May 2007: "Cardiovascular Effects of Pulmonary Exposure to Single-Wall Carbon Nanotubes", Zheng Li et al.
Environmental Health Perspectives 115 (2007) 377-382 [doi: 10.1289/ehp.9688]
June 2007a: "Cellular Interaction of Different Forms of Aluminium Nanoparticles in Rat Alveolar Macrophages", Andrew J. Wagner et al.
J. Phys. Chem. B 111 (2007)7353-7359 [doi: 10.1021/jp068938n]
June 2007b: "Stabilization of C60 Nanoparticles by Protein Adsorption and Its Implications for Toxicity Studies", Shigeru Deguchi et al.
Chem. Res. Toxicol. 20(6) (2007) 854-858 [doi: 10.1021/tx6003198]
July 2007: "A comparison of dispersing media for various engineered carbon nanoparticles", Mary C Buford et al.
Particle and Fibre Toxicology 4(6) (2007) [doi: 10.1186/1743-8977-4-6]
Sept 2007: "The pro-inflammatory effects of low-toxicity low-solubility particles, nanoparticles and fine particles, on epithelial cells in vitro", Claire Monteilleret al.
Occup and Environ Med 64 (2007) 609–615 [doi: 10.1136/oem.2005.024802]
October 2007: "Pulmonary and Systemic Immune Response to Inhaled Multiwalled Carbon Nanotubes", Leah A. Mitchell et al.
Toxicological Sciences 100(1) (2007) 203-214 [doi:10.1093/toxsci/kfm196]
November 2007:"Tissue distribution and toxicity of intravenously administered titanium dioxide nanoparticles in rats", E. Fabian et al.
Archives of Toxicology 82(3) (2008) 151-157 [doi: 10.1007/s00204-007-0253-y]
December 2007: "DNA damage induced by multiwalled carbon nanotubes in mouse embryonic stem cells", L. Zhu
Nano Letters 7(12) (2007) 3592-3597 [doi: 10.1021/nl071303v]
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 May 2007: "Cardiovascular Effects of Pulmonary Exposure to Single-Wall Carbon Nanotubes", Zheng Li et al.
Environmental Health Perspectives 115 (2007) 377-382 [doi: 10.1289/ehp.9688] |
General comments
In this study, the authors examined the effects of Single-Wall Carbon Nanotubes (SWCNT) intratracheally instilled in mice in the development of atherosclerosis.
They found that SWCNT was associated with accelerated atherosclerosis and oxidative blood vessel alterations, without modifying lipid levels in blood and without inducing systemic inflammation.
This is an interesting study that demonstrates that respiratory exposure to manufactured nanomaterials can induce cardiovascular alterations. In this sense, this study is close to those demonstrating a positive association between air pollution (i.e. ultrafine particulates) and adverse cardiovascular outcomes, particularly in high risk individuals such as those with preexisting chronic pulmonary and cardiovascular diseases (refs).
However, although interesting this study has some limitations. The first is related to the intratracheal instillation of the SWCNT. This was of administration is not as representative of an accidental human exposure as exposure to an aerosol of CNT. Furthermore, intratracheal instillation was associated with formation of aggregates of SWCNT in the lung. This aggregates can influence the local and/or cardiovascular effects of SWCNT. Another limitation is that the authors did not measure SWCNT n the blood and in the atherosclerotic plaques. Therefore it is difficult to know if individual or aggregated SWCNT translocate from lung to blood. In spite of these criticisms, the study is of valuable interest.
Methods
Materials : SWCNT (CNI, Houston, TX) produced by the high pressure carbon monoxide proportionation process (HiPco) technique and purified by acid treatment to remove metal contaminants Mice received a single intratracheal instillation of SWCNT in doses of 10-40 µg/mouse by single intrapharyngeal instillation and were sacrificed at time points including 1, 7, 28, and 56 days after exposure. Aortic mitochondrial alterations were analyzed by oxidative stress assays, including quantitative polymerase chain reaction of mitochondrial (mt) DNA. Plaque formation was quantified by morphometric analysis.
