Physicochemical predictors of Multi-Walled Carbon Nanotube-induced pulmonary histopathology and toxicity one year after pulmonary deposition of 11 different Multi-Walled Carbon Nanotubes in mice

Abstract

Multi-walled carbon nanotubes (MWCNT) are widely used nanomaterials that cause pulmonary toxicity upon inhalation. The physicochemical properties of MWCNT vary greatly, which makes general safety evaluation challenging to conduct. Identification of the toxicity-inducing physicochemical properties of MWCNT is therefore of great importance. We have evaluated histological changes in lung tissue 1 year after a single intratracheal instillation of 11 well-characterized MWCNT in female C57BL/6N BomTac mice. Genotoxicity in liver and spleen was evaluated by the comet assay. The dose of 54 μg MWCNT corresponds to three times the estimated dose accumulated during a work life at a NIOSH recommended exposure limit (0.001 mg/m³ ). Short and thin MWCNT were observed as agglomerates in lung tissue 1 year after exposure, whereas thicker and longer MWCNT were detected as single fibres, suggesting biopersistence of both types of MWCNT. The thin and entangled MWCNT induced varying degree of pulmonary inflammation, in terms of lymphocytic aggregates, granulomas and macrophage infiltration, whereas two thick and straight MWCNT did not. By multiple regression analysis, larger diameter and higher content of iron predicted less histopathological changes, whereas higher cobalt content significantly predicted more histopathological changes. No MWCNT-related fibrosis or tumours in the lungs or pleura was found. One thin and entangled MWCNT induced increased levels of DNA strand breaks in liver; however, no physicochemical properties could be related to genotoxicity. This study reveals physicochemical-dependent difference in MWCNT-induced long-term, pulmonary histopathological changes. Identification of diameter size and cobalt content as important for MWCNT toxicity provides clues for designing MWCNT, which cause reduced human health effects following pulmonary exposure.

Keywords: biodistribution; carbon nanotubes; granuloma; in vivo; lymphocytic aggregate; macrophage infiltration.

Figure 1

Lung sections from mice one year after instillation of 54 μg/animal of MWCNT: (A) NM‐403 and (B) NRCWE‐006 (Mitsui MWNT‐7). The most common histopathological findings were foreign material granuloma (Ai), macrophage infiltrates (Aii, green arrows) and lymphocytic aggregates (Aiii, blue arrows). MWCNT were observed as black aggregates in granuloma (Ai) and in macrophages (Aii), except for NRCWE‐006 (and NM‐401, not shown) which were distributed as single fibres throughout the lung, detectable with enhanced darkfield (Bi‐Bii, white arrows). H&E stain

Figure 2

Lung sections one year after instillation of 0 (A) or 54 μg/animal (B) of MWCNT NRCWE‐042: Vehicle control showed normal lung structure (Ai‐Aiii). NRCWE‐042 exposure gave lymphocytic aggregates (Bi, blue arrows). MWCNT were observed as black aggregates in macrophages (Bii, Biii, green arrows). H&E stain

Figure 3

Enhanced darkfield of liver sections one year after instillation of MWCNT: NRCWE‐006 (Mitsui MWNT‐7) (A) and NM‐401 (B). MWCNT were distributed as single fibres throughout the liver (white arrows). H&E stain

Figure 4

Rare histopathological findings: Fibrosis and tumours: (A,B) Immature connective tissue in NM‐400 exposed mouse (Sirius Red–stained lung) and (C) adenoma in NM‐401 exposed mouse (H&E‐stained lung)