Time course of pulmonary inflammation and trace element biodistribution during and after sub-acute inhalation exposure to copper oxide nanoparticles in a murine model

Abstract

Background: It has been shown that copper oxide nanoparticles (CuO NPs) induce pulmonary toxicity after acute or sub-acute inhalation exposures. However, little is known about the biodistribution and elimination kinetics of inhaled CuO NPs from the respiratory tract. The purposes of this study were to observe the kinetics of pulmonary inflammation during and after CuO NP sub-acute inhalation exposure and to investigate copper (Cu) biodistribution and clearance rate from the exposure site and homeostasis of selected trace elements in secondary organs of BALB/c mice.

Results: Sub-acute inhalation exposure to CuO NPs led to pulmonary inflammation represented by increases in lactate dehydrogenase, total cell counts, neutrophils, macrophages, inflammatory cytokines, iron levels in bronchoalveolar lavage (BAL) fluid, and lung weight changes. Dosimetry analysis in lung tissues and BAL fluid showed Cu concentration increased steadily during exposure and gradually declined after exposure. Cu elimination from the lung showed first-order kinetics with a half-life of 6.5 days. Total Cu levels were significantly increased in whole blood and heart indicating that inhaled Cu could be translocated into the bloodstream and heart tissue, and potentially have adverse effects on the kidneys and spleen as there were significant changes in the weights of these organs; increase in the kidneys and decrease in the spleen. Furthermore, concentrations of selenium in kidneys and iron in spleen were decreased, pointing to disruption of trace element homeostasis.

Conclusions: Sub-acute inhalation exposure of CuO NPs induced pulmonary inflammation, which was correlated to Cu concentrations in the lungs and started to resolve once exposure ended. Dosimetry analysis showed that Cu in the lungs was translocated into the bloodstream and heart tissue. Secondary organs affected by CuO NPs exposure were kidneys and spleen as they showed the disruption of trace element homeostasis and organ weight changes.

Keywords: Copper oxide nanoparticles (CuO NPs); Cytokines; Inflammation; Inhalation; Nanomaterial; Pulmonary toxicity; Trace elements.

Fig. 1

Particle size distribution of CuO NP aerosols measured by a scanning mobility particle sizer (SMPS) from the nose-only port of the exposure system during inhalation exposure. GM = geometric mean, GSD = geometric standard deviation

Fig. 2

Lactate dehydrogenase enzyme levels in BAL fluid (Red-highlighted area indicated time during CuO exposure). Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. Data are expressed as mean ± SD (n = 5). *** P < 0.001, **P < 0.01, *P < 0.05

Fig. 3

Total cell count in BAL fluid (Red-highlighted area indicated time during CuO exposure). Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. Data are expressed as mean ± SD (n = 5). ****P < 0.0001, *** P < 0.001, **P < 0.01

Fig. 4

Inflammatory cell numbers in BAL fluid of mice exposed to CuO NPs in aerosols. a Neutrophils, b Macrophages, c Lymphocytes, and d Eosinophils (Red-highlighted area indicated time during CuO exposure). Statistical analysis was performed using Kruskal–Wallis test. Data are expressed as mean ± SD (n = 5). *** P < 0.001, **P < 0.01, *P < 0.05

Fig. 5

Micrographs of leukocytes in BAL fluid from mice exposed to CuO NPs and necropsied on Days 3, 12 and 27 and control mice. Aggregated CuO NPs are visible on Day 3 inside the macrophages as well as free between the cells (thin black arrows). Recruitment of neutrophils is present on Day 3–Day 27 (thick black arrows). The appearance of macrophages (thick red arrows) changes overtime; becoming progressively more activated and enlarged from Day 3 to Day 27. Cells were stained with Protocol® HEMA 3 stain set

