Valerian E. Kagan Valerian E. Kagan Macrophage Response to Single - PowerPoint PPT Presentation

Valerian E. Kagan Valerian E. Kagan Macrophage Response to Single Walled Carbon Nanotubes: Macrophage Response to Single Walled Carbon Nanotubes: Oxidative Stress and Inflammatory Consequences. Oxidative Stress and Inflammatory Consequences. Center For Free Radical and Antioxidant Health, Department of Environmental and Center For Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh, Occupational Health, University of Pittsburgh,

Single Walled Carbon Nanotubes Handling nanotube material ‘ Tangles ’ of nanotubes and nanoropes Nanotubes Nanoropes Raw SWCNT material Catalyst particles Raw single walled carbon nanotube material. Courtesy of Andrew Maynard

Transmission Electron Transmission Electron Metal Components of SWCNT Metal Components of SWCNT Microscopy of SWCNT Microscopy of SWCNT (  g/gram) (  g/gram) Component Component Aluminum 233 Molybdenum 1070 Calcium 164 Sodium 8750 Cadmium 23.4 Nickel 8,750 Chromium 13.1 Palladium 28 Copper 2,530 Selenium <2.001 Iron 239, 000 Titanium 6.92 Original magnification x 145,000 Zinc 85.9 EPR spectra of partially-purified SWCNT EPR spectra of partially-purified SWCNT manufactured by high-pressure CO conversion manufactured by high-pressure CO conversion (HiPco ™ ) technology as compared to purified (HiPco ™ ) technology as compared to purified SWCNT additionally treated with an iron chelator, SWCNT additionally treated with an iron chelator, deferoxamine (DFO). deferoxamine (DFO). 330G Note that partially-purified SWCNT displayed a broad signal with g value 2.0 and half-width of 640G, the signal Partially-Purified Purified was not detectable in purified DFO-treated SWCNT. SWCNT SWCNT+DFO

Raw Samples of Raw Samples of Carbon Carbon Nanotubes Nanotubes Contain Contain Redox-Active Redox-Active Iron Iron

Proteins O 2 Fe 2+ Fe 3+ Lipids SOD O 2 + + Fe 3+ DNA Fe 2+ Catalase H 2 O 2 Peroxidase Fe 2+ Fe 3+ No HO (RO) Enzymatic Control

Iron-Catalyzed Decomposition of H 2 O 2 Forms A Potent Oxidant – Hydroxyl Radical H 2 O 2 Fe 2+ Fe 3+ OH . Ascorbate + + Asc . + HOH +

EPR spectra of ascorbate radicals generated by EPR spectra of ascorbate radicals generated by partially purified SWCNT. partially purified SWCNT. Ascorbate Ascorbate Ascorbate+partially purified SWCNT, 5 min RAW264.7+ Ascorbate Ascorbate+partially purified SWCNT+DFO RAW264.7 + Ascorbate+partially Ascorbate+partially purified purified SWCNT + 10 G SWCNT, 5 min DFO, 5 min 20 G Model system RAW264.7 macrophages Conditions: Zymosan (2.5 mg/ml)-stimulated RAW264.7 Conditions: Ascorbate (10 mM) in PBS (pH 7.4); partially macrophages (20x106 cells/ml); partially purified SWCNT (2.5 wt% purified SWCNT (0.12 mg/ml, 2.5 wt% of iron); of iron, 0.12 mg/ml) desferioxamine, DFO (0.2 mM); EPR conditions: microwave power, 20 mW; modulation amplitude, 1.0 G; time constant, 1.3 sec; conversion time, 0.6 sec.

Carbon Carbon Nanotubes Nanotubes Directly Directly Damage Damage Broncho-Epithelial Broncho-Epithelial Cells Cells

Particles, Nanotubes Epithelial Cell

Engulfment of SWCNT by BEAS- -2B Cells 2B Cells Engulfment of SWCNT by BEAS x 29,000 SWCNT SWCNT x 87,000 x 11,600

Fenton Reaction Fenton Reaction Fe 3+ + HO - + . OH Fe 2+ + H 2 O 2 DMPO Adduct Formation DMPO Adduct Formation H Me Me . + OH H OH + Me N Me N . - O O 5,5-Dimethyl-1-Pyrroline-N-Oxide (DMPO)

DMPO ESR spectra of ESR spectra of DMPO+SWCNT DMPO adducts of DMPO adducts of free radicals formed free radicals formed by partially purified by partially purified SWCNT in the SWCNT in the DMPO+SWCNT +H2O2 presence of presence of BEAS-2B cells. BEAS-2B cells. DMPO+SWCNT+ H 2 O 2 +Catalase Conditions: BEAS-2B (2x105 cells/ml) in PBS (pH 7.4); DMPO+SWCNT 100 mM DMPO; partially purified SWCNT (2.5 wt% of +DTPA iron, 0.12 mg/ml) H 2 O 2 (1 mM); catalase (20 U/ml); DTPA (0.2 mM). ESR conditions: microwave power, 20 mW; modulation 20 Gauss amplitude, 1.0 G; time constant, 1.3 sec; conversion time, 0.6 sec.

