Oxygen consumption and electron spin resonance studies of free radical production by alveolar cells exposed to anoxia: inhibiting effects of the antibiotic ceftazidime.
By EPR spectroscopy, we investigated free radical production by cultured human alveolar cells subjected to anoxia/re-oxygenation (A/R), and tested the effects of ceftazidime, an antibiotic previously demonstrated to possess antioxidant properties. Two A/R models were performed on type II pneumocytes (A549 cell line), either on cells attached to culture dishes (monolayer A/R model; 3.5 h of anoxia, 30 min of re-oxygenation) or after cell detachment (suspension A/R model; 1 h of anoxia, 10 min of re-oxygenation). Ceftazidime and selective inhibitors (SOD, Tiron, L-NMMA) were added before anoxia. Free radical production was assessed by the EPR spin trapping technique. Oxygen consumption was monitored, in parallel with EPR studies, in the suspension A/R model. The production of free radical species was demonstrated by the generation of PBN-radical adducts: (a(N) = 15.2 G) in the monolayer A/R model and a six-line EPR spectrum (a(N) = 15.7 G and a(H) = 2.7 G) in the suspension A/R model. A kinetic study performed by oximetry, in parallel with EPR spectroscopy, demonstrated marked alterations of the cell respiratory function and that the free radical production started during anoxia and increased during re-oxygenation. In the suspension A/R model, the amplitude of EPR spectra were decreased upon the addition of 200 U/ml SOD (37% inhibition), 0.1 mM Tiron (67% inhibition) and 1 mM L-NMMA (43% inhibition). Addition of 1 mM ceftazidime decreased the amplitude of EPR spectra (37% inhibition) in both A/R models. Complementary in vitro EPR studies demonstrated that CAZ scavenged the hydroxyl radical (produced by the Fenton reaction). The protective effect of ceftazidime in the cell model could thus be linked to its ability to scavenge superoxide anions, nitrogen-derived species and hydroxyl radicals.
Centre for Oxygen Research and Development, Institut de Chimie, B6a, Domaine Universitaire du Sart Tilman, 4000 Liège, Belgium. amouithys@ulg.ac.be
Ange
A
Mouithys-Mickalad
Marianne
M
Mathy-Hartert
Guanadu
G
Du
Francis
F
Sluse
Carol
C
Deby
Maurice
M
Lamy
Ginette
G
Deby-Dupont
D000230:Adenocarcinoma; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002442:Ceftazidime / Q000494:pharmacology*; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms; D010101:Oxygen Consumption / Q000187:drug effects; D011650:Pulmonary Alveoli / Q000187:drug effects / Q000502:physiology*; D014407:Tumor Cells, Cultured; D019323:omega-N-Methylarginine / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D005609:Free Radicals; D019323:omega-N-Methylarginine; D002442:Ceftazidime