利用核磁共振光譜儀探討塑膠微粒暴露對小鼠肝臟與小腸之代謝體影響

Abstract

塑膠微粒(microplastics, MPs)泛指尺寸小於 5 毫米的塑膠顆粒。塑膠材料進入環境後，受到物理、化學及生物性作用影響，可持續破碎形成尺寸較小的塑膠微粒，並進一步產生粒徑低於 1000 奈米的次微米塑膠微粒(sub-microplastics)，甚至形成小於 100 奈米的奈米塑膠微粒(nanoplastics)。由於塑膠微粒具有高度持久性且廣泛存在於各類環境介質中，已普遍分布於海洋、河川、土壤及大氣等環境。人類可能經由受污染的飲用水、食物及空氣等途徑接觸塑膠微粒。近年研究顯示，部分塑膠微粒可能跨越生物屏障並分布於不同組織與器官，進而與氧化壓力、發炎反應及代謝失衡等生物效應相關。然而，不同粒徑塑膠微粒對體內代謝體的影響仍有待釐清，尤其缺乏同時考量粒徑、性別、器官及暴露時間之系統性研究。 本研究選用聚苯乙烯次微米與奈米塑膠微粒作為暴露物質，並透過核磁共振(nuclear magnetic resonance, NMR)代謝體學分析，探討其對小鼠小腸與肝臟代謝體之影響。實驗使用八週齡雄性與雌性小鼠，分別暴露於 50 nm 與 500 nm 聚苯乙烯粒子懸浮液，並以二次水作為對照。小鼠每週以胃管灌食兩次，劑量為 10 mL/kg 體重，相當於 10 mg/kg 體重之暴露劑量，暴露時間分為一個月與三個月。暴露結束後採集小腸及肝臟組織進行 NMR 代謝體分析，以比較不同粒徑、暴露時間與性別下的代謝反應。 研究結果顯示，各組相對體重均未呈現顯著差異，但部分暴露組的相對器官重量有所改變。暴露三個月後，50 nm 組雌性小鼠的相對小腸重量顯著降低；肝臟方面，暴露一個月後，50 nm 與 500 nm 組雌性小鼠，以及 500 nm 組雄性小鼠的相對肝臟重量皆顯著低於相應對照組。NMR 代謝體分析進一步顯示，50 nm 與 500 nm 聚苯乙烯粒子暴露後，小腸與肝臟呈現不同程度的代謝變化，且其反應模式隨粒徑、性別及暴露時間而異。在小腸中，代謝反應以雄性小鼠較為明顯，尤其在三個月暴露後，主要涉及能量代謝、核苷酸代謝、腸道菌相相關代謝物及膽汁酸相關代謝。相較之下，雌性小鼠的小腸代謝變化較少，主要呈現與腸道菌相來源有機酸相關的變化。肝臟方面，雄性小鼠的代謝反應相對有限，主要涉及能量與嘌呤相關代謝；雌性小鼠則在一個月與三個月暴露後皆呈現較廣泛的代謝變化，其中以 500 nm 組暴露一個月後的變化較為明顯，主要涉及葡萄糖與能量代謝、胺基酸代謝、膽鹼與磷脂代謝及核苷酸代謝。整體而言，50 nm 與 500 nm 粒子在不同器官中呈現不同的代謝反應模式，顯示聚苯乙烯次微米與奈米塑膠微粒暴露所產生的代謝變化可能受到粒徑與組織類型等因素影響。 綜合本研究結果，聚苯乙烯次微米與奈米塑膠微粒暴露所伴隨的代謝反應具有器官、粒徑、性別及暴露時間上的差異，並未呈現單一且一致的反應模式。儘管相對體重與器官重量的變化相對有限，NMR代謝體分析仍可辨識不同暴露條件下的代謝差異。這些變化主要涉及葡萄糖與能量代謝、胺基酸代謝、核苷酸代謝、膽鹼與磷脂代謝，以及腸道菌相相關代謝過程。未來若能結合脂質體學、腸道菌相分析及其他多體學方法，並延長暴露時間，將有助於進一步釐清聚苯乙烯次微米與奈米塑膠微粒暴露相關的生物作用機制，並提供評估其潛在人類健康影響之科學依據。

Microplastics (MPs) are generally defined as plastic particles smaller than 5 mm. Environmental weathering through physical, chemical, and biological processes can progressively fragment plastic materials into smaller particles, including sub-microplastics (<1000 nm) and nanoplastics (<100 nm). Because of their environmental persistence and widespread occurrence, MPs are present across diverse environmental compartments, including marine and freshwater systems, terrestrial environments, and the atmosphere. Human exposure can therefore occur through multiple routes, including the consumption of contaminated food and drinking water and the inhalation of airborne particles. Recent evidence further indicates that some MPs may cross biological barriers and become distributed in different tissues and organs, where they have been associated with oxidative stress, inflammatory responses, and metabolic alterations. Nevertheless, the metabolic responses associated with different particle sizes remain insufficiently characterized, particularly with respect to the combined influences of particle size, biological sex, target organ, and exposure duration. In this study, polystyrene sub-microplastics and nanoplastics were investigated using nuclear magnetic resonance (NMR)-based metabolomics to characterize metabolic responses in the small intestine and liver of mice. Eight-week-old male and female mice received 50 nm or 500 nm polystyrene particle suspensions (0.1%, w/v) by oral gavage twice weekly at 10 mL/kg body weight, corresponding to a dose of 10 mg/kg body weight. Control animals received double-distilled water. Exposure periods of 1 and 3 months were evaluated. At the end of each exposure period, small intestinal and liver tissues were collected for NMR-based metabolomic profiling to compare metabolic responses across particle sizes, exposure durations, and sexes. The results showed that relative body weight remained comparable across treatment groups, while changes in relative organ weights were observed only in specific groups. After three months of exposure, female mice receiving 50 nm particles showed a lower relative intestinal weight than controls. Lower relative liver weights were also found in female mice exposed to either particle size for one month and in male mice exposed to 500 nm particles for one month. Despite these relatively limited changes in conventional physiological measures, NMR-based metabolomic profiling revealed substantial alterations in the small intestine and liver, with distinct response patterns according to particle size, sex, and exposure duration. In the small intestine, metabolic changes were more evident in male mice, especially after three months of exposure, and were associated mainly with energy and nucleotide metabolism, gut microbiota-related metabolism, and bile acid-related pathways. Female mice showed comparatively fewer intestinal metabolic changes, primarily involving microbiota-derived organic acids. In the liver, male mