亞慢性暴露微米與次微米聚苯乙烯塑膠微粒對小鼠肝毒性與腎毒性研究

Hepatotoxicity and Nephrotoxicity induced by subchronic exposure to polystyrene microplastics in mice

林庭安(Ting-An Lin)

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(2022年)

https://doi.org/10.6342/NTU202202669

摘要

塑膠製品已是人類生活不可或缺的一部份，然而過度使用，導致環境內積累大量的塑膠垃圾卻難以被分解。目前研究已知塑膠產品會透過物理、化學以及生物性的方式降解成塑膠碎片，小於5毫米的塑膠碎片被定義為微米級塑膠微粒，介於1毫米到5毫米之間的塑膠碎片被稱為次微米級塑膠微粒。塑膠微粒可藉由攝入、吸入以及皮膚接觸進入體內，目前研究認為攝入是人類主要的暴露途徑。
先前我們實驗室研究經胃管灌食暴露次微米級塑膠微粒四週後，發現跟控制組相比，暴露組小鼠24小時排尿量減少、腎臟發炎反應以及肝臟重量減少。此外，近期研究指出塑膠微粒暴露後會導致肝臟脂質代謝異常、胰島素調節失常以及天門冬胺酸轉胺酵素 (Aspartate Transaminase, AST) 與丙胺酸轉胺酵素 (Alanine aminotransferase, ALT) 指數上升。少數論文也發現塑膠微粒對腎臟的毒性，包括腎臟重量下降、促發炎因子含量上升、腎小管損傷等。然而，至今塑膠微粒引起肝毒性及腎毒性的確切機制仍不明確，此外上述研究多為急性或亞急性研究，所以本研究探討亞慢性暴露塑膠微粒後引起的肝、腎毒性，進一步瞭解腸道菌群與肝毒性之間的關聯，亦會分析微米與次微米級塑膠微粒對肝、腎臟毒性之差異。
所有的動物實驗皆通過國立臺灣大學實驗動物照護及使用委員會審查（同意書編號：20201287）。本研究使用次微米（0.5 微米）以及微米（5微米）之尼羅紅螢光聚苯乙烯塑膠微粒 (Polystyrene microplastic, PS-MPs)，每週胃管灌食小鼠兩次，每次0.3毫克，共12週。暴露之前、中以及後利用代謝籠收集小鼠的尿液以及糞便共5次。在實驗終點，小鼠將斷食4小時，並且用愛福寧麻醉犧牲小鼠，收集周邊血液、肝臟、腎臟以及腸道內容物以 -80度保存待後續分析。將代謝籠收集之尿液進行基本檢測，以及用Elisa kit檢測腎臟早期損傷生物標誌物，包括N-acetyl-β-D-glucosaminidase (NAG)、Kidney Injury Molecule-1 (KIM-1)、Neutrophil gelatinase-associated lipocalin (NGAL) 以及Tissue inhibitor of metalloproteinases-2 (TIMP-2) 與Insulin like Growth Factor Binding Protein 7 (IGFBP7)。