Release of the lipid peroxidation marker 8‐epi‐prostaglandin F 2α 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; 1552-8618
• DOI: 10.1897/07-510.1

Abstract The aim of the present study was to evaluate oxidative injury in gill pavement cells (GPCs) from fathead minnow ( Pimephales promelas ) using F 2 ‐isoprostane (F 2 ‐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 F 2 α (8‐epi‐PGF2α) were measured by incubating GPCs in physiological buffer (106 cells/ml) and enzyme immunoassay. After 60 min, the amount of immunoreactive 8‐epi‐PGF 2α ( ir 8‐epi‐PGF 2α ) in control medium ranged from 1,374 to 5,515 pg/ml. Lead nitrate, 0.6 to 120 μM, did not influence ir 8‐epi‐PGF 2α release, whereas FeCl 3 stimulated release at 500 μM but not at 5 μM. 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‐PGF 2α 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 2α . 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 2 ‐iPs by fish gills. In summary, F 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.