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MILPAAHEL-Miljøpåvirkning og helse

Inflammatory, genotoxic and tumorigenic effects of multi-walled carbon nanotubes with emphasis on the interleukin-1 family

Awarded: NOK 3.0 mill.

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Project Period:

2011 - 2015


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1) In vivo animal studies in IL1-KO mice We have been working on the 28 days exposure study performed in the IL1-WT and IL1-KO mice last year. We have extracted total RNA from the blood, lungs, liver and pleura. We have investigated the mRNA levels in pools from lung, pleura and blood on a Mouse Fibrosis PCR array to find genes with an extraordinary regulation. 13 genes involved in fibrosis were choosen for further work. We have made primers for several of these genes and are currently running them on qpCR on individual samples from each mouse. In addition we have run serum samples from these mice on a cytokine array to detect protein levels by Luminex technology. Samples from the different tissues were also taken for histology and we are currently investigating these. The work on this part of the project has been delayed due to prioritization of the long-term tumorigenesis mouse study. If the finances allow for it, a PCR array for miRNA will be used to investigate the expression of miRNA in the samples. Finally, the data from qPCR, protein levels in serum and histology will be summed up in a manuscript that will be published. In may 2015 we injected 192 mice with control media or two doses of two types of CNTs for the long-term tumorigenesis study. 8-10 week old wildtype (N=91) and IL1-KO (N=91) were divided into three groups for each genotype. Group 1 (N=38) were injected in the pleura with either 5 µg/mouse (n=19) or 50 µg/mouse (n=19) of Mitsui-7 CNTs; Group 2 (N=38) were injected in the pleura with either 5 µg/mouse (n=19) or 50 µg/mouse (n=19) of NM401 CNTs and Group 3 (N=15) were injected in the pleura with only the dispersion solution. The mice were observed several times per week and upon any signs of disease/discomfort they were sacrificed for the experiment. Blood and samples from spleen, liver, kidney, pleura, lungs and heart were taken from each mouse for RNA extraction and for histology. After six months most of the mice injected with the high dosage (50 ug) showed signs of disease and it was decided to stop the experiment. During November all animals were sacrificed and samples taken. Experience and data from the short-term experiment will aid in analysis of the samples from the long-term study. This will eventually lead to a publication. 2) In vitro exposure of IL1-KO mouse cells We used mouse cells from the Balb/c IL1-KO mice lacking both IL1 genes. Upon exposure to the two different CNTs the IL1-KO cells were less sensitive than the IL1-WT cells. We also observed that the amount of cells undergoing apoptosis was dependent on the presence of IL1. In addition the levels of the mitogen-activated protein kinases extracellular regulated kinase (ERK) and c-Jun N terminal kinase (JNK) were regulated differently. The paper showing these results was accepted for publication in Cytokine and was published in May 2015. We have continued working on gap junctional intercellular communication in the same cell types. The scrape loading technique showed that intercellular communication decreased after CNT exposure and that this was independent of IL1 or the type of CNT. The three different connexins Cx43 (Gja1 in mice), Cx26 (Gjb2 in mice) and Cx32 (Gjb1 in mice) were regulated in a different manner on the mRNA and protein level as investigated by qPCR, western blotting and immunofluorescence staining followed by laser scanning microscopy. As both CNTs are cytotoxic to the cells used in this study and reduce cell survival, we hypothesize that the reduction in intercellular communication may be important for inhibiting transfer of cell survival signals between cells. These data have been summed up in a manuscript which is submitted to Nanotoxicology. 3) Exposure of human lung epithelial cells We have obtained data on the toxicity of n-Tec and Mitsui-7 and have further investigated the gene and protein expression of the connexin genes Cx43, Cx26 and Cx32 which are important for intercellular communication. The scrape loading technique and laser scanning microscopy have been used to detect communication between the cells. The data obtained require verification until a manuscript can be written. We have also made and verified a stable IL1-knock down human lung cell line. For these cells we are planning to do COMET assay after exposure to the CNTs. However, the work on the human lung cells has been delayed due to prioritization of the mouse long-term tumorigenesis in vivo experiment. We are, however, planning to complete these experiments in 2016.

The engineered nanomaterials (ENM), including carbon nanotubes, are becoming a part of our daily life as they are used in manufacturing of hundreds of products in food packaging, sporting, cosmetics, clothing, electronics, aerospace, and for medical purpo ses. The ENM may also enter the environment through various routs such as waste release by production industries or by personal and health care consumer products. The exposure of humans to ENM may occur through inhalation, ingestion, dermal contact and sy stemic administration. There is growing concern about the risk and hazards posed by engineered carbon nanotubes (CNT) as these are the most common ENMs found in consumer products. Potential human adverse health effects include lung fibrosis, cancer, cardi ovascular diseases and enhanced allergy where inflammation plays a key role. The main goal of the proposed project is to investigate the hazard posed by the engineered multi-walled CNT (MWCNT) as these are widely manufactured and used in several nanoprodu cts. In aim I, we will specifically study MWCNT related 1) inflammation 2) genotoxicity and 3) tumorigenesis in a mouse model lacking the proinflammatory gene interleukin-1. In aim I we will use two MWCNTs to expose the IL-1 KO and WT mice in order to stu y: Tumor induction, inflammatory biomarkers, genotoxic/mutagenic effects and non-genotoxic effects effects. In aim II we will expose WT and IL-1 deficient human lung cells to study cellular and molecular mechanisms including 1) Genotoxic/mutagenic effetcs such as gene and miRNA expression changes, DNA damage /DNA adducts, mutations/chromosome aberrations, protein modifications, inflammatory biomarkers 2) Non-genotoxic effects such as DNA methylation, histone acetylation and cell-cell communication. Togeth er, the results generated by the proposed project will generate useful knowledge for safe handling of products and materials containing MWCNT and may have positive effects on decisions by regulatory authorities.

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MILPAAHEL-Miljøpåvirkning og helse