| Classroom teaching with boards and chalks, one of the cheapest methods of teaching, still dominates many parts of worldwide. During classroom teaching with chalks, a large amount of chalk dust PM10(particulate matter with aerodynamic diameters lower than 10 μm) and PM2.5 (particulate matter with aerodynamic diameters lower than 2.5 μm) are produced. These inhalable particles, especially PM2.5 can deposit in bronchiole, pulmonary alveoli and then be transferred into the blood circulation. So chalk dust may exert adverse health effects on teachers and students. The hypothesis is supported by the facts that short-term exposure to chalk dust impaired the function of respiratory tract and long-term exposure to chalk dust can result in respiratory diseases. Nevertheless, the toxicities on chalk dust have not been investigated. Regarding the close link of toxicity of PM to its physicochemical characteristics, a novel, single-particle analytical technique, called electron probe X-ray microanalysis (EPMA), was used to determine the chalk dust particles’components. Corresponding, in vitro and vivo toxicities to chalk dust were also investigated in order to clarify the biological effects of chalk dust on human and to provide toxicological basis for respiratory diseases induced by chalk dust.The morphology and chemical composition were analyzed by energy dispersive-EPMA technology. The results showed that chalk dust PM10, PM2.5 and PM1 were produced after writing or wiping. Also, these particles appeared to be sphere, cuboid, square or irregular shape. EPMA results showed that these particles composed of CaSO4, CaCO3/CaMg(CO3)2, CaSiO3, SiO2 and organic adhesives.Regarding alveolar macrophage (AM) is an important immune cell in lung and dysfunction of AMs can induce respiratory diseases, the ability to produce ROS/RNS in rat AMs exposed to chalk dust particles was investigated using a luminol-enhanced chemiluminescence (CL) method and the effects of various inhibiters on CL in AMs exposed to particles were also examined. The resulted showed:(1) chalk dust PM2.5 and PM10 dose-dependently induced CL in AMs, which was inhibited by 0.5μM or 1 mM L-NAME, indicating that chalk dust PM2.5 and PM10 triggered respiratory burst in AMs. (2) CaSO4/CaCO3 PM2.5 or PM10 at the level of 5-200μg/106 cells induced CL in a dose-dependent manner. With regard to CaSO4 or CaCO3, the smaller the particle size, the stronger CL. Also, CaCO3 particles significantly induced the stronger CL in AMs than CaSO4. (3) CL in AMs exposed to CaSO4 or CaCO3 were inhibited significantly by antimycin A, SOD, DPI and L-NAME. These results indicated that chalk dust-induced ROS in AMs may derive from NADPH oxidase in cytoplasmic membrane and complex III in mitochondria and RNS may result from NOS activation in AMs. Furthermore, chalk dust-induced ROS/RNS in AMs is closely related to the presence of CaSO4 and CaCO3 in chalk.In order to understand whether ROS/RNS cause further damage to AMs, the potential to generate NO, ACP、Na+K+ATP, Ca2+Mg2+ATP, oxidative stress markers (SOD、GSH、CAT and MDA) and inflammatoty cytokines (TNF-a, IL-6 and TGF-β1) mRNA expression in AMs and cytotoxicity elicited by chalk dust PM2.5 and PM10 were also examined 4 h following the treatment of AMs with differing dosages of chalk dust particles, respectively. The results showed:(1) Exposure to chalk dust PM2.5 at all doses significantly decreased intracellular SOD content in AMs while PM10 at the concentration of 300μg/mL reduced SOD and GSH content. Also, these particles at high dose increased CAT and MDA content. It suggested that the accumulation of ROS/RNS in the respiratory burst of AM resists the defensive effects of cellular antioxidant enzymes, e.g. SOD and GSH. Hydrogen peroxide originated from disproportionation of 02-· is degraded to oxygen and water by CAT, thus the decrease of SOD activity in