Transitory metabolic disruption and cytotoxicity elicited by benzo[a]pyrene in two cell lines from rainbow trout liver.

Two cell lines, RTL-W1 and R1, from rainbow trout liver were used to investigate the effects of benzo[A]pyrene (BaP). BaP induced a catalytic measure of CYP1A, 7-ethoxyresorufin-O-deethylase (EROD) activity, in the rainbow trout liver cell line RTL-W1 but not in R1. Geldanamycin inhibited EROD induction by BaP. Potential BaP metabolites, BaP-7,8-dihydrodiol (BDP) and 6,12-BaP quinone (BQ) also induced EROD activity in RTL-W1. Very low BaP concentrations slightly stimulated cell proliferation in both cell lines. Higher BaP concentrations caused cytotoxicity in RTL-W1 but not in R1. Cytotoxicity was detected in a cell viability assay with 5-carboxyfluorescein diacetate acetoxymethyl ester, and as a decline in cell number. In both cell lines, BaP exposure impaired the reduction of the redox dye, alamar Blue (AB). After BaP removal, AB reduction recovered. Similar results were observed with BQ. As AB monitors metabolic activity, this novel phenomenon was termed transitory metabolic disruption. This decline in AB readings that was caused by BaP was ameliorated in RTL-W1 by alpha-naphthoflavone and geldanamycin, which suggests a role for CYP1A, and in R1 by indomethacin, which suggests involvement of prostaglandin-H-synthase. The significance of the response to BaP that is detected with AB and whether other PAHs cause it will be interesting future questions.

Ability of 16 priority PAHs to be photocytotoxic to a cell line from the rainbow trout gill.

Sixteen polycyclic aromatic hydrocarbons (PAHs) were screened for their ability to be photocytotoxic to a cell line from the rainbow trout gill, RTgill-W1. PAHs could be divided into one of three groups: incapable of being photocytotoxic, able to be both photocytotoxic and directly cytotoxic, or capable of being only photocytotoxic. Photocytotoxicity was distinct from direct cytotoxicity in that EC50 values were lower with the neutral red assay immediately after the PAH/UV treatment than with alamar Blue or CFDA-AM, indicating a more specific action on lysosomes. As well, in photocytotoxicity but not in direct cytotoxicity, the three assays showed increased impairment 24 h after treatment. Most PAHs were found to be strictly photocytotoxic; however, only six compounds were photocytotoxic at concentrations theoretically achievable in water. When photocytotoxic PAHs were ranked relative to fluoranthene to establish fluoranthene equivalent factors (FEFs), benzo[a]pyrene and benzo[g,h,i]perylene were found to be most potent. However, when the water solubility of each compound was taken into account in order to calculate the potential environmental photocytotoxic potency (PEPP), fluoranthene and pyrene appeared to have the most potential to impact fish through photocytotoxicity.

Methodology for demonstrating and measuring the photocytotoxicity of fluoranthene to fish cells in culture.

Methodology was developed for quantifying the photocytotoxicity of fluoranthene to a gill cell line from rainbow trout for future use in screening polycyclic aromatic hydrocarbons for their relative photocytotoxicity to fish. Solubilization in a modified culture medium was achieved with and without foetal bovine serum (FBS) and with and without dimethyl sulfoxide (DMSO). FBS caused most of the fluoranthene 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 Blue() and 5-carboxyfluorescein diacetate acetoxymethyl ester 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 mumol UV-B/m(2)/sec (UV-A:UV-B, 1.5) or 1.1 mumol UV-B/m(2)/sec (UV-A:UV-B, 9.7), both dyes indicated increasing loss of viability with increasing doses of fluoranthene. EC(50) values ranged from 18 to 44 ng/ml (89-217 nM), with the alamar Blue assay being slightly more sensitive.