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

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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.

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SLIDE 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,

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,

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SLIDE 2

Raw single walled carbon nanotube material. Nanotubes Nanoropes Catalyst particles

Single Walled Carbon Nanotubes

Courtesy of Andrew Maynard ‘Tangles’ of nanotubes and nanoropes

Handling nanotube material

Raw SWCNT material

slide-3
SLIDE 3

Metal Components of SWCNT Metal Components 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

Transmission Electron Transmission Electron Microscopy of SWCNT Microscopy of SWCNT

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). 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).

330G

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. Partially-Purified SWCNT Purified SWCNT+DFO

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SLIDE 4

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

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SLIDE 5

O2

Fe2+ Fe3+

O2

Proteins Lipids DNA

No

Enzymatic Control

HO (RO)

Fe2+ Fe3+

H2O2

+ +

Fe3+ Fe2+

SOD

Catalase Peroxidase

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SLIDE 6

Iron-Catalyzed Decomposition of H2O2 Forms A Potent Oxidant – Hydroxyl Radical

OH. H2O2

Fe2+ Fe3+

+ + Ascorbate Asc. + + HOH

slide-7
SLIDE 7

RAW264.7 macrophages

10 G Ascorbate RAW264.7 + Ascorbate+partially purified SWCNT, 5 min RAW264.7+ Ascorbate Conditions: Zymosan (2.5 mg/ml)-stimulated RAW264.7 macrophages (20x106 cells/ml); partially purified SWCNT (2.5 wt%

  • f iron, 0.12 mg/ml)

20 G

EPR spectra of ascorbate radicals generated by partially purified SWCNT. EPR spectra of ascorbate radicals generated by partially purified SWCNT.

Model system

Ascorbate Ascorbate+partially purified SWCNT, 5 min Ascorbate+partially purified SWCNT + DFO, 5 min Ascorbate+partially purified SWCNT+DFO 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); EPR conditions: microwave power, 20 mW; modulation amplitude, 1.0 G; time constant, 1.3 sec; conversion time, 0.6 sec.

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SLIDE 8

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

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SLIDE 9

Particles, Nanotubes

Epithelial Cell

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SLIDE 10

SWCNT

SWCNT

Engulfment of SWCNT by BEAS Engulfment of SWCNT by BEAS-

  • 2B Cells

2B Cells

x 11,600 x 87,000 x 29,000

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SLIDE 11

5,5-Dimethyl-1-Pyrroline-N-Oxide (DMPO)

Fe2+ + H2O2 Fe3+ + HO- + .OH

N H Me Me O

+ OH .

  • +

.

N OH Me Me O H

Fenton Reaction Fenton Reaction DMPO Adduct Formation DMPO Adduct Formation

slide-12
SLIDE 12

ESR spectra of DMPO adducts of free radicals formed by partially purified SWCNT in the presence of BEAS-2B cells. ESR spectra of DMPO adducts of free radicals formed by partially purified SWCNT in the presence of BEAS-2B cells.

20 Gauss

DMPO DMPO+SWCNT DMPO+SWCNT +H2O2 DMPO+SWCNT+ H2O2+Catalase DMPO+SWCNT +DTPA 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). ESR conditions: microwave power, 20 mW; modulation amplitude, 1.0 G; time constant, 1.3 sec; conversion time, 0.6 sec.

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SLIDE 13

Total Antioxidant Reserve in BEAS-2B Cells Following Exposure to SWCNT

0.06 0.12 0.24

*

*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;

* ** * ** ***

0.00 SWCNT concentration, mg/ml

Peroxyl radicals scavenged by cells homogenates, nmol/mg protein

20 40 60 80 100 120 140

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SLIDE 14

0.06 0.12 0.24

* * ** * ** ***

GSH and Protein Thiols in BEAS-2B Cells Following Exposure to SWCNT

GSH

*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;

