Development of in vitro toxicity assays with a rainbow trout gill cell line and their use in determining the cytotoxicity and photocytotoxicity of polycyclic aromatic hydrocarbons individually and in a complex mixture

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in aquatic environments. However, studying their impact on fish is problematic. They are difficult to study because they can occur singly or as complex mixtures and can be modified by ultraviolet (UV) radiation. They are costly to study because of the expense associated with the exposure of fish to PAHs and subsequently with the disposal of large volumes of contaminated water. To overcome these problems, in vitro methods have been developed in this thesis to study the toxicity of individual PAHs and a PAH mixture to fish cells and to examine the influence of UV on toxicity.

Methodology was developed for quantifying the photocytotoxicity of fluoranthene to the rainbow trout gill cell line, RTgill-W1, for future use in screening PAHs for their relative photocytotoxicity to fish. Solubilization in a modified culture medium was achieved with and without fetal bovine serum (FBS) and with and without dimethyl sulfoxide (DMSO). FBS caused most of the flouranthene to remain in solution and blocked photocytotoxicity if present during UV irradiation. DMSO had little effect on fluoranthene distribution in cell cultures but caused cells to be slightly more sensitive to the phototoxicity of fluoranthene. The indicator dyes, alamar BlueTM and 5-carboxyfluorescein diacetate acetoxynethyl ester (CFDA-AM), were used to quantify cytotoxicity in two different ways: singly in two separate assays, and mixed together in a novel single assay, which saved time and material. With UV irradiation for 2 hr at a photon fluence rate of either 1.4 umol m-2s-1 UV-B (UV-A : UV-B, 1.5) or 1.1 umol m-2s-1 UV-B (UV-A : UV-B, 9.7), both dyes indicated increasing loss of viability with increasing doses of fluoranthene. EC50s ranged from 18 to 44 ng/ml (89 to 217 nM), with the alamar Blue assay being slightly more sensitive.