140 cells homogenates, nmol/mg protein *p < 0.05 vs control cells; ** p < 0.05 vs cells exposed to Total 120 Peroxyl radicals scavenged by 0.06 mg/ml SWCNT; *** p < 0.05 vs cells exposed to 0.12 mg/ml SWCNT; Antioxidant 100 * ** Reserve in * 80 BEAS-2B Cells *** 60 ** * Following 40 Exposure to 20 SWCNT 0 0.00 0.06 0.12 0.24 SWCNT concentration, mg/ml

GSH and Protein Thiols in BEAS-2B Cells Following Exposure to SWCNT 60 80 Protein Thiols GSH 70 Thiols, nmol/mg protein 50 * 60 * 40 50 ** * 30 40 *** ** * 30 20 20 10 10 0 0 0.00 0.06 0.12 0.24 0.00 0.06 0.12 0.24 SWCNT concentration, mg/ml SWCNT concentration, mg/ml *p < 0.05 vs control cells; ** p < 0.05 vs cells exposed to 0.06 mg/ml SWCNT; *** p < 0.05 vs cells exposed to 0.12 mg/ml SWCNT;

Exposure to SWCNT Induced Apoptotic Cell Death SWCNT (0.24 mg/ml) Control BEAS-2B cells were stained with TUNEL reagent. Apoptotic cells exhibited yellow-green fluorescence, while normal cells counter-stained with propidium iodide fluoresced red. Original magnification x 40.

Epithelial cells ulmonary Toxicants Arachidonic Acid (particles, fibers, Metabolism Amplification microbes) of Pulmonary Chemokine and Toxicity Endotoxin or Metals Cytokine Release Attachment to and Spreading on Direct Toxic Extracellular Matrix Effects Activation of Vascular Macrophages PMNs Lung Injury, Cell Proliferation and Release of Fibrogenic Factors Upregulation of Reorganization of Adhesion Molecules Cytoskeleton Release of Oxidants and PMN Motility & Attachment PMN Recruitment in Proteolytic Enzymes the Lung Courtesy of M. Luster

Aggregates of Carbon Aggregates of Carbon Nanotubes Nanotubes Are Recognized and Are Recognized and Sequestered by Sequestered by Macrophages Macrophages

Days post Exposure Day 0 1 3 7 28 60 C57BL/6 Histopathology Pulmonary injury Lung functions Aspiration with: Collagen morphometry CNT (10, 20, 40  g/mouse) Oxidative stress UFCB (40  g/mouse) Cytokines Silica (40  g/mouse & 2.5 mg/mouse) Single Walled Carbon Nanotube Toxicity - purified SWCNT

Histopathology of lung of C57BL/6J mice after pharyngeal Histopathology of lung of C57BL/6J mice after pharyngeal aspiration of partially purified SWCNT aspiration of partially purified SWCNT 1 Day Control 7 Day 3 Day Partially purified SWCNT (2.5 wt% of iron, 0.5 mg/kg b. w.).

Cell differential in BAL fluid of C57BL/6J mice after Cell differential in BAL fluid of C57BL/6J mice after treatment with partially purified SWCNT. treatment with partially purified SWCNT. 35 600 Lymphocytes Total Cell Counts 0.8 PMNs 30 500 Cell Number, x10 6 Cell Number, x10 3 Cell Number, x10 3 25 0.6 400 20 300 0.4 15 200 10 0.2 100 5 0 0 0 1 3 7 1 3 7 7 1 3 Days after exposure Days after exposure Days after exposure Control Partially purified SWCNT (2.5 wt% of iron, 0.5 mg/kg b.w.) SWCNT

Macrophages in BAL fluid of C57BL/6J mice after treatment with partially purified SWCNT. 700 Control 600 SWCNT Cell Number, x10 6 500 400 300 200 100 0 1 3 7 28 60 Days Post Exposure Partially purified SWCNT (40  g per mouse)

Damage or Damage or Pro- Pro -apoptotic apoptotic signals signals P S P S PS P S PSX S P SWCNT

Non-Aggregated Non-Aggregated Carbon Nanotubes Carbon Nanotubes Do NOT Significantly Do NOT Significantly Damage Damage Macrophages Macrophages

Carbon Nanotubes Carbon Nanotubes Do Do Stress and Damage of Stress and Damage of Stress and Damage of Macrophages Macrophages Macrophages Carbon Carbon ? ? Nanotubes Nanotubes Induce Induce Apoptosis Apoptosis in in Macrophages? Macrophages?

Fluorescent micrographs of RAW 264.7 macrophages Fluorescent micrographs of RAW 264.7 macrophages incubated in the presence of partially purified SWCNT. incubated in the presence of partially purified SWCNT. Control SWCNT, 6h A Note nuclear condensation and fragmentation as revealed by Hoechst 33342 staining (shown for clarity in green pseudo-color). Partially purified SWCNT (2.5 wt% of iron, 0.12 mg/ml).

Apoptosis induced by partially purified Apoptosis induced by partially purified SWCNT in RAW 264.7 macrophages . SWCNT in RAW 264.7 macrophages . 250 100 Zymosan 200 Caspase 3 activity, Apoptosis, % AU/mg protein 150 50 100 SWCNT 50 0 0 4 h 6 h Control 0 10 20 30 SWCNT Time, h Partially purified SWCNT (2.5 wt% of iron, 0.1 mg/ml).

Electron micrographs illustrating effects of partially Electron micrographs illustrating effects of partially purified SWCNT exposure on RAW 264.7 purified SWCNT exposure on RAW 264.7 macrophages. macrophages. 1 h Control 6 h B C A SWCNT SWCNT SWCNT SWCNT SWCNT SWCNT x3,800 x3,800 x3,800 Partially purified SWCNT exposure (2.5 wt% of iron, 0.12 mg/ml)