title: Hypoxia-independent apoptosis in neural cells exposed to carbon monoxide in vitro.
abstract: The neurotoxic effects of carbon monoxide (CO) are well known. Brain hypoxia due to the binding of CO to hemoglobin is a recognized cause of CO neurotoxicity, while the direct effect of CO on intracellular targets remains poorly understood. In the present study, we have investigated the pathways leading to neural cell death induced by in vitro exposure to CO using a gas exposure chamber that we have developed. Mouse hippocampal neurons (HT22) and human glial cells (D384) were exposed to concentrations of CO ranging from 300 to 1000 ppm in the presence of 20% oxygen. Cytotoxicity was observed after 48 h exposure to 1000 ppm, corresponding to approximately 1 microM CO in the cultured medium, as measured by gas chromatography. CO induced cell death with characteristic features of apoptosis. Exposed cells exhibited loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, nuclei with chromatin condensation, and exposure of phosphatidyl serine on the external leaflet of the plasma membrane. CO also triggered activation of caspase and calpain proteases. Pre-incubation with either the pancaspase inhibitor Z-VAD-fmk (20 microM) or the calpain inhibitor E64d (25 microM) reduced by 50% the occurrence of apoptosis. When pre-incubating the cells with the two inhibitors together there was an additional reduction in the number of cells with apoptotic nuclei. These data suggest that CO causes apoptosis via activation of parallel proteolytic pathways involving both caspases and calpains. Furthermore, pre-treatment with the antioxidant MnTBAP (100 microM) significantly reduced the number of apoptotic nuclei, pointing to a critical role of oxidative stress in CO toxicity.
authors.affiliation: Institute of Environmental Medicine, Division of Toxicology and Neurotoxicology, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: R
authors.initials: R
authors.lastname: Tofighi
authors.forename: N
authors.initials: N
authors.lastname: Tillmark
authors.forename: E
authors.initials: E
authors.lastname: Daré
authors.forename: A M
authors.initials: AM
authors.lastname: Aberg
authors.forename: J E
authors.initials: JE
authors.lastname: Larsson
authors.forename: S
authors.initials: S
authors.lastname: Ceccatelli
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism; D002249:Carbon Monoxide Poisoning / Q000473:pathology*; D002352:Carrier Proteins / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects; D002467:Cell Nucleus / Q000648:ultrastructure; D003470:Culture Media; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006624:Hippocampus / Q000473:pathology; D006801:Humans; D002534:Hypoxia, Brain / Q000473:pathology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects; D009474:Neurons / Q000187:drug effects / Q000473:pathology*; D010718:Phosphatidylserines / Q000494:pharmacology; D011419:Propidium; D015398:Signal Transduction / Q000187:drug effects; D014343:Trypan Blue; D013049:Spectrin
chemical_list: D017304:Annexin A5; D002154:Calpain; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D020169:Caspases; D003470:Culture Media; D045304:Cytochromes c; D004791:Enzyme Inhibitors; D008840:Microfilament Proteins; D010718:Phosphatidylserines; D011419:Propidium; D014343:Trypan Blue; D013049:Spectrin