Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation

Abstract

BackgroundCarbon nanotube (CNT) is used for various industrial purposes, but exhibits carcinogenic effects in experimental animals. Chronic inflammation in the respiratory system may participate in CNT-induced carcinogenesis. 8-Nitroguanine (8-nitroG) is a mutagenic DNA lesion formed during inflammation. We have previously reported that multi-walled CNT (MWCNT) induced 8-nitroG formation in lung epithelial cells and this process involved endocytosis. To clarify the mechanism of CNT-induced carcinogenesis, we examined the role of Toll-like receptor (TLR) 9, which resides in endosomes and lysosomes, in 8-nitroG formation in human lung epithelial cell lines.MethodsWe performed immunocytochemistry to examine 8-nitroG formation in A549 and HBEpC cells treated with MWCNT with a length of 1-2 μm (CNT-S) or 5-15 μm (CNT-L) and a diameter of 20-40 nm. We examined inhibitory effects of endocytosis inhibitors, small interfering RNA (siRNA) for TLR9, and antibodies against high-mobility group box-1 (HMGB1) and receptor for advanced glycation end-products (RAGE) on 8-nitroG formation. The release of HMGB1 and double-stranded DNA (dsDNA) into the culture supernatant from MWCNT-treated cells was examined by ELISA and fluorometric analysis, respectively. The association of these molecules was examined by double immunofluorescent staining and co-immunoprecipitation.ResultsCNT-L significantly increased 8-nitroG formation at 0.05 μg/ml in A549 cells and its intensity reached a maximum at 1 μg/ml. CNT-L tended to induce stronger cytotoxicity and 8-nitroG formation than CNT-S. Endocytosis inhibitors, TLR9 siRNA and antibodies against HMGB1 and RAGE largely reduced MWCNT-induced 8-nitroG formation. MWCNT increased the release of HMGB1 and dsDNA from A549 cells into culture supernatant. The culture supernatant of MWCNT-exposed cells induced 8-nitroG formation in fresh A549 cells. Double immunofluorescent staining and co-immunoprecipitation showed that TLR9 was associated with HMGB1 and RAGE in lysosomes of MWCNT-treated cells.ConclusionsMWCNT induces injury or necrosis of lung epithelial cells, which release HMGB1 and DNA into the extracellular space. The HMGB1-DNA complex binds to RAGE on neighboring cells and then CpG DNA is recognized by TLR9 in lysosomes, leading to generation of nitric oxide and 8-nitroG formation. This is the first study demonstrating that TLR9 and related molecules participate in MWCNT-induced genotoxicity and may contribute to carcinogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0127-7) contains supplementary material, which is available to authorized users.

The role of high‐mobility group protein box 1 in lung cancer

“…[60][61][62][63][64] In addition, TLR9 could recognize DNA, and is generally associated with HMGB1-DNA complex induced nucleotide immunity, while RAGE could amplify the TLR9dependent signal transduction cascade. [65][66][67] However, HMGB1-TLR9 promotes mitochondrial biogenesis in hepatocellular carcinoma to induce tumor growth mediated by hypoxia. 68…”

“…HMGB1-TLR9 could promote mitochondrial biogenesis in hepatocellular carcinoma to induce tumor growth mediated by hypoxia. 20,[60][61][62][63][64][65][66][67][68] TIM-3 Inhibits nucleic acid-mediated antitumor immunity in DCs. 19…”

Establishment of an in vivo simulating co‐culture assay platform for genotoxicity of multi‐walled carbon nanotubes

“…It has recently been shown that MWCNT is incorporated into A549 cells via endocytosis, and leads to inflammatory reactions and induces RNOS . However, Hiraku et al demonstrate that MWCNT induces RNOS and 8‐nitro‐dG, but not in A549 cells, and indicate that the cell culture conditions may alter the balance of the generation of hydroxyl radicals and nitric oxide. It should be considered, therefore, that ROS‐originated and RNOS‐originated oxidative stresses are both involved in the (geno)toxicity of MWCNT and ENM in general.…”

“…The G to A transition is commonly observed in spontaneously‐occurring and chemically‐induced mutants, and the deamination of 5‐methylcytosine or the alkylation of guanine are its potential origin . Nitric oxide induces DNA damage by producing dinitrogen trioxide and then diazonium ions lead to the DNA deamination and 8‐nitro‐dG formation, and 8‐nitro‐dG preferentially causes the G:C to A:T transition through production of an apurinic site . However, nitric oxide is produced by activated macrophages in inflamed organs in vivo.…”

<p>Toxicity of Carbon Nanotubes as Anti-Tumor Drug Carriers</p>

“…Excessive ROS levels can trigger DNA strand breakage, 90 protein-peptide chain disruption, 91 lipid peroxidation 92 and other macromolecular damage, eventually leading to cell death. 93,94 The main targets of CNT cytotoxicity are cell membrane, lysosome, mitochondria, nucleus and cytoskeleton (such as actin). Damage to these structures induces loss of phagocytic ability, release of ROS, and injury to normal tissues.…”