Valerian E. Kagan - ianano.org · Valerian E. Kagan Macrophage Response ... staining (shown for clarity in green pseudo-color). ... during incubation of xanthine oxidase/xanthine

Valerian E. KaganMacrophage 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. KaganMacrophage 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,

Raw single walled carbon nanotubematerial.

Nanotubes

Nanoropes

Catalyst particles

Single Walled Carbon Nanotubes

Courtesy of Andrew Maynard

‘Tangles’of nanotubes and nanoropes

Handling nanotube material

Raw SWCNT material

Metal Components of SWCNTMetal Components of SWCNT

Component (g/gram) Component (g/gram)

Aluminum 233 Molybdenum 1070Calcium 164 Sodium 8750Cadmium 23.4 Nickel 8,750Chromium 13.1 Palladium 28Copper 2,530 Selenium <2.001Iron 239, 000 Titanium 6.92

Zinc 85.9 Original magnification x 145,000

Transmission ElectronTransmission ElectronMicroscopy of SWCNTMicroscopy of SWCNT

EPR spectra of partially-purified SWCNTmanufactured 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 SWCNTmanufactured by high-pressure CO conversion(HiPco™) technology as compared to purified

SWCNT additionally treated with an iron chelator,deferoxamine (DFO).

330GNote that partially-purified SWCNT displayed a broad

signal with g value 2.0 and half-width of 640G, the signalwas not detectable in purified DFO-treated SWCNT.

Partially-PurifiedSWCNT

PurifiedSWCNT+DFO

Raw Samples ofCarbon

NanotubesContain

Redox-ActiveIron

Raw Samples ofCarbon

NanotubesContain

Redox-ActiveIron

O2Fe2+

Fe3+

O2

ProteinsLipids

DNA

NoEnzymatic

ControlHO (RO)

Fe2+

Fe3+

H2O2

++ Fe3+

Fe2+

SOD

CatalasePeroxidase

Iron-Catalyzed Decomposition of H2O2

Forms A Potent Oxidant –Hydroxyl Radical

OH.

H2O2 Fe2+

Fe3+

++ Ascorbate

Asc. ++ HOH

RAW264.7 macrophages

10 G

Ascorbate

RAW264.7 +Ascorbate+partially purified

SWCNT, 5 min

RAW264.7+Ascorbate

Conditions: Zymosan (2.5 mg/ml)-stimulated RAW264.7macrophages (20x106 cells/ml); partially purified SWCNT (2.5 wt%of iron, 0.12 mg/ml)

20 G

EPR spectra of ascorbate radicals generated bypartially purified SWCNT.

EPR spectra of ascorbate radicals generated bypartially purified SWCNT.

Model system

Ascorbate

Ascorbate+partially purifiedSWCNT,

5 min

Ascorbate+partiallypurified SWCNT +

DFO, 5 min

Ascorbate+partiallypurified SWCNT+DFO

Conditions: Ascorbate (10 mM) in PBS (pH 7.4); partiallypurified 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.

CarbonNanotubes

DirectlyDamage

Broncho-EpithelialCells

CarbonNanotubes

DirectlyDamage

Broncho-EpithelialCells

Particles, Nanotubes

Epithelial Cell

SWCNT

SWCNT

Engulfment of SWCNT by BEASEngulfment of SWCNT by BEAS--2B Cells2B Cells

x 11,600 x 87,000

x 29,000

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

Fe2+ + H2O2Fe3+ + HO- + .OH

N

HM e

M e

O

+ OH.

-

+

.N

OH

M e

M e

O

H

Fenton ReactionFenton Reaction

DMPO Adduct FormationDMPO Adduct Formation

ESR spectra ofDMPO adducts of

free radicals formedby partially purified

SWCNT in thepresence of

BEAS-2B cells.



SWCNT in thepresence 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% ofiron, 0.12 mg/ml) H2O2 (1 mM); catalase (20 U/ml);DTPA (0.2 mM).