0.00 SWCNT concentration, mg/ml

Thiols, nmol/mg protein

10 20 30 40 50 60

0.06 0.12 0.24

* Protein Thiols

10 20 30 40 50 60 70 80

0.00 SWCNT concentration, mg/ml

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SLIDE 15

Exposure to SWCNT Induced Apoptotic Cell Death

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. SWCNT (0.24 mg/ml) Control

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SLIDE 16

Courtesy of M. Luster Release of Oxidants and Proteolytic Enzymes PMN Recruitment in the Lung

Attachment to and Spreading on Extracellular Matrix Arachidonic Acid Metabolism Chemokine and Cytokine Release Activation of Vascular PMNs Upregulation of Adhesion Molecules Reorganization of Cytoskeleton PMN Motility & Attachment

ulmonary Toxicants (particles, fibers, microbes)

Endotoxin or Metals

Lung Injury, Cell Proliferation and Release of Fibrogenic Factors

Direct Toxic Effects Amplification

  • f Pulmonary

Toxicity

Macrophages Epithelial cells

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SLIDE 17

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

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SLIDE 18

Day 0 Aspiration with:

CNT (10, 20, 40 g/mouse) UFCB (40 g/mouse) Silica (40 g/mouse & 2.5 mg/mouse)

1 Days post Exposure Histopathology Pulmonary injury Lung functions Collagen morphometry Oxidative stress Cytokines 3 7 28 60

C57BL/6

Single Walled Carbon Nanotube Toxicity - purified SWCNT

slide-19
SLIDE 19

3 Day 1 Day Histopathology of lung of C57BL/6J mice after pharyngeal aspiration of partially purified SWCNT Histopathology of lung of C57BL/6J mice after pharyngeal aspiration of partially purified SWCNT 7 Day Control

Partially purified SWCNT (2.5 wt% of iron, 0.5 mg/kg b. w.).

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SLIDE 20

1 3 7 Days after exposure PMNs 100 200 300 400 500 600 Cell Number, x103 Days after exposure 1 3 7 Lymphocytes 5 10 15 20 25 30 35 Cell Number, x103

Cell differential in BAL fluid of C57BL/6J mice after treatment with partially purified SWCNT. Cell differential in BAL fluid of C57BL/6J mice after treatment with partially purified SWCNT.

Cell Number, x106 Total Cell Counts 0.2 0.4 0.6 0.8 1 3 7 Days after exposure Control SWCNT

Partially purified SWCNT (2.5 wt% of iron, 0.5 mg/kg b.w.)

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SLIDE 21

100 200 300 400 500 600 700 1 3 7 28 60

Macrophages in BAL fluid of C57BL/6J mice after treatment with partially purified SWCNT.

Partially purified SWCNT (40 g per mouse)

Days Post Exposure SWCNT Control Cell Number, x106

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SLIDE 22

Damage or Damage or Pro Pro-

  • apoptotic

apoptotic signals signals

P S P S PS P S P S

PSX

SWCNT

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SLIDE 23

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

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SLIDE 24
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SLIDE 25

Do Do Carbon Carbon Nanotubes Nanotubes Induce Induce Apoptosis Apoptosis in in Macrophages? Macrophages?

Carbon Nanotubes Carbon Nanotubes

Stress and Damage of

Macrophages

Stress and Damage of Stress and Damage of

Macrophages Macrophages

? ?

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SLIDE 26

SWCNT, 6h

A

Control Note nuclear condensation and fragmentation as revealed by Hoechst 33342 staining (shown for clarity in green pseudo-color).

Fluorescent micrographs of RAW 264.7 macrophages incubated in the presence of partially purified SWCNT. Fluorescent micrographs of RAW 264.7 macrophages incubated in the presence of partially purified SWCNT.

Partially purified SWCNT (2.5 wt% of iron, 0.12 mg/ml).

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SLIDE 27

Apoptosis induced by partially purified SWCNT in RAW 264.7 macrophages . Apoptosis induced by partially purified SWCNT in RAW 264.7 macrophages .

Partially purified SWCNT (2.5 wt% of iron, 0.1 mg/ml).

