次微米塑膠微粒在小鼠經由腸胃道的吸收分佈及毒性研究

The distribution and toxicity of submicron plastic particles through the gastrointestinal tract in mice

郭子瑄(Tzu-Hsuan Kuo)

指導教授 ： 鄭尊仁

國立臺灣大學/公共衛生學院/環境與職業健康科學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202101281

摘要

塑膠微粒為近年來興起的全球議題，其被定義為粒徑小於5毫米的聚合物，廣泛的分佈在環境中，已在各大海洋、湖泊及冰川檢測到塑膠微粒，因其不易降解的特性，將隨著食物鏈累積在生物體，除了海洋生物，已有研究顯示哺乳類，例如海豚、海龜及海鷗都有檢測出塑膠微粒。暴露到塑膠微粒的途徑有皮膚吸收、呼吸道吸入及食道攝入這三種途徑，其中，攝入(Ingestion)被認為是人類主要暴露塑膠微粒的途徑。
相關研究過去多著重於微米級塑膠微粒(>1 µm)，顯示塑膠微粒在攝入後將會被腸胃道吸收並且累積在肝臟、脾臟及腎臟等器官，並且將會造成氧化壓力及發炎反應，但結果並不一致，對於哺乳類毒性的研究也很有限。且已有研究顯示越小的塑膠微粒在生物體內累積後的清除效率越低，而在體外細胞實驗，在相同重量濃度下較小的塑膠微粒其因為有較大的總表面積，會產生較大的細胞毒性。為進一步瞭解次微米級塑膠微粒(<1 µm)的健康效應，本研究探討次微米塑膠微粒在小鼠腸道的吸收、在體內的分佈及毒理效應。
本研究使用粒徑800 nm與200 nm的Nile Red螢光聚苯乙烯微珠，以胃管灌食6週大C57BL/6母鼠，為比較200 nm與800 nm微粒在組織的分佈，每次灌食109顆，每週三次連續灌食四週。暴露後24小時尿液以全代謝籠於暴露期間隔週收集，周邊血液於犧牲後由心臟收集，同時採集腸道及其他器官，包括肝臟、脾臟、腎臟及肺臟。使用螢光顯微鏡及全景組織細胞分析儀(TissueFAXS)觀察塑膠微粒在組織的分佈及累積，及使用流式細胞儀(Flow cytometry)來檢測血液、尿液及將器官組織消化過後的組織消化液，以獲得單位微粒數目濃度。
另外檢測生物指標，將部份尿液做四種全身性氧化壓力(Oxidative stress)的濃度檢測，包括8-OHdG、8-NO2Gua、8-IsoPF2a及HNE-MA，及將肺臟及腎臟組織檢測四種發炎因子(Cytokine)的含量，包括Interferon- γ (IFNγ)、Interleukin- 1β (IL-1β)、Interleukin-6 (IL-6)與Tumor Necrosis Factor- α (TNFα)。
在以胃管灌食小鼠四週後，次微米塑膠微粒會穿越腸胃道屏障進入血液循環，並進一步累積在肝臟、脾臟及腎臟，平均每個器官將會有102 ~ 103的微粒累積，並且肺臟在兩者次微米塑膠微粒皆是最主要微粒累積的器官，平均每個肺(右肺)將會有104 ~ 105的微粒累積。比較200 nm與800 nm的微粒累積，200 nm在各器官的平均微粒總累積量是800 nm的10倍左右。在24小時尿液當中，在第二週及第四週從部份800 nm暴露組小鼠尿液中檢測到塑膠微粒，在200 nm微粒暴露組則皆低於偵測極限。另外將肺部切片在螢光顯微鏡底下觀察，觀察到相比800 nm暴露組，200 nm微粒的暴露組肺臟累積的微粒含量要來得多，且大部分的微粒皆被巨噬細胞吞噬而聚集。
暴露組的肝臟重量顯著比控制組輕，且800 nm組別要比200 nm組別要來的輕，顯示較多的800 nm微粒累積在腸道將會導致菌群紊亂，肝臟重量下降。另外尿液中活性氧化物(ROS)為系統性氧化壓力，可以看到暴露組在8-OHdG、8-NO2Gua、8-IsoPF2a及HNE-MA的濃度均顯著比控制組要來得高(p<0.05)，且其中HNE-MA在200 nm微粒暴露組顯著比800 nm微粒暴露組來得高。此外，發炎因子的結果顯示在800 nm暴露組的在腎臟的IFNγ、IL-1β、IL-6與TNFα皆顯著高於控制組(p<0.05)，顯示800 nm微粒在腎臟排出的過程中可能造成了腎臟的發炎損傷，導致總排尿量顯著下降，肺臟發炎因子相較控制組則是沒有顯著變化。
上述實驗結果顯示相比800 nm微粒，200 nm微粒更能穿越腸胃道屏障累積在肝、肺、腎及脾臟當中，其中肺臟為主要累積的器官。部分800 nm微粒將會經由腎臟排泄進入尿液，造成腎臟發炎因子的上升，而200 nm微粒傾向累積在小鼠體內而不易被排出，並且兩者粒徑的微粒皆會引起全身性氧化壓力。本實驗在四週的重複暴露後，顯示較小的塑膠微粒較容易穿越腸胃道屏障並累積在哺乳類動物體內，產生氧化壓力及發炎反應，後續應繼續關注在長期重複暴露次微米或奈米塑膠微粒的毒性研究。