ESR conditions: microwave power, 20 mW; modulationamplitude, 1.0 G; time constant, 1.3 sec; conversion time, 0.6 sec.

TotalAntioxidantReserve in

BEAS-2B CellsFollowing

Exposure toSWCNT

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;

***

******

0.00SWCNT concentration, mg/ml

Per

oxyl

radi

cals

scav

enge

dby

cells

hom

ogen

ates

,nm

ol/m

gpr

otei

n

0

20

40

60

80

100

120

140

0.06 0.12 0.24

*

***

******

GSH and Protein Thiols in BEAS-2B CellsFollowing 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;


Thi

ols,

nmol

/mg

prot

ein

0

10

20

30

40

50

60

0.06 0.12 0.24

*

Protein Thiols

0

10

20

30

40

50

60

70

80


Exposure to SWCNT Induced Apoptotic Cell Death

BEAS-2B cells were stained with TUNEL reagent. Apoptotic cells exhibited yellow-greenfluorescence, while normal cells counter-stained with propidium iodide fluoresced red.Original magnification x 40.

SWCNT (0.24 mg/ml)Control

Courtesy of M. Luster

Release of Oxidants andProteolytic Enzymes

PMN Recruitment inthe Lung

Attachment to andSpreading on

Extracellular Matrix

Arachidonic AcidMetabolism

Chemokine andCytokine Release

Activation of VascularPMNs

Upregulation ofAdhesion Molecules

Reorganization ofCytoskeleton

PMN Motility & Attachment

Pulmonary Toxicants(particles, fibers,

microbes)Endotoxin or Metals

Lung Injury, Cell Proliferationand Release of Fibrogenic

Factors

Direct Toxic

Effects

Amplificationof Pulmonary

Toxicity

Macrophages

Epithelial cells

Aggregates of CarbonNanotubes

Are Recognized andSequestered byMacrophages

Aggregates of CarbonNanotubes

Are Recognized andSequestered byMacrophages

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

HistopathologyPulmonary injuryLung functionsCollagen morphometryOxidative stressCytokines

3 7 28 60

C57BL/6

Single Walled Carbon Nanotube Toxicity - purified SWCNT

3 Day

1 Day

Histopathology of lung of C57BL/6J mice after pharyngealaspiration of partially purified SWCNT

Histopathology of lung of C57BL/6J mice after pharyngealaspiration of partially purified SWCNT

7 Day

Control

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

1 3 7

Days after exposure

PMNs

0

100

200

300

400

500

600

Cel

lNum

ber,

x103

Days after exposure

1 3 7

Lymphocytes

0

5

10

15

20

25

30

35

Cel

lNum

ber,

x103

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

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

Cel

lNum

ber,

x106

Total Cell Counts

0

0.2

0.4

0.6

0.8

1 3 7

Days after exposure

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

0

100

200

300

400

500

600

700

1 3 7 28 60

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

Partially purified SWCNT (40 g per mouse)

Days Post Exposure

SWCNT

Control

Cel

lNum

ber,

x106

Damage orDamage or

ProPro--apoptoticapoptotic

signalssignals

PS

PS

PS

PS

PS PSX

SWCNT

Non-AggregatedCarbon Nanotubes

Do NOT SignificantlyDamage

Macrophages

Non-AggregatedCarbon Nanotubes

Do NOT SignificantlyDamage

Macrophages

DoDo

CarbonCarbon

NanotubesNanotubes

InduceInduce

ApoptosisApoptosis

inin

Macrophages?Macrophages?

Carbon NanotubesCarbon Nanotubes

Stress and Damage ofMacrophages

Stress and Damage ofStress and Damage ofMacrophagesMacrophages

??

SWCNT, 6h

AAControl

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

Fluorescent micrographs of RAW 264.7 macrophagesincubated 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).

Apoptosis induced by partially purifiedSWCNT in RAW 264.7 macrophages .Apoptosis induced by partially purifiedSWCNT in RAW 264.7 macrophages .

