Multi-walled carbon nanotube-induced genotoxic, inflammatory and pro-fibrotic responses in mice: Investigating the mechanisms of pulmonary carcinogenesis.

The International Agency for Research on Cancer has classified one type of multi-walled carbon nanotubes (MWCNTs) as possibly carcinogenic to humans. However, the underlying mechanisms of MWCNT- induced carcinogenicity are not known. In this study, the genotoxic, mutagenic, inflammatory, and fibrotic potential of MWCNTs were investigated. Muta™Mouse adult females were exposed to 36±6 or 109±18μg/mouse of Mitsui-7, or 26±2 or 78±5μg/mouse of NM-401, once a week for four consecutive weeks via intratracheal instillations, alongside vehicle-treated controls. Samples were collected 90days following the first exposure for measurement of DNA strand breaks, lacZ mutant frequency, p53 expression, cell proliferation, lung inflammation, histopathology, and changes in global gene expression. Both MWCNT types persisted in lung tissues 90days post-exposure, and induced lung inflammation and fibrosis to similar extents. However, there was no evidence of DNA damage as measured by the comet assay following Mitsui-7 exposure, or increases in lacZ mutant frequency, for either MWCNTs. Increased p53 expression was observed in the fibrotic foci induced by both MWCNTs. Gene expression analysis revealed perturbations of a number of biological processes associated with cancer including cell death, cell proliferation, free radical scavenging, and others in both groups, with the largest response in NM-401-treated mice. The results suggest that if the two MWCNT types were capable of inducing DNA damage, strong adaptive responses mounted against the damage, resulting in efficient and timely elimination of damaged cells through cell death, may have prevented accumulation of DNA damage and mutations at the post-exposure time point investigated in the study. Thus, MWCNT-induced carcinogenesis may involve ongoing low levels of DNA damage in an environment of persisting fibres, chronic inflammation and tissue irritation, and parallel increases or decreases in the expression of genes involved in several pro-carcinogenic pathways.

[1]  H. C. Budtz,et al.  Acute and subacute pulmonary toxicity and mortality in mice after intratracheal instillation of ZnO nanoparticles in three laboratories. , 2015, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[2]  U. Vogel,et al.  Validation of freezing tissues and cells for analysis of DNA strand break levels by comet assay , 2013, Mutagenesis.

[3]  Cationic nanoparticles directly bind angiotensin-converting enzyme 2 and induce acute lung injury in mice , 2015, Particle and Fibre Toxicology.

[4]  Vicki Stone,et al.  Nuclear translocation of Nrf2 and expression of antioxidant defence genes in THP-1 cells exposed to carbon nanotubes. , 2010, Journal of biomedical nanotechnology.

[5]  S. Fukushima,et al.  Genotoxicity and Cytotoxicity of Multi‐wall Carbon Nanotubes in Cultured Chinese Hamster Lung Cells in Comparison with Chrysotile A Fibers , 2010, Journal of occupational health.

[6]  Kurt Straif,et al.  Carcinogenicity of fluoro-edenite, silicon carbide fibres and whiskers, and carbon nanotubes. , 2014, The Lancet. Oncology.

[7]  A. Pant,et al.  Multi-walled carbon nanotubes induce oxidative stress and apoptosis in human lung cancer cell line-A549 , 2011, Nanotoxicology.

[8]  Per Axel Clausen,et al.  Multi-walled carbon nanotube physicochemical properties predict pulmonary inflammation and genotoxicity , 2016, Nanotoxicology.

[9]  M Methner,et al.  Field Application of the Nanoparticle Emission Assessment Technique (NEAT): Task-Based Air Monitoring During the Processing of Engineered Nanomaterials (ENM) at Four Facilities , 2012, Journal of occupational and environmental hygiene.

[10]  U. Vogel,et al.  Time-Dependent Subcellular Distribution and Effects of Carbon Nanotubes in Lungs of Mice , 2015, PloS one.

[11]  G. Douglas,et al.  Characterisation of Muta™Mouse λgt10-lacZ transgene: evidence for in vivo rearrangements , 2010, Mutagenesis.