關鍵字

次微米塑膠微粒 ； 胃管灌食 ； 小鼠 ； 吸收效率 ； 組織分佈 ； 流式細胞儀 ； 氧化壓力 ； 發炎因子

並列摘要

Microplastics (MPs) are a global issue that has emerged in recent years, and defined as polymer particles <5 mm in diameter. MPs are widely distributed in the environment, such oceans, lakes and glaciers. Because of the characteristic of undegradable, they are easily accumulated in organisms along the food chain. In addition to marine organisms, there are studies have shown that mammals, such as dolphins, sea turtles and seagulls, have detected plastic particles. Routes to expose plastic particles are ingestion, inhalation and skin contact. Among them, ingestion is considered the major route of human exposure to microplastics.
Most studies in the past focused on micron level plastic particles (>1 µm). These studies have shown that MPs were absorbed by the gastrointestinal tract, accumulated in the liver, spleen, kidneys and caused oxidative stress and inflammation after ingested. However, the results are controversial, and the research on the toxicity of mammals is also very limited. In addition, studies have shown that the smaller the plastic particles, the lower the removal efficiency in the organism. Because of the larger total surface area, the smaller the plastic particles at the same weight concentration, the greater cytotoxicity they will produce in vitro cell experiments. In order to further understand the toxicity of submicron plastic particles (<1 µm), this study investigate the distribution and toxicity of submicron plastic particles through the gastrointestinal tract in mice.
In my research, the 6-weel-old c57BL/6 female mice were treated with plastic particles of 800 nm and 200 nm of Nile Red fluorescent polystyrene particles by oral gavage. In order to compare the distribution of different particle sizes in the tissue, 109 particles for one gavage were given. Oral gavage was performed 3 times a week for 4 weeks. Also, Urine was collected 24 hours after exposure in metabolic cages during the exposure. After sacrificing, the peripheral blood was collected from the heart, and the intestines and other organs, including liver, spleen, kidney, lungs were collected. Fluorescence microscope and TissueFAXS were used to observe the distribution and accumulation of plastic particles in tissues section. In addition, Flow Cytometer (FCM) was used to detect the number concentration of particles in the blood, urine and digestion of tissues.
In addition, I analyzed the level of biological indicators. Part of the urine was analyzed for the level of four kinds of systemic oxidative stress, including 8-OHdG, 8-NO2Gua, 8-IsoPF2a and HNE-MA. The lung and kidney tissues are analyzed for the level of inflammatory factors (Cytokine), including Interferon-γ (IFNγ), Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and Tumor Necrosis Factor-α (TNFα).
After oral gavage the polystyrene for four weeks, the submicroplastics will cross the gastrointestinal barrier, enter the blood circulation, and accumulate in the liver, spleen, kidneys. On average, each organ will have hundreds to thousands of particles accumulated. Among them, the most particles were accumulated in the lungs both in two sizes of submicroplastics. On average, each lung (right lung) will accumulate tens of thousands to hundreds of thousands of particles. The total particle content of 200 nm particles in mice is 10 times more than the number of 800 nm particles in mice. In the 800 nm exposure group, particles in the 24- hour urine of several mice were detected in the second and fourth weeks. However, in the urine of 200 nm particle exposure group, all particles content were below the detection limit. In addition, the lung sections were observed under a fluorescent microscope. The 200 nm particle exposure group had a much higher amount of particles accumulated in the lungs as comparing with the 800 nm particles exposure group, and most of the particles were aggregated by the phagocytosis.
The liver weight of the exposed group was significantly decreased than control group, and the 800 nm group was decreased more than the 200 nm group. Therefore, more 800 nm particles accumulated in the intestinal mucosa would cause microbiota disorders and reduce liver weight. In addition, the reactive oxygen species (ROS) in urine is a systemic oxidative pressure. The 8-OHdG、8-NO2Gua、8-IsoPF2a and HNE-MA level of exposure group is significantly higher than the control group (p<0.05), and HNE-MA level of 200 nm particles group was significantly higher than the 800 nm particle exposure group. In addition, the results of inflammatory factors showed that the four inflammatory factors of kidneys in the 800 nm exposure group were significantly higher than those in the control group (p<0.05). The results indicates that 800 nm particles caused inflammation and damage by the process of clearing particles through the kidney, leading to urine volume decreased significantly. However, the lung inflammation factors did not change significantly comparing with the control group.
The results of the above results show that 200 nm particles can cross the gastrointestinal barrier and accumulate in organs as comparing to the 800 nm particles. Among them, the most particles were accumulated in the lungs both in two sizes of submicroplastics. Furthermore, part of the 800 nm particles will be excreted through the kidneys into the urine, causing an increase in kidney inflammatory factors. However, 200 nm particles tend to accumulate in tissue instead of being excreted. In addition, the particles of both sizes caused systemic oxidative stress. In this experiment, it was shown that smaller plastic particles were easier to cross the gastrointestinal barrier and accumulate in mice. Moreover, submicroplastics will cause oxidative stress and inflammation. Therefore, future studies are needed to focus on the toxicity studies of submicroplastics or nanoplastics in long-term repeated exposure.

