471065 High Dimension Biological Analysis of Carbon Nanotube Toxicity
The immunological study included the evaluation of levels of CD3+CD95+, CD3+CD25+, CD3+CD126+ cells using the BD FACS CANTO II flow cytometer (BD Biosciences, USA). Mitochondrial membrane potential (MMP) was measured by the MitoProbe™ JC-1 Assay Kit using the BD FACS CANTO II flow cytometer. MMP was measured in leukocytes (MMPL) and mature СD3+ T lymphocytes (MMP СD3+). The percentage of cells with low MMP was reckoned on the basis of the measurement results.
Biochemical analyses were used to assess the levels of primary products of lipid peroxidation (LPO) – lipid hydroperoxides (LHP) and the intermediate products of LPO – diene conjugates (DC), ketodienes (KD), conjugated trienes (CT), compounds containing isolated double bonds (CCIDB). The LPO products have different wavelength absorption maxima (λLHP = 233 nm, λDC = 232 nm, λKD+CT = 278 nm, λCCIDB =220 nm), and they are differently extracted by heptane and isopropanol. The heptane phase contains the products of neutral lipid peroxidation; the isopropanol phase includes the products of phospholipid peroxidation. We used different ratios of heptane/isopropanol mixtures for the extraction. For LHP the ratio 2:1, v/v was used; for DC, KD, CT and CCIDB two mixtures in sequence were used at different heptane/isopropanol ratios, namely 1:1 and 3:7, v/v respectively.
A549 cells from a human Caucasian lung adenocarcinoma with the alveolar type II phenotype were obtained from ECACC (Sigma–Aldrich, Milan, Italy). The cells were cultured in Dulbecco’s modified Eagle medium (DMEM), 2 mM l-glutamine, 50 IU/ml penicillin, and 50 μg/ml streptomycin, in a humidified atmosphere containing 5% CO2 at 37 0C and grown to 80% confluence. Exposure to purified and unpurified MWCNTs was done on sub-confluent cells. HaCaT cells were cultured in calcium-free DMEM, with 10% chelexed FBS, 4 mM L-Glutamine, and supplemented with calcium chloride at 0.03 mM or 2.8 mM final concentration. FBS was calcium-depleted by incubation with Chelex 100 resin for 1 h at 4 °C according to the BioRad protocol. The Chelex was subsequently removed using a 50 mL Millipore 0.22 um filter unit system. Microarray data analysis identified differentially expressed genes, applying different statistical filters to allow for only the statistically and biologically significant probes to be evaluated. The results of microarray analysis were validated using quantitative real-time polymerase chain reaction (PCR).
All analyses were done in triplicate. Data were presented as median and 25% to 75% interquartile range. Statistica 10.0 (Statsoft) software was used for the statistical analysis. The differences between the groups were analyzed using the Mann-Whitney test. A value of р ≤0.05 was considered significant. Significant probe sets were evaluated for relevance to canonical pathway, molecular function and biological function using the Protein ANalysis THrough Evolutionary Relationships (PANTHER) Classification System (http://www.pantherdb.org). Statistically significant over- and under-represented annotation categories were determined by binomial statistics, using the observed number of expressed genes versus the numbers expected by chance within a certain annotation group. Categories with p-values > 10-2 were rejected.
Our study showed that the MWCNTs with lower content of metallic impurities (Type 1) affected lipid peroxidation parameters inducing pre-inflammatory effects. Oxidative disorders were manifested in the form of increase in the content of the primary products of lipid peroxidation (LPO) and redistribution of the intermediate products (CCIDB, DC, KD+CT) in neutral lipids and phospholipids of blood cell membranes. Type 1 MWCNTs increased the levels of LHP in blood by 1.8 times (р<0.05). The load test by the MWCNTs with the higher amount of metallic impurities (Type 2) on the whole blood caused a significant increase in LHP levels as compared to the purified MWCNTs (1.5 times, р<0.05) and 2.6 times as compared to the untreated blood. This may be due to the high content of metal ions in the unpurified MWCNTs that originate from Fe–Co catalyst and can be washed away by water. Peroxidation induction is consistent with other studies; it is presumably caused by the pro-oxidant action of iron ions (Fe2+), which generate hydroxyl and alcoxy lipid radicals. The initiation of new lipid peroxidation chain reactions leads to an increase in the levels of ketodienes and conjugated trienes. Exposure to MWCNTs with a minimal content of impurities caused an increase in the percentage of immune cells with reduced mitochondrial membrane potential. The number of mature T lymphocytes with reduced potential underwent a more than three-fold increase. At the same time, upregulation of the pre-apoptotic CD95 marker on CD3+ cells was observed. Purified MWCNTs activated the early phase of T-cell response and caused an increase in the percentage of the functionally active T lymphocytes expressing interleukin-6 (IL-6 receptor (CD3+CD126+). The damaging effect of unpurified MWCNTs was manifested in the significant increase in the percentage of lymphocytes and CD3+ cells with reduced mitochondrial membrane potential (MMPL and MMP СD3+ respectively). The increased levels of CD3+CD95+, CD3+CD25+ cells were not significantly different from the levels corresponding to exposure to the purified MWCNTs. Whole genome transcriptomics in the lung epithelium (A549) and keratinocytes (HaCaT), which were selected as in vitro models in this study showed that gene expression significantly differs after exposure to purified and unpurified MWCNTs. 48 hours exposure resulted in a significant number of genes differentially expressed compared to the control; approximately 500 genes were expressed differentially in the case of MWCNTs with different levels of impurities. Similar results were found when we analyzed the data isolating the 25 genes involved in the molecular pathway associated to oxidative stress. The picture changes slightly in terms of modulated gene expression, when focusing on the genes involved in inflammation mediated by chemokine and cytokine signaling.
Analysis of the effects of different types of MWCNTs on gene expression showed that impurities influence significantly the induction of key toxicity pathways such as inflammation mediated by chemokine and cytokine signaling. Pathway analysis showed the modulation of genes related to the NFkB pathway, which is more evident for unpurified MWCNTs.
Our study showed that the main mechanism of effect for both types of MWCNTs is oxidative stress induction. Its intensity is directly related to the amount of metallic impurities in the MWCNTs. The subsequent development of inflammatory responses and enhanced peroxidation cause damage to mitochondria in immunocompetent cells. Unpurified MWCNTs generate more oxidation reactions than the purified CNTs, inducing significant mitochondrial dysfunction. Transcriptome analysis showed that activation of the NFkB pathway is the key biological process initiating a cascade of effects. This may cause a perturbation of the IL-6 pathway that aims to regulate the inflammatory processes and compensate apoptotic changes. Overall the immunological responses related to MWCNT exposure are considered as the result of the synergistic effect of systemic (mediated by cells of the routes of exposure) and local inflammation (blood cells).