Particle_Risk – Six Months Report, Year 1
WP4.4. CardioVascular Model (NIOH – Hakan Wallin)
The ability of NEST particles to activate platelets determined by closure time with a PFA-100 machine. It has earlier been reported that in the scientific literature that diesel exhaust particulate material (DEP) (National Institute of Standards, Reference material 2975) activate platelets with this technique in human blood ex vivo, and in the blood of hamsters that have
been given DEP particles. We have repeatedly tried to reproduce these results with diesel particles but have been unsuccessful. In our attempts we have had many discussions with the group who have published these results, and we have visited them to discuss this. We have also tested NEST particles and they have not produced platelet activation by the technique. We have also tested the feasibility of alternative methods for studying platelet aggregation by nanosized particles. Master student Anne Mette Boisen has studied expression of CD62P and PAC-1 by diesel particles and the CdTe quantum dots in human platelets in an collaboration with Ken Donaldson. The results was presented in a masters thesis in March 2007. (Principal researcher, Master student, Anne Mette Boisen.)
The toxicity, the DNA damaging activity and mutagenicity of the NEST particle has was tested and compared with a quarts reference particle in a cell line. We determined the mutant
frequency following eight repeated 72 h incubations with 75 μg/ml carbon black ?(Printex 90) or 100 μg/ml quartz (SRM1878a) particles in the FE1 MutaMouse lung
epithelial cell line. For carbon black exposed cells, the mutant frequency was 1.40-fold (95 % CI: 1.22-1.58) for cII and 1.23-fold (95 % CI: 1.10-1.37) for lacZ compared to
identically passaged untreated cells. Quartz did not significantly affect the mutant frequency. Carbon black also induced DNA strand breaks (p=0.02) and oxidized
purines (p=0.008), as measured by the comet assay. Quartz induced marginally more oxidized purines, but no change in strand breaks. We detected five (phenanthrene, flouranthene, pyrene, benzo[a]anthracene and chrysene) of the 16 EPA priority PAHs in an extract of carbon black. The detected PAHs are only weakly mutagenic compared to benzo[a]pyrene, and were present in very low amounts. It was demonstrated that carbon
black was weakly mutagenic and clearly able of damaging DNA. A paper is in press in Environmental and Molecular Mutagenesis). The fullerenes, and SWCNT were similarily toxic to the FE1 cells as the carbon black, and the CdTe quantum dots were about one hundred-fold more toxic to the cells. None of these particles were mutagenic to the FE1 cells upon eight repeated treatments.
We tested the ability of other NEST particles to induce cell death, DNA damage and mutations in a lung epithelial cell line. The reactive oxygen species generating ability and its implications are discussed. The inflammatory potential was, additionally, determined in an in vivo pulmonary exposure setup. In the Particle Risk project we
developed a long-term sub culture exposure model to determine the mutagenic effects of the tested particles as well as shown that apolipoprotein E knockout mice are a sensitive model for evaluating particle driven inflammation.
The main conclusions based on the publications included in the thesis of Nicklas Raun Jacobsen are that quantum dots were the most toxic particle tested. Quantum dots were followed by diesel exhaust particles, carbon black, single-walled carbon nanotubes, quartz and with C-fullerener and gold being the least toxic. All particles (except gold) 60
tested by instillation were capable of inducing pulmonary inflammation. Instillation of single-walled carbon nanotubes caused an inflammation which was short in duration but also extremely strong. The response appeared different than we observed for other
Particle_Risk – Six Months Report, Year 1
comparable tested materials and make single-walled carbon nanotubes interesting for future studies.
Carbon black and diesel exhaust particles increased the level of mutations in the lung epithelial cell line and mechanistic tests showed a possible link between the production of reactive oxygen species, DNA damage and mutations. In general we detected good correlation between in vitro and in vivo results, but validation, including more doses,
more time points and more end points are needed prior to substituting in vivo
experiments for in vitro.
