title: Chloropicrin-induced toxic responses in human lung epithelial cells.
abstract: Chloropicrin is a slowly evaporating toxic irritant that is known to cause damage in the respiratory system. Here we used a lung epithelial cell line (A549) to study the molecular responses underlying chloropicrin toxicity. Glutathione (GSH), synthetic peptide and 2'-deoxyguanosine were used as in vitro trapping agents to identify early markers of chloropicrin toxicity. Microscopy of the cells revealed massive vacuolization by chloropicrin exposure (80-100μM). The number of apoptotic cells increased with the chloropicrin concentration as assessed by flow cytometry. Immunoblotting analysis revealed increases in the amount of four proteins (p53, p21, p27 and phospho-Erk1/2) that are involved in DNA-damage, cell cycle regulation and apoptosis. Chloropicrin evoked a dose-dependent increase in levels of reactive oxygen species within one hour of exposure. The treatment triggered also the formation of disulphide bonds between the model thiol-containing peptides as analysed by LC/MS. Chloropicrin did not form stable adducts with the model peptides or 2'-deoxyguanosine. N-acetyl-cysteine (1mM NAC) fully prevented the vacuoles and chloropicrin-induced cytotoxicity. The results suggest that an oxidative insult, particularly modification of free sulfhydryl groups in proteins is involved in the acute toxicity evoked by chloropicrin in airway epithelial cells. The protective effect of NAC as a potential antidote in chloropicrin intoxication will require further investigation.
authors.affiliation: Research and Development Department, Centre for Military Medicine, Finnish Defence Forces, Helsinki, Finland; Faculty of Health Sciences, School of Pharmacy/Toxicology, University of Eastern Finland, Kuopio, Finland. Electronic address: Maija.Pesonen@uef.fi.
authors.forename: Maija
authors.initials: M
authors.lastname: Pesonen
authors.affiliation: Faculty of Health Sciences, School of Pharmacy/Toxicology, University of Eastern Finland, Kuopio, Finland.
authors.forename: Merja
authors.initials: M
authors.lastname: Häkkinen
authors.affiliation: Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.
authors.forename: Kirsi
authors.initials: K
authors.lastname: Rilla
authors.affiliation: Faculty of Health Sciences, School of Pharmacy/Toxicology, University of Eastern Finland, Kuopio, Finland.
authors.forename: Risto
authors.initials: R
authors.lastname: Juvonen
authors.affiliation: Faculty of Health Sciences, School of Pharmacy/Toxicology, University of Eastern Finland, Kuopio, Finland.
authors.forename: Tapio
authors.initials: T
authors.lastname: Kuitunen
authors.affiliation: Faculty of Health Sciences, School of Pharmacy/Toxicology, University of Eastern Finland, Kuopio, Finland.
authors.forename: Markku
authors.initials: M
authors.lastname: Pasanen
authors.affiliation: Faculty of Health Sciences, School of Pharmacy/Toxicology, University of Eastern Finland, Kuopio, Finland.
authors.forename: Kirsi
authors.initials: K
authors.lastname: Vähäkangas
mesh_terms: D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D019610:Cytoprotection; D003849:Deoxyguanosine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006843:Hydrocarbons, Chlorinated / Q000633:toxicity*; D007509:Irritants / Q000633:toxicity*; D008168:Lung / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D010766:Phosphorylation; D055503:Protein Multimerization; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D000975:Antioxidants; C493956:CDKN1A protein, human; C436291:CDKN1B protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004220:Disulfides; D006843:Hydrocarbons, Chlorinated; D007509:Irritants; D017382:Reactive Oxygen Species; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D050760:Cyclin-Dependent Kinase Inhibitor p27; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D003849:Deoxyguanosine; D005978:Glutathione; C100187:chloropicrin
keywords: Disulfide bonds; N-acetyl-cysteine; Vacuoles; p21; p27; p53