Pulmonary toxicity of synthetic amorphous silica – effects of porosity and copper oxide doping

Figures & data

Table 1. Representative electron microscopy images and physico-chemical properties of the used test-materials.

Name and description	Representative electron microscopy image	CuO (wt.%)	Water-loss/ Methylation (wt.%)	Specific surface area (m^2/g)	ζ-average (nm) mean and SD of 6 measurements	PDI mean and SD of 6 measurements	ζ--potential (mV)	pH
NRCWE#065 porous silica 300 nm		n.a.	0.72 / 2.1	828	128 µg (2.56 g/L)^a :	128 µg:	−36.5	7.68
					337.7 ± 4.9	0.297 + 0.031
					43 µg 0.86 g/L):	43 µg:
					335.8 ± 9.1	0.377 + 0.032
					14 µg (0.28 g/L):	14 µg:
					281.1 ± 37.3	0.387 + 0.038
NRCWE#066 solid silica 300 nm		n.a.	5.94 / 2.4	10	128 µg (2.56 g/L):	128 µg:	−32.5	7.48
					379.2 ± 61.45	0.464 ± 0.047
					43 µg (0.86 g/L):	43 µg:
					327.5 ± 128.2	0.562 ± 0.054
					14 µg (0.28 g/L):	14 µg:
					316.3 ± 168.8	0.619 ± 0.097
NRCWE#067 porous silica 100 nm		n.a.	0.19 / 1.6	844	128 µg (2.56 g/L):	128 µg:	−32.5	7.59
					247.6 ± 40.44	0.541 ± 0.065
					43 µg (0.86 g/L):	43 µg:
					188.2 ± 31.16	0.524 ± 0.065
					14 µg (0.28 g/L):	14 µg:
					326.6 ± 127.5	0.513 ± 0.082
NRCWE#068 solid silica 100 nm		n.a.	4.35 / 2.8	22	128 µg (2.56 g/L):	128 µg:	−31.1	7.85
					155.2 ± 2.06	0.222 ± 0.014
					43 µg (0.86 g/L):	43 µg:
					163.2 ± 5.64	0.281 ± 0.012
					14 µg (0.28 g/L):	14 µg:
					149.8 ± 7.93	0.248 ± 0.036
NRCWE#069 porous silica 300 nm-CuO doping		4.6	0.26 / 3.0	862	43 µg (0.86 g/L):	43 µg:	−38.6	7.50
					268.8 ± 3.81	0.265 ± 0.023
					14 µg (0.28 g/L):	14 µg:
					254.0 ± 5.98	0.293 ± 0.025
					4.7 µg (0.10 g/L):	4.7 µg:
					218.9 ± 5.31	0.399 ± 0.023
NRCWE#070 porous silica 100 nm-CuO doping		5.3	1.10 / 2.4	747	43 µg (0.86 g/L):	43 µg:	−31.7	7.48
					215.6 ± 62.95	0.465 ± 0.055
					14 µg (0.28 g/L):	14 µg:
					235.3 ± 77.57	0.466 ± 0.061
					4.7 µg (0.10 g/L):	4.7 µg:
					204.7 ± 100.4	0.457 ± 0.023
NRCWE#071 solid silica 300 nm-CuO doping		9.3	4.43 / 6.4	9	14 µg (0.28 g/L):	14 µg:	−50.0	10.30
					587.8 ± 45.92	0.496 ± 0.165
					4.7 µg (0.10 g/L):	4.7 µg:
					466.8 ± 60.91	0.762 ± 0.111
					1.5 µg (0.03 g/L):	1.5 µg
					417.4 ± 148.9	0.731 ± 0.146
NRCWE#072 CuO nanoparticles. 9.8 nm		89.2	4.55 / 0.0	75	4.7 µg (0.10 g/L):	4.7 µg:	−27.2	8.14
					135.4 ± 21.44	0.364 ± 0.036
					1.5 µg (0.03 g/L):	1.6 µg:
					174.9 ± 91.93	0.400 ± 0.055
					0.5 µg (0.01 g/L):	0.5 µg:
					497.5 ± 401	0.673 ± 0.186
Printex 90 Carbon black		N/A	N/A	295 m^2/g in (Saber et al. 2005)	162 µg (3.24 g/L): 93.64 ± 0.69	0.228 ± 0.01	N/A	N/A
					/ 80.05 ± 0.44	/ 0.188 ± 0.013
					(dosed on 2 different days)	(dosed on 2 different days)

PDI is an abbreviation of poly-dispersity index. Scale bar SEM image (NRCWE#71): 1 µm; Scale bar TEM-images (all particles except NRCWE#71): 100 nm; Arrows in NRCWE-069 and NRCWE-070 illustrate the presence of CuO in these samples. CuO is not immediately recognized in the SEM-image of NRCWE-071. ζ-average and PDI are from the suspensions dosed to the animals. Other data are from (Sahlgren et al. in preparation).

^aThe instillation volume was 50 µl.

Table 2. Study doses.