血液進行血清生化檢測，檢測項目包括，白蛋白 (Albumin, ALB)、ALT、AST、總膽固醇 (Total cholesterol, TCH)、三酸甘油酯 (Triglyceride, TG)、血糖 (Glucose, GLU)、高密度脂蛋白 (High-density lipoprotein, HDL)、低密度脂蛋白 (Low-density lipoprotein, LDL)、血清尿素氮 (Blood urea nitrogen, BUN) 與血清肌肝酸 (Serum creatinine, sCr)。肝臟與腎臟切片以H E染色與Oil red O染色進行病理分析。以Elisa kit 檢測腎臟與肝臟消化液中促發炎因子，包括Interferon-γ (IFN-γ)、Interleukin-1β (IL-1β)、Interleukin-6 (IL-6) 及Tumor necrosis factor-α (TNF-α)。以Elisa kit 檢測肝臟與腎臟消化液中氧化壓力指標，包括Superoxide Dismutase (SOD)、8-Oxo-2'-deoxyguanosine (8-OhdG)、8-nitroguanine (8-NO2Gua)、4-hydroxy-2-nonenal-mercapturic acid (HNE-MA) 以及Prostaglandin F2α (PGF2a)。此外，全景組織細胞分析儀 (TissueFAXS) 與流式細胞儀 (Flow cytometry, FCM)，為檢測 PS-MPs進入肝臟與腎臟後的分佈。最後使用盲腸內容物進行 16S rRNA 基因測序分析檢測菌群豐度改變。
對於肝臟的研究結果表示，0.5 µm暴露組在TissueFAXS發現PS-MPs 累積，在 FCM 檢測下，平均有 105 顆 PS-MPs 累積在 1 克的肝臟組織中。此外，血清檢測肝功能指標 AST 與 ALT 以及代謝指標 TG 及 GLU ，在 0.5 µm暴露組顯著高於控制組。生物指標氧化壓力包括 SOD 與 HNE-MA 及促發炎因子 IL-6 在 0.5 µm 暴露組顯著高於控制組。而後根據組織病理學的檢測，發現經過Oil red O 染色後脂肪油滴表現量在0.5 µm暴露組顯著高於控制組。5 µm暴露組發現肝臟重量指數顯著高於控制組，在血清生化檢測，HDL顯著高於控制組。腎臟結果雖未觀察到PS-MPs的累積或是組織學變化，然而在氧化壓力 SOD 以及促發炎因子 IL-6 與IFN-γ，0.5 µm暴露組表現量顯著高於控制組。此外，相關性分析結果顯示，放線菌門以及脫鐵桿菌門的改變對 ALT、AST 與 TG 變化有高度正相關。上述實驗結果表明，亞慢性暴露次微米PS-MPs後，可能藉由進入小鼠肝臟內累積或改變腸道菌群豐度，直接與間接的引起小鼠肝臟氧化壓力、發炎反應以及血糖與脂質的代謝異常。腎臟中雖未觀察到塑膠微粒的累積，但仍可在次微米級塑膠微粒暴露組別發現氧化壓力與發炎反應的增加。
總體來說，經過12週重複暴露 PS-MPs 後，次微米 PS-MPs 對肝臟與腎臟的毒性反應大於微米級，且對肝臟代謝功能有較大的毒性，後續應繼續探討次微米塑膠微粒長期暴露對肝臟代謝機制的影響。

