Valerian E. Kagan Macrophage Response to Single Walled Carbon Nanotubes: Oxidative Stress and Inflammatory Consequences.

Transcription

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

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

3 Metal Components of SWCNT Transmission Electron Microscopy of SWCNT Component ( g/gram) Component ( g/gram) 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 Zinc 85.9 Original magnification x 145,000 Partially-Purified SWCNT 330G Purified SWCNT+DFO EPR spectra of partially-purified SWCNT manufactured by high-pressure CO conversion (HiPco ) technology as compared to purified SWCNT additionally treated with an iron chelator, deferoxamine (DFO). Note that partially-purified SWCNT displayed a broad signal with g value 2.0 and half-width of 640G, the signal was not detectable in purified DFO-treated SWCNT.

4 Raw Samples of Carbon Nanotubes Contain Redox-Active Iron

5 O2 Fe 2+ Proteins Fe 3+ O2 + Fe 3+ SOD Lipids H2O2 Fe 2+ Fe 2+ Fe 3+ HO (RO) Catalase Peroxidase DNA No Enzymatic Control

6 Iron-Catalyzed Decomposition of H2O2 Forms A Potent Oxidant H2O2 Fe 2+ Fe 3+ Hydroxyl Radical OH. + Ascorbate Asc. + HOH

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

8 Carbon Nanotubes Directly Damage Broncho-Epithelial Cells

9 Epithelial Cell Particles, Nanotubes

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

11 Fenton Reaction 3+ + HO- +.OH 2+ Fe Fe + H2O2 DMPO Adduct Formation Me H Me. + OH N+ O- 5,5-Dimethyl-1-Pyrroline-N-Oxide (DMPO) H Me OH Me N O.

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

13 Peroxyl radicals scavenged by cells homogenates, nmol/mg protein *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; * ** * *** ** * SWCNT concentration, mg/ml Total Antioxidant Reserve in BEAS-2B Cells Following Exposure to SWCNT

14 GSH and Protein Thiols in BEAS-2B Cells Following Exposure to SWCNT Thiols, nmol/mg protein 60 GSH * Protein Thiols * 50 ** * *** ** * 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;

15 Exposure to SWCNT Induced Apoptotic Cell Death Control SWCNT (0.24 mg/ml) 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.

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

17 Aggregates of Carbon Nanotubes Are Recognized and Sequestered by Macrophages

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

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

20 Cell differential in BAL fluid of C57BL/6J mice after treatment with partially purified SWCNT Days after exposure Control SWCNT 7 Lymphocytes PMNs Cell Number, x103 Total Cell Counts Cell Number, x103 Cell Number, x Days after exposure 1 3 Days after exposure Partially purified SWCNT (2.5 wt% of iron, 0.5 mg/kg b.w.) 7

21 Cell Number, x106 Macrophages in BAL fluid of C57BL/6J mice after treatment with partially purified SWCNT. 700 Control 600 SWCNT Days Post Exposure Partially purified SWCNT (40 g per mouse)

22 Damage or ProPro-apoptotic signals PS PS PS PS PS SWCNT PS X

23 Non-Aggregated Carbon Nanotubes Do NOT Significantly Damage Macrophages

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

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

27 Apoptosis induced by partially purified SWCNT in RAW macrophages Apoptosis, % Caspase 3 activity, AU/mg protein Zymosan SWCNT Time, h 30 Control Partially purified SWCNT (2.5 wt% of iron, 0.1 mg/ml). 4h 6h SWCNT

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

29 DHE-positive cells, % of total DHE-positive RAW macrophages: zymosan and SWCNT. 60 * Control Zymosan 0.25 mg/ml 0.1 mg/ml 0.5 mg/ml SWCNT (pure) 0.5 mg/ml 0.1 mg/ml SWCNT Macrophages 0.3 x 106/well) were pre-incubated with DHE (10 mm for 10 min at 37oC). Then RAW macrophages were stimulated by zymosan or SWCNT (for 30 min at 37oC). * - p<0.05 vs. control cells

30 DAF2-positive RAW macrophages: zymosan and SWCNT. DAF2-positive cells, % of total 20 * LPS- 15 LPS Control Zymosan 0.25 mg/ml 0.1 mg/ml 0.5 mg/ml SWCNT (pure) 0.1 mg/ml 0.5 mg/ml SWCNT Naïve macrophages (0.3 x 106/well) and macrophages stimulated by LPS (0.1 mg/ml for 6 h at 37oC) macrophages were pre-incubated with DAF2DA (2 mm for 1 h at 37oC). Then RAW macrophages were stimulated by zymosan or SWCNT (for 2h at 37oC). * - p<0.05 vs. control cells

31 Zymosan Reduction of iron Fe3+ Fe2+ OH. O2 O2. H2O2. O2. O2 NADPH oxidase Activation Cell Death Fe-SWCNT Fe-SWCNT Fe-SWCNT Apoptosis Oxidative Stress Necrosis

