h/106 cells). From this value, hepatic clearance can be calculated which is then used to predict the in vivo BCF value.
- The findings form the first project show further that a number of intrinsic and extrinsic factors clearly influence in vitro CLint values of rainbow trout hepatocytes. These factors include – season: in vitro clearance rates are higher in cell isolates prepared during summer months than in cell isolates prepared during winter months
- cell density: in vitro clearance rates decrease with increasing cell concentration in the assay
- substrate concentration: in vitro clearance rates decrease with increasing substrate concentration
Ongoing work examine sthe role of bioavailability and incubation temperature.
Conclusions and Relevance for 3R
In regulatory applications, the toxicological test procedure usually applied to determine bioconcentration in aquatic organisms is the in vivo fish test according to OECD Test Guideline 305, which is associated with high animal use. It has been estimated that with the implementation of REACH approximately 300 000 additional fishes will be used for additional bioconcentration testing, provided that alternatives are not available. This situation highlights the urgent need for establishing valid alternative testing approaches. The present project evaluates the ability of the fish hepatocyte assay to be used for generation of metabolic rate data in an alternative strategy that replaces or reduces in vivo bioaccumulation testing with fish.
References
Braunbeck T, Segner H. 2000. Isolation and cultivation of teleost hepatocytes. In: Berry MN, Edwards AM (eds). The Hepatocyte Review. Kluwer Academic Publishers, Dordrecht, NL. pp. 49-72.
Dyer SD, Bernhard MJ (2004). Follow up to feasibility study on in vitro biotransformation.systems.. Determining uptake, loss and bioconcentration of two surfactants. ERASM Report. Brussles, Belgium
ECETOC (European Centre for Ecotoxicology and Toxicology of Chemicals) (2005) Alternative Testing Approaches in Environmental Safety Assessment. Technical Report No. 97. Brussels, December 2005.
Han X, Nabb DL, Mingoi RT, Yang CY (2007). Determination of xenobiotic intrinsic clearance in freshly isolated hepatocytes form rainbow trout and rat and its application in bioaccumulation assessment. Environ Sci Technol 41:3269-3276.
Heringa MB, Schreurs RHMM, Busser F, van der Saag PT, van der Burg B, Hermns JLM (2004). Toward more useful in vitron toxicity data with measured free concentration. Environ Sci Technol 38:6263-6270.
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.
Figures
Figure 1: Freshly isdolated fish hepatocytes in suspension.
Figure 2: Morphology of fish hepatocytes, 24 hours after seeding.
| Last modified 2010/08/29 |  |