The development of a hybrid fish gill cell-polymer PAMPA assay system to replace bioconcentration factor tests for highly hydrophobic compounds
Analytical Chemistry
Public Health, Healthcare Research, Social and Occupational Medicine
Final Report Abstract
The accumulation of xenobiotics in fish and other aquatic organisms has been a significant concern to industry, government regulators, the academic community, and the general public for a number of years. The information on bioaccumulation of chemicals is an important requirement for environmental risk assessment and for regulatory regimes such as the EU Regulation No 1907/2006 concerning the Registration, Evaluation, Authorization, and restriction of CHemicals (REACH). Studies required to assess bioaccumulation use a large number of fish and are costly and are extremely difficult for hydrophobic compounds due to lack of solubility in water and volatility. Previous work has assessed the uptake of hydrophobic compounds across synthetic membranes, but this lacks biological relevance. Hence, the aim of the project was to develop a hybrid in vitro fish gill cell-polymer parallel artificial membrane permeability (PAMPA) assay system to replace the use of fish in uptake studies for highly hydrophobic compounds. In this system primary gill cells or the cell line are grown on inserts to mimic the external and internal environments. Polydimethylsiloxane membranes (donor disks) are loaded with the hydrophobic test compounds (chrysene, pyrene, benzo(α)pyrene and fluoranthene) are placed on one side of the cell epithelia and a membrane disk is place on the over side (acceptor disk). This set up allows for a constant release of the test compound from the donor disk, which then interacts with the epithelium and the quantity that crosses the epithelium accumulates on the acceptor disk. An important determinant of hydrophobic chemical uptake is the lipid content of the membrane and this was also measured. Results showed that the cells remained confluent over the testing period and no cytotoxicity was observed when exposed to the test chemicals. The dosing was not a rate limiting step as the depletion of chemicals from the donor disks was negligible and uptake was measurable into the acceptor disk when serum was present in the medium. In serum-free medium experiments, the detection of chemicals in the acceptor disks were inconsistent. The project demonstrated that an in vitro system has the potential to replace fish uptake studies for highly hydrophobic compounds.
