Nose-only inhalations of high-dose alumina nanoparticles/hydrogen chloride gas mixtures induce strong pulmonary pro-inflammatory response: a pilot study

Figures & data

Figure 1. Nose-only inhalation exposure system. Al₂O₃ NPs aggregates suspension in water and HCl_g bottle were connected to the system on two parallel paths that joined to create the mixture. Mass flow meters allowed to generate and calibrate the air flow in order to avoid re-inhalation phenomena of the air exhaled by the animals (dynamic air circulation, 10 L/min). During exposures, rats were placed in individual containment tubes positioned on the oro-nasal inhalation tower. Al₂O₃ NPs and HCl_g mixture reached the inhalation chamber at a flow rate of 20 L/min. Bubblers and samplers connected to the inhalation tower allowed the collection of samples during the exposures in order to characterize the generated aerosol. A release neutralization system (bubbler with soda and desiccant) was set up at the inhalation tower exit.

Table 1. Novotec scoring and corresponding pulmonary lesions.

Lesion score	Observed lesions
0	No lesion or physiological lesions
1	Vascular lesions
2	Limited to moderate vascular and alveolar lesions, with or without bronchial lesion
3	Significant vascular and alveolar lesions, with or without bronchial lesion

Figure 2. Bulk powder and aerosols alumina nanoparticles physico-chemical characterization. Transmission electron microscopy (TEM) images of Al₂O₃ NPs in bulk powder (A) and generated aerosol (C) (respective scale bars = 200 nm and 500 nm). Crystalline phases (γ and δ) identification using X-ray diffraction (XRD) on bulk Al₂O₃ NPs (B). NPs elementary chemical composition (in %) using energy-dispersive X-ray (EDX) spectroscopy on Al₂O₃ NPs aggregates retrieved in the generated aerosol (D). Table E summarizes Al₂O₃ NPs representative particles size distribution in the generated aerosols measuring using Sioutas cascade impactor (mg/m³ for each size class in μm).

Table 2. Generated aerosols alumina and HCl_g contents.

A
Aerosol	Al (µg/filter)^a	Al concentration (mg/m^3)^b	Al2O3 concentration (mg/m^3)^c	Al2O3 concentration mean ± SD (mg/m^3)
Al2O3	137	9.1	17.3	22.1 ± 4.5
	183	12.2	23.0
	207	13.8	26.1
Al2O3 + HClg	185	12.3	23.3	20.0 ± 2.9
	147	9.8	18.5
	144	9.6	18.1
B
Aerosol	Particulate fraction (filters)		Free fraction (bubblers)		Cl^− total concentrations (µg/m^3)^B	Cl^− total concentrations mean ± SD (µg/m^3)
	Cl^− (µg/filter)	Cl^− (µg/m^3)^A	Cl^− (µg/bubbler)	Cl^− (µg/m^3)^A
HClg	–	–	32.2	2145	2145	791 ± 1178
	–	–	3.4	228	228
	–	–	0	0	0
Al2O3 + HClg	8.9	596	0	0	596	617 ± 32
	9.0	601	0	0	601
	9.8	653	0	0	653

^aAfter white deduction.

^bSampling at 1 Lpm for 15 min (/0.015).

^c× 1.889.

Table A shows alumina (Al₂O₃) mean mass concentrations in the generated aerosols. Aluminum mass was measured on three different filters (µg/filter). Based on these results, aluminum and alumina concentrations in aerosols (mg/m³) were calculated and alumina mean mass concentrations (mg/m³) were determined for each experimental aerosol. Table B shows chlorine mean concentrations in the generated aerosols. Chlorine particulate and free fractions were, respectively, measured on three filters and bubbler samples (μg/filter or μg/bubbler). Corresponding chlorine mass concentrations were calculated (µg/m³) and were added to determine total chlorine levels (μg/m³). Total chlorine mean mass concentrations (μg/m³) were calculated for each experimental aerosol. Bold values refer to chlorine mass concentrations in μg/m³.

^ASampling at 1 Lpm for 15 min (/0.015).

^BParticulate fraction + free fraction.

