The Cellular and Molecular Toxicity of Low Solubility Nanoparticles Prof Vicki Stone Centre for Health and Environment Napier University, Edinburgh http://www.lifesciences.napier.ac.uk/research/che1.htm Safety of nanomaterials Interdisciplinary Research Centre http://www.snirc.org/
PM 10 Carbon based particles Traffic and industrial Many ultrafine Transition metals *** *** Sulphates/Nitrates Traffic/photochemical Mainly ultrafine 500nm Wind blown dust Mainly coarse Biological components Spores Pollen Mainly fine and course
Lung Defence Mechanisms mucus cilia Airway Alveolus Antioxidants Particles phagocytosed and cleared Gas exchange Particles trapped and cleared on mucociliary escalator Macrophage Blood vessel Particles
The Ultrafine Particle Hypothesis Particles Blood vessel Release of mediators by macrophages and epithelial cells INFLAMMATION Impaired movement Particles cross the epithelial barrier Seaton al. 1995 The Lancet 345: 176-178
Ultrafine particle toxicology Neutrophils in bronchoalveolar lavage fluid X 10 5 100 80 60 40 20 21 nm TiO 2 250 nmtio 2 Ferin et al 1992 Am.J.Respir.Cell.Mol.Biol. 6: 535-542 0 0 10 20 30 40 50 60 70 Time from start of exposure (weeks)
Accidental versus engineered nanoparticles Nanoparticles Source Exposure Toxicology Accidental Fossil fuel combustion E.g. Traffic, cooking. Low exposure to everyone Lots e.g. diesel, CB, welding fume etc Purposeful Bulk use of nanoparticles in industry e.g. carbon black Nanotechnology High exposure to workers followed by low exposure to everyone Virtually none Adapted from Ken Donaldson
Model nanoparticles 14 nm 254 m 2 /g 535nm CB 100nm 202nm 260 nm 8 m 2 /g 64nm 100nm Carbon black 500nm Polystyrene beads Low solubility, low toxicity materials.
Factors involved in NP toxicity 1. SIZE 10μm 1 μm 0.1μm Cilia 0.25μm diameter. 1.0 μm 0.1μm Bronchial epithelium
Factors involved in NP toxicity 2. PARTICLE NUMBER There are thousands more ultrafine particles / mg of dust than larger, respirable particles. 3. SURFACE AREA Particle surface area / mg is much greater for ultrafine particles than for larger, respirable particles. Fine carbon black 7.9 m 2 /g Ultrafine carbon black 253.9 m 2 /g
Particle surface area and surface reactivity Duffin et al. (2002) Ann.Occ.Hyg.46;242-245 Neutrophils in BAL (x10 6 ) 10 5 Quartz 0 Relatively low toxicity particles 0 200 400 600 800 1000 Surface Area instilled (cm 2 ) Low toxicity low solubility particles straight line relationship between SA and neutrophils. Highly pathogenic particles highly reactive surface (eg quartz). do not sit on same line as low toxicity particles.
Nanoparticles, ROS and oxidative stress.
Reactive oxygen species production by carbon particles Supercoiled band intensity x 10 3 12 10 8 260 nm CB 14 nm CB 6 4 * * 2 0 1 5 10 Particle dose / 290 μl plasmid Stone et al. 1998 Toxicol In Vitro 12: 649-659.
Reactive oxygen species produced by nanoparticles DCF Fluorescence Intensity 150 100 50 14 nm CB *** *** ** 260 nm CB 0 0.5 1.0 1.5 2.0 2.5 Particle dose (mg/ml) DCF Fluorescence Intensity 55 45 35 25 15 5-5 *** 64 nm 202 nm 535 nm Polystyrene particle diameter Wilson et al. 2002 TAP 184: 172-179. Brown et al. 2001 TAP 175: 191-199.
Effect of carbon particles on intracellular antioxidant content of A549 cells 120 110 100 GSH (% of control) 90 80 70 60 50 40 0.78 μg/mm 2 * 0 2 4 6 8 Time (hours) 14 nm CB 260 nm CB Control Stone et al. 1998 TIV 12: 649-659.
Effects of Nanoparticles and PM 10 on Intracellular Signalling Pathways
Effect of particles on Ca 2+ signalling in human MonoMac 6 cells [Ca 2+ ] c 160 140 120 100 80 60 40 20 0 Control ** 14 nm CB 260 nm CB Stone et al. 2000 Eur Resp J 15: 297-303. 2000 sec
The response to thapsigargin in human MM6 cells in the presence of CB and verapamil 260 nm CB 14 nm CB Ca 2+ Ca 2+ THAPSIGARGIN *** Ca 2+ VERAPAMIL Control Particles Particles + verapamil 0 200 400 600 800 1000 1200 [Ca 2+ ] c nm after treatment with thapsigargin Stone et al. 2000 Eur Resp J 15: 297-303. 2000 sec
The effect of polystyrene nanoparticles on calcium signalling in macrophages Polystyrene bead diameter 535 nm 202 nm 64 nm Control 0 100 200 300 400 500 600 700 800 900 [Ca 2+ ]c nm after treatment with thapsigargin 1500 sec Stone et al. 2000 Inhal Tox 12 (suppl 3): 345-351. ***
Calcium Signalling Inhibitors Calcium channel Ca 2+ Endoplasmic reticulum [mm] [nm] BAPTACa 2+ Ca 2+ Ca chellator W-7 Calmodulin Signalling Verapamil Ca 2+ channel blocker
The role of Ca 2+ in the induction of TNFα expression by carbon nanoparticles TNFα pg/ml 250 200 150 100 50 *** Rat alveolar macrophages 4 hour treatments 0 Control 14nm CB 260nm CB 14nm CB Verap Verap 14nm CB BAPTA BAPTA Brown et al. 2004 AJP 286; L344-L353
The role of Ca 2+ in the induction of TNFα mrna expression by CB nanoparticles GAPDH TNFα TNFα mrna (% GAPDH) 30 25 20 15 10 Control 14nm CB 5 0 *** Control 14nm CB 260nm CB 14 CB Verap 260nm14nm CB CB Verap Verap 14 CB BAPTA BAPTA ** Verap 14nm CB BAPTA LPS BAPTA Brown et al. 2004 AJP 286; L344-L353
The effect of antioxidants on CB induced TNFα expression TNFα pg/ml 250 200 150 100 50 0 ** Control 14nm CB 14nm CB Nacystelin Nacystelin TNFα mrna (% GAPDH) 30 25 20 15 10 5 *** *** 0 Control14nm 14nm Nacystelin CB CB Nacystelin Brown et al. 2004 AJP 286; L344-L353
Particle induced TNFα expression role of calcium and oxidants TNFα pg/ml 200 150 ** Human monocyte derived macrophages 4 hour treatments 100 50 0 Control 14 nm CB CB CB CB CB + verap + BAPTA + W-7 + Trolox Brown et al. 2004 AJP 286; L344-L353
Activation of NFκB and promoter binding Degraded IκB NFκB P IκB P IκB P U DNA NFκB Phosphorylation NFκB mrna NFκB Promoter TRANSCRIPTION Nuclear membrane
Immunofluorescent staining of the p65 subunit of NFκB in human monocyte/macrophages 14 nm CB Control Brown et al. 2004 AJP 286; L344-L353
Nuclear localisation of the p65 subunit of NFκB in human monocytes Nuclear p65 intensity (% control) 160 * 120 80 40 Control CB CB CB CB CB CB + verap + W-7 + BAPTA + Nac + Trolox Brown et al. 2004 AJP 286; L344-L353
Nanoparticles, calcium and oxidants Ultrafine, nanoparticles Macrophages Oxidative Calcium stress signalling Activation of NF-κB Production of pro-inflammatory proteins
J774 macrophages exposed to 14nm CB CONTROL Wilson et al., manuscript in prep 14 nm CB
Measurement of cytoskeletal stiffness using ferromagnetic particles Step twist Θ Twisting field B TW Magnetic particles within phagosomes 6 Hz Macrophage Fluxgate sensor Magnetic twisting device Aligned ferromagnetic microparticles ingested by macrophages, Detection by an array of magnetic fluxgate sensors (Förster device).
