In vitro-in vivo correlations of pulmonary inflammogenicity and genotoxicity of MWCNT

Abstract

Background Multi-walled carbon nanotubes (MWCNT) have received attention due to extraordinary properties, resulting in concerns for occupational health and safety. Costs and ethical concerns of animal testing drive a need for in vitro models with predictive power in respiratory toxicity. The aim of this study was to assess pro-inflammatory response (Interleukin-8 expression, IL-8) and genotoxicity (DNA strand breaks) caused by MWCNT with different physicochemical properties in different pulmonary cell models and correlate these to previously published in vivo data. Seven MWCNT were selected; two long/thick (NRCWE-006/Mitsui-7 and NM-401), two short/thin (NM-400 and NM-403), a pristine (NRCWE-040) and two surface modified; hydroxylated (NRCWE-041) and carboxylated (NRCWE-042). Carbon black Printex90 (CB) was included as benchmark material. Human alveolar epithelial cells (A549) and monocyte-derived macrophages (THP-1a) were exposed to nanomaterials (NM) in submerged conditions, and two materials (NM-400 and NM-401) in co-cultures of A549/THP-1a and lung fibroblasts (WI-38) in an air-liquid interface (ALI) system. Effective doses were quantified by thermo-gravimetric-mass spectrometry analysis (TGA-MS). To compare genotoxicity in vitro and in vivo, we developed a scoring system based on a categorization of effects into standard deviation (SD) units (< 1, 1, 2, 3 or 4 standard deviation increases) for the increasing genotoxicity. Results Effective doses were shown to be 25 to 53%, and 21 to 57% of the doses administered to A549 and THP-1a, respectively. In submerged conditions (A549 and THP-1a cells), all NM induced dose-dependent IL-8 expression. NM-401 and NRCWE-006 caused the strongest pro-inflammatory response. In the ALI-exposed co-culture, only NM-401 caused increased IL-8 expression, and no DNA strand breaks were observed. Strong correlations were found between in vitro and in vivo inflammation when doses were normalized by surface area (also proxy for diameter and length). Significantly increased DNA damage was found for all MWCNT in THP-1a cells, and for short MWCNT in A549 cells. A concordance in genotoxicity of 83% was obtained between THP-1a cells and broncho-alveolar lavaged (BAL) cells. Conclusion This study shows correlations of pro-inflammatory potential in A549 and THP-1a cells with neutrophil influx in mice, and concordance in genotoxic response between THP-1a cells and BAL cells, for seven MWCNT.

Multiwalled Carbon Nanotubes Induce Fibrosis and Telomere Length Alterations

“…Moreover, NM-401-exposed HBEC-3KT cells revealed alteration in expression of several genes involved in fibrosis and inflammation, such as tumour necrosis factor ( TNF ), interleukin 8 ( IL8 ), C-X-C motif chemokine 2 ( CXCL2 ), gelatinase ( MMP2 ), and C-C motif chemokine 3 ( CCL3 ). In agreement with our results, IL8 , which is an early pro-inflammatory biomarker, was highly expressed during nanomaterial-induced inflammation [ 10 , 25 ]. Previous studies reported that exposure to MWCNT induced persistent and chronic inflammation and fibrotic lesions in lungs, which coincide with deregulation of a number of genes associated with hallmarks of cancer [ 3 , 25 , 26 ].…”

“…In agreement with our results, IL8 , which is an early pro-inflammatory biomarker, was highly expressed during nanomaterial-induced inflammation [ 10 , 25 ]. Previous studies reported that exposure to MWCNT induced persistent and chronic inflammation and fibrotic lesions in lungs, which coincide with deregulation of a number of genes associated with hallmarks of cancer [ 3 , 25 , 26 ]. However, while NM-401-exposure provided similar effects in animals and HBEC-3KT cells, no effects were observed in the mesothelial Met-5A MWCNT-exposed cells, except for an upregulation of IL8 .…”

“…Critically short telomers result in activation of DNA damage response proteins, such as the phosphorylated forms of ataxia telangiectasia mutated (ATM), H2A histone family member X (H2A.X), and RAD17 checkpoint clamp loader component (RAD17) [ 34 ]. While many studies have shown that exposure to MWCNT leads to increased levels of DNA strand breaks and increased DNA repair activity and DNA damage [ 9 , 19 , 25 ], other studies did not observe genotoxicity following MWCNT-exposure [ 5 , 35 , 36 ]. In this study, we did not observe a regulation of genes involved in DNA damage or repair (i.e., ATM , ATR , RAD17 , NIEL3 , XPA , ALKBH1 , ALKBH5 , or OGG1 ) in HBEC-3KT cells exposed to NM-401.…”

Genotoxicity of Particles From Grinded Plastic Items in Caco-2 and HepG2 Cells

“…However, it should also be noted that we have used dispersion protocols with high-energy sonication that favors stable particle suspensions, whereas the sedimentation rate of particles might be lower. Using carbon-based nanomaterials, we have observed that approximately 10% of the administered dose deposits on cells at the bottom of the cell culture wells ( 90 , 91 ). Longer incubation time increases the deposition, but it does not necessarily increase the level of DNA damage after particle exposure, which may be due to concurrent repair of DNA lesions.…”

TFEB-lysosome pathway activation is associated with different cell death responses to carbon quantum dots in Kupffer cells and hepatocytes

“…A recent study found that carbon-based nanomaterials can induce chronic inflammation, interfering with endocrine functioning and xenobiotic metabolism [ 22 ], and causing fibrosis and carcinogenesis. The toxicity of carbon-based materials is mainly attributed to the generation of reactive oxygen species (ROS) triggered by carbon-based materials [ 23 ], resulting in DNA damage [ 24 ], mitochondrial dysfunction, inflammation, and apoptosis [ 25 ]. Cell damage eventually leads to different types of cell death, of which apoptosis is the primary mode and considered an important target of anti-tumor therapy [ 26 ].…”