50 100 150 200 250 10 20 30 Time, h Caspase 3 activity, AU/mg protein

Zymosan SWCNT 6 h SWCNT

50 100

4 h Control

Apoptosis, %

slide-28
SLIDE 28

C

SWCNT

SWCNT

6 h

x3,800

B

SWCNT SWCNT

SWCNT SWCNT

1 h

x3,800

Electron micrographs illustrating effects of partially purified SWCNT exposure on RAW 264.7 macrophages. Electron micrographs illustrating effects of partially purified SWCNT exposure on RAW 264.7 macrophages.

A

Control

x3,800

Partially purified SWCNT exposure (2.5 wt% of iron, 0.12 mg/ml)

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SLIDE 29

DHE-positive RAW 264.7 macrophages: zymosan and SWCNT. DHE-positive RAW 264.7 macrophages: zymosan and SWCNT.

10 20 30 40 50 60

Control Zymosan 0.25 mg/ml 0.1 mg/ml

DHE-positive cells, % of total

0.1 mg/ml 0.5 mg/ml 0.5 mg/ml SWCNT (pure) SWCNT

*

Macrophages 0.3 x 106/well) were pre-incubated with DHE (10 mM for 10 min at 37oC). Then RAW 264.7 macrophages were stimulated by zymosan or SWCNT (for 30 min at 37oC). * - p<0.05 vs. control cells

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SLIDE 30

DAF2-positive RAW 264.7 macrophages: zymosan and SWCNT. DAF2-positive RAW 264.7 macrophages: zymosan and SWCNT.

5 10 15 20 DAF2-positive cells, % of total

LPS- LPS+ Control Zymosan 0.25 mg/ml 0.1 mg/ml 0.1 mg/ml 0.5 mg/ml 0.5 mg/ml SWCNT (pure) 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 DAF- 2DA (2 mM for 1 h at 37oC). Then RAW 264.7 macrophages were stimulated by zymosan or SWCNT (for 2h at 37oC). * - p<0.05 vs. control cells

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SLIDE 31

Necrosis Apoptosis

Cell Death

H2O2 Fe2+ Fe3+ OH•

Reduction

  • f iron

O2 .

O2

.

O2

.

O2

.

NADPH oxidase Activation

Fe-SWCNT

Fe-SWCNT

Oxidative Stress

Fe-SWCNT

Zymosan Zymosan

slide-32
SLIDE 32

Superoxide-DMPO Adduct

RAW 264.7 macrophages plus partially purified SWCNT

EPR spectra of DMPO radical adducts generated during incubation of xanthine

  • xidase/xanthine and

zymosan-stimulated RAW 264.7 macrophages by partially purified SWCNT in the absence or presence of partially purified SWCNT. EPR spectra of DMPO radical adducts generated during incubation of xanthine

  • xidase/xanthine and

zymosan-stimulated RAW 264.7 macrophages by partially purified SWCNT in the absence or presence of partially purified SWCNT.

15 G

RAW 264.7 macrophages 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.

Hydroxyl Radical-DMPO Adduct

slide-33
SLIDE 33

15 G Control Zymosan Zymosan+ SWCNT

ESR spectra of DMPO adducts of free radicals formed by partially purified SWCNT in the presence of RAW 264.7 macrophages ESR spectra of DMPO adducts of free radicals formed by partially purified SWCNT in the presence of RAW 264.7 macrophages

Hydroxyl Radical-DMPO Adduct

slide-34
SLIDE 34 CH2 H2C CH2 H2C CH2 HC CH2 H2C C CH2 H2C CH2 H2C HC HC CH CH CH C CH2 H2C CH2 H2C CH2 H2C CH2 H2C CH2 H2C CH2 H2C CH2 H2C CH2 H3C HC C H2 CH O O CH2 O O H C H2C O P O O O CH2 CH COO NH3