Impact of particles on the macrophage phagosome transport Relaxation b5 (drug/control) 1.6 * 1.4 * 1.2 1 0.8 0.6 0.4 0.2 Relaxation b5 (drug/control) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 * $ $ $ 0 14nm CB 320 μg/million cells EC90 EC90 DEP UD 0 EC90 Verap BAPTA W7 Moeller et al., 2003 Manuscript submitted
Effects of nanoparticles and PM 10 on migration and phagocytosis
Chemotaxis Chamber Macrophages (J774.2) Filter (5 µm pore) Direction of migration Chemoattractant Actual Filter
Chemotaxis of J774 macrophages in response to epithelial cell conditioned medium Chemotaxis % of Control Particles & 200 Cells Particles Only * 150 100 50 ZAS 250 nm TiO 2 Barlow et al. 2005 Particle Fibre Toxicol 2:11. 20 nm TiO 2 260 nm CB 14 nm CB
Chemotactic response of J774 macrophages to epithelial cell conditioned medium % of Control 200 180 160 140 120 100 ZAS >30 10-30 5-10 <5 Molecular Weight Fraction of Conditioned Medium kda Barlow et al. 2005 Particle Fibre Toxicol 2:11.
Chemotactic response of J774 macrophages to serum treated with particles 210 190 *** % of Negative Control 170 150 130 110 *** 90 ZAS 1 1.5 5 1 1.5 5 1 1.5 5 1 1.5 5 mg/ml 14nm CB 260nm CB 21nm TiO 2 250nm TiO 2 Barlow et al. 2005 Toxicol Lett 155:397-401.
Nanoparticle uptake by macrophages
Phagocytosis Barlow et al. 2006 Submitted.
Effect of TiO 2 and CB on phagocytosis % of cells that phagocytose >2 beads 8h exposure Renwick et al. 2001 Toxicol Applied Pharmacol. 172: 119-127
Non-phagocytic cells Renwick et al. 2001 Toxicol Applied Pharmacol. 172: 119-127
Cambridge multiwalled carbon nanotube
Nottingham Multiwalled carbon nanotubes
Control Carbon nanotubes Cambridge Commercial Nottingham
Effects of nanotubes on cell viability Apoptosis Necrosis 40 30 % Total Cells 20 10 40 35 30 25 20 15 10 5 % total cells 00 Control Control E21 3-01 PR19 1-03 9-01 UfCB LFA Triton TNF Nanotubes/fibres Nanotube Treatment 15.625ug/ml Nanotubes CB LFA Triton TNFα Necrotic Apoptotic 16μg/ml 40 30 % Total Cells 20 10 40 35 30 25 20 15 10 5 % total cells 00 Control Control E21 3-01 PR19 1-03 9-01 UfCB LFA Triton TNF Nanotubes/fibres Nanotube treatment 31.25ug/ml nanotubes Necrotic Apoptotic CB LFA Triton TNFα 31μg/ml
Effect of nanotubes and nanofibres on phagocytosis 90 Phagocytosis (% Total) 80 70 60 50 40 30 20 10 15.625ug/m l 31.25ug/m l 62.5ug/m l 0 Medium E21 PR19 Sample 9-01 Sample 3-01 Sample 1-03 Uf CB LFA Treatment All treatments induced a significant (p<0.05) inhibition
Summary Low toxicity, low solubility NP: Inflammation is related to surface area dose. Generate ROS leading to oxidative stress in epithelial and macrophage cells. Induce Ca 2+ influx in macrophages in vitro resulting in; activation of NFκB and AP1 up-regulation of pro-inflammatory cytokine expression Inhibition of phagosome transport
Summary Low toxicity, low solubility NP: Stimulate production of chemotaxins by epithelial cells. Rapidly taken up into macrophages. Inhibit subsequent phagocytosis of larger particles MWCNT: Morphology determines uptake by macrophages. Inhibit subsequent phagocytosis of E.Coli.
Napier University Dr David Brown Dr Martin Wilson Marieke Tuinman Dr Suzanne Patterson Janis Vamvakopolous Dr Peter Barlow Martin Clift University of Edinburgh Prof Ken Donaldson University of Cambridge Prof Alan Windle Dr Ian Kinloch UIF Dusseldorf Dr Rodger Duffin Dr Roel Schins Dr Paul Borm Dr Markus Denhart GSF Munich Dr Winfried Moeller Nottingham University Dan Walters THE COLT FOUNDATION