Multi-walled carbon nanotube physicochemical properties predict pulmonary inflammation and genotoxicity

Figures & data

Table 1. Overview of selected physicochemical characteristics of the studied MWCNT and reference materials.

MWCNT group	Code	Type	Source	Length# nm (±SD)	Diameter# nm (±SD)	OH (mmol/g)	BET (m^2/g)	Endotoxin EU/mg	Fe2O3* content	CoO* content	NiO* content	MgO* content	MnO* content
GROUP I	NRCWE-040	PRISTINE	Cheaptubes	518.9 (±598)	20.56 (±6.94)	0.35	150	0.01	0.2	0.001	0.56	0.01	0.002
	NRCWE-041	OH	Cheaptubes	1005 (±2948)	26.38 (±11.08)	1.69	152	0.18	0.13	0.001	0.31	0.02	0.001
	NRCWE-042	COOH	Cheaptubes	723.2 (±971.9)	20.5 (±5.32)	4.09	141	–	0.08	0	0.21	0.03	0.001
GROUP II	NRCWE-043	PRISTINE	Cheaptubes	771.3 (±3471)	26.73 (±6.88)	0.18	82	0.26	0.008	0.001	1.2	0.01	–
	NRCWE-044	OH	Cheaptubes	1330 (±2454)	32.55 (±14.4)	0.23	74	0.25	0.004	0.002	1.04	0.02	–
	NRCWE-045	COOH	Cheaptubes	1553 (±2954)	28.07 (±13.85)	0.63	119	0.27	1.17	0.25	1.34	0.02	0.002
GROUP III	NRCWE-046	PRISTINE	Cheaptubes	717.2 (±1214)	17.22 (±5.77)	0.63	223	0.34	0.008	0.25	0.0045	0.22	0.3
	NRCWE-047	OH	Cheaptubes	532.5 (±591.9)	12.96 (±4.44)	0.26	216	0.19	0.007	0.25	0.0043	0.22	0.3
	NRCWE-048	COOH	Cheaptubes	1604 (±5609)	15.08 (±4.69)	0.58	185	0.01	0.007	0.24	0.0037	0.19	0.28
	NRCWE-049	NH2	Cheaptubes	731.1 (±1473)	13.85 (±6.09)	0.33	199	0.03	0.004	0.25	0.0038	0.19	0.29
Ref. Mat.	Printex 90	–	Evonik	ND	9	ND	182	0.05	0.006	–	0.0003	–	–
	Crocidolite	–	Leibniz IUF	90% < 4500£	90% < 460£	ND	5.24	0.37	7.23$	-$	-$	0.19$	0.03$

Detailed data published in Jackson et al. (2015). *Determined by WDXRF. Chemical composition data were calculated wt% of the oxides of the elements determined. All Fe is calculated as Fe³⁺. #Determined by computerized image analysis of SEM micrographs. $: Meassured in 2% serum; £: Detailed data published by Muhle et al. (1987); –: Not detected; ND: Not determined.

Table 2. Pearson’s Correlations of physicochemical parameters.

	Length	Diameter	BET	Mn	Mg	Ni	Co	Fe	OH
Length	1	0.3245	−0.3235	0.1299	0.0413	0.0351	−0.0242	−0.0501	0.0738
Diameter	0.3245	1	−0.8758	−0.8904	−0.8390	0.9095	−0.8786	0.4318	0.0272
BET	−0.3235	−0.8758	1	0.9431	0.7794	−0.8584	0.7648	−0.1349	0.2524
Mn	0.1299	−0.8904	0.9431	1	0.9492	−0.9803	0.9968	−0.8854	−0.6741
Mg	0.0413	−0.8390	0.7794	0.9492	1	−0.9801	0.9881	−0.6326	−0.022
Ni	0.0351	0.9095	−0.8584	−0.9803	−0.9801	1	−0.9805	0.5421	0.0190
Co	−0.0242	−0.8786	0.7648	0.9968	0.9881	−0.9805	1	−0.6471	0.0188
Fe	−0.0501	0.4318	−0.1349	−0.8854	−0.6326	0.5421	−0.6471	1	0.5952
OH	0.0738	0.0272	0.2524	−0.6741	−0.022	0.0190	0.0188	0.5952	1

All variables were log-transformed. Pairwise correlated parameters with correlation coefficiencies more than 0.75 or less than −0.75 are highlighted in bold.

Figure 1. H&E stained histopathological lung sections of mice exposed to 0 or 54 μg MWCNT. (A–F) Representative images of the most predominant histological changes. (G–L) Representative images of Group I MWCNT on day 28 (G–I) and 92 (J–L). Lymphocytic aggregates (Lycy) and foreign material-containing alveolar macrophages where found in all groups of mice exposed to 54 μg MWCNT and are commonly marked with blue and red arrows, respectively, across all images. Objectives are 10× unless otherwise stated. (A) Negative control on day 92. (B) NRCWE-041 on day 92. MWCNT are mainly localized in alveolar macrophages (red arrows). Objective 20×. (C) NRCWE-040 on day 92. Non-dust granuloma (black arrow). Non-collagenous, slight thickening of alveolar ducts and alveolar walls, with hyperplasia of type II cells (green arrows). Objective 20×. (D) NRCWE-040 on day 92. Small CNT-laden (foreign body) granulomas (black arrow). Small perivascular Lycy (blue arrow). (E and F) NRCWE-045 on day 28. Small area of perivascular Lycy (blue arrows). Focal alveolitis (green arrows). For image E the objective was 2×. (G) NRCWE-040 on day 28. (H) NRCWE-041 a on day 28. Slight focal alveolitis, with inflammatory cells and debris in alveoli (green arrow). (I) NRCWE-042 on day 28. (J) NRCWE-040 on day 92. (K) NRCWE-041 on day 92. (L) NRCWE-042 on day 92.