Oxidation

CH 2 H 2C CH 2 H 2C CH 2 HC CH H 2C C CH 2 H 2C CH 2 H 2C CH HC CH CH CH C CH 2 H 2C CH 2 H 2C CH 2 H 2C CH 2 H 2C CH 2 H 2C CH 2 H 2C CH 2 H 2C CH 2 H 3C HC C H2 CH O O CH 2 O O H C H 2C O P O O O CH 2 CH COO NH 3

HO O

Cells

ROS Generation Phospholipid Oxidation Apoptosis

1

NL FFA PE CL PC SPH LPC PS PI Origin

2

2D-HPTLC

CH 2 H 2C CH 2 H 2C CH 2 HC CH H 2C C CH 2 H 2C CH 2 H 2C CH HC CH CH CH C CH 2 H 2C CH 2 H 2C CH 2 H 2C CH 2 H 2C CH 2 H 2C CH 2 H 2C CH 2 H 2C CH 2 H 3C HC C H2 CH O O CH 2 O O H C H 2C O P O O O CH 2 CH COO NH 3

HOO

C CH2 H2C CH2 H2C CH2 H2C CH2 H2C CH2 H2C CH2 H2C CH2 H2C CH2 H3C O O C H2 OH H C H2C O P O O O CH2 CH COO NH3 CH 2 H 2C CH 2 H 2C CH 2 H C CH H 2C HO O C CH 2 H 2C CH 2 H 2C CH H C CH CH CH H C C H 2 CH

HO O

+

Phospholipase A2

FA-OOH Lyso-PL PL-OOH

Phosholipid Spot + PLA2

H O C O H H3 C O O N H O O O N

N-Acetyl-3,7-Dihydroxyphenoxazine (Amplex Red) 7-Hydroxy-3H-Phenoxazine-3-one (Resorufine) FA-OOH

MP11 Amplex Red + Microperoxidase 11

200 400 600 800 2 4 6 8 10 Resorufin or 9SHpODE, pmol Fluorescence a.u. Resorufin 9SHpODE

100 200 300 2 4 6

Time, min Fluorescence, a.u.

Resorufin

Fluorescence

HPLC

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SLIDE 35

Partially purified SWCNT induce lipid peroxidation in RAW 264.7 macrophages . Partially purified SWCNT induce lipid peroxidation in RAW 264.7 macrophages .

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

Control Zymosan Zymosan +SWCNT

*

1.0 2.0 3.0

Lipid hydroperoxides, nmol/106 cells

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SLIDE 36

Effect of partially purified SWCNT exposure on GSH content in RAW 264.7 macrophages. Effect of partially purified SWCNT exposure on GSH content in RAW 264.7 macrophages. 10 20 30 40 50

GSH, nmol/mg protein

Zymosan Zymosan +SWCNT Control

slide-37
SLIDE 37

20 40 60 80 100 120 Zymosan Cytokines released, % of zymosan

Production of cytokines by zymosan-stimulated RAW 264.7 macrophages and SWCNT treated RAW 264.7 macrophages. Production of cytokines by zymosan-stimulated RAW 264.7 macrophages and SWCNT treated RAW 264.7 macrophages.

Zymosan- 0.25 mg/ml; SWCNT 0.1 mg/ml.

TGF IL-10 SWCNT Anti-inflammatory IL-1 TNF Pro-inflammatory

slide-38
SLIDE 38
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SLIDE 39

Recognition Recognition and and digestion digestion

  • f
  • f

Cells Cells

B16 melanoma cells B16 melanoma cells (green) (green) and and dendritic dendritic cells cells

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SLIDE 40

Can Macrophages Be Forced to Recognize and Digest Carbon Nanotubes Can Macrophages Be Forced to Recognize and Digest Carbon Nanotubes

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SLIDE 41

Phosphatidylserine (PS) as an “eat-me” signal in phagocytosis

  • f apoptotic cells

Phosphatidylserine Phosphatidylserine (PS) as an (PS) as an “ “eat eat-

  • me

me” ” signal in signal in phagocytosis phagocytosis

  • f apoptotic cells
  • f apoptotic cells

Damage or Damage or Pro Pro-

  • apoptotic

apoptotic signals signals

P S P S PS P S P S

PSX?

