Transition metal-induced apoptosis in lymphocytes via hydroxyl radical generation, mitochondria dysfunction, and caspase-3 activation: an in vitro model for neurodegeneration.
BACKGROUND
Redox transition metals have been implicated as crucial players in pathogenesis of neurodegenerative diseases. Intracellular signaling mechanism(s) responsible for oxidative stress and death in single-cell model exposed to metals has not yet been fully elucidated. The objective of the study was to determine the mechanism by which metals induced apoptosis in human peripheral blood lymphocytes (PBL).


METHODS
PBL were exposed to 50, 100, 250, 500, and 1,000 microM (Fe2+), (Mn2+), (Cu2+), and (Zn2+)-(SO4). Apoptotic/necrotic morphology was assessed with acridine orange/ethidium bromide staining. Further evaluations comprised production of H2O2, generation of hydroxyl radical (.OH), disruption of mitochondrial transmembrane potential (DeltaPsim), caspase-3 activation, and activation of NF-kappaB and p53 transcriptional factors.


RESULTS
Morphologic analysis showed that 500 microM provoked maximal percentage of apoptosis (22-30% AO/EB) and minimal necrosis (3-7%), whereas low concentrations were innocuous but 1,000 microM induced mainly necrosis (>40% AO/EB). Metals generated both H2O2 and (.OH) by Fenton reaction. Hydroxyl scavengers protected PBL from metal-induced apoptosis. All metals induced mitochondrial depolarization (17-62% nonfluorescent cells) and activated caspase-3 concomitantly with apoptotic morphology (25-32% AO/EB) at 24 h, and neither NF-kappaB nor p53 transcription factor showed activation.


CONCLUSIONS
This study provides evidence that redox-active (Fe2+), (Mn2+), (Cu2+), and (Zn2+) ion-induced apoptosis in PBL by (H2O2)/(.OH) generation, resulting in mitochondria depolarization, caspase-3 activation, and nuclear fragmentation independent of NF-kappaB and p53 transcription factors activation. Our data highlight the potential use of lymphocytes as a model to screen antioxidant strategies designed to remove H2O2/.OH associated with metal-catalyzed reactions in neurodegenerative disorders.
Jiménez Del Río
Marlene
M
Facultad de Medicina, Departamento de Medicina Interna, Programa de Investigación en Neurociencias, Universidad de Antioquia, Medellín, Colombia. mdelrio@quimbaya.udea.edu.co
Vélez-Pardo
Carlos
C
D000328:Adult; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D004789:Enzyme Activation; D005609:Free Radicals; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017665:Hydroxyl Radical; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism; D007477:Ions; D007501:Iron / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism / Q000473:pathology*; D008274:Magnesium / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D009336:Necrosis; D019636:Neurodegenerative Diseases / Q000473:pathology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D000975:Antioxidants; D005609:Free Radicals; D007477:Ions; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D017665:Hydroxyl Radical; D003300:Copper; D006861:Hydrogen Peroxide; D007501:Iron; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D008274:Magnesium; D015032:Zinc