Results
A single intrapharyngeal instillation of SWCNTs induced activation of heme oxygenase-1 (HO-1), a marker of oxidative insults, in lung, aorta, and heart tissue in HO-1 reporter transgenic mice. Furthermore, mice, exposed to SWCNT (10 and 40 µg/mouse), developed aortic mtDNA damage at 7, 28, and 60 days after exposure. mtDNA damage was accompanied by changes in aortic mitochondrial glutathione and protein carbonyl levels. Because these modifications have been related to cardiovascular diseases, the authors evaluated whether repeated exposure to SWCNTs (20 µg/mouse once every other week for 8 weeks) stimulates the progression of atherosclerosis in ApoE-/- transgenic mice. Although SWCNT exposure did not modify the lipid profiles of these mice, it resulted in accelerated plaque formation in ApoE-/- mice fed an atherogenic diet. Plaque areas in the aortas, measured by the en face method, and in the brachiocephalic arteries, measured histopathologically, were significantly increased in the SWCNT-treated mice. This response was accompanied by increased mtDNA damage but not inflammation.
Conclusions
Taken together, the findings are of sufficient significance to warrant further studies to evaluate the systemic effects of SWCNT under workplace or environmental exposure paradigms. |
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June 2007a: "Cellular Interaction of Different Forms of Aluminium Nanoparticles in Rat Alveolar Macrophages", Andrew J. Wagner et al.
J. Phys. Chem. B 111 (2007)7353-7359 [doi: 10.1021/jp068938n] |
General comments
In this focused study, the authors examined the interaction of nanosized aluminium oxide (Al-ox-NP) and aluminium (Al-NP)with phagocytitic cells (rat alveolar macrophages - NR8383). The reported effects on cell viability and on the phagocytitic capacity of the macrophages are referred to the particle size and the particle’s chemical composition.
Although all studied particles have the same chemical composition (Al-oxide) in contact with the cells in cultures the toxic effects (cytotoxicity and the reduction of the phagocytic capacity) of the AL-NP are significant larger than those of the “pure” Al-ox-NP.
On the basis of the data of the producer the sizes of (chemically) different particles do not overlap, but due to the “good practice” of the researchers several physical parameters, among which also the size, were assessed. It was found that the different particles did overlap in size, allowing conclusions linking biological effects to both composition and size.
Methods
Materials: The materials, aluminium oxide nanoparticles (30 nm, 40 nm) and aluminium nanoparticles with a 2-3 nm oxide coat (50 nm, 80 nm, 120 nm), were synthesized NovaCentrix, Austin, TX.
The physical parameters such as size (of the primary materials and in dispersion) and zeta potential of the different materials in different solutions (conc, media) were characterised, using Transmission Electron Microscopy (TEM), Dynamic Light Scattering Analysis (DLS) and Laser Doppler Velocimetry (LDV).
The viabililty of the NR8383 macrophages was assessed with the MTT assay, the inhibition of the phagcytotic capacity was scored as the PI after incubation with 2 µm latex beads.
Results
All used nanomaterials agglomerated in suspension. The larger the particles (and the higher the concentration) the larger the agglomerates. The viability of the NR8383 cells remains high in the presence of Al-ox-NP (25 – 100 µg/ml) while Al-NP already show toxicity at 25 µg/ml). Phagocytosis is more inhibited by Al-NP compared to Al-ox-NP.
Conclusions
The reason why we choose this paper is because it describes not only biological effect but also some physico-chemical characteristics were studied, this is the type of research needed to build a comprehensive database on health and/or toxic effects of nanomaterials. The authors try to link chemical composition and size to the final biological effects. |
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June 2007b: "Stabilization of C60 Nanoparticles by Protein Adsorption and Its Implications for Toxicity Studies", Shigeru Deguchi et al.
Chem. Res. Toxicol. 20(6) (2007) 854-858 [doi: 10.1021/tx6003198] |
General comments
The stability of a dispersion can be considered as an important parameter in health effect studies. In this study the dispersion stability of nanoparticles (C60) under condition simulating the physiological environment was examined. Adding proteins (1 mg/ml or higher) to a phosphate buffered saline (PBS) solution resulted in a stable dispersion of fullerenes. Dynamic light scattering (DLS) measurements showed that the human serum albumin (HAS) adsorbs onto the surfaces of the fullerenes, forming an expanded layer which prevents salt-induced coagulation.
Although this work only shows stabilisation of one type of nanomaterial (C60), at a relative low concentration (9.3 10-6 M), it has the merit to test, in a relative simple way, the potential of a nanomaterial to be present as a mono-disperse particle in a biological fluid. This observation can possibly have an serious impact for all health and/or therapeutic related studies using nanomaterials.
Methods
Materials: C60 molecules (99.9 % purity) were purchased from Tokyo Kasie Kogyo.
Methods: Simple and well described dispersion protocols. DLS measurement to assess the hydrodynamic diameter.