[12]  Vincent Castranova,et al.  Extrapulmonary transport of MWCNT following inhalation exposure , 2013, Particle and Fibre Toxicology.

[13]  Douglas E. Evans,et al.  Exposure and emissions monitoring during carbon nanofiber production--Part I: elemental carbon and iron-soot aerosols. , 2011, The Annals of occupational hygiene.

[14]  R. Marcos,et al.  Multi-walled carbon nanotubes (NM401) induce ROS-mediated HPRT mutations in Chinese hamster lung fibroblasts. , 2016, Environmental research.

[15]  Heinrich Ernst,et al.  The carcinogenic effect of various multi-walled carbon nanotubes (MWCNTs) after intraperitoneal injection in rats , 2014, Particle and Fibre Toxicology.

[16]  Nianqiang Wu,et al.  Acute pulmonary dose–responses to inhaled multi-walled carbon nanotubes , 2012, Nanotoxicology.

[17]  D. Kufe,et al.  Mucins in cancer: function, prognosis and therapy , 2009, Nature Reviews Cancer.

[18]  Yi Cao,et al.  Vascular effects of multiwalled carbon nanotubes in dyslipidemic ApoE-/- mice and cultured endothelial cells. , 2014, Toxicological sciences : an official journal of the Society of Toxicology.

[19]  Y. Cho,et al.  S-adenosylmethionine reduces airway inflammation and fibrosis in a murine model of chronic severe asthma via suppression of oxidative stress , 2016, Experimental & Molecular Medicine.

[20]  Penny Nymark,et al.  In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes , 2016, Nanotoxicology.

[21]  F. Nesslany,et al.  Lung inflammation and lack of genotoxicity in the comet and micronucleus assays of industrial multiwalled carbon nanotubes Graphistrength© C100 after a 90-day nose-only inhalation exposure of rats , 2015, Particle and Fibre Toxicology.

[22]  Dale W Porter,et al.  Genotoxicity of multi-walled carbon nanotubes at occupationally relevant doses , 2014, Particle and Fibre Toxicology.

[23]  M. Birch.  Exposure and emissions monitoring during carbon nanofiber production--Part II: polycyclic aromatic hydrocarbons. , 2011, The Annals of occupational hygiene.

[24]  A. T. Saber,et al.  Biodistribution of Carbon Nanotubes in Animal Models , 2017, Basic & clinical pharmacology & toxicology.

[25]  Maumita Bandyopadhyay,et al.  Multi-walled carbon nanotubes (MWCNT): induction of DNA damage in plant and mammalian cells. , 2011, Journal of hazardous materials.

[26]  I. Kennedy,et al.  Novel lanthanide-labeled metal oxide nanoparticles improve the measurement of in vivo clearance and translocation , 2013, Particle and Fibre Toxicology.

[27]  L. Garfinkel,et al.  Scar cancer of the lung. Increase over a 21 year period , 1979, Cancer.

[28]  J. Kanno,et al.  Dose-dependent mesothelioma induction by intraperitoneal administration of multi-wall carbon nanotubes in p53 heterozygous mice , 2012, Cancer science.

[29]  K. Wakabayashi,et al.  Genotoxicity of multi-walled carbon nanotubes in both in vitro and in vivo assay systems , 2013, Nanotoxicology.

[30]  G. Bae,et al.  Exposure assessment of carbon nanotube manufacturing workplaces , 2010, Inhalation toxicology.

[31]  M. Copin,et al.  Induction of MUC2 and MUC5AC Mucins by Factors of the Epidermal Growth Factor (EGF) Family Is Mediated by EGF Receptor/Ras/Raf/Extracellular Signal-regulated Kinase Cascade and Sp1* , 2002, The Journal of Biological Chemistry.

[32]  H. van Steeg,et al.  Mechanisms of non-genotoxic carcinogens and importance of a weight of evidence approach. , 2009, Mutation research.

[33]  Hai Lin,et al.  Lessons Learned from Whole Exome Sequencing in Multiplex Families Affected by a Complex Genetic Disorder, Intracranial Aneurysm , 2015, PloS one.