Researcher Nicklas Raun Jacobsen has visited Wolfgang Kreyling at GSF and establish common procedures for instillation of particles in the mouse lung. Experiments with instillations with NEST particles will take place within the next weeks. We have investigated different vehicles for producing well-suspended dispersion of the NEST particles for instillations. We have arrived at that it is best to suspend the NEST particles in diluted mouse lung lining fluid for instillations in the mouse lung.
Master student Lise Vesterdal investigated the effects of the NEST particles on aorta endothelial physiology in January 2007. She investigated the association between intraperitoneal injection of pristine C60 fullerenes and vasomotor dysfunction in the aorta of 11-13 and 40-42 weeks old apolipoprotein E knockout mice (apoE-/-) with different degree of atherosclerosis. The aged apoE-/-mice had lower endothelium-dependent vasorelaxation elicited by acetylcholine in aorta segments mounted in myographs and the phenylephrine-dependent vasoconstriction response was increased. One hour after an intraperitoneal injection of 0.05 or 0.5 mg/kg of C60 fullerenes, the young apoE-/- mice had slightly reduced maximal endothelium-dependent vasorelaxation. A similar tendency was observed in the old apoE-/- mice. Hampered endothelium-independent vasorelaxation was also observed as slightly increased EC50 of sodium nitroprusside-induced vasorelaxation response in young apoE-/- mice. Treatment with C60 fullerenes affected mainly the response to vasorelaxation in young apoE-/- mice, whereas the vasomotor dysfunction in old apoE-/- mice with more advanced atherosclerosis was less affected by acute C60 fullerene treatment. These findings represent an important step in the hazard characterization of C60 fullerenes by showing that intraperitoneal administration is associated with a moderate decrease in the vascular function of mice with atherosclerosis. Relaxation and contractility of aorta rings from ApoE mice and wild type mice that have been treated with NEST particles will be investigated. The revised version 5 was submitted for publication in November 2008. Lise Vesterdal successfully defended her master thesis at the University of Copenhagen in May 2008.
PhD student Janne K. Folkman studied C60 fullerenes and single-walled carbon nanotubes (SWCNT) oxidative damage to DNA by the pre-mutagenic 8-oxo-7,8-dihydro-2’-
deoxyguanosine (8-oxodG) in the colon mucosa, liver and lung of rats after intragastric administration of pristine C60 fullerenes or SWCNT at two low doses. The particles were given in saline solution to one group of mice and in corn oil in another. The regulation of DNA repair systems toward 8-oxodG in liver and lung tissue was investigated. Both doses of SWCNT increased the levels of 8-oxodG in liver and lung. Administration of C60 fullerenes increased the hepatic level of 8-oxodG, whereas only the high dose generated 8-oxodG in the lung. We detected no effects on 8-oxodG in the colon mucosa. Suspension of particles in saline solution or corn oil yielded similar extent of genotoxicity, whereas corn oil per se generated more
genotoxicity the particles. Whilst there was increased mRNA expression of 8-oxoguanine DNA glycosylase (OGG1) in the liver of C60 fullerene-treated rats, there was no significant increase in repair activity. The paper 6 has been submitted for publication.
Particle_Risk – Six Months Report, Year 1
4. Jacobsen, Møller, P., Jensen, K.A., Vogel U., Ladefoged, O., Loft, L., Wallin, H., Lung inflammation and genotoxicity following pulmonary exposure to nanoparticles in ApoE-/- mice. Submitted for publication
5. Vesterdal, L.K., Folkmann, J.F., Jacobsen, N.R., Sheykhzade, M., Wallin, H., Loft, S.,
Møller, P., Modest vasomotor dysfunction induced by low doses of C60 fullerenes in apolipoprotein E knockout mice with different degree of atherosclerosis, submitted for
6. Folkmann, J.F., Vesterdal, L.K., Jacobsen, N.R., Wallin, H., Loft, S., Møller, Oxidatively
Damaged DNA in Rats Exposed by Oral Gavage to C60 Fullerenes and Single-walled Carbon Nanotubes, submitted for publication
Janne K Folkmann, Lotte Risom, Nicklas R Jacobsen, Håkan Wallin, Steffen Loft and Peter Møller”