Results
C60 molecules aggregate over time in PBS. Adding 1mg/ml – or more – HSA prevents this aggregation. Albumin stabilized C60 molecules have a larger hydrodynamic diameter, compared to albumin free C60, this diameter is only slightly dependent on the HSA concentration.
Conclusions
This paper studies the effect of serum albumin on the dispersion of C60 molecules in phosphate buffered saline. Relative low concentrations of C60 (9.3 10-6 M) is stable in a buffer enriched with 1 mg/ml albumin.
The finding is certainly interesting because it allows the study of “monodisperse” nanoparticles in e.g cell cultures, but it also introduces a layer on the material, changing the the biological interactions – depending on the objectives of the study this can be an advantage or an disadvantage. |
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July 2007: "A comparison of dispersing media for various engineered carbon nanoparticles", Mary C Buford et al.
Particle and Fibre Toxicology 4(6) (2007) [doi: 10.1186/1743-8977-4-6] |
General comments
This well conducted study suggest that the optimal dispersion of CNP in a biological system is one of the key critical step to be considered for effective relevant in vitro toxicity studies and effective CNP distribution through the tissue.
This observation suggests that various dispersions may modify CNP biological activity. The paper shows in fact that no “perfect” solution exists for the problem because we need to disperse the material, but any dispersion will change the biological activity.
Methods
Particles:
C60CS from SES Research (Houston, TX) and from Bucky USA (Houston, TX);
SWNT from SES Research (Houston, TX) and from nanotechnologies Inc (Houston, TX); MWNT from SES Research and from nanolab (Newton, MA).
Suspension media:
Various media were used including, cell culture media RPMI with fetal calf serum (FCS), FCS alone, phosphate buffered saline (PBS), dimethylsulfoxide (DMSO), 1%Tween80 in PBS, delipidated FCS, 7.5% bovine serum albumin (BSA)
Dispersion analysis:
Light microscopy at 400 x magnification
In vivo studies:
Particle instillation intratracheally in Balb/c mice (12 weeks of age) at a dose of 250 µg per mouse.
Results
CNP suspended in serum produced particle suspension with the fewest large agglomerates, and the most uniform distribution in mouse lungs. In addition, no apparent clearance of instilled CNP took place from lungs even after seven days.
Conclusions
This work demonstrates that CNP agglomerates are present in all dispersing vehicles to some degree. The source of CNP is also a factor in the degree of particle agglomeration within the same vehicle. |
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Sept 2007: "The pro-inflammatory effects of low-toxicity low-solubility particles, nanoparticles and fine particles, on epithelial cells in vitro", Claire Monteilleret al.
Occup and Environ Med 64 (2007) 609–615 [doi: 10.1136/oem.2005.024802] |
Background & objectives
In occupational setting particles exposure is still regulated on a mass basis, but this might not be the most appropriate metric. It is, based on several former studies, hypnotized that for a range of LSLTP surface area might be a better descriptor of the exposure.
Methods
In a human alveolar A549 cells, interleukin (IL)-8 mRNA, IL8 protein release and glutathione (GSH) depletion were measured as markers of pro-inflammatory effects and oxidative stress. The cells were treated with several LSLTP (fine and nanoparticles) and DQ12 quartz, a particle with a highly reactive surface.
Results
All nanoparticle preparations (TiO2 and carbon black) produced stronger responses than the same mass dose of fine particles. Two ultra-fine metal dusts (cobalt and nickel) produced GSH depletion similar to TiO2-np, for a similar surface area.
Conclusion
The findings are consistent with the hypothesis for a range of LSLTP in vitro and in vivo, consequently the high surface area of nanoparticles is considered to be a key factor in their inflammogenicity. The authors suggest that a threshold for surface area of around 1–10 cm2/cm2 exists.
DQ12 quartz was in this study used as a positive control showed already a clear response under this threshold, probably indicating that beside surface area the intrinsic characteristics of the compound also plays a role. |
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October 2007: "Pulmonary and Systemic Immune Response to Inhaled Multiwalled Carbon Nanotubes", Leah A. Mitchell et al.
Toxicological Sciences 100(1) (2007) 203-214 [doi:10.1093/toxsci/kfm196] |
General comments
This study was conducted to assess short-term pulmonary and systemic immune response to inhaled MWCNTs as a function of dose and time, in mice.
The authors found that exposure to 0.3-5 mg/m3 for 7 or 14 days induced no significant lung inflammation or damage but caused systemic immune functions alterations.