32 Superoxide-DMPO Adduct RAW macrophages 15 G RAW macrophages plus partially purified SWCNT EPR spectra of DMPO radical adducts generated during incubation of xanthine oxidase/xanthine and zymosan-stimulated RAW macrophages by partially purified SWCNT in the absence or presence of partially purified SWCNT. Hydroxyl Radical-DMPO Adduct Incubation system contained: xanthine oxidase (0.1U/mL), xanthine (1 mm), zymosan (2.5 mg/ml)-stimulated RAW264.7 macrophages (20x106 cells/ml) in PBS (ph 7.4) plus 100 mm DMPO; partially purified SWCNT (2.5 wt% of iron, 0.12 mg/ml); EPR conditions: microwave power, 20 mw; modulation amplitude, 1.0 G; time constant, 1.3 sec; conversion time, 0.6 sec.

33 Hydroxyl Radical-DMPO Adduct Control Zymosan Zymosan+ SWCNT 15 G ESR spectra of DMPO adducts of free radicals formed by partially purified SWCNT in the presence of RAW macrophages

34 NH3 Cells NL O O P O Apoptosis 1 FFA HC HC PE PC CL C H2 SPH O O P O CH HC ROS Generation Phospholipid Oxidation H C O C O CH CH CH HC NH3 CH COO O O C O H3C HC CH HC HC C H2 N H3 NH3 PI O P O 2D-HPTLC Origin 2 O H C O HOOCH Phosholipid Spot + PLA2 Fluorescence a.u. Fluorescence, a.u HC CH HC 100 HC Resorufin 800 OH 600 O C O CH H C 2 H3C O C O C H2 C H2 C H2 + C H2 Phospholipase A2 C H2 H3C PL-OOH Resorufin C H2 HOOC CH HOOCH CH HC CH HC H C SHpODE Amplex Red + Microperoxidase Resorufin or 9SHpODE, pmol C H2 C H2 C H2 Lyso-PL FA-OOH 0 4 Time, min N FA-OOH MP11 HO 2 O C N 6 Fluorescence HPLC O OH N-Acetyl-3,7-Dihydroxyphenoxazine (Amplex Red) HO CH CH HC O C O C O CH CH CH CH H C 2 C H2 H3C O C H 2 C H C OO O P O O H C C H2 O CH COO LPC PS H C O C O CH CH CH HOOCH Oxidation CH COO O O O 7-Hydroxy-3H-Phenoxazine-3-one (Resorufine)

35 Partially purified SWCNT induce lipid peroxidation in RAW macrophages. Lipid hydroperoxides, nmol/106 cells * Control Zymosan Zymosan +SWCNT Stimulation of macrophages with zymosan (0.25 mg/ml) cause a slight increase in lipid peroxidationm further enhanced by partially purified iron-containing SCWNT (0.1 mg/ml). Lipid peroxidation was assessed by our newly developed fluorescence HPLC-based protocol for lipid hydroperoxides

36 Effect of partially purified SWCNT exposure on GSH content in RAW macrophages. GSH, nmol/mg protein Control Zymosan Zymosan +SWCNT

37 Production of cytokines by zymosan-stimulated RAW macrophages and SWCNT treated RAW macrophages. Cytokines released, % of zymosan 120 SWCNT Zymosan TGF IL-10 Anti-inflammatory IL-1 TNF Pro-inflammatory Zymosan mg/ml; SWCNT 0.1 mg/ml.

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39 Recognition and digestion of Cells B16 melanoma cells (green) and dendritic cells

40 Can Macrophages Be Forced to Recognize and Digest Carbon Nanotubes

41 Phosphatidylserine (PS) as an eat -me signal in phagocytosis eat-me of apoptotic cells Damage or ProPro-apoptotic signals PS PS PS PS PS SWCNT PS X?

42 RAW macrophages effectively phagocytose PS-containing liposomes but not PC-containing liposomes. PS-containing liposomes PC-containing liposomes RAW macrophages (105 cells/ml) were incubated for 6 h with liposomes (0.33 mm) composed of a mixture of PC:PS (with fluorescently labeled PS) or PC (with fluorescently labeled PC). After incubation, macrophages were fluorescently labeled with Hoechst 3343 (nuclei, blue fluorescence), and Cell Tracker Orange (cytosol, red fluorescence). Liposomes fluorescently labeled with with NBD-phospholipids (green fluorescence) PS-containing liposomes were prepared by sonication of a mixture of PC:PS 1:1 with the addition of 10 mol% of NBD-PS). PCcontaining liposomes were prepared by sonication of a mixture of PC with the addition of 10 mol% of NBD-PC.

43 PS-labeled SWCNT PC-labeled SWCNT RAW macrophages effectively phagocytose PS-labeled SWCNT but not PC labeled SWCNT. RAW264.7 macrophages (105 cells/ml) were incubated for 6h with fluorescently labeled SWCNT (0.1 mg/ml).

