Changes in DNA Methylation in Mouse Lungs after a Single Intra-Tracheal Administration of Nanomaterials

<div><p>Aims</p><p>This study aimed to investigate the effects of nanomaterial (NM) exposure on DNA methylation.</p><p>Methods and Results</p><p>Intra-tracheal administration of NM: gold nanoparticles (AuNPs) of 5-, 60- and 250-nm diameter; single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) at high dose of 2.5 mg/kg and low dose of 0.25 mg/kg for 48 h to BALB/c mice. Study showed deregulations in immune pathways in NM-induced toxicity <i>in vivo</i>. NM administration had the following DNA methylation effects: AuNP 60 nm induced CpG hypermethylation in <i>Atm</i>, <i>Cdk</i> and <i>Gsr</i> genes and hypomethylation in <i>Gpx</i>; <i>Gsr</i> and <i>Trp53</i> showed changes in methylation between low- and high-dose AuNP, 60 and 250 nm respectively, and AuNP had size effects on methylation for <i>Trp53</i>.</p><p>Conclusion</p><p>Epigenetics may be implicated in NM-induced disease pathways.</p></div

Bronchoalveolar lavage (BAL) fluid analysis in mice exposed to nanomaterial.

<p>a): total cell count; b): differential cell count.; c): uptake/association by/with BAL macrophages. For clarity of presentation in panel b, significant groups are not annotated. In panel b, macrophages count was significant in following exposure groups: AuNPs 5nm 50μg and AuNPs 60nm 50μg compared to the vehicle; AuNP 5nm 50μg compared to AuNP 250nm 5μg; and AuNPs 60 nm 50μg compared to the AuNP 250nm 5μg dose categories. Neutrophils count was significant in following exposure groups: SWCNT 50μg and MWCNTs 50μg compared to vehicle. Lymphocytes count was significant in following exposure groups: SWCNT 50μg and MWCNTs 50μg compared to vehicle. In panel c, 100 macrophages were randomly counted for the microscopic presence or absence of NM aggregated inside the cytoplasm at 1000X magnification. Representative images of macrophage d): vehicle; e): AuNPs 5nm; f): AuNPs 60nm; g): AuNPs 250 nm; h): SWCNTs; i): MWCNTs. In panel a and c; the box plot describes the median (line across the box), inter-quartile range and maximum and minimum values (whiskers). Outliers are shown as colored circles outside the ends of whiskers. Data in panel b is represented as median ±SD. Asterisk sign (*) shows significance levels at <i>p</i> = 0.05 (dunn’s statistics). Gold nanoparticles: AuNPs; single-walled- and multi-walled carbon nanotubes: SWCNTs and MWCNTs.</p

Effect of nanomaterial (NM) exposure variables on methylation of promoters of genes.

<p>Effect of nanomaterial (NM) exposure variables on methylation of promoters of genes.</p

Effect of nanomaterial (NM) exposure on gene promoter methylation.

<p>Bars connect exposure groups with significant methylation difference, a-d): effects of gold nanoparticles (AuNPs) exposure on promoter methylation levels of <i>Atm</i> (a), <i>Cdk</i> (b), <i>Gp x</i>(c), and <i>Gsr</i> (d) genes in lungs. Effect of single and multi-walled carbon nanotubes (SWCNTs, MWCNTs) exposure on gene promoter methylation levels of <i>Atm</i> (e) gene in lungs. In panels, box plot describes the median (line across the box), inter-quartile range and maximum and minimum values (whiskers). Outliers are shown as colored circles outside the ends of whiskers. Asterisk sign (*) shows significance levels at <i>p</i> = 0.5 (dunn’s statistics). <i>Atm</i>: ataxia telangiectasia mutated; <i>Cdk</i>; cyclin-dependent kinase; <i>Gsr</i>: glutathione reductase; <i>Gpx</i>: glutathione peroxidase.</p