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

0

50

100

150

200

250

0 10 20 30Time, h

Cas

pase

3ac

tivi

ty,

AU

/mg

prot

ein

Zymosan

SWCNT

6 hSWCNT

0

50

100

4 hControl

Apo

ptos

is,%

C

SWCNTSWCNT

6 h

x3,800

BSWCNT

SWCNT

SWCNT

SWCNT

1 h

x3,800

Electron micrographs illustrating effects of partiallypurified SWCNT exposure on RAW 264.7

macrophages.

Electron micrographs illustrating effects of partiallypurified SWCNT exposure on RAW 264.7

macrophages.

AControl

x3,800

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

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

0

10

20

30

40

50

60

Control Zymosan0.25 mg/ml

0.1 mg/ml

DH

E-p

osit

ive

cells

,%of

tota

l

0.1 mg/ml0.5 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 stimulatedby zymosan or SWCNT (for 30 min at 37oC). * - p<0.05 vs. control cells

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

0

5

10

15

20

DA

F2-

posi

tive

cells

,%of

tota

l

LPS-

LPS+

Control Zymosan0.25 mg/ml

0.1 mg/ml 0.1 mg/ml0.5 mg/ml 0.5 mg/mlSWCNT (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

NecrosisApoptosis

CellDeath

H2O2Fe2+

Fe3+

OH•

Reductionof iron

O2.

O2.

O2.

O2.

NADPH oxidaseActivation

Fe-SWCNT

Fe-SWCNT

Oxidative Stress

Fe-SWCNT

ZymosanZymosan

Superoxide-DMPO Adduct

RAW 264.7macrophages pluspartially purified

SWCNT

EPR spectra of DMPOradical adducts generated

during incubation of xanthineoxidase/xanthine and

zymosan-stimulated RAW264.7 macrophages by

partially purified SWCNT inthe absence or presence ofpartially purified SWCNT.

EPR spectra of DMPOradical adducts generated

during incubation of xanthineoxidase/xanthine and

zymosan-stimulated RAW264.7 macrophages by

partially purified SWCNT inthe absence or presence ofpartially purified SWCNT.

15 G

RAW 264.7macrophages

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, 20mW; modulation amplitude, 1.0 G; time constant, 1.3 sec; conversion time, 0.6 sec.

