Weight of evidence analysis for assessing the genotoxic potential of carbon nanotubes

<p>Carbon nanotube (CNT) is a nanomaterial that has received interest because of its high-tensile strength and low weight. Although CNTs differ substantially in physico-chemical properties, they share high aspect ratio which resembles that of asbestos and other fibers causing lung cancer and mesothelioma. One type of multi-walled CNTs (i.e. MWCNT-7) has been classified as possibly carcinogenic to humans by IARC (Group 2B) based on experimental animal data, whereas other types of MWCNTs and single-walled CNTs (SWCNT) could not be classified due to lack of data from epidemiologic studies and insufficient mechanistic evidence. Damage to DNA is considered to be a key mechanistic step in the development of fiber-induced cancer. Thus, the genotoxic potential can be a cornerstone in the evaluation of hazards of CNTs. The present study used a weight of evidence (WoE) analysis to evaluate the genotoxicity of different types of CNTs. Genotoxicity endpoints close to cancer (mutations and chromosome aberrations) and animal models had highest weight in the WoE analysis. Eight CNT materials out of 130, which had been assessed in several studies, were evaluated in the WoE analysis. The results demonstrated that MWCNT-7 has strongest WoE for a genotoxic hazard among the MWCNTs. Two types of SWCNTs have a similar WoE for genotoxicity as MWCNT-7. Several reference materials from the Joint Research Centre have less WoE for genotoxicity. The WoE analysis demonstrates a difference in genotoxicity for CNTs, but further research is required to unravel the physico-chemical characteristics that govern the differences in genotoxic hazard.</p

Baseline-adjusted SB (A) and FPG sites (B) in A549 cell cultures exposed 24 hours to SRM1650, SRM2975 and ASPM

The data are obtained from figure 4 and table 1. Data points are mean ± SEM (SRMs, n = 9; ASPM, n = 6). There was no significant difference between the particle's ability to induce SB and FPG sites at any doses (> 0.05, Kruskal-Wallis test).<p><b>Copyright information:</b></p><p>Taken from "DNA damage and cytotoxicity in type II lung epithelial (A549) cell cultures after exposure to diesel exhaust and urban street particles"</p><p>http://www.particleandfibretoxicology.com/content/5/1/6</p><p>Particle and Fibre Toxicology 2008;5():6-6.</p><p>Published online 8 Apr 2008</p><p>PMCID:PMC2323018.</p><p></p

8-oxodG formation in calf thymus DNA measured with HPLC/ED after 30 minutes exposure to A) SRM2975 and B) SRM1650 and C) ASPM; ■ filter 1, ▲ filter 2, △ filter 3, ○ filter 4, ◆ filter 5

For SRM2975 and SRM1650 dots represents mean ± SEM; n = 3. The DNA was incubated at 37°C with 5 mM HO. For ASPM, the DNA was incubated at 37°C with 10 μM HO.<p><b>Copyright information:</b></p><p>Taken from "DNA damage and cytotoxicity in type II lung epithelial (A549) cell cultures after exposure to diesel exhaust and urban street particles"</p><p>http://www.particleandfibretoxicology.com/content/5/1/6</p><p>Particle and Fibre Toxicology 2008;5():6-6.</p><p>Published online 8 Apr 2008</p><p>PMCID:PMC2323018.</p><p></p

DNA damage measured by the comet assay in A549 cells exposed to SRM2975 and SRM1650 for 3, 24 or 48 hours

(A) SB, SRM2975, (B) FPG sites, SRM2975, (C) SB, SRM1650 and (D) FPG sites, SRM1650. Mean ± SEM, n = 9. * Dose where all exposure times are statistically significant compared to control (< 0.01, ANOVA, except for 3 hours exposure to 100 μg/ml where SRM1650 is tested with Kruskal-Wallis test, < 0.05). Dose where a single time point is statistically significant compared to the respective control (ANOVA): (A) SRM2975, SB, 2.5 μg/ml, 48 h, < 0.05, (C) SRM1650, SB, 2.5 μg/ml, 3 h, < 0.05 and SRM1650, SB, 25 μg/ml, 48 h, < 0.01, (D) SRM1650, FPG, 2.5 μg/ml, 3 h, < 0.05 and SRM1650, FPG, 25 μg/ml, 24 h, < 0.05.<p><b>Copyright information:</b></p><p>Taken from "DNA damage and cytotoxicity in type II lung epithelial (A549) cell cultures after exposure to diesel exhaust and urban street particles"</p><p>http://www.particleandfibretoxicology.com/content/5/1/6</p><p>Particle and Fibre Toxicology 2008;5():6-6.</p><p>Published online 8 Apr 2008</p><p>PMCID:PMC2323018.</p><p></p

Endothelial cell activation, oxidative stress and inflammation induced by a panel of metal-based nanomaterials

<div><p></p><p>The importance of composition, size, crystal structure, charge and coating of metal-based nanomaterials (NMs) were evaluated in human umbilical vein endothelial cells (HUVECs) and/or THP-1 monocytic cells. Biomarkers of oxidative stress and inflammation were assessed because they are important in the development of cardiovascular diseases. The NMs used were five TiO₂ NMs with different charge, size and crystal structure, coated and uncoated ZnO NMs and Ag which were tested in a wide concentration range. There were major differences between the types of NMs; exposure to ZnO and Ag resulted in cytotoxicity and increased gene expression levels of <i>HMOX1</i> and <i>IL8</i>. The intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) expression were highest in TiO₂ NM-exposed cells. There was increased adhesion of THP-1 monocytic cells onto HUVECs with Ag exposure. None of the NMs increased the intracellular ROS production. There were no major effects of the coating of ZnO NMs. The TiO₂ NMs data on ICAM-1 and VCAM-1 expression suggested that the anatase form was more potent than the rutile form. In addition, the larger TiO₂ NM was more potent than the smaller for gene expression and ICAM-1 and VCAM-1 expression. The toxicological profile of cardiovascular disease-relevant biomarkers depended on composition, size and crystal structure of TiO₂ NMs, whereas the charge on TiO₂ NMs and the coating of ZnO NMs were not associated with differences in toxicological profile.</p></div