Figure 3. Pulmonary aluminum deposition after nose-only exposures. Rats were exposed to air, HCl_g, Al₂O₃ or to the mixture Al₂O₃ + HCl_g. Clear blue points symbolize single exposures (SE) and dark blue points symbolize repeated exposures (RE) to each aerosol. Mean aluminum quantities (μg/g of lung) ± SD were measured in lungs 24 h after the last exposure using inductively coupled plasma optical emission spectrometry (ICP-OES). One-way ANOVA and Tukey’s post-test (n = 3, ** p-value < 0.01, **** p-value < 0.0001, α risk = 0.05) were performed to compare each experimental condition to the control within the same exposure scenario and Two-way ANOVA and Sidak’s post-test (n = 3, ¤¤¤p-value < 0.001, ¤¤¤¤ p-value < 0.0001, α risk = 0.05) were performed to compare the two exposure scenarios for the same aerosol.

Table 3. Rats body weight changes during the experimental period.

A			4 h (AE)	28 h (BS)
Single exposure (SE)	Air	Mean (g) ± SD	−16.1 ± 4.6	−1.2 ± 5.7
		SA
	HClg	Mean (g) ± SD	−16.5 ± 4.8	−3.4 ± 3.7
		SA	ns	Ns
	Al2O3	Mean (g) ± SD	−13.1 ± 3.8	−4.5 ± 4.1
		SA	ns	*
	Al2O3 + HClg	Mean (g) ± SD	−15.2 ± 2.8	−3.9 ± 2.7
		SA	ns	Ns
B			4 h (AE)	24 h (BE)	28 h (AE)	48 h (BE)	52 h (AE)	72 h (BE)	76 h (AE)	100 h (BS)
Repeated exposure (RE)	Air	Mean (g) ± SD	−15.7 ± 2.9	−5.0 ± 4.0	−21.2 ± 3.0	−9.0 ± 4.0	−23.6 ± 3.9	−9.9 ± 5.2	−21.1 ± 4.1	−10.0 ± 5.2
		SA
	HClg	Mean (g) ± SD	−18.6 ± 3.5	−1.5 ± 2.1	−20.9 ± 3.1	−3.5 ± 2.6	−22.5 ± 4.2	−4.4 ± 3.5	−22.5 ± 4.4	−2.8 ± 4.5
		SA	ns	ns	ns	**	Ns	**	ns	****
	Al2O3	Mean (g) ± SD	−12.8 ± 2.1	−4.5 ± 3.4	−15.3 ± 4.6	−7.5 ± 6.0	−18.6 ± 6.0	−9.6 ± 6.8	−21.8 ± 5.1	−8.7 ± 8.3
		SA	ns	ns	**	Ns	**	Ns	ns	ns
	Al2O3 + HClg	Mean (g) ± SD	−15.3 ± 3.3	−2.6 ± 2.1	−15.3 ± 2.6	−2.4 ± 2.7	−15.2 ± 3.7	−2.9 ± 3.3	−13.9 ± 5.0	−3.2 ± 4.3
		SA	ns	ns	**	***	****	****	****	***

Rats were exposed once or repeatedly to air, HCl_g, Al₂O₃ or to the mixture Al₂O₃ + HCl_g. Rats body weight change (in gram, mean ± SD) was measured before and after each aerosol exposure. Obtained results during single exposures (SE) and during repeated exposures (RE) are, respectively, presented in Tables A and B. Tables show variations from initial body weight (t₀, before the first exposure) before each exposure (BE, 24 h (B), 48 h (B), 72 h (B)), after each exposure (AE, 4 h, 28 h (B), 52 h (B), 76 h (B)) and before sacrifice (BS, 28 h (A) and 100 h (B)). Two-way ANOVA and Dunnett’s post-test (n = 12, *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001, α risk = 0.05) were performed as statistical analysis (SA) to compare body weights variation compared with initial body weights (t0) in each experimental condition with the control group (air). AE: after exposure; BE: before exposure; BS: before sacrifice; SA: statistical analysis; ns: not significant.

Figure 4. Total proteins, LDH and immune cells populations in bronchoalveolar lavage fluids (BALF). Rats were exposed to air, HCl_g, Al₂O₃ or to the mixture Al₂O₃ + HCl_g. Clear blue points symbolize single exposures (SE) and dark blue points symbolize repeated exposures (RE) to each aerosol. Total proteins (g/L) (A) and lactate dehydrogenases (LDH, U/L) (B) concentrations were measured in rats BALF 24 h after the last exposure. Total cells, polymorphonuclear neutrophils (PMN), macrophages and lymphocytes (cells × 10⁶) were counted on BALF cytospins (respectively C, D, E, and F). Results are expressed as mean values ± SD. One-way ANOVA and Tukey’s post-test (n = 6, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001, α risk = 0.05) were performed to compare each experimental condition to the control within the same exposure scenario and two-way ANOVA and Sidak’s post-test (n = 6, ¤ p-value < 0.05, ¤¤ p-value < 0.01, ¤¤¤ p-value < 0.001, ¤¤¤¤ p-value < 0.0001, α risk = 0.05) were performed to compare the two exposure scenarios for the same aerosol.

Figure 5. Aerosols pro-inflammatory potential on rat lungs after nose-only inhalations. Rats were exposed to air, HCl_g, Al₂O₃ or to the mixture Al₂O₃ + HCl_g. Clear blue points symbolize single exposures (SE) and dark blue points symbolize repeated exposures (RE) to each aerosol. Pro-inflammatory cytokines concentrations (pg/mL) of TNF-α (A), IL-1β (B), GRO/KC (C) and MIP-2 (D) were measured in rats BALF 24 h after the last exposure by ELISA multiplex. Results are expressed as mean values ± SD. One-way ANOVA and Tukey’s post-test (n = 6, * p-value < 0.05, ** p-value < 0.01, **** p-value < 0.0001, α risk = 0.05) were performed to compare each experimental condition to the control within the same exposure scenario and two-way ANOVA and Sidak’s post-test (n = 6, ¤¤¤¤ p-value < 0.0001, α risk = 0.05) were performed to compare the two exposure scenarios for the same aerosol.

Figure 6. Pulmonary histopathological lesions observations following inhalation exposures. Rats lung parenchyma stained in HPS were observed in optical microscopy for histopathological analysis. Images show, respectively, lung parenchyma 24 h after single exposure (SE) to air (A), HCl_g (C), Al₂O₃ (E) and Al₂O₃ + HCl_g (G) and 24 h after repeated exposures (RE) to air (B), HCl_g (D), Al₂O₃ (F) and Al₂O₃ + HCl_g (H). Scale bar = 200 μm. Pictures legends: a = alveoli; b = bronchioles or bronchi; v = blood vessel; c = vascular congestion; blue arrows = vascular edema; red arrows = bronchial (pre)-exfoliation; i = inflammatory area; black squares = velamentous epithelium and black circles = interalveolar septum edema.

Figure 7. Pulmonary histopathological lesions scoring. Rats were exposed to air, HCl_g, Al₂O₃ or to the mixture Al₂O₃ + HCl_g. Scoring was performed on HPS stained lungs slices of three rats of each group. Lesion scores (mean ± SD, n = 3) were estimated for each experimental aerosol and exposure scenario based on the presence and the gravity of vascular, alveolar and bronchial lesions observed in rats lungs 24 h after the last exposure. Empty and striped bars represent, respectively, single exposures (SE) and repeated exposures (RE) results.

Figure 8. Oxidative stress induction by experimental aerosols inhalation exposures. Immunofluorescence was performed to detect 8-OHdG (8-hydroxy-2'-deoxyguanosine, green fluorescence) on rat lungs slices 24 h after the last exposure. DNA was stained by DAPI, which emits in blue fluorescence. Pictures represent obtained results following single exposures (SE) to air (A), HCl_g (B), Al₂O₃ (C) and Al₂O₃ + HCl_g (D). Scale bars = 50 μm. Pictures legends: a = alveoli; b = bronchioles or bronchi. Red blood cells could also emit in green fluorescence, but red arrows and red circles point out specific 8-OHdG staining. 8-Isoprostane concentrations (pg/mL) were measured in rats BALF 24 h after the last exposure (E). Clear blue points symbolize single exposures (SE) and dark blue points symbolize repeated exposures (RE) to each aerosol. Results are expressed as mean values ± SD. One-way ANOVA and Tukey’s post-test (n = 3, * p-value < 0.05, ** p-value < 0.01, **** p-value < 0.0001, α risk = 0.05) were performed to compare each experimental condition to the control within the same exposure scenario and two-way ANOVA and Sidak’s post-test (n = 3, α risk = 0.05) were performed to compare the two exposure scenarios for the same aerosol.