微/奈米塑膠顆粒物之生物安全性評估

Biosafety Assessment of Microplastics and Nanoplastics

陳怡君(Yi-Chun Chen)

指導教授 ： 陳谷汎

共同指導教授 ： 林家驊(Chia Hua Lin)

國立暨南國際大學/科技學院/土木工程學系/博士(2021年)

https://doi.org/10.6837/ncnu202100182

摘要

有鑑於環境塑膠顆粒物污染的日趨嚴重，生物體可能經由不同暴露途徑(例如食入及呼吸)攝入塑膠顆粒物，並使大量塑膠顆粒物累積於生物體中。目前相關研究大多以塑膠顆粒物對環境生物之暴露風險檢測為主，並沒有太多研究針對人類健康危害進行評估。因此本研究將利用環境中最常見的聚苯乙烯(polystyrene, PS)塑膠微米(microplastics, MPs)及奈米顆粒物(nanoplastics, NPs)來進行人體肺部及腎臟之生物毒性評估，並進一步釐清塑膠顆粒物於不同細胞內之毒性作用機制。此外，本研究以塑膠顆粒物實際環境濃度計算出人體不同器官之可能暴露劑量，並以其為實驗中不同細胞之反應濃度。此將使本研究能更真實地評估塑膠顆粒物對人類肺部及腎臟細胞所造成的危害及引發相關疾病的風險。
由PSMPs (3.53 μm)和PSNPs (160 nm)暴露於人類肺上皮BEAS-2B細胞及腎臟胚胎HEK293細胞之觀察結果顯示，PSMPs和PSNPs皆會貼附於BEAS-2B細胞和HEK293細胞表面，並進一步攝入及累積於細胞中。PSMPs之暴露會使BEAS-2B細胞存活率下降，此細胞毒性效應與PSMPs可於細胞內誘發大量活性氧分子生成有關。PSMPs亦可誘發BEAS-2B細胞內發炎反應，並抑制BEAS-2B細胞內緊密連接蛋白(Zonula occluden, ZO)及α1-抗胰蛋白酶(α1- Antitrypsin, AAT)等相關蛋白之生成。此現象將使細胞失去細胞屏障之保護機制，進而提高誘發慢性肺阻塞疾病的罹患風險。為了更真實評估PSNPs對人類肺部的影響，本研究在PSNPs實驗中使用較低劑量之實際環境濃度為細胞暴露濃度。根據實驗結果發現，PSNPs之暴露會使BEAS-2B細胞透過細胞凋亡效應導致存活率下降，此細胞毒性效應亦與PSNPs於細胞內誘發大量活性氧分子生成有關。低濃度PSNPs (1 ng/cm2)可於BEAS-2B細胞內誘發ROCK-1、NF-κB及NLRP3等炎症因子之表現，然而在高濃度PSNPs (1000 ng/cm2)下相關發炎因子卻無顯著表達。研判主要原因應為大量細胞凋亡之誘發，導致部分發炎因子表現被抑制。PSNPs之暴露並不會導致BEAS-2B細胞內ZO蛋白之表現量下降，且AAT蛋白之表現量也沒有顯著降低。此結果雖顯示短時間暴露於環境中PSNPs並不會顯著提升慢性肺阻塞疾病之罹患風險，然而長時間暴露的風險我們亦不能排除。此外，PSMPs之環境濃度暴露會使HEK293細胞透過自噬和細胞凋亡效應導致存活率下降，該細胞毒性效應亦與PSMPs誘發大量活性氧分子生成有關。PSMPs雖然沒有於HEK293細胞內誘發發炎反應，但卻抑制HEK293細胞內緊密連接蛋白(ZO)及α1-抗胰蛋白酶(AAT)等相關蛋白之生成，此將提高誘發慢性腎臟疾病的罹患風險。由上述所有研究結果顯示，長期暴露於塑膠顆粒物污染環境中，確實可能對人體之肺部及腎臟存在危害之風險。

關鍵字

塑膠微米顆粒 ； 塑膠奈米顆粒 ； 發炎反應 ； 細胞凋亡 ； 慢性肺阻塞 ； 慢性腎臟病

並列摘要

Due to the increasing pollution of plastic particles in the environment, organisms may ingest plastic particles through different exposure routes (e.g. ingestion and inhalation) and accumulate large amounts of plastic particles. At present, most of the related studies focus on the detection of the exposure risk of plastic particles to environment and organisms, and there have not been many studies to evaluate health hazards on human. Therefore, in this study, the most common polystyrene plastic microparticles (PSMPs) and nanoparticles (PSNPs) in the environment were used to evaluate the biological toxicity of human lungs and kidneys and to further clarify the toxic mechanism of plastic particles in different cells. In addition, the actual environmental concentrations of plastic particles were used to calculate the possible exposure doses to different organs of the human body, which were used as the response concentration of different cells in the experiment. This study would enable a more realistic assessment of the impact of plastic particles on human lung and kidney cells and the risk of related diseases.
The exposure of PSMPs and PSNPs to epithelial BEAS-2B cells of human lung BEAS-2B and renal embryonic HEK293 cells showed that both PSMPs and PSNPs adhered to the surface of BEAS-2B cells and HEK293 cells and were further ingested and accumulated in the cells. Exposure to PSMPs decreased the survival rate of BEAS-2B cells, and the cytotoxic effect of PSMPs on BEAS-2B cells was related to its ability to induce a large number of reactive oxygen molecules (ROS) in the cells. PSMPs also induced the BEAS-2B cell inflammatory reaction and inhibited the production of tight junction proteins (Zonula occluden, ZO) in BEAS-2B cells, α1-antitrypsin proteins (AAT) and others. This phenomenon would make the cell lose the protective mechanism of cell barrier, then increase the risk of inducing chronic obstructive pulmonary disease (COPD). To more realistically assess the effects of PSNPs on human lung, the present study used a lower dose of actual environmental concentration as cell exposure concentration in the PSNPs experiment. According to the experimental results, the PSNPS could decrease the survival rate of BEAS-2B cells through apoptosis, and the cytotoxic effect was related to the generation of ROS induced by PSNPs. Low concentration of PSNPs (1 ng/cm2) could induce the expression of inflammatory factors such as ROCK-1, NF-κB and NLRP3 in BEAS-2B cells, but not in high concentration of PSNPs (1000 ng/cm2). This study suggested that the main reason was due to the induction of apoptosis, which resulted in the inhibition of some inflammatory factors. The exposure of PSNPs did not result in a decrease of expression of ZO protein in BEAS-2B cells, and the expression of AAT protein was only slightly decreased (there was no significant difference). Although short-term environmental exposure to PSNPs did not significantly increase the risk of COPD, long-term exposure cannot be ruled out. In addition, the survival rate of HEK293 cells was decreased by the autophagy and apoptosis induced by PSMPs. The cytotoxic effect of PSMPs was also related to the production of ROS. Although PSMPs did not induce inflammatory response in HEK293 cells, it inhibited the production of the protein ZO and AAT-associated proteins in HEK293 cells, which would increase the risk of chronic kidney disease. The results of all these studies suggested that long-term exposure to plastic particulate pollution may pose risks to the lungs and kidneys.

並列關鍵字

Microplastics ； Nanoplastics ； Inflammation ； Cell apoptosis ； Chronic Obstructive Pulmonary Disease (COPD) ； Chronic Kidney Disease (CKD)