IL-13 is a central mediator of chemical-induced airway hyperreactivity in mice

<div><p>Background</p><p>While the importance of the Th2 cytokine IL-13 as a central mediator of airway hyperreactivity (AHR) has been described in allergic protein-induced asthma, this has never been investigated in chemical-induced asthma.</p><p>Objective</p><p>We examined the importance of IL-13 in a mouse model of chemical-induced AHR, using toluene-2,4-diisocyanate (TDI).</p><p>Methods</p><p>In a first set-up, wild type (WT) and <i>IL-13</i> knockout (KO) C57Bl/6 mice were dermally treated on days 1 and 8 with 1% TDI or vehicle (acetone/olive oil) on both ears. On day 15, mice received an intranasal instillation with 0.1% TDI or vehicle. In a second set-up, WT mice sensitized with 1% TDI or vehicle, received i.v. either anti-IL-13 or control antibody prior to the intranasal challenge.</p><p>Results</p><p>TDI-sensitized and TDI-challenged WT mice showed AHR to methacholine, in contrast to TDI-sensitized and TDI-challenged <i>IL-13</i> KO mice, which also showed lower levels of total serum IgE. TDI-sensitized and TDI-challenged <i>IL-13</i> KO mice had lower numbers of T-cells in the auricular lymph nodes. TDI-treated WT mice, receiving anti-IL-13, showed no AHR, in contrast to those receiving control antibody, despite increased levels of IgE. Anti-IL-13 treatment in TDI-treated WT mice resulted in lower levels of serum IL-13, but did not induce changes in T- and B-cell numbers, and in the cytokine production profile.</p><p>Conclusion and clinical relevance</p><p>We conclude that IL-13 plays a critical role in the effector phase of chemical-induced, immune-mediated AHR. This implicates that anti-IL-13 treatment could have a beneficial effect in patients with this asthma phenotype.</p></div

Effect of shapes of single and multiwalled carbon nanotubes (SWCNTs, MWCNTs) upon exposure on <i>Atm</i> gene methylation.

<p>Bars connect exposure groups with significant methylation difference. In panels, box plot describes the median (line across the box), inter-quartile ranges and maximum and minimum values (whiskers). Outliers are shown as colored circles outside the ends of whiskers. Asterisk sign (*) shows significance levels at <i>p</i> = 0.5 (dunn’s statistics). <i>Atm</i>: ataxia telangiectasia mutated; <i>Trp53</i>: tumor protein P53.</p

Global DNA methylation (5mC) and hydroxymethylation (5hmC) in lungs.

<p>a): no significant effects (Wilcoxon test) of gold nanoparticle (AuNPs) and single-walled and multi-walled carbon nanotubes (SWCNTs and MWCNTs) were observed on 5mC (<i>p</i> = 0.667 and 0.284 respectively). b): also no significant effect of AuNPs exposure on lung 5hmC were observed (<i>p</i> = 0.107). However, CNTs exposure showed significant effect on 5hmC (<i>p</i> = 0.024) levels by Wilcoxon statistics, while no group remained significant after multiple comparisons (Dunn all pairs post-hoc). In panel a and b; box plot describes the median (line across the box), inter-quartile range and maximum and minimum values (whiskers). Outliers are shown as colored circles outside the ends of whiskers.</p

Effect of nanoparticles (NPs) dose and size on gene promoter methylation upon exposure.

<p>Bars connect exposure groups with significant methylation difference, a-d): effects of gold NPs (AuNPs) exposure dose on promoter methylation levels of <i>Gsr</i> (a, and b), <i>Trp53</i> (c) in lungs, and <i>Pparg</i> (d) genes in blood. AuNPs size effect on CpG methylation of <i>Trp53</i> gene was observed between 60 nm and 250 nm AuNPs. In panels, box plot describes the median (line across the box), inter-quartile range and maximum and minimum values (whiskers). Outliers are shown as colored circles outside the ends of whiskers. Asterisk sign (*) shows significance levels at <i>p</i> = 0.5 (dunn’s statistics). <i>Gsr</i>: glutathione reductase; <i>Trp53</i>: tumor protein P53; <i>Pparg</i>: peroxisome proliferator-activated receptor gamma.</p

Additional file 2: Table S1. of Forced expiration measurements in mouse models of obstructive and restrictive lung diseases

Complete overview of all lung function parameters, measured at baseline using the flexivent FX. (DOCX 23 kb

Airway and immune responses in <i>IL-13</i> KO mice.

<p>(A) Dose-response curves of R_n to methacholine (0–20 mg/mL). (B) Individual values and group means of the corresponding area under the curve (AUC) of airway hyperreactivity. (C) Total serum immunoglobulin E (IgE) and (D) IL-13 levels. (E) Lymphocyte subpopulations from auricular lymph nodes, stained with anti-CD3⁺, anti-CD3⁺CD4⁺, anti-CD3⁺CD4⁺CD25⁺ and anti-CD3⁺CD8⁺ or with anti-CD19⁺. Associated cytokine release of (F) IL-13, (G) IFN-γ and (H) IL-10. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to own corresponding control group (WT AOO/AOO or <i>IL-13</i> KO AOO/AOO group). ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to WT TDI/TDI group. N.D., non-detectable levels. n = 5–8 per group.</p