| There are a large number of epidemiological studies show that the concentration of atmospheric fine particulate matter(PM2.5) is closely related to the morbidity and mortality of crowd cardiopulmonary disease, especially for the elderly, infants, young children, and persons preexisting chronic diseases. As the epidemiological studies cannot expound the real reasons about how atmospheric PM2.5 particles affect people’s health, toxicology studies of atmospheric PM2.5 particles in vitro and in vivo are important means to discover the pathogenic mechanism of PM2.5 particles.The diameter of PM2.5 particles is so small that it can turn into alveolus by respiration and enter into lung capillaries, even circulatory system by the processing of gas exchanging, which can make adverse effects to organisms, especially the respiratory system and cardiovascular system. Firstly, this research use two typical cell lines, namely, human lung epithelial A549 cells and rat myocardial H9C2 cells, as the cell toxicity evaluation models to study the toxicological effects of atmospheric PM2.5 particles and its different fractions for vitro study; Secondly, this research use the Balb/c mice, which are one of the international typical and common experimental animals, as the animal toxicity evaluation models to study the toxicological effects of atmospheric PM2.5 related particulate matter by the treatment way of aerosols entering into tracheas for vivo study.Objectives 1. To get the different fractions of PM2.5 associated with atmospheric PM2.5 particles and detect their chemical and biological compositions to lay the foundation for future research about their biological toxicological effects. 2. To investigate the toxic effects and verify the dose response relationship in vitro toxicity evaluation models by treating A549 cells and H9C2 cells with atmospheric PM2.5 particles and its different fractions from Beijing urban areas. 3. To investigate acute toxic effects and verify the dose response relationship in vivo toxicity evaluation models by treating Balb/c mice with aerosols of atmospheric PM2.5 particles and its pure solid particles from Beijing urban areas by the treatment of aerosols entering into tracheas.Contents and methods Atmospheric PM2.5 particles were collected from one of Beijing urban areas, and original particles of PM2.5, water-soluble PM2.5, fat-soluble PM2.5 and pure solid particles of PM2.5(designated OPP2.5, WSP2.5, FSP2.5 and PPP2.5, respectively) were prepared subsequently. After analyzing, the concentration of 8 kinds of water-soluble ions, 16 kinds of “acid extraction” elements, 10 kinds of “water extraction” elements and 16 kinds of PAHs, endotoxin as well as the copies of bacterial and fungal genomes in the above four PM2.5 samples were detected. The SEM was used to observe the surface features of OPP2.5 and PPP2.5, and A549 cells were treated with different concentration gradients(10, 50, 100, 200, 400μg/m L) of OPP2.5, WSP2.5, FSP2.5 and PPP2.5. The MTS method was used to test the cell viability of A549 cells at 6h, 10 h, 24 h, 48 h and 72 h post-treatment, the contents of LD and the activities of LDH and SOD were detected at 24 h post-treatment, meanwhile ELISA and RT-QPCR were employed to detect the production of inflammatory cytokines IL-6 and TNF-α and the AP-sites counting was conducted to determine the levels of intracellular DNA damage at 24 h post-treatment. H9C2 cells were treated with those four PM2.5 samples, and the concerned items and detected methods of different items are similar to the treatment of A549 cells, except for the same treatment dosages with corresponding A549 cells in testing the cell viability by the MTS methods, the treatment dosages of other items of H9C2 cells were only detected when the cells were treated in solvent control and the concentration of 10μg/m L from those four PM2.5 samples. The Balb/c mice were treated with aerosols of OPP2.5 and PPP2.5 by the treatment of aerosols entering into tracheas, and they were killed after picking their eyes to take blood at 24 h post-treatment. Except for a small amount of whole blood was retained to detect the changes of blood routine, the majority of the whole blood was used to prepare serum to detect the changes of biochemical and inflammatory factors, Broncho alveolar lavage fluid(BALF) of these mice were prepared to detect the changes of biochemical and inflammatory factors, while heart, liver and lungs were fixed for histopathological assessment and lung homogenates were prepared to detect the changes of inflammatory factors and oxidative damage-related indicators.Results The water-soluble ions accounted for 67.71%, 0.91%, 33.37% and 0.09% of the mass fractions in WSP2.5, FSP2.5, OPP2.5 and PPP2.5, respectively. The “acid extraction” elements accounted for 4.84%, 2.29%, 1.86% and 0.78% of the mass fractions in these four PM2.5 samples, respectively. The polycyclic aromatic hydrocarbon accounted for 0. 