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Abstract
Background:
Recent years, there occurs heavy haze pollution in northern China during wintertime. The potential influence of airborne particulate matter (PM) on human health attracts great concern. The fuel-derived PM in the inhalable size range is dominated by aggregates of nanoparticles of Carbon black (CB). However, there are still lack of evidences especially regarding long-term exposure to explain the chronic effects of nanoscaled CB and the relative mechanism.
Purpose:
The objective of this study was to identify the potential mechanism of chronic effects of nanoscale CB. The systemic toxicity, immune suppression or activity and local toxicity were evaluated.
Methods:
32 rats were divided into 2 groups: 30 mg/m3 CB exposure (nose only, 90 d, 6h/d) and control (clean air). Half of rats were scarified after exposure and another half of rats recovered for 14 days. Eight rats in each group were executed the lung function tests using a ventilated bias flow whole body plethysmograph (WBP). SDS-PAGE protocol was used to detect the deposition and retention of CB in lung of rats. HE staining was used to observe the changes of histopathology. Cell apoptosis was examined by TUNEL assay or flow cytometry. The levels of IL-6, IL-8, IL-17 and TNF-α in serum and lung tissue were evaluated with commercially available ELISA kit. The peripheral blood cell counts were detected by Auto 5-diff hematology analyzer.
Results:
The lung burden of CB was 16 mg in lung of rats after a 90-day exposure by MPPD. Fourteen percentages of the amount of CB accumulated at the end of the exposure period was cleared from the lung during the 14 dys recovery period. The lung function was significantly decreased and could not recover after a short time recovery. The fibroblasts and granuloma formation were found in lung. The levels of apoptosis and DNA damages were significantly increased in lung cells after CB inhalation. The cytokines levels in lung but not in serum were significantly increased in CB exposure group. The cell counts of WBC, monocytes and neutrophils had 1.72, 3.13, and 2.73-fold increases after CB exposure, respectively. The percentages of CD4+ lymphocytes and the rates of CD4+/CD8+ were statistically increased after CB exposure. The stimulation indexes of the peripheral blood lymphocytes were significantly decreased after CB exposure. In the CB exposure group, the disrupted histomorphology of thymus and spleen were found as well as the early apoptotic thymocytes had a 2.36-fold increase.
Conclusion:
CB induced the localized or direct toxicity and systemic immune toxicity. The direct and systemic immune responses had a combined effect on the lung damages caused by CB.
Acknowledgments
This work is supported by Natural Science Foundation of China (81573190, 91643108), Natural Science Foundation of Hebei Province of China (H2015206326), and Natural Science Foundation of Education Department of Hebei Province of China (ZD2015008).
Highlights
CB could deposit in the lung and could not clearance within a short time.
CB inhalation induces granulomas and fibrosis in the lung of rats.
CB inhalation triggers the inflammation both in lung and immune organ.
Both direct and systemic immune responses are interlinked on the toxicity of CB.
Novelty Statement
This work studied the localized or direct, and systemic immune toxicity induced by chronic carbon black inhalation in rats. We evaluated the deposition and clearance rates of CB in the lung by a rapid and sensitive method. It was clear from the present study that CB inhalation induced chronic inflammation, formation of granulomas, DNA damage, apoptosis in lung and development of fibrosis in the lung interstitium, blood-gas barrier and then lung function reduction. The above damages in the lung might partly relate to the damages of thymocytes and the immune depression.
Abbreviations
BSA, Bull Serum Albumin; CB, Carbon black; CD4+, Cluster of differentiation 4; CD8+, Cluster of differentiation 8; CRP, C-reactive protein; DE, diesel exhaust; DEPs, Diesel exhaust particles; ELISA, Enzyme-linked immunosorbent assay; FBS, Fetal bovine serum; FITC, Fluorescein isothiocyanate; IgE, Immunoglobulin E; IHC, Immunohistochemical staining; IL-1β, Interleukin- 1β; IL-6, Interleukin-6; IL-8, Interleukin-8; IL-17, Interleukin-17; MCP-1, Monocyte chemoattractant protein-1; MPPD, Multiple path particle dosimetry; NK cells, Natural killer cell; OD, Optical density; SI, Stimulation index; OTM, Olive tail moment; PE, Phycoerythrin; PHA, Phytohaemagglutinin; PM, Particulate matter; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; SPF, Specific-pathogen-free; TEM, Transmission electron microscope; TNF-α, Tumor necrosis factor alpha; Th17, T helper 17 cells; WBC, White blood cell.
Ethical standards
The animal use protocol has been reviewed and approved by the Laboratory Animal Ethical and Welfare Committee of Hebei Medical University, Shijiazhuang, China. Approval No. is IACUC-Hebmu-20160047.
Disclosure
The authors report no conflicts of interest in this work.
Supplementary materials
Table 1 Summary of significant histopathological findings in the lung of rats after CB exposure
Table 2 The cytokines levels in the lung supernatant of rats (pg/mg protein)
Table 3 The lung function indexes after CB exposure in rats
Table 4 The changes of peripheral blood cell counts and lymphocyte subsets in rats after Carbon black (CB) inhalation for 90 days
Table S1 The organ coefficients after CB exposure in rat (g/g body weight).
Table S2 The lymphocyte phenotype of spleen (%).
Figure S1 Environmental monitoring of the exposure chamber.
Notes: A: The temperature, humidity, concentration of CO2 and O2 in the exposure chamber during the inhalation; B: The concentration of CB aerosol in the exposed chamber during the inhalation.
Figure S2 Changes of the body weight of rats during the inhalation and recovery.
Notes: From Day 21, statistically significant differences of body weight were found between CB exposure group and the control group. In the recovery group, the body weight of rats still significant lower than the control. Data were shown as the mean ± SD (standard deviation). n=8.
Figure S3 Representative histopathology of the pulmonary lymph node in rats by HE staining (100×).
Notes: A higher magnification of the lung tissue (400×).
Figure S4 The DNA damages in lung tissue of rats by Comet assay.
Notes: The number of rats in each group was 6 and data were shown as the mean ± SD (standard deviation). Multi-group comparisons of the means were carried out by a one-way analysis of variance test followed by SNK’s multiple comparison tests. *P<0.05 compared with the control group. #P<0.05 compared with the CB exposure group.
Figure S5 Representative TEM images of blood-gas barrier in rats (6000×).
Notes: A higher magnification of the lung tissue (12000×). The arrows indicate fibrosis of blood-gas barrier.
Figure S6 The cytokine levels of lung tissue by IHC.
Notes: A: The representative graphs of the cytokines expression in lung tissue (200×). The cytokines positive cells displayed brownish yellow granules. In lung cells, they were located mainly in the cytoplasm and karyon. B: The statistic analysis of the cytokine levels. The number of rats in each group was 6 and data were shown as the mean ± SD (standard deviation). Multi-group comparisons of the means were carried out by a one-way analysis of variance test followed by SNK’s multiple comparison tests. *P<0.05 compared with the control group. #P<0.05 compared with the CB exposure group.
Figure S7 The cytokine levels of thymus tissue by IHC.
Notes: A: The representative graphs of the cytokines expression in thymus tissue (400×). The cytokines positive cells displayed brownish yellow granules. In thymus cells, they were located mainly in the cytoplasm and karyon. B: The statistic analysis of the cytokine levels. The number of rats in each group was 6 and data were shown as the mean ± SD (standard deviation). Multi-group comparisons of the means were carried out by a one-way analysis of variance test followed by SNK’s multiple comparison tests. *P<0.05 compared with the control group. #P<0.05 compared with the CB exposure group.