[34]  Akihiko Hirose,et al.  Induction of mesothelioma by a single intrascrotal administration of multi-wall carbon nanotube in intact male Fischer 344 rats. , 2009, The Journal of toxicological sciences.

[35]  Vicki Stone,et al.  Review of fullerene toxicity and exposure--appraisal of a human health risk assessment, based on open literature. , 2010, Regulatory toxicology and pharmacology : RTP.

[36]  Gwi-Nam Bae,et al.  Monitoring Multiwalled Carbon Nanotube Exposure in Carbon Nanotube Research Facility , 2008 .

[37]  Michael A. Hollingsworth,et al.  Mucins in cancer: protection and control of the cell surface , 2004, Nature Reviews Cancer.

[38]  M. Jordana,et al.  A Mouse Model of Airway Disease: Oncostatin M-Induced Pulmonary Eosinophilia, Goblet Cell Hyperplasia, and Airway Hyperresponsiveness Are STAT6 Dependent, and Interstitial Pulmonary Fibrosis Is STAT6 Independent , 2011, The Journal of Immunology.

[39]  Peter Morfeld,et al.  Response to the Reply on behalf of the ‘Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area’ (MAK Commission) by Andrea Hartwig Karlsruhe Institute of Technology (KIT) , 2015, Particle and Fibre Toxicology.

[40]  U. Vogel,et al.  Modest effect on plaque progression and vasodilatory function in atherosclerosis-prone mice exposed to nanosized TiO2 , 2011, Particle and Fibre Toxicology.

[41]  Andrew Williams,et al.  Subchronic Oral Exposure to Benzo(a)pyrene Leads to Distinct Transcriptomic Changes in the Lungs That Are Related to Carcinogenesis , 2012, Toxicological sciences : an official journal of the Society of Toxicology.

[42]  P. Jeggo,et al.  DNA repair, genome stability and cancer: a historical perspective , 2015, Nature Reviews Cancer.

[43]  Masanobu Komatsu,et al.  Muc4/sialomucin complex, an intramembrane modulator of ErbB2/HER2/Neu, potentiates primary tumor growth and suppresses apoptosis in a xenotransplanted tumor , 2001, Oncogene.

[44]  U. Vogel,et al.  Intratracheally instilled titanium dioxide nanoparticles translocate to heart and liver and activate complement cascade in the heart of C57BL/6 mice , 2015, Nanotoxicology.

[45]  Yong Qian,et al.  Multiwalled Carbon Nanotube-Induced Gene Signatures in the Mouse Lung: Potential Predictive Value for Human Lung Cancer Risk and Prognosis , 2012, Journal of toxicology and environmental health. Part A.

[46]  V. Skaug,et al.  Detection of Multi-walled Carbon Nanotubes and Carbon Nanodiscs on Workplace Surfaces at a Small-Scale Producer. , 2015, The Annals of occupational hygiene.

[47]  M. J. Silva,et al.  Evaluation of the cytotoxic and genotoxic effects of benchmark multi-walled carbon nanotubes in relation to their physicochemical properties. , 2016, Toxicology letters.

[48]  I. Lambert,et al.  Detailed review of transgenic rodent mutation assays. , 2005, Mutation research.

[49]  Brad T. Sherman,et al.  Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists , 2008, Nucleic acids research.

[50]  P. Clavien,et al.  Blocking the path to death: anti-apoptotic molecules in ischemia/reperfusion injury of the liver. , 2006, Current pharmaceutical design.

[51]  John B. Shoven,et al.  I , Edinburgh Medical and Surgical Journal.

[52]  Craig A. Poland,et al.  Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. , 2008, Nature nanotechnology.

[53]  Antonio Marcomini,et al.  Genotoxicity, cytotoxicity, and reactive oxygen species induced by single‐walled carbon nanotubes and C60 fullerenes in the FE1‐Muta™Mouse lung epithelial cells , 2008, Environmental and molecular mutagenesis.

[54]  Joana M. Xavier,et al.  The role of p53 in apoptosis. , 2010, Discovery medicine.

[55]  J. Kanno,et al.  Induction of mesothelioma in p53+/- mouse by intraperitoneal application of multi-wall carbon nanotube. , 2008, The Journal of toxicological sciences.

