Abstract
The aim of the present study was to evaluate oxidative injury in gill pavement cells (GPCs) from fathead minnow (Pimephales promelas) using F2-isoprostane (F2-iP) release as an index of lipid peroxidation. Cells were isolated from pooled gill tissue by collagenase treatment, mechanical sieving, and Percoll density gradient centrifugation. Baseline levels of 8-epi-prostaglandin F2 alpha (8-epi-PGF2 alpha) were measured by incubating GPCs in physiological buffer (10(6) cells/ml) and enzyme immunoassay. After 60 min, the amount of immunoreactive 8-epi-PGF2 alpha (ir8-epi-PGF2 alpha) in control medium ranged from 1,374 to 5,515 pg/ml. Lead nitrate, 0.6 to 120 microM, did not influence ir8-epi-PGF2 alpha release, whereas FeCl3 stimulated release at 500 microM but not at 5 microM. Incubation medium was extracted for acidic lipids and analyzed by liquid chromatography/mass spectrometry/electrospray ionization. A compound in the medium exhibited a retention time on reverse-phase high-performance liquid chromatography nearly identical to that of synthetic 8-epi-PGF2 alpha The mass spectrum taken from the total ion chromatogram from 14.8 to 15.1 min contained a prominent ion at m/z 353, as expected for the molecular ion of 8-epi-PGF2 alpha. Similar results were obtained with tissue subjected to base hydrolysis. Mass spectra of extracted ion chromatograms obtained with gill extracts and authentic standard showed a close correspondence of fragment ions, providing definitive evidence for production and storage of F2-iPs by fish gills. In summary, F2-iP release occurs during lipid peroxidation injury to fish gill epithelium, and its measurement may facilitate aquatic toxicology studies of metallic and nonmetallic contaminants.
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