Figure 2. Total number of neutrophils in the BAL fluid after exposure to MWCNT and reference materials. Error bars indicate SD. (A) Day 1. (B) Day 28. (C) Day 92. *p < 0.05, **p < 0.01, ***p < 0.001 compared to vehicle controls. †: 54 > 6 μg/ml; ‡: 54 > 6 and 18 μg/ml; x: 18 > 6 μg/ml; ^: higher than the –OH form; ♦: higher than Group I; ♠: higher than Group II; ♣: higher than Group III.

Figure 3. The relationship between total number of neutrophils in the BAL fluid and the total instilled surface area on day 1. Individual data points are the average total neutrophil numbers for each exposure group. The linear relationship, the R² and the p value for the correlation are shown for all correlations. (A) All MWCNT types and doses. (B) All MWCNT types at dose 6 μg/mouse. (C) All MWCNT types at dose 18 μg/mouse. (D) All MWCNT types at dose 54 μg/mouse.

Table 3. Multiple regression analyses of MWCNT physicochemical properties as predictors of neutrophil influx and DNA damage.

Neutrophil influx						DNA damage
In bronchoalveolar lavage						In bronchoalveolar lavage
Day	Exposure variable	Multiplicative effect	LowerCL	UpperCL	Probt	Day	Exposure variable	Multiplicative effect	LowerCL	UpperCL	Probt
1	Per doubling in dose	2.763	2.286	3.199	<0.0001	1	Per doubling in dose	1.013	0.988	1.037	0.307
	Per doubling in Fe2O3	0.927	0.858	1.002	0.0562		Per doubling in Fe2O3	1.006	0.994	1.019	0.309
	Per doubling in OH	0.869	0.718	1.053	0.151		Per doubling in OH	1.028	0.998	1.060	0.069
	Per doubling in length	1.164	0.776	1.746	0.462		Per doubling in length	1.051	0.989	1.117	0.108
	Per 25% difference in BET	1.212	1.070	1.373	0.003		Per 25% difference in BET	0.884	0.864	0.904	<.0001
28	Per doubling in dose	4.342	3.360	5.611	<0.0001	In lung tissue
	Per doubling in Fe2O3	0.951	0.832	1.086	0.457
	Per doubling in OH	0.394	0.278	0.558	<0.0001	1	Per doubling in dose	0.999	0.965	1.034	0.948
	Per doubling in length	3.537	1.782	7.020	0.0003		Per doubling in Fe2O3	0.999	0.982	1.018	0.999
	Per 25% difference in BET	1.908	1.528	2.383	<0.0001		Per doubling in OH	1.057	1.010	1.106	0.016
							Per doubling in length	0.870	0.792	0.954	0.003
92	Per doubling in Fe2O3	0.657	0.503	0.858	0.003		Per 25% difference in BET	0.910	0.884	0.937	<.0001
	Per doubling in OH	1.214	0.650	2.266	0.538
	Per doubling in length	2.176	0.603	7.850	0.230	28	Per doubling in dose	1.022	0.989	1.056	0.196
	Per 25% difference in BET	3.053	1.942	4.800	<0.0001		Per doubling in Fe2O3	1.009	0.992	1.026	0.324
							Per doubling in OH	1.052	1.008	1.097	0.020
							Per doubling in length	1.094	1.003	1.193	0.044
							Per 25% difference in BET	0.962	0.935	0.989	0.006

Left: Physicochemical parameters and their influence on neutrophil influx in BAL after intratracheal exposure to MWCNT in a multiple regression analysis. Right: Physicochemical parameters and their influence on DNA damage in BAL and lung tissue after intratracheal exposure to MWCNT in a multiple regression analysis. Significant p values (p ≤ 0.01) are highlighted in bold.

Figure 4. DNA strand breaks in the lung tissue after exposure to MWCNT and reference materials. Error bars indicate SD. (A) Day 1. (B) Day 28. (C) Day 92. *p < 0.05, ***p < 0.001 compared to vehicle controls. #: higher than the pristine form; ♦: higher than Group I; ♣: higher than Group III.

Jackson P, Kling K, Jensen KA, Clausen PA, Madsen AM, Wallin H, Vogel U. 2015. 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. Environ Mol Mutagen 56:183–203

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Muhle H, Pott F, Bellmann B, Takenaka S, Ziem U. 1987. Inhalation and injection experiments in rats to test the carcinogenicity of MMMF. Ann Occup Hyg 31:755–64

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