Treatment	Doses	Amount of CuO dosed
Intratracheal instillation study
Control vehicle	0 µg/mouse (0 mg/kg bw)	n/a
NRCWE#065 porous Silica 300 nm	14, 43 and 128 µg/mouse	n/a
	∼0.7, 2.2, and 6.4 mg/kg bw
NRCWE#066 solid silica 300 nm	14, 43 and 128 µg/mouse	n/a
	∼0.7, 2.2, and 6.4 mg/kg bw
NRCWE#067 porous silica 100 nm	14, 43 and 128 µg/mouse	n/a
	∼0.7, 2.2, and 6.4 mg/kg bw
NRCWE#068 solid silica 100 nm	14, 43 and 128 µg/mouse	n/a
	∼0.7, 2.2, and 6.4 mg/kg bw
NRCWE#069 porous silica 300 nm-CuO doping	4.7, 14 and 43 µg/mouse	∼0.01, 0.03 and 0.09 mg/kg bw
	∼0.2, 0.7, and 2.2 mg/kg bw
NRCWE#070 porous silica 100 nm-CuO doping	4.7, 14 and 43 µg/mouse	∼0.01, 0.03 and 0.09 mg/kg bw
	∼0.2, 0.7, and 2.2 mg/kg bw
NRCWE#071 solid silica 300 nm-CuO doping	1.6, 4.7 and 14 µg/mouse	∼0.007, 0.02 and 0.06 mg/kg bw
	∼0.08, 0.2, and 0.7 mg/kg bw
NRCWE#072 CuO nanoparticles	0.5, 1.6 and 4.7 µg/mouse	∼0.02, 0.06 and 0.18 mg/kg bw
	∼0.03, 0.08, and 0.2 mg/kg bw
Positive control: carbon black (CB) Printex 90	162 µg/mouse ∼8.1 mg/kg bw	No copper was detected in a previous study (Jacobsen et al. 2008)
Oropharyngeal aspiration study
NRCWE#065 porous Silica 300 nm	43 and 128 µg/mouse	n/a
	∼2.2, and 6.4 mg/kg bw
NRCWE#066 solid silica 300 nm	43 and 128 µg/mouse	n/a
	∼2.2, and 6.4 mg/kg bw
NRCWE#067 porous silica 100 nm	43 and 128 µg/mouse	n/a
	∼2.2, and 6.4 mg/kg bw
NRCWE#068 solid silica 100 nm	43 and 128 µg/mouse	n/a
	∼2.2, and 6.4 mg/kg bw

Based on a pilot study the intratracheal instillation and oropharyngeal aspiration doses in mice were as shown below.

Figure 1. Neutrophil numbers in bronchoalveolar lavage fluid of mice exposed to the nanomaterials. The upper panel show data from 24 h of exposure and the lower panel from 28 days of exposure. The columns represent means and bars SD. ***, ** and * designates p values of <0.001, <0.01 and <0.05 respectively of one way ANOVA with Holm-Sidak’s multiple comparisons test in case of data approaching normality and not having a highly different variation. In the case of carbon black ***, ** and * designates p values of <0.001, <0.01 and <0.05 respectively vs. vehicle of t-test. ^## and ^# designate Bonferroni-corrected (11 comparisons) p values of <0.01 and <0.05 of unpaired t-test or Mann-Whitney test.

Figure 2. Neutrophil numbers in bronchoalveolar lavage fluid of mice exposed to the nanomaterials by oropharyngeal aspiration. The plots represent neutrophil recruitment to the airways 24 h, 3 and 7 days after the treatment with vehicle or nanomaterial dispersions. Data are mean and bars represent SD. ****, ***, ** and * designates p values of <0.0001, <0.001, <0.01 and <0.05 respectively of one way ANOVA with Holm-Sidak’s multiple comparisons test in case of data approaching normality and not having a highly different variation. In the case of carbon black ***, ** and * designates p values of <0.001, <0.01 and <0.05 respectively vs. vehicle of t-test. ^### and ^## designate Bonferroni-corrected (4 comparisons) p values of <0.001 and <0.01, respectively, of unpaired t-test or Mann-Whitney test.

Figure 3. Neutrophil numbers in bronchoalveolar lavage fluid of mice as function of dosed BET surface area of each nanomaterial. The data on neutrophil numbers from Figure 1. (intratracheal instillation) are here depicted as function of dosed BET surface area per mouse. The upper panel represents data from 24 h of exposure of porous and non-porous particles; and the lower panel data from 24 h of exposure of CuO nanoparticles, porous silica 300 nm CuO and porous silica 300 nm to illustrate the effect of CuO doping. Some error bars are not depicted, because the exponential axis does not accommodate negative values.

Figure 4. Saa3 mRNA level in lungs of mice exposed to silica nanomaterials. Mice were administered the silica materials by intratracheal instillation and 24 h (upper panel), and 28 days (lower panel) later the lungs were recovered, and the Saa3 mRNA level was measured by quantitative real time PCR. Data are mean and bars represent SD. ****, ***, ** and * designates p values of <0.0001, <0.001, <0.01 and <0.05 respectively, of one way ANOVA with Holm-Sidak’s multiple comparisons test in case of data approaching normality and not having a highly different variation. In the case of carbon black ****, and ** designate p values of <0.0001 and <0.01, respectively, vs. vehicle of t-test.

Figure 5. Saa3 mRNA levels in lungs of mice as function of dosed BET surface area of each nanomaterial. The data on Saa3 from Figure 4. are here depicted as function of dosed BET surface area per mouse. The upper panel represents data from 24 h of exposure of porous and non-porous particles; and the lower panel data from 24 h of CuO nanoparticles, porous silica 300 nm CuO and porous silica 300 nm to illustrate the effect of CuO doping. Some error bars are not depicted, as the exponential axis does not accommodate negative values.

Figure 6. Saa3 mRNA levels in lungs, depicted as function of the dosed amount of CuO (current data) or ZnO (previous data). The amount of CuO in the CuO particle is described in Tables 1 and 2. The amount of ZnO dosed was calculated as a percentage of 100 of 0.2, 0.7 or 2 µg/mouse equal to ∼0.01, 0.035, and 0.1 mg ZnO/kg bw. The ZnO data were previously published (Hadrup et al. 2019). Carbon black (CB) data are from the current study.

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