218?, 49.125?, 0.403? and 0.007? of the mass fractions in these four PM2.5 samples, respectively. The endotoxin content was 0.0547 EU, 0.0184 EU, 0.4333 EU and 0.0419 EU per unit mass(milligram) for these four PM2.5 samples, respectively. The number of the bacterial and fungal genomes copies were(2.572±0.954)×108 and(4.269±0.916)×108 per unit mass(gram) in OPP2.5, respectively. After A549 cells were treated with those four PM2.5 samples, the results showed that WSP2.5 had limited effect on inhibit cell growth and induced inflammatory damages and DNA base deletion; FSP2.5, PPP2.5 and OPP2.5 at high concentrations showed the inhibitory effect to cell growth across treatment, and when cells were treated with low concentrations of FSP2.5, PPP2.5 or OPP2.5, the inhibition of cell growth occurred within a short period and then disappeared over time; those four PM2.5 samples had effects on increasing intracellular contents of LD, intracellular activities of SOD and intracellular or supernatant activities of LDH, because more cells were dead when treated with high concentrations of PM2.5 samples, the changes of supernatant contents of LD and activities of SOD were not regular, and supernatant contents of LD and activities of SOD decreased in the highest concentration of PM2.5 samples; FSP2.5, PPP2.5 and OPP2.5 treatment significantly induced the production of IL-6 at both m RNA and protein levels, while WSP2.5, PPP2.5 and OPP2.5 treatment significantly induced the m RNA expression of TNF-α. FSP2.5, PPP2.5 and OPP2.5 treatment also induced the considerable DNA base deletion. After H9C2 cells were treated with those four PM2.5 samples, the results show that OPP2.5 and PPP2.5 can significantly inhibit cells growth, and even lead to death of all cells whitin a low exposure concentration(50μg/m L) and a short exposure time(within 24h), except for the highest exposure concentration(400μg/m L) of FSP2.5, WSP2.5 and FSP2.5 had weak, or even no inhibitory effects on cells growth; those four PM2.5 samples significantly decreased the intracellular activities of LDH and SOD; there no proteins of IL-6 and TNF-α was detected in supernatant post-treatment, OPP2.5 treatment significantly induced the production of intracellular m RNA of TNF-α, while FSP2.5 treatment significantly induced the production of intracellular m RNA of IL-6 and TNF-α; however, there no DNA base deletion was detected post-treatment. The in vivo exposure experiments show that at the systemic level, after the Balb/c mice were treated with aerosols of OPP2.5 and PPP2.5 for 24 hours, the percentage of intermediate cells in the whole blood from mice increased slightly; the contents of GLOB in serum from mice treated with OPP2.5 and PPP2.5 and the contents of TP in serum from mice treated with the high concentration of OPP2.5 are increased; the contents of IL-6 in serum from mice treated with OPP2.5 and PPP2.5 and the contents of IL-1α and CRP in serum from mice treated with the high concentration of OPP2.5 are increased. At the organic level, no significant abnormality can be observed in liver from mice treated with OPP2.5 and PPP2.5; the slight level of inflammation in heart from mice treated with OPP2.5 can be observed; there is obvious inflammatory cells infiltration in lung from mice treated with OPP2.5 and PPP2.5; the activities of AST and LDH in BALF from mice treated with OPP2.5 and PPP2.5 significantly increased; the contents of Eotaxin, IL-1β, IL-6, LIF, MCP-1, MIP-1α, MIP-2, VEGF, TNF-α, IL-8 and CRP in BALF from mice treated with OPP2.5 and PPP2.5 significantly increased; the contents of IL-1α, IL-1β, IL-6, LIF, MCP-1, MIP-1α, MIP-2 and TNF-α in lung homogenates from mice treated with OPP2.5 and PPP2.5 significantly increased, besides, some inflammatory factors in BALF from mice treated with the low concentration of PPP2.5 are higher than those treated with the same concentration of OPP2.5, such as Eotaxin, MCP-1, MIP-1α, MIP-2, IL-8, etc.; while the activities of SOD in lung homogenates from mice treated with OPP2.5 and PPP2.5 decreased.Conclusions The methods that prepare for OPP2.5, WSP2.5, FSP2.5, and PPP2.5 in these studies are appropriate, and could achieve the expected goals. After A549 cells were treated with those four PM2.5 samples, the results show that WSP2.5 has a relative limited effect on inhibiting cell growth, inducing inflammatory damages and DNA base deletion, while PPP2.5 could show significantly toxic effects in all aspects to A549 cells. After A549 cells were treated with those four PM2.5 samples, the results show that the unsolvable particulate samples, namely OPP2.5 and PPP2.5 have significant inhibitory effects on cells growth. The in vivo exposure experiments show that when causing systemic inflammatory response, OPP2.5 might has stronger toxic effects than PPP2.5 to mice; while when causing lung inflammation, PPP2.5 might have stronger toxic effects than OPP2.5 do. In summary, not only the complex components adsorbed on the solid core granules of PM2.5, but also the solid core granules themselves contributed to the toxic effects. |
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