response to chalk dust PM2.5 can reduce the capacity against lipid peroxidation. PM2.5 at the same level resulted in more MDA content in AMs than PM10 did as a result of its stronger capacity to decrease SOD, indicating PM2.5 exerted more oxidative damage to AMs than PM10. (2) Chalk dust PM2.5 at all doses significantly increased the content of Ca2+Mg2+-ATP. The contents of Na+K+-ATP, Ca2+Mg2+-ATP or ACP were down-regulated in AMs exposed to chalk dust PM10 at the level of 300μg/mL. But there is no statictical difference between PM2.5 and PM10. (3) Activities of NO in AMs were notably increased by chalk dust PM2.5 and PM10 at the level of 100μg/mL or 300μg/mL, and the ability to release NO is stronger for PM10 than PM2.5. (4) RT-PCR results showed that the expressions of IL-6 mRNA were markedly up-regulated by PM2.5 at all doses while the level of TNF-a mRNA was increased by PM10 at high dose. According, TGF-β1 mRNA level was markedly increased by PM2.5 and PM10 at high dose. (5) Chalk dust PM2.5 and PM10 at the dose of 300μg/mL induced leakage of lactate dehydrogenase (LDH) and reduction of the cell viability, but there is no statistical difference in chalk dust PM2.5 and PM10. In a word, chalk dust PM2.5 and PM10 exerted oxidative stress and inflammatory effects and chalk dust PM2.5 could make more oxidative damages on AMs.Then the toxicities of chalk dust PM2.5 were also confirmed by in vivo test. Lung tissue pathology was assessed by HE staining and the mRNA expression of these genes (TNF-a, IL-6, iNOS, ICAM, VEGF-A, TF, TGF-β PDGF-D and Coll Iα) in inflammatory responses and fibrosis and protein level (p38, ERKs, and JNK MAPK, Smad-3 and STAT-3) were examined by RT-PCR and western blot methods. The results showed that (1) TNF-a mRNA level and IL-6 mRNA level were significantly up-regulated in rat lung exposed to 8 mg/kg b.w. or 32 mg/kg b.w. chalk dust PM2.5. (2) The expression iNOS mRNA was notably in rat lung exposed to 8 mg/kg b.w. and 32 mg/kg b.w. chalk dust PM2.5. (3) Corresponding, PM2.5 at all doses increased ICAM mRNA expression in rat lung, revealing that chalk dust PM2.5 can make inflammatory damage to lung tissue. (4) HE staining showed that chalk dust PM2.5 increased inflammatory cell infiltration and hyperemia. (5) The notable dose-dependent increase of VEGF-A, Collα1 and STAT-3 mRNA occurred in rats exposed to chalk dust PM2.5 at all the doses. The increases of TGF-β, PDGF-D/PDGFR-β and Smad-3 mRNA in rat exposed to 8 mg/kg b.w. or 32 mg/kg b.w. were also observed. However, there is no statistical difference for TF mRNA, STAT-3 and Smad-3 protein relative to the control. (6) Furthermore, PM2.5 markedly increased HO-1 mRNA level and decreased Nrf2 mRNA expression in lung exposed to 8 mg/kg b.w. or 32 mg/kg b.w. PM2.5. (7) P38 and ERKs protein were up-regulated by 8 mg/kg b.w. or 32 mg/kg b.w. chalk dust PM2.5, suggesting that chalk dust PM2.5 induced inflammatory responses and elevated the gene transcriptional level in fibrosis by p38 and ERKs MAPK signal pathway. (8) Instead, NAC treatment attenuated the expression of these genes in oxidative stress, inflammatory response and fibrosis, indicating that oxidative stress plays an important role in the toxicity of PM2.5 to rat lung.In summary, inhalable chalk dust particles triggered respiratory burst in AMs and induced ROS/RNS generation. Excessive ROS/RNS decreased the antioxidases activities and resulted in lipid perixidation. Also, these particles reduced ACP content in AMs and elevated NO release in AMs. Corresponding, they induce inflammatory responses in AMs. Also, chalk dust PM2.5 induced more oxidative injury than PM10 did. In addition, fine chalk dust particles increased in vivo toxicity by enhancing inflammatory and fibrosis. Instead, NAC decreased the damage in rat lung exposed to chalk dust PM2.5. |
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