Release Of The Lipid Peroxidation Marker 8-Epi-Prostaglandin F2alpha From Isolated Gill Pavement Cells

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 F2alpha (8-epi-PGF2,) were measured by incubating GPCs in physiological buffer (106 cells/ml) and enzyme immunoassay. After 60 min, the amount of immunoreactive 8-epi-PGF2alpha (ir8-epi-PGF2alpha) in control medium ranged from 1,374 to 5,515 pg/ml. Lead nitrate, 0.6 to 120 muM, did not influence ir8-epi-PGF2alpha release, whereas FeCl3 stimulated release at 500 muM but not at 5 muM. Incubation medium was extracted for acidic lipids and analyzed by liquid chromatography/mass spectrometry/electrosprayionization. A compound in the medium exhibited a retention time on reverse-phase high-performance liquid chromatography nearly identical to that of synthetic 8-epi-PGF2alpha. The mass spectrum taken from the total ion chromatogram from 14.8 to 15.1 min contained a prominent ion at mlz 353, as expected for the molecular ion of 8-epi-PGF2alpha. 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 FZ-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.