44 Thanks To My Collaborators: Kagan s Lab: A. A. Arroyo Arroyo N. N. Belikova Belikova G. G. Borisenko Borisenko J. J. Jiang Jiang K. K. Kawai Kawai V. V. Kini Kini S.-X. S.-X. Liu Liu T. T. Matsuura Matsuura A. A. Osipov Osipov A. A. Potapovich Potapovich B. B. Serinkan Serinkan V. V. Tyurin Tyurin Y. Y. Tyurina Tyurina Q. Q. Zhao Zhao NIOSH: A. Shvedova (Morgantown) V. Castranova (Morgantown) R. Mercer (Morgantown) E. Kisin (Morgantown) A. Maynard (Cincinnati)

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47 Reduction of iron Fe2+ OH Fe-SWCNT. O2 Fe3+ O2. H2O2 Oxid ative. O2. O2 NADPH oxidase Activation Recruitment of new M Cell Death Stre ss Production of pro-inflammatory cytokines Fe-SWCNT Fe-SWCNT Microbial Infection lam f n I to ma r e yr s se n o p Necrosis Apoptosis Production of anti-inflammatory cytokines PS PS PS oxidation/ Apoptotic externalization cell Ap op Fe-SWCNT to sis

48 Superoxide production, ethidium positive cells, % of total Effect of apoptotic cells and PS on superoxide generation in zymosan-stimulated RAW macrophages. Zymosan * 20 * 10 0 Control Zymosan Naïve cells Apoptotic cells PS enriched cells PC enriched cells PS nmol/106 cells (30 min at 37oC); PC nmol/106 cells (30 min at 37oC); Zymosan mg/ml (1h at 37oC); DHE - 10 µm. Apoptosis in Jurkat cells was induced by anti-fas (250ng/106 cells, 4h at 37oC.)

49 Production of TNFa by zymosan-stimulated (0.25mg/ml) RAW macrophages. Effect of apoptotic cells on formation of NO in LPS-induced zymosan-stimulated RAW macrophages Zymosan 40 * Control Zymosan Control cells Apoptotic cells TNF-, ng/ml DAF(+) cells, % of total Zymosan Control Zymosan Control cells Apoptotic cells Zymosan mg/ml (1h at 37oC). Apoptosis in Jurkat cells was induced by anti-fas (250ng/106 cells, 4h at 37oC.)

50 Carbon Nanotubes in Interstitial Space Collagen CNT Elastin TEM of carbon nanotubes in interstitial space. Micrograph shows carbon nanotubes intermixed with normal connective tissue matrix of the lungs.

51 Addressing occupational impact Exposure routes Exposure routes Exposure Exposure Characterization Characterization Education Dose Dose Risk Health Effects Health Effects Control Knowledge Level Poor Toxicity Reduced risk/impact Reduced risk/impact Good

52 Pharyngeal Aspiration of CNT in Mice Caused: o Rapid development of granulomatous bronchointerstitial pneumonia o Inflammation evolving with time from neutrophilic to granulomatous o Morphologic alterations localized to the interstitial of the bronchiolar walls, alveolar ducts and adjacent alveoli o Hypocellular eosinophilic material consistent with fibrous connective tissue observed within granulomas 20 microns 20 microns 1 day post exposure 40 g/mouse CNT 28 days post exposure 40 g/mouse CNT

53 SWCNT 330G SWCNT+DFO EPR spectra of partially-purified SWCNT (0.5 mg/ml, 2.5wt% iron) manufactured by highpressure CO conversion (HiPco ) technology as compared to purified SWCNT additionally treated with an iron chelator, deferoxamine (DFO). Note that partially-purified SWCNT displayed a broad signal with g value 2.0 and half-width of 640G, the signal was not detectable in purified DFO-treated SWCNT.

54 Levels of GSH in BAL of C57BL/6J mice 7 days after exposure to partially purified SWCNT. GSH, nmol/ml 0.2 * Control SWCNT Partially purified SWCNT (2.0 mg/kg b.w.). *p<0.05 vs. control

55 Single walled carbon nanotubes (SWCNT) aggregate and form ropes, bundles, and bird s nests

56 Phosphatidylserine (PS) as an eat -me signal in phagocytosis eat-me of apoptotic cells Damage or ProPro-apoptotic signals PS PS PS PS PS PS X?

57 Vitamin E Level in BEAS-2B Cells Following Exposure to SWCNT Vitamin E, pmol/mg protein *p < 0.05 vs control cells; ** p < 0.05 vs cells exposed to 0.06 mg/ml SWCNT; * * * ** SWCNT concentration, mg/ml

58 Electron Microscopy of BEAS-2B Cells Exposed to SWCNT Control SWCNT, 18h Original magnification x200 Original magnification x 4500 SWCNT, 0.24 mg/ml