關鍵字

塑膠微粒 ； 胃管灌食 ； 流式細胞儀 ； 肝毒性 ； 腎毒性 ； 氧化壓力 ； 發炎因子 ； 代謝異常

並列摘要

Plastic products have become useful in many aspects of human life because of their excellent chemical and physical properties. However, over-used plastic products and slow biodegradation rate, resulting in the accumulation the plastic litters in the environment. Practically, plastic products are degraded into microplastic through chemical and physical degradation. Microplastic and submicron plastic are a kind of plastic particle with a diameter of 1 µm to 5 mm and less than 1 µm, respectively. These plastic particle fragments can enter the human body through ingestion, inhalation and dermal contact. Ingestion is considered the major exposure route.
Our previous research investigated the distribution and toxicity of submicron plastic particles in mice by oral gavage. We found that 24-hour urine was decreased, inflammatory factors of kidney were increased and liver weight was decreased as compared to control group after 4 weeks exposure. Recent studies have indicated that microplastics could accumulate in liver tissue, and alter the composition and diversity of gut microbiota, thus causing hepatic lipid disorder, insulin resistance, and abnormal liver enzymes test Aspartate Transaminase (AST), Alanine aminotransferase (ALT). Furthermore, a few articles also found some toxicological effects on kidney in mice, including decreased kidney weight, increased proinflammatory cytokines, and tubular injury and albumin leakage in urine sample. However, the exact mechanisms remain unclear. In addition, most studies are acute or subacute toxicity tests. Therefore, I would like to investigate the nephrotoxicity and hepatotoxicity induced by subchronic exposure to polystyrene microplastics in mice, and further study the association between hepatotoxicity and gut microbiota. The toxicity between micron and submicron plastic particles will also be compared.
All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC Approval No: 20201287). In my study, 6 weeks old C57BL/6 female mice were administered with 0.3 mg of 0.5 µm (submicron) and 5 µm (micron) Nile red fluorescent polystyrene microplastics (PS-MPs) twice a week for 12 weeks by oral gavage. Mouse urine were collected in metabolic cages for 24 hours before, during and after the exposure experiment. At the end of the experiment, all mice were fasted for 4 hours, anesthetized with isoflurane and sacrificed. After sacrificing, the peripheral blood, liver tissue, kidney tissue and cecum content were collected and stored in -80 ℃ refrigerator. Urine were analyzed for basic urinalysis and early molecular biomarkers for kidney dysfunction by Elisa kit, including N-acetyl-β-D-glucosaminidase (NAG), Kidney Injury Molecule-1 (KIM-1), Neutrophil gelatinase-associated lipocalin (NGAL), and Tissue inhibitor of metalloproteinases-2 (TIMP-2) and Insulin like Growth Factor Binding Protein 7 (IGFBP7). Serum biochemistry were used to test kidney and liver damage by microplastic, including Albumin (ALB), ALT, AST, Total cholesterol (TCH), Triglyceride (TG), Glucose (GLU), High-density lipoprotein (HDL), Low-density lipoprotein (LDL), Blood urea nitrogen (BUN), and serum creatinine (sCr). Liver and kidney tissue were stained for light microscopy examination and Oil red O test for pathological changes. Furthermore, proinflammatory factors of Interferon-γ (IFN-γ), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Tumor necrosis factor α (TNFα) and markers of oxidative stress of Superoxide Dismutase (SOD), 8-Oxo-2'-deoxyguanosine (8-OhdG), 8-nitroguanine (8-NO2Gua), 4-hydroxy-2-nonenal-mercapturic acid (HNE-MA) and Prostaglandin F2α (PGF2a) were measured by ELISA kits. TissueFAXS and Flow Cytometry (FCM) were carried out for realizing the distribution and determining the number of particles in the liver and kidney, respectively. To determine the main constituents of the gut microbiome, cecum content were processed with 16S rRNA gene sequencing analysis, and colon content.
The study showed 0.5 µm PS-MPs could accumulate in the liver section of exposure group. On average, liver tissue have 105 particles/g accumulated. Furthermore, we found AST, ALT, TG and GLU level were significantly higher than control group in serum biochemistry analysis. Markers of Oxidative stress and proinflammation factors analysis in liver, we found SOD, HNE-MA level and IL-6 level of 0.5 µm PS-MPs exposure group were significantly higher than the control group. The result of pathological examination showed that the expression of lipid droplets in 0.5 µm PS-MPs exposure group were significantly higher than control group after Oil red O staining. Although no accumulation of 0.5 µm PS-MPs or histological changes were observed in the kidney, the SOD level and IL-6, IFN-γ level were significantly higher in the 0.5 µm PS-MPs exposure group than in the control group in kidney. The 5 µm PS-MPs exposure group found that the liver weight index was significantly higher than that of the control group, and in serum biochemical tests, HDL was significantly higher than that of the control group. In addition, correlation analysis indicated that the alterations of Actinobacteria and Deferriobacteria were positively correlatied with the changes of ALT, AST and TG level in serum.
The results above indicated that subchronic exposure to submicron PS-MPs may directly and indirectly induced oxidative stress, increased inflammatory cytokines, insulin resistance and lipid metabolism disorder by PS-MPs accumulation in liver and the altered gut microbiota in mice. Although we did not observe PS-MPs in kidney, the oxidative stress and inflammation cytokines were increased after exposure to submicron microplastics. After continuous exposure to PS-MPs for 12 weeks, submicron PS-MPs can cause severe toxicity effects than micron PS-MPs in liver and kidney. Long-term effects of submicron plastics on mice liver are worth noting in the future.

並列關鍵字

microplastics ； oral gavage ； flow cytometry ； hepatotoxicity ； nephrotoxicity ； oxidative stress ； cytokine ； metabolic disorder

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