[56]  N. Chatterjee,et al.  Lung cancer risk following detection of pulmonary scarring by chest radiography in the prostate, lung, colorectal, and ovarian cancer screening trial. , 2008, Archives of internal medicine.

[57]  Per Axel Clausen,et al.  Characterization of genotoxic response to 15 multiwalled carbon nanotubes with variable physicochemical properties including surface functionalizations in the FE1‐Muta(TM) mouse lung epithelial cell line , 2015, Environmental and molecular mutagenesis.

[58]  Canu,et al.  Carcinogenicity of fluoro-edenite, silicon carbide fibres and whiskers, and carbon nanotubes , 2022 .

[59]  Tao Chen,et al.  Genotoxicity of titanium dioxide nanoparticles , 2014, Journal of food and drug analysis.

[60]  Nianqiang Wu,et al.  Mouse pulmonary dose- and time course-responses induced by exposure to multi-walled carbon nanotubes. , 2010, Toxicology.

[61]  S. Toyokuni,et al.  Intraperitoneal administration of tangled multiwalled carbon nanotubes of 15 nm in diameter does not induce mesothelial carcinogenesis in rats , 2013, Pathology international.

[62]  Paul B. Tchounwou,et al.  Study of hepatotoxicity and oxidative stress in male Swiss-Webster mice exposed to functionalized multi-walled carbon nanotubes , 2011, Molecular and Cellular Biochemistry.

[63]  Dongmei Wu,et al.  Transcriptomic Analysis Reveals Novel Mechanistic Insight into Murine Biological Responses to Multi-Walled Carbon Nanotubes in Lungs and Cultured Lung Epithelial Cells , 2013, PloS one.

[64]  M. Pino,et al.  Proliferative and Nonproliferative Lesions of the Rat and Mouse Respiratory Tract , 2009, Toxicologic pathology.

[65]  Thomas A. Wynn,et al.  Integrating mechanisms of pulmonary fibrosis , 2011, The Journal of experimental medicine.

[66]  S. Toyokuni,et al.  Diameter and rigidity of multiwalled carbon nanotubes are critical factors in mesothelial injury and carcinogenesis , 2011, Proceedings of the National Academy of Sciences.

[67]  Jiong Mei,et al.  Femur performed better than tibia in autologous transplantation during hemipelvis reconstruction , 2014, World Journal of Surgical Oncology.

[68]  N. Kayani,et al.  Case control study of novel prognostic markers and disease outcome in pregnancy/lactation-associated breast carcinoma. , 2003, Pathology, research and practice.

[69]  A. MacKinnon,et al.  The molecular and cellular biology of lung cancer: identifying novel therapeutic strategies. , 2010, British medical bulletin.

[70]  B. van Ravenzwaay,et al.  Inhalation toxicity of multiwall carbon nanotubes in rats exposed for 3 months. , 2009, Toxicological sciences : an official journal of the Society of Toxicology.

[71]  Maria João Silva,et al.  Genotoxicity evaluation of nanosized titanium dioxide, synthetic amorphous silica and multi-walled carbon nanotubes in human lymphocytes. , 2014, Toxicology in vitro : an international journal published in association with BIBRA.

[72]  Yong Qian,et al.  System-based identification of toxicity pathways associated with multi-walled carbon nanotube-induced pathological responses. , 2013, Toxicology and applied pharmacology.

[73]  Gilles Patriarche,et al.  Carbon nanotube translocation to distant organs after pulmonary exposure: insights from in situ (14)C-radiolabeling and tissue radioimaging. , 2014, ACS nano.

[74]  X. Cui,et al.  Improved statistical tests for differential gene expression by shrinking variance components estimates. , 2005, Biostatistics.

[75]  Dale W Porter,et al.  Promotion of lung adenocarcinoma following inhalation exposure to multi-walled carbon nanotubes , 2014, Particle and Fibre Toxicology.

[76]  Nicklas Raun Jacobsen,et al.  Mutation spectrum in FE1‐MUTATMMouse lung epithelial cells exposed to nanoparticulate carbon black , 2011, Environmental and molecular mutagenesis.

