title: Early mitochondrial dysfunction, superoxide anion production, and DNA degradation are associated with non-apoptotic death of human airway epithelial cells induced by Pseudomonas aeruginosa exotoxin A.
abstract: It has been shown that bacterial exoproducts may induce airway epithelium injury. During the epithelial repair process, the respiratory epithelial cells no more establish tight junctional intercellular complexes and may be particularly susceptible to bacterial virulence factors. In this study, we analyzed the effect of Pseudomonas aeruginosa exotoxin A (ETA) at different periods of time and concentrations on 16 HBE 14o(-) human bronchial epithelial cells in culture conditions inducing a phenotype of repairing cells. ETA treatment for 24 and 48 h led to the killing of 40.0 +/- 5.7% and 79.0 +/- 1.4% of the cells, respectively, as determined by the dimethylthiazole 2,5 diphenyl tetrazolium bromide assay. At 1,000 ng/ml, ETA led to the killing of 25.2 +/- 6.6, 59.4 +/- 5.9, and 82.3 +/- 3.7% of the cells, after treatment periods of 7, 24, and 48 h, respectively. Cell death could not be inhibited by z-VAD-fmk, a broad spectrum caspase inhibitor. By transmission electron microscopy, ultrastructural characteristics described in apoptosis were not detected in ETA-treated cells. Instead, the mitochondria of cells treated for 24 and 48 h with ETA at 100 and 1,000 ng/ml were highly condensed. Human nasal polyp epithelial cells in primary culture exposed to ETA at 1,000 ng/ml did not exhibit characteristic features of apoptotic cells either. Cytofluorometric analysis of ETA-treated 16 HBE 14o(-) cells labeled with DiOC(6)(3) and hydroethidine showed a time- and dose-dependent reduction of the mitochondrial transmembrane potential, detected 7 h after ETA treatment, and an increase in superoxide production, detected at 24 h, respectively. By a photometric assay, DNA degradation was also detected 7 h after cell treatment with ETA at 100 and 1,000 ng/ml. Taken together, our results show that ETA-induced death of epithelial respiratory cells was preceded by early mitochondrial dysfunction and superoxide anion production, but was not followed by the classically described apoptotic pathways.
authors.affiliation: Department of Microbiology and Immunology, FCM/UERJ, Rio de Janeiro, Brazil. mcplot@uerj.br
authors.forename: Maria-Cristina
authors.initials: MC
authors.lastname: Plotkowski
authors.forename: Helvécio C C
authors.initials: HC
authors.lastname: Póvoa
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Zahm
authors.forename: Gérard
authors.initials: G
authors.lastname: Lizard
authors.forename: Geraldo M B
authors.initials: GM
authors.lastname: Pereira
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Tournier
authors.forename: Edith
authors.initials: E
authors.lastname: Puchelle
mesh_terms: D036002:ADP Ribose Transferases / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D001427:Bacterial Toxins; D001980:Bronchi; D061945:Caspase Inhibitors; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism* / Q000648:ultrastructure; D005098:Exotoxins / Q000633:toxicity*; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D009298:Nasal Polyps / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D009336:Necrosis; D020545:Respiratory Mucosa / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D013481:Superoxides / Q000378:metabolism*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D061945:Caspase Inhibitors; D004247:DNA; D004791:Enzyme Inhibitors; D005098:Exotoxins; D013481:Superoxides; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A