A Clearance Period after Soluble Lead Nanoparticle Inhalation Did Not Ameliorate the Negative Effects on Target Tissues Due to Decreased Immune Response

The inhalation of metal (including lead) nanoparticles poses a real health issue to people and animals living in polluted and/or industrial areas. In this study, we exposed mice to lead(II) nitrate nanoparticles [Pb(NO3)2 NPs], which represent a highly soluble form of lead, by inhalation. We aimed to uncover the effects of their exposure on individual target organs and to reveal potential variability in the lead clearance. We examined (i) lead biodistribution in target organs using laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS) and atomic absorption spectrometry (AAS), (ii) lead effect on histopathological changes and immune cells response in secondary target organs and (iii) the clearance ability of target organs. In the lungs and liver, Pb(NO3)2 NP inhalation induced serious structural changes and their damage was present even after a 5-week clearance period despite the lead having been almost completely eliminated from the tissues. The numbers of macrophages significantly decreased after 11-week Pb(NO3)2 NP inhalation; conversely, abundance of alpha-smooth muscle actin (α-SMA)-positive cells, which are responsible for augmented collagen production, increased in both tissues. Moreover, the expression of nuclear factor κB (NF-κB) and selected cytokines, such as tumor necrosis factor alpha (TNFα), transforming growth factor beta 1 (TGFβ1), interleukin 6(IL-6), IL-1α and IL-1β , displayed a tissue-specific response to lead exposure. In summary, diminished inflammatory response in tissues after Pb(NO3)2 NPs inhalation was associated with prolonged negative effect of lead on tissues, as demonstrated by sustained pathological changes in target organs, even after long clearance period.

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[3] Mitchell, L. A., Gao, J., Wal, R. V., Gigliotti, A., Burchiel, S. W., & McDonald, J. D. (2007). Pulmonary and systemic immune response to inhaled multiwalled carbon nanotubes. Toxicol Sci, 100(1), 203-214.   We evaluated at least 8-10 slides per organ and assessed alterations in histopathological changes as follows: inflammatory cell infiltrate peribronchiolar, inflammatory cell infiltrate perivascular, atelectasis, bronchiolitis, thickened septa with congested capillaries, alveolar emphysema, hemorrhage, and bronchiectasis; co1 -co5 control animals, Pb1 -Pb5 exposed animals, cl1 -cl5 animals after a 5-week clearance period at designated time-points. The increased level of phenotype was labelled by increased number of + symbols, where "+" means mild phenotype, "++" moderate phenotype, and "+++" severe phenotype in relevant type of alteration in organ. Data are presented as mean ± SD; analyses were performed with five mice per each group. Number of CD68+ macrophages was evaluated from 4 slides (10 images/1 slide) of each animal. The values of CD68+ macrophages were counted per square millimeter; *p < 0.05, **p < 0.01 compared with the corresponding control group (ctr) by unpaired t-test. Data were obtained from five animals per every group. As reference values were used values of female mice crl:CD-1 (ICR) BR of different strains of female mice according to Serfilippi et al. (2003).
Reference biochemical values were count to our used units.   We evaluated at least 8-10 slides per organ and assessed alterations in histopathological changes as follows: mononuclear cell infiltrate, focal necrosis (degenerating hepatocytes), polynuclear hepatocytes, macrovesicular steatosis, hemostasis, hepatic remodeling, hypertrophic hepatocytes, infiltrates in portal area, hepatocyte dystrophy, vacuoles in hepatocyte nuclei, presence of megakaryocytes; co1 -co5 control animals, Pb1 -Pb5 exposed animals, cl1 -cl5 animals after a 5-week clearance period at designated time-points.
The increased level of phenotype was labelled by increased number of + symbols, where "+" means mild phenotype, "++" moderate phenotype, and "+++" severe phenotype in relevant type of alteration in organ. Data are presented as mean ± SD; analyses were performed with five mice per each group. Number of CD68+ cells was evaluated from 2 slides (10 images/1 slide) of each animal, the values of cells were counted per square millimeter; ***p < 0.001 compared with the control group, and *p < 0.05 compared with the Pb(NO3)2 NP group by unpaired t-test.   Figure S1. Weight of mice after Pb(NO3)2 NP inhalation. Lung, kidney, liver and spleen weight coefficient at different time points (2, 6 and 11 weeks).
The graphs values indicate average ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the corresponding control group (ctr), † p < 0.05 compared with the previous control by unpaired t-test. The graphs values denote average ± SD for 5 mice/group, ** p < 0.01, *** p < 0.001 compared with the corresponding control group (or between adjacent time points), and † † p < 0.01 compared with the corresponding Pb(NO3)2 NP group by unpaired t-test. Limit of detection (LOD) for Pb in the lungs was 26 ng/g.  A) Distribution of selected elements (Na, K and Fe) in kidney samples using LA-ICP-MS after Pb(NO3)2 NP inhalation. Na and K were observed in similar manner in control and Pb(NO3)2 NPexposed kidneys. The extent of the Fe was slightly increased after Pb(NO3)2 NP inhalation compared to the control. Numbers in parentheses show maximal value (µ g/g) of element on a scale. Scale bar in all panels = 3 mm. B) The graphs of Na and K in kidney at designated time points. The graph values denote average ± SD for 5 mice/group. Figure S5. The distribution of selected metals at designated time points after Pb(NO3)2 NP inhalation in the liver. A) Distribution of selected elements (Na, K, Fe and Zn) in liver samples using LA-ICP-MS after Pb(NO3)2 NP inhalation. Numbers in parentheses show maximal value (µ g/g) of element on a scale. Scale bar in all panels = 4 mm. B) The graphs of Na and K in liver at designated time points. The graph values denote average ± SD for 5 mice/group.