並列關鍵字

submicroplastics ； oral gavage ； mice ； uptake ； distribution ； flow cytometry ； oxidative stress ； cytokine

參考文獻

1. Abarghouei, S., Hedayati, A., Raeisi, M., Hadavand, B. S., Rezaei, H., Abed-Elmdoust, A. (2021). Size-dependent effects of microplastic on uptake, immune system, related gene expression and histopathology of goldfish (Carassius auratus). Chemosphere, 276, 129977. doi:10.1016/j.chemosphere.2021.129977

2. Abu-Shanab, A., Quigley, E. M. (2010). The role of the gut microbiota in nonalcoholic fatty liver disease. Nat Rev Gastroenterol Hepatol, 7(12), 691-701. doi:10.1038/nrgastro.2010.172

3. Anselmo, A. C., Gupta, V., Zern, B. J., Pan, D., Zakrewsky, M., Muzykantov, V., Mitragotri, S. (2013). Delivering nanoparticles to lungs while avoiding liver and spleen through adsorption on red blood cells. ACS Nano, 7(12), 11129-11137. doi:10.1021/nn404853z

4. Avery-Gomm, S., Provencher, J. F., Liboiron, M., Poon, F. E., Smith, P. A. (2018). Plastic pollution in the Labrador Sea: An assessment using the seabird northern fulmar Fulmarus glacialis as a biological monitoring species. Mar Pollut Bull, 127, 817-822. doi:10.1016/j.marpolbul.2017.10.001

5. Ayala, A., Munoz, M. F., Arguelles, S. (2014). Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxid Med Cell Longev, 2014, 360438. doi:10.1155/2014/360438