Hydroxyl Radical-DMPO Adduct

15 G

Control

Zymosan

Zymosan+SWCNT



SWCNT in thepresence of RAW

264.7 macrophages



SWCNT in thepresence of RAW

264.7 macrophages

Hydroxyl Radical-DMPO Adduct

CH2H2C

CH2H2C

CH2

HCCH2 H2C

CCH2

H2CCH2

H2C

HC

HC CH

CHCH

CCH2

H2CCH2

H2CCH2

H2CCH2

H2CCH2

H2CCH2

H2CCH2

H2CCH2

H3C

HC

CH2

CH

O

OCH2

OO

HCH2C

OPO

OOCH2 CH COO

NH3

OxidationCH2

H2CCH2

H2CCH2

HCCH H2C

CCH2

H2CCH2

H2C

CH

HC CH

CHCH

CCH2

H2CCH2

H2CCH2

H2CCH2

H2CCH2

H2CCH2

H2CCH2

H2CCH2

H3C

HC

CH2

CH

O

OCH2

OO

HCH2C

OPO

OOCH2 CH COO

NH3

HOO

Cells

ROS GenerationPhospholipid Oxidation

Apoptosis1

NL

FFAPE

CL

PC

SPH

LPCPS

PI Origin

22D-HPTLC CH2

H2CCH2

H2CCH2

HCCH H2C

CCH2

H2CCH2

H2C

CH

HC CH

CHCH

CCH2

H2CCH2

H2CCH2

H2CCH2

H2CCH2

H2CCH2

H2CCH2

H2CCH2

H3C

HC

CH2

CH

O

OCH2

OO

HCH2C

OPO

OOCH2 CH COO

NH3

HOO

CC H2

H2CC H2

H2CC H2

H2CC H2

H2CC H2

H2CC H2

H2CC H2

H2CC H2

H3C

O

OC H2

OH

HCH2C

OPO

OOC H2 C H C OO

N H3

CH2H2C

CH2H2C

CH2

HCCH H2C

HOOCCH2

H2CCH2

H2C

CH

HC CH

CHCH

HC

CH2

CH

HOO+Phospholipase A2

FA-OOHLyso-PLPL-OOH

Phosholipid Spot+ PLA2

H O

C

O H

H3 C O

O

N

H O OO

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

0 2 4 6 8 10Resorufin or

9SHpODE, pmol

Flu

ores

cenc

ea.

u. Resorufin

9SHpODE

0

100

200

300

0 2 4 6Time, min

Flu

ores

cenc

e,a.

u. Resorufin

Fluorescence

HPLC

Partially purified SWCNT induce lipid peroxidation inRAW 264.7 macrophages .

Partially purified SWCNT induce lipid peroxidation inRAW 264.7 macrophages .

Stimulation of macrophages with zymosan (0.25 mg/ml) cause a slight increase in lipid peroxidationm furtherenhanced 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

*

0

1.0

2.0

3.0

Lip

idhy

drop

erox

ides

,nm

ol/1

06ce

lls

Effect of partially purified SWCNT exposure on GSH contentin RAW 264.7 macrophages.

Effect of partially purified SWCNT exposure on GSH contentin RAW 264.7 macrophages.

0

10

20

30

40

50G

SH,n

mol

/mg

prot

ein

Zymosan Zymosan+SWCNT

Control

0

20

40

60

80

100

120

Zymosan

Cyt

okin

esre

leas

ed,

%of

zym

osan

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

macrophages.

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

macrophages.

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

TGF IL-10

SWCNT

Anti-inflammatoryIL-1 TNF

Pro-inflammatory

RecognitionRecognitionandand

digestiondigestionofof

CellsCells

B16 melanoma cellsB16 melanoma cells(green)(green)

andand dendriticdendritic cellscells

Can MacrophagesBe Forced

to Recognize andDigest

CarbonNanotubes

Can MacrophagesBe Forced

to Recognize andDigest

CarbonNanotubes

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

of apoptotic cells

PhosphatidylserinePhosphatidylserine (PS) as an(PS) as an““eateat--meme””signal insignal in phagocytosisphagocytosis

of apoptotic cellsof apoptotic cellsDamage orDamage or


signalssignals

PS

PS

PS

PS

PS PSX?

SWCNT

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

RAW 264.7 macrophages effectively phagocytose PS-containingliposomes 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 fluorescentlylabeled 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 (greenfluorescence) 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-containingliposomesPS-containing

liposomes

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

RAW 264.7 macrophages effectivelyphagocytose PS-labeled SWCNT but

not PC labeled SWCNT.

RAW 264.7 macrophages effectivelyphagocytose PS-labeled SWCNT but

not PC labeled SWCNT.

PS-labeled SWCNT PC-labeled SWCNT

Kagan’s Lab:

A. ArroyoN. BelikovaG. BorisenkoJ. JiangK. KawaiV. KiniS.-X. LiuT. MatsuuraA. OsipovA. PotapovichB. SerinkanV. TyurinY. TyurinaQ. Zhao

Kagan’s Lab:

A. ArroyoN. BelikovaG. BorisenkoJ. JiangK. KawaiV. KiniS.-X. LiuT. MatsuuraA. OsipovA. PotapovichB. SerinkanV. TyurinY. TyurinaQ. 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)

Fe-SWCNT

Fe-SWCNT

NecrosisApoptosis

CellDeath

Production ofpro-inflammatory

cytokines

Recruitmentof new M

Oxidative Stress

H2O2Fe2+

Fe3+

OH•

Reductionof iron

O2.