SWCNT

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SLIDE 42

RAW 264.7 macrophages effectively phagocytose PS-containing liposomes but not PC-containing liposomes. RAW 264.7 macrophages effectively phagocytose PS-containing liposomes but not PC-containing liposomes.

RAW 264.7 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). PC- containing liposomes were prepared by sonication of a mixture of PC with the addition of 10 mol% of NBD-PC. PC-containing liposomes PS-containing liposomes

slide-43
SLIDE 43

RAW264.7 macrophages (105 cells/ml) were incubated for 6h with fluorescently labeled SWCNT (0.1 mg/ml).

RAW 264.7 macrophages effectively phagocytose PS-labeled SWCNT but not PC labeled SWCNT. RAW 264.7 macrophages effectively phagocytose PS-labeled SWCNT but not PC labeled SWCNT.

PS-labeled SWCNT PC-labeled SWCNT

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SLIDE 44

Kagan’s Lab:

A. Arroyo

  • N. Belikova
  • G. Borisenko
  • J. Jiang
  • K. Kawai
  • V. Kini

S.-X. Liu

  • T. Matsuura
  • A. Osipov
  • A. Potapovich
  • B. Serinkan
  • V. Tyurin
  • Y. Tyurina
  • Q. Zhao

Kagan’s Lab:

A. Arroyo

  • N. Belikova
  • G. Borisenko
  • J. Jiang
  • K. Kawai
  • V. Kini

S.-X. Liu

  • T. Matsuura
  • A. Osipov
  • A. Potapovich
  • B. Serinkan
  • V. Tyurin
  • Y. Tyurina
  • Q. Zhao

Thanks To My Collaborators: Thanks To My Collaborators: Thanks To My Collaborators:

NIOSH:

  • A. Shvedova (Morgantown)
  • V. Castranova (Morgantown)
  • R. Mercer (Morgantown)
  • E. Kisin (Morgantown)
  • A. Maynard (Cincinnati)

NIOSH:

  • A. Shvedova (Morgantown)
  • V. Castranova (Morgantown)
  • R. Mercer (Morgantown)
  • E. Kisin (Morgantown)
  • A. Maynard (Cincinnati)
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SLIDE 45
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SLIDE 46
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SLIDE 47

Fe-SWCNT

Fe-SWCNT

Necrosis Apoptosis

Cell Death

Production of pro-inflammatory cytokines Recruitment

  • f new M

O x i d a t i v e S t r e s s

H2O2 Fe2+ Fe3+ OH•

Reduction

  • f iron

O2 .

O2

.

O2

.

O2

.

NADPH oxidase Activation

Fe-SWCNT

Inflammatory Response

Microbial Infection

PS oxidation/ externalization

A p

  • p

t

  • s

i s

PS PS Apoptotic cell Fe-SWCNT

Production of anti-inflammatory cytokines

slide-48
SLIDE 48

Effect of apoptotic cells and PS on superoxide generation in zymosan-stimulated RAW 264.7 macrophages. Effect of apoptotic cells and PS on superoxide generation in zymosan-stimulated RAW 264.7 macrophages.

PS - 150 nmol/106 cells (30 min at 37oC); PC - 150 nmol/106 cells (30 min at 37oC); Zymosan - 0.25 mg/ml (1h at 37oC); DHE - 10 µM. Apoptosis in Jurkat cells was induced by anti-FAS (250ng/106 cells, 4h at 37oC.)

Zymosan

10 20 30 40

Control

* *

Zymosan

Superoxide production, ethidium positive cells, % of total

PC enriched cells PS enriched cells Apoptotic cells Naïve cells

slide-49
SLIDE 49

Production of TNFa by zymosan-stimulated (0.25mg/ml) RAW 264.7 macrophages. Production of TNFa by zymosan-stimulated (0.25mg/ml) RAW 264.7 macrophages.

