title: Release of the lipid peroxidation marker 8-epi-prostaglandin F2 alpha from isolated gill pavement cells.
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.
authors.affiliation: Department of Chemistry and Biochemistry, Rider University, Lawrenceville, New Jersey 08648-3099, USA. espokas@rider.edu
authors.forename: Eric G
authors.initials: EG
authors.lastname: Spokas
authors.forename: Scott
authors.initials: S
authors.lastname: Harshman
authors.forename: Glenn M
authors.initials: GM
authors.lastname: Cohen
authors.forename: Chen
authors.initials: C
authors.lastname: Jiang
authors.forename: Jaime M
authors.initials: JM
authors.lastname: Levine
authors.forename: Ana R
authors.initials: AR
authors.lastname: Rodriguez
authors.forename: Jon
authors.initials: J
authors.lastname: Foglein
authors.forename: Bernd W
authors.initials: BW
authors.lastname: Spur
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D002712:Chlorides; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003530:Cyprinidae; D015237:Dinoprost / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005290:Ferric Compounds / Q000633:toxicity*; D005880:Gills / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006868:Hydrolysis; D007854:Lead / Q000633:toxicity*; D015227:Lipid Peroxidation / Q000187:drug effects*; D013058:Mass Spectrometry; D009566:Nitrates / Q000633:toxicity*; D012680:Sensitivity and Specificity
chemical_list: D015415:Biomarkers; D002712:Chlorides; D005290:Ferric Compounds; D009566:Nitrates; C075750:8-epi-prostaglandin F2alpha; D007854:Lead; C017461:lead nitrate; D015237:Dinoprost; C024555:ferric chloride