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

• Published in: Environmental toxicology and chemistry Vol. 27; no. 7; pp. 1569 - 1575
• Main Authors: Spokas, Eric G, Harshman, Scott, Cohen, Glenn M, Jiang, Chen, Levine, Jaime M, Rodriguez, Ana R, Foglein, Jon, Spur, Bernd W
• Format: Journal Article
• Language: English
• Published: 01.07.2008
• ISSN: 0730-7268

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.