10 20 30 40

Control Zymosan

TNF-, ng/ml

Zymosan Control cells Apoptotic cells Zymosan - 0.25 mg/ml (1h at 37oC). Apoptosis in Jurkat cells was induced by anti-FAS (250ng/106 cells, 4h at 37oC.)

Effect of apoptotic cells on formation of NO• in LPS-induced zymosan-stimulated RAW 264.7 macrophages. Effect of apoptotic cells on formation of NO• in LPS-induced zymosan-stimulated RAW 264.7 macrophages.

10 20 30 40 50

Zymosan

DAF(+) cells, % of total *

Control Zymosan Control cells Apoptotic cells

slide-50
SLIDE 50

Carbon Nanotubes in Interstitial Space

Collagen Elastin CNT

TEM of carbon nanotubes in interstitial space. Micrograph shows carbon nanotubes intermixed with normal connective tissue matrix of the lungs.

slide-51
SLIDE 51

Dose Exposure Toxicity Risk Control Reduced risk/impact Exposure routes Health Effects Characterization Dose Exposure Toxicity Risk Control Reduced risk/impact Exposure routes Health Effects Characterization Poor Good

Knowledge Level

Education

Addressing occupational impact

slide-52
SLIDE 52

1 day post exposure 1 day post exposure 40

40  g/mouse CNT g/mouse CNT

  • Rapid development of granulomatous bronchointerstitial pneumonia
  • Inflammation evolving with time from neutrophilic to granulomatous
  • Morphologic alterations localized to the interstitial of the bronchiolar walls, alveolar ducts

and adjacent alveoli

  • Hypocellular eosinophilic material consistent with fibrous connective tissue observed within

granulomas

20 microns

28 days post exposure 28 days post exposure 40

40  g/mouse CNT g/mouse CNT

20 microns

Pharyngeal Aspiration of CNT in Mice Caused:

slide-53
SLIDE 53

SWCNT+DFO

EPR spectra of partially-purified SWCNT (0.5 mg/ml, 2.5wt% iron) manufactured by high- pressure CO conversion (HiPco™) technology as compared to purified SWCNT additionally treated with an iron chelator, deferoxamine (DFO). EPR spectra of partially-purified SWCNT (0.5 mg/ml, 2.5wt% iron) manufactured by high- pressure CO conversion (HiPco™) technology as compared to purified SWCNT additionally treated with an iron chelator, deferoxamine (DFO).

330G SWCNT

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.

slide-54
SLIDE 54

Levels of GSH in BAL of C57BL/6J mice 7 days after exposure to partially purified SWCNT. Levels of GSH in BAL of C57BL/6J mice 7 days after exposure to partially purified SWCNT.

Partially purified SWCNT (2.0 mg/kg b.w.). *p<0.05 vs. control

0.1 0.2 Control SWCNT

*

GSH, nmol/ml

slide-55
SLIDE 55

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

slide-56
SLIDE 56

Phosphatidylserine (PS) as an “eat-me” signal in phagocytosis

  • f apoptotic cells

Phosphatidylserine Phosphatidylserine (PS) as an (PS) as an “ “eat eat-

  • me

me” ” signal in signal in phagocytosis phagocytosis

  • f apoptotic cells
  • f apoptotic cells

Damage or Damage or Pro Pro-

  • apoptotic

apoptotic signals signals

P S P S PS P S P S

PSX?

slide-57
SLIDE 57

0.00 SWCNT concentration, mg/ml

Vitamin E, pmol/mg protein

Vitamin E Level in BEAS-2B Cells Following Exposure to SWCNT

*p < 0.05 vs control cells; ** p < 0.05 vs cells exposed to 0.06 mg/ml SWCNT;

10 20 30 40 50 60 70

0.06 0.12 0.24

** * * *

slide-58
SLIDE 58

Electron Microscopy of BEAS Electron Microscopy of BEAS-

  • 2B Cells

2B Cells Exposed to SWCNT Exposed to SWCNT

SWCNT, 0.24 mg/ml

SWCNT, 18h

Original magnification x 4500

Control

Original magnification x200