[77]  Andrew Williams,et al.  MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs. , 2015, Toxicology and applied pharmacology.

[78]  Miss A.O. Penney.  (b) , 1974, The New Yale Book of Quotations.

[79]  Absence of in vivo mutagenicity of multi-walled carbon nanotubes in single intratracheal instillation study using F344 gpt delta rats , 2017, Genes and Environment.

[80]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[81]  J Vijg,et al.  Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutations in vivo. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[82]  K. Shimoi,et al.  Estrogen- and stress-induced DNA damage in breast cancer and chemoprevention with dietary flavonoid , 2017, Genes and Environment.

[83]  Makoto Ohnishi,et al.  Lung carcinogenicity of inhaled multi-walled carbon nanotube in rats , 2015, Particle and Fibre Toxicology.

[84]  Hao Wu,et al.  MAANOVA: A Software Package for the Analysis of Spotted cDNA Microarray Experiments , 2003 .

[85]  P. Baron,et al.  Inhalation vs. aspiration of single-walled carbon nanotubes in C57BL/6 mice: inflammation, fibrosis, oxidative stress, and mutagenesis. , 2008, American journal of physiology. Lung cellular and molecular physiology.

[86]  Mark R. Miller,et al.  Cardiovascular health effects of oral and pulmonary exposure to multi-walled carbon nanotubes in ApoE-deficient mice. , 2016, Toxicology.

[87]  Andrew Williams,et al.  IL-1 Receptor Regulates microRNA-135b Expression in a Negative Feedback Mechanism during Cigarette Smoke–Induced Inflammation , 2013, The Journal of Immunology.

[88]  Martha W. Betz,et al.  Distribution and accumulation of 10 nm silver nanoparticles in maternal tissues and visceral yolk sac of pregnant mice, and a potential effect on embryo growth , 2016, Nanotoxicology.

[89]  Julia Catalán,et al.  Genotoxicity of short single-wall and multi-wall carbon nanotubes in human bronchial epithelial and mesothelial cells in vitro. , 2013, Toxicology.

[90]  Yung‐Chie Lee,et al.  Overexpression of MUC5 genes is associated with early post‐operative metastasis in non‐small‐cell lung cancer , 1996, International journal of cancer.

[91]  U. Gozalan,et al.  Classification of patients with breast cancer according to Nottingham Prognostic Index highlights significant differences in immunohistochemical marker expression , 2014, World Journal of Surgical Oncology.

[92]  Wojciech Zareba,et al.  Ambient fine particulate air pollution triggers ST-elevation myocardial infarction, but not non-ST elevation myocardial infarction: a case-crossover study , 2014, Particle and Fibre Toxicology.

[93]  H. Patel,et al.  Multi-walled carbon nanotube-induced inflammatory response and oxidative stress in a dynamic cell growth environment , 2012, Journal of biological engineering.

[94]  Ivan Rusyn,et al.  Key Characteristics of Carcinogens as a Basis for Organizing Data on Mechanisms of Carcinogenesis , 2015, Environmental health perspectives.

[95]  Y. Takeshima,et al.  Expression of MUC1, MUC2, MUC5AC, and MUC6 in atypical adenomatous hyperplasia, bronchioloalveolar carcinoma, adenocarcinoma with mixed subtypes, and mucinous bronchioloalveolar carcinoma of the lung. , 2004, American journal of clinical pathology.

[96]  Kyunghee Choi,et al.  Pro-inflammatory and potential allergic responses resulting from B cell activation in mice treated with multi-walled carbon nanotubes by intratracheal instillation. , 2009, Toxicology.

[97]  J. Schlager,et al.  Comparative study of the clastogenicity of functionalized and nonfunctionalized multiwalled carbon nanotubes in bone marrow cells of Swiss‐Webster mice , 2010, Environmental toxicology.

[98]  D. Lilley,et al.  DNA Repair , 1998, Nucleic Acids and Molecular Biology.

[99]  Nicklas Raun Jacobsen,et al.  Increased mutant frequency by carbon black, but not quartz, in the lacZ and cII transgenes of muta™mouse lung epithelial cells , 2007, Environmental and molecular mutagenesis.