Monocyte adhesion induced by multi-walled carbon nanotubes and palmitic acid in endothelial cells and alveolar–endothelial co-cultures

<p>Free palmitic acid (PA) is a potential pro-atherogenic stimulus that may aggravate particle-mediated cardiovascular health effects. We hypothesized that the presence of PA can aggravate oxidative stress and endothelial activation induced by multi-walled carbon nanotube (MWCNT) exposure <i>in vitro</i>. We investigated the interaction between direct exposure to MWCNTs and PA on THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs), as well as on indirect exposure in an alveolar–endothelial co-culture model with A549 cells and THP-1-derived macrophages exposed in inserts and the effect measured in the lower chamber on HUVECs and THP-1 cells. The exposure to MWCNTs, including a short (NM400) and long (NM402) type of entangled fibers, was associated with elevated levels of reactive oxygen species as well as a decrease in the intracellular glutathione concentration in HUVEC and A549 monocultures. Both effects were found to be independent of the presence of PA. MWCNT exposure significantly increased THP-1 monocyte adhesion to HUVECs, and co-exposure to PA aggravated the NM400-mediated adhesion but decreased the NM402-mediated adhesion. For the co-cultures, the exposure of A549 cells did not promote THP-1 adhesion to HUVECs in the lower chamber. When THP-1 macrophages were present on the cell culture inserts, there was a modest increase in the adhesion and an increase in interleukin-6 and interleukin-8 levels in the lower chamber whereas no tumor necrosis factor was detected. Overall, this study showed that direct exposure of HUVECs to MWCNTs was associated with oxidative stress and monocyte adhesion and the presence of PA increased the adhesion when exposed to NM400.</p

mRNA expression of <i>GCLM</i>, <i>HMOX1</i> and <i>VCAM1</i> in HUVECs after exposure to CB for 3 h.

<p>The symbols represent the mean and SEM of three independent experiments. ^#P<0.05 (linear regression analysis).</p

Carbon Black Nanoparticles Promote Endothelial Activation and Lipid Accumulation in Macrophages Independently of Intracellular ROS Production

<div><p>Exposure to nanoparticles (NPs) may cause vascular effects including endothelial dysfunction and foam cell formation, with oxidative stress and inflammation as supposed central mechanisms. We investigated oxidative stress, endothelial dysfunction and lipid accumulation caused by nano-sized carbon black (CB) exposure in cultured human umbilical vein endothelial cells (HUVECs), THP-1 (monocytes) and THP-1 derived macrophages (THP-1a). The proliferation of HUVECs or co-cultures of HUVECs and THP-1 cells were unaffected by CB exposure, whereas there was increased cytotoxicity, assessed by the LDH and WST-1 assays, especially in THP-1 and THP-1a cells. The CB exposure decreased the glutathione (GSH) content in THP-1 and THP-1a cells, whereas GSH was increased in HUVECs. The reactive oxygen species (ROS) production was increased in all cell types after CB exposure. A reduction of the intracellular GSH concentration by buthionine sulfoximine (BSO) pre-treatment further increased the CB-induced ROS production in THP-1 cells and HUVECs. The expression of adhesion molecules ICAM-1 and VCAM-1, but not adhesion of THP-1 to HUVECs or culture dishes, was elevated by CB exposure, whereas these effects were unaffected by BSO pre-treatment. qRT-PCR showed increased <i>VCAM1</i> expression, but no change in <i>GCLM</i> and <i>HMOX1</i> expression in CB-exposed HUVECs. Pre-exposure to CB induced lipid accumulation in THP-1a cells, which was not affected by the presence of the antioxidant N-acetylcysteine. In addition, the concentrations of CB to induce lipid accumulation were lower than the concentrations to promote intracellular ROS production in THP-1a cells. In conclusion, exposure to nano-sized CB induced endothelial dysfunction and foam cell formation, which was not dependent on intracellular ROS production.</p></div

ICAM-1 and VCAM-1 surface expression after 24 h CB exposure in mono-cultures of HUVECs or THP-1a cells, co-cultures with 90% HUVECs and 10% THP-1a cells or 50% HUVECs and 50%THP-1a cells with number of seeded cells indicated (A and B) and effect of BSO pre-treatment on ICAM-1 and VCAM-1 surface expression in HUVECs (C).

<p>Data are expressed as percentage of absorbance of unexposed control cells and bars are means ±SEM of at least three independent experiments. *P<0.05 compared with unexposed cells.</p

THP-1 adhesion to HUVEC cells or culture dishes assessed by BrdU labeling after 24 h CB exposure (A).

<p>In a separate experiment, HUVECs were exposed to 100 µM of BSO for 24 h before they were exposed to CB in co-culture with THP-1 cells (B). Data are expressed as percentage of adherent THP-1 cells compared with the total number of THP-1 cells (cell culture+supernatant) and bars are means ±SEM of three independent experiments *P<0.05 compared with unexposed cells, ^#P<0.05 compared with co-cultures where the HUVECs were not pre-treated with BSO.</p