Release of the Lipid Peroxidation Marker 8-Epi-Prostaglandin F sub(2Alpha) 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
• Subjects: Freshwater; Pimephales promelas
• ISSN: 0730-7268; 1552-8618
• DOI: 10.1897/07-510.1

The aim of the present study was to evaluate oxidative injury in gill pavement cells (GPCs) from fathead minnow (Pimephales promelas) using F sub(2)-isoprostane (F sub(2)-iP) release as an index of lipid peroxidation. Cells were isolated from pooled gill tissue by collagenase treatment, mechanical sieving, and Percoll[reg.] density gradient centrifugation. Baseline levels of 8-epi-prostaglandin F sub(2alpha) (8-epi-PGF sub(2alpha)) were measured by incubating GPCs in physiological buffer (10 super(6) cells/ml) and enzyme immunoassay. After 60 min, the amount of immunoreactive 8-epi- PGF sub(2alpha) ( sub(ir)8-epi-PGF sub(2alpha)) in control medium ranged from 1,374 to 5,515 pg/ml. Lead nitrate, 0.6 to 120 mu M, did not influence sub(ir)8-epi- PGF sub(2alpha) release, whereas FeCl sub(3) stimulated release at 500 mu M but not at 5 mu M. 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-PGF sub(2alpha.) 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-PGF sub(2alpha). 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 F sub(2)-iPs by fish gills. In summary, F sub(2)-iP release occurs during lipid peroxidation injury to fish gill epithelium, and its measurement may facilitate aquatic toxicology studies of metallic and nonmetallic contaminants.