O2.

O2.

O2.

NADPH oxidaseActivation

Fe-SWCNT

Inflammatory Response

MicrobialInfection

PS oxidation/externalization

Apoptosis

PS

PS

Apoptoticcell

Fe-SWCNT

Production ofanti-inflammatory

cytokines

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

Effect of apoptotic cells and PS on superoxide generation inzymosan-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

0

10

20

30

40

Control

**

Zymosan

Supe

roxi

depr

oduc

tion

,et

hidi

umpo

siti

vece

lls,%

ofto

tal

PC enrichedcells

PS enrichedcells

Apoptotic cellsNaïve cells

Production of TNFa byzymosan-stimulated

(0.25mg/ml) RAW 264.7macrophages.

Production of TNFa byzymosan-stimulated

(0.25mg/ml) RAW 264.7macrophages.

0

10

20

30

40

Control

Zymosan

TN

F-

,ng/

ml

Zymosan Controlcells

Apoptoticcells

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 onformation of NO•in LPS-inducedzymosan-stimulated RAW 264.7

macrophages.

Effect of apoptotic cells onformation of NO•in LPS-inducedzymosan-stimulated RAW 264.7

macrophages.

0

10

20

30

40

50Zymosan

DA

F(+

)cel

ls,%

ofto

tal

*

Control Zymosan Controlcells

Apoptoticcells

Carbon Nanotubes in Interstitial Space

Collagen

ElastinCNT

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

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

1 day post exposure1 day post exposure 4040g/mouse CNTg/mouse CNT

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

and adjacent alveolio Hypocellular eosinophilic material consistent with fibrous connective tissue observed within

granulomas

20 microns

28 days post exposure28 days post exposure 4040g/mouse CNTg/mouse CNT

20 microns

Pharyngeal Aspiration of CNT in Mice Caused:

SWCNT+DFO

EPR spectra of partially-purifiedSWCNT (0.5 mg/ml, 2.5wt%iron) manufactured by high-

pressure CO conversion(HiPco™) technology as

compared to purified SWCNTadditionally treated with an ironchelator, deferoxamine (DFO).

EPR spectra of partially-purifiedSWCNT (0.5 mg/ml, 2.5wt%iron) manufactured by high-

pressure CO conversion(HiPco™) technology as

compared to purified SWCNTadditionally treated with an ironchelator, deferoxamine (DFO).

330G

SWCNT

Note that partially-purified SWCNT displayed a broad signal with g value 2.0 and half-width of 640G, thesignal was not detectable in purified DFO-treated SWCNT.

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

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

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

0

0.1

0.2

Control SWCNT

*G

SH,n

mol

/ml

Single walled carbonnanotubes

(SWCNT) aggregate andform

ropes, bundles, andbird’s nests

Single walled carbonnanotubes

(SWCNT) aggregate andform

ropes, bundles, andbird’s nests

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

of apoptotic cells

PhosphatidylserinePhosphatidylserine (PS) as an(PS) as an““eateat--meme””signal insignal in phagocytosisphagocytosis

of apoptotic cellsof apoptotic cellsDamage orDamage or


signalssignals

PS

PS

PS

PS

PS PSX?


Vit

amin

E,p

mol

/mg

prot

ein

Vitamin E Level in BEAS-2B Cells FollowingExposure to SWCNT

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

0

10

20

30

40

50

60

70

0.06 0.12 0.24**

** *

Electron Microscopy of BEASElectron Microscopy of BEAS--2B Cells2B CellsExposed to SWCNTExposed to SWCNT

SWCNT, 0.24 mg/ml

SWCNT, 18h

Original magnification x 4500

Control

Original magnification x200

Documents

Valerian E. Kagan - ianano.org · Valerian E. Kagan Macrophage Response ... staining (shown for clarity in green pseudo-color). ... during incubation of xanthine oxidase/xanthine