title: Silica-induced apoptosis in alveolar macrophages: evidence of in vivo thiol depletion and the activation of mitochondrial pathway.
abstract: Studies have shown that silica induces apoptosis through mechanisms that also regulate the inflammatory responses of lung cells to silica exposure. Although implicated in cell culture studies, the major in vivo pathway through which silica induces apoptosis has not been characterized. The present study is to study the role of mitochondria in silica-induced oxidative stress and apoptosis in vivo. Rats were intratracheally instilled with saline or silica (20 mg/kg) and sacrificed at 3 days post-exposure unless otherwise specified. Alveolar macrophages (AM) were harvested by bronchoalveolar lavage and measured for apoptosis and secretion of inflammatory mediators in the presence or absence of appropriate inhibitors. Concurrent studies were carried out to determine the presence of intracellular reactive oxygen species (ROS) via confocal microscopy, mitochondrial trans-membrane potential by flow cytometry, mitochondrial release of cytochrome c, and the activation of caspase activities in AM by Western blot analysis. Silica was shown to induce elevated levels of intracellular ROS, resulting in a marked decrease in intracellular glutathione (GSH) and cysteine and a sustained presence of apoptotic AM in silica-exposed rats up to two weeks post-exposure. The apoptotic AM were characterized by decreased mitochondrial trans-membrane potential, increased mitochondrial release of cytochrome c, activated caspase 9 (but not caspase 8) and caspase 3 activities, and PARP degradation, comparing to cells from the saline control. Silica induced AM production of IL-1 and TNF-alpha, which may be inhibited by ex vivo treatment of cells with N-acetylcysteine (NAC) or microtubule modifiers such as tetrandrine and taxol. NAC was shown to prevent intracellular GSH depletion and silica-induced production of IL-1beta and TNF-alpha but not apoptosis in AM from silica-exposed rats. These results show that silica-induced apoptosis is mediated through the mitochondrial pathway but not through cellular production of inflammatory cytokines, ROS generation, however, induces both apoptosis and cellular secretion of inflammatory mediators.
authors.affiliation: School of Pharmacy, West Virginia University, Morgantown, WV 26506, USA.
authors.forename: Shuiying
authors.initials: S
authors.lastname: Hu
authors.forename: Hongwen
authors.initials: H
authors.lastname: Zhao
authors.forename: Nabil H
authors.initials: NH
authors.lastname: Al-Humadi
authors.forename: Xuejun J
authors.initials: XJ
authors.lastname: Yin
authors.forename: Joseph K H
authors.initials: JK
authors.lastname: Ma
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D044182:Benzylisoquinolines / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D003545:Cysteine / Q000096:biosynthesis; D045304:Cytochromes c / Q000096:biosynthesis; D004195:Disease Models, Animal; D005978:Glutathione / Q000096:biosynthesis; D018836:Inflammation Mediators / Q000276:immunology; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000276:immunology*; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D018384:Oxidative Stress; D017239:Paclitaxel / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000032:analysis / Q000276:immunology; D012822:Silicon Dioxide / Q000009:adverse effects*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D000470:Alkaloids; D000893:Anti-Inflammatory Agents; D051017:Apoptosis Regulatory Proteins; D044182:Benzylisoquinolines; D018836:Inflammation Mediators; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D014409:Tumor Necrosis Factor-alpha; C009438:tetrandrine; D012822:Silicon Dioxide; D045304:Cytochromes c; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione; D003545:Cysteine; D017239:Paclitaxel; D000111:Acetylcysteine