This study is very interesting because it is one of the first dealing with inhalation of carbon nanotubes. The absence of pulmonary effects, even at relatively high exposure concentrations contrasts with many of the findings (granulomas, fibrosis, inflammation) associated with the pulmonary toxicity of CNTs administered by intratracheal instillation. The systemic immune function alterations observed, characterised by a suppressed splenic T-cell-dependant antigen response and a decreased splenocyte Natural Killer (NK) activity, have never been observed for CNTs, although already described after inhalation of carbon-based particles.
Another point of the study is the development and characterization of a CNT-aerosol, suitable for rodent exposure. A point that could be criticised is that the animals were exposed in whole-body inhalation exposure chambers and so could have ingested some CNTs present in their hair-coat. It is therefore not evident that the systemic effects are really due to inhaled CNTs.
Despite of this point, the study is of valuable interest and opens the field to numerous investigations, including the assessment of long-term toxicity of inhaled CNTs, the comparison of various CNTs and also the determination of the mechanism of MWCNT-induced immune suppression.
Materials and Methods
MWCNTs (Shenzen nanotech Port Co, China) were produced by chemical vapor deposition (CVD) process.
Nanotube aerosols were produced by mechanical agitation/resuspension of MWCNTs using a jet mill coupled to a dry chemical screw feeder. Aerosols were in the respirable size for rodents (< 3 µm). Mice were exposed in whole-body inhalation exposure chambers for 7 or 14 consecutive days, at doses of 0, 0.3 ± 0.1, 1.0 ± 0.1, 5.3 ± 0.6 mg/m3 and sacrificed the following morning.
Results
Aerosol characterization : Electron microscopy showed that the aerosolised MWCNT were more or less agglomerate, including some single tubes. The size of agglomerates was about 0.7-1 µm for the 0.3-and 1-mg/m3 concentrations and 1.8 µm for the 5-mg/m3 concentration.
Lung response: analysis of BAL showed numerous macrophages with MWCNTs inside, but there was no inflammation and no change in the BAL cellularity and cell number compared to the controls. Histopathology confirmed that the majority of CNT were engulfed by macrophages. No change was observed in the expression of IL-6, IL-10 and NQO1 mRNA.
Spleen response: Mice exposed to MWCNTs for 14 days show alterations of the systemic immune function.
Discussion - Conclusion
According to the authors, the absence of effect in the lung, which contrasts with results obtained by intratracheal instillation, may be attributed to the type of CNTs used, the dose and the route of administration. Instillation of CNT suspensions is more likely to lead to large concentration of material collected in small area of the lung, supporting the development of "foreign-body" type response (ie granulomas). In parallel, as for many other materials such silica, the response to CNTs may manifest over several months, arguing for the development of chronic inhalation studies.
The splenic immune suppression observed is very intriguing and warrants further studies to place these results in context and to further elucidate a mechanism for this response.
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November 2007:"Tissue distribution and toxicity of intravenously administered titanium dioxide nanoparticles in rats", E. Fabian et al.
Archives of Toxicology 82(3) (2008) 151-157 [doi: 10.1007/s00204-007-0253-y] |
General comments
This paper describes the toxicity and the time dependent tissue distribution of intravenously administered nanoparticles in the situation of 100 % bioavailability. For these investigations, the authors administered nanoparticular TiO2 (TiO2 >80 wt% at <100 nm size) into the systemic circulation of rats at a dose level of 5 mg/kg bw. The tissue content of TiO2 was determined 1, 14, and 28 days later. Biochemical parameters and antigens in serum were also assessed. The measured TiO2 levels were highest in the liver, followed in decreasing order by the levels in the spleen, lung, and kidney, and highest on day 1 in all organs. TiO2 levels were retained in the liver, whereas there was a slight decrease in TiO2 levels in the spleen over the study period of 28 days.there was a return to control levels by day 14 in the lung and kidney. There were no changes in the cytokines and enzymes measured in blood samples, indicating that there was no detectable inflammatory response or organ toxicity. Overall, rats exposed to TiO2 nanoparticles by a route that allows immediate systemic availability showed expected tissue distribution, no obvious toxic health effects, no immune response, and no change in organ function over the complete study period of 28 days.
Methods
Test material: Titanium dioxide [TiO2; CAS no. 13463-67-7], consisting of both anatase and rutile forms (70/30), without surface coating, particle size range: 20–30 nm, BET: 48.6 m2/g
Species: Male Wistar rats
Treatment: 1 intravenous administration
Dose: 5 mg/kg bw.
Examination: Clinical observation, clinical chemistry and haematology, cytokines in blood samples, TiO2 residues in organs and tissues
Results
The tissue distribution and toxicity of intravenously administered nanoparticles of TiO2 was investigated because of the fundamental importance to obtain information on the kinetics of this widely used nanoparticle in a situation of 100% bioavailability. 9 male Wistar rats were treated with single intravenous injections of a suspension of TiO2 (5 mg/kg body weight), and the tissue content of TiO2 was determined 1, 14, and 28 days later in blood cells, plasma, lymph nodes, liver, kidney, lung, spleen, and brain by ICP-AES after appropriate sample work up. Biochemical parameters and antigens in serum were also assessed to determine potential pathological changes. There were no detectable levels of TiO2 in blood cells, plasma, brain, or lymph nodes. The TiO2 levels were highest in the liver, followed in decreasing order by the levels in the spleen, lung and kidney, and highest on day 1 in all organs. Control TiO2 levels in these organs ranged from 0.2 – 1.7 µg/g. TiO2 levels were retained in the liver for 28 days, there was a slight decrease in TiO2 levels in the spleen from day 1 to days 14 and 28, and a return to control levels by day 14 in the lung and kidney. There were no changes in the cytokines and enzymes measured in blood samples. The parameters measured to assess inflammatory responses were divided into four classes: (1) chemokines that are attractants for monocytes, Th lymphocytes, eosinophils, basophils, and neutrophils; (2) indicators of cell proliferation or inhibition; (3) indicators of inflammation (e.g., cytokines); and (4) indicators of immune modulation. All these parameters were unchanged, indicating that there was no detectable inflammatory response or organ toxicity over the study period of 28 days.
Conclusion
Overall, rats exposed to TiO2nanoparticles by a route that allows immediate systemic availability showed expected tissue distribution, no obvious toxic health effects, no immune response, and no change in organ function. Therefore, even with 100% bioavailability of the 5 mg/kg TiO2 dose afforded by the intravenous route of administration, there were no remarkable toxic effects evident in the experimental animals. These results indicate that TiO2 nanoparticles could be used safely in low doses. |
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December 2007: "DNA damage induced by multiwalled carbon nanotubes in mouse embryonic stem cells", L. Zhu
Nano Letters 7(12) (2007) 3592-3597 [doi: 10.1021/nl071303v] |
General comments
This paper assessed the DNA damage in mouse embryonic stem (ES) cells upon exposure to MWNTs and moved to the molecular effects. This is clearly the strength of the study.
It remains difficult to put these data into perspective. Are the effects seen in this in vitro system comparable to the in vivo effects?
We (reviewers from Nanosafe) would have liked a little more on physico-chemical characterization of the material because that would help to link it to the biological response. Important measures could be: Size of the dosed clusters of material; Are any monodisperse MWNT present; Effect of HCl treatment on surface characteristics, zeta potential, etc.
Methods
Materials: Commercially available MWNTs, from Tsinghua and Nanfeng Chemical Group Cooperation, China.
Material was further purified (see supplement: http://pubs.acs.org/subscribe/journals/nalefd/suppinfo/nl071303v/nl071303v.pdf).
Results
The authors found that MWNTs can accumulate and induce apoptosis (programmed cell death) in mouse ES cells.
At the expression level an activation of the tumor suppressor protein p53 was found within 2 h of exposure. Moreover, an increase in the expression of base excision repair protein 8-oxoguanine-DNA glycosylase 1 (OGG1) was found and two double strand break repair proteins: Rad 51 and X-ray cross-complementation group 4 (XRCC4).
Beside these changes in expression of typical proteins also an increased phosphorylation of H2AX histone (serine 139) and SUMO modification of XRCC4 was found following the treatment with MWNTs.
In a third group of experiments (mutagenesis study) an endogenous molecular marker was used (adenine phosphoribosyltransferase (Aprt)). This study showed that MWNTs increased the mutation frequency by 2-fold compared with the spontaneous mutation frequency in mouse ES cells.
Conclusions
Taking together all these studies, the authors concluded that care must be taken in using nanomaterials in e.g. medical applications. The clear genotoxic effects of MWNTs in this study, together with previous studies demonstrating limited or no toxicity of these materials at the cellular level in vitro, indicates that the toxicity of nanomaterials varies dramatically from cellular to molecular levels when monitoring only in the acute phase. |
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POM 2007