Fig. 6

Levels of inflammatory cytokines/chemokines: a KC, b G-CSF, c GM-CSF, d MCP-1, e MIP-1α, f MIP-1β, g eotaxin, h RANTES, i IL-1α, j IL-6, k IL-10, and l IL-12(Pp0) during (red-highlighted area) and after exposure. Statistical analysis of results for KC, MIP-1α, MIP-1β, eotaxin, RANTES, IL-1α, IL-6, IL-10, and IL-12(p40) was performed using one-way ANOVA with Dunnett’s post hoc test, while statistical analysis of results for G-CSF, GM-CSF, and MCP was performed by Kruskal–Wallis test (compare to the control group). Data are expressed as mean ± SD (n = 5). ****P < 0.0001, *** P < 0.001, **P < 0.01, *P < 0.05

Fig. 7

Micrographs of lung sections stained with H&E. Development of progressive perivascular aggregates (black arrows), mainly lymphocytes, indicating chronic inflammation was observed from Day 3 – 27. Occasional neutrophils (red arrows) were seen mainly at earlier time points (Day 3 and Day 12) indicating an acute inflammation. Control mice showed no lesions with clear alveolar spaces and no overt recruitment of lymphocytes or neutrophils. Similarly, as seen in BAL fluid, macrophages became more enlarged and activated with foamy vacuoles (black circles). All images were taken at the same magnification

Fig. 8

The concentration versus time profile of Cu in lung tissue. Log scale during (red-highlighted area) and after exposure. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test (compared to the control group). Data are expressed as mean ± SD (n = 5). ****P < 0.0001, *** P < 0.001

Fig. 9

The concentration versus time profile of copper and iron in BAL fluid during (red-highlighted area) and after exposure. Statistical analysis of results for Fe in BAL fluid was performed using one-way ANOVA with Dunnett’s post hoc test, while statistical analysis of results for Cu in BAL fluid was performed by Kruskal–Wallis test (compare to the control group). Data are expressed as mean ± SD (n = 5). ****P < 0.0001, *** P < 0.001, **P < 0.01

Fig. 10

The concentration versus time profile in whole blood of Cu and Mn during (red-highlighted area) and after exposure. Statistical analysis of Mn was performed using one-way ANOVA with Dunnett’s post hoc test, while that of Cu was performed by Kruskal–Wallis test (compare to the control group). Data are expressed as mean ± SD (n = 5). ****P < 0.0001, *** P < 0.001, **P < 0.01, *P < 0.05

Fig. 11

The concentration of each element in brain, heart, spleen, liver, kidneys, and lung; a Mg, b K, c Ca, d Mn, e Fe, f Cu, g Zn, and h Se. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test if the data were normally distributed. If not, Kruskal–Wallis test was used (compared to the control group). Data are expressed as mean ± SD (n = 5). ****P < 0.0001, *** P < 0.001, **P < 0.01, *P < 0.05

Fig. 12

Blood concentration of a Fe, b Cu and c ratio of Fe/Cu. Black dots indicate outliers

Fig. 13

Cu concentration (µg/mL) in pooled urine samples during (red-highlighted area) and after exposure

Fig. 14

Serum hemoglobin levels (mg/mL) during (red-highlighted area) and after exposure. Statistical analysis was performed by Kruskal–Wallis test (compared to the control group). There was no significant difference between the control and exposed mice. Data are expressed as mean ± SD (n = 5)

Fig. 15

Serum ceruloplasmin level (mU/mL) during (red-highlighted area) and after exposure. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. There was no significant difference between the control and exposed mice. Data are expressed as mean ± SD (n = 5)

Fig. 16

Body weight as a relative percentage of starting weight ("0%") during (red-highlighted area) and after exposure

Fig. 17

Organ dry weight change during (red-highlighted area) and after exposure. Statistical analysis of results for liver, kidney, heart, spleen, and lung was performed using one-way ANOVA with Dunnett’s post hoc test, while statistical analysis of results for brain was performed by Kruskal–Wallis test (compared to the control group). Data are expressed as mean ± SD (n = 5). ****P < 0.0001, *** P < 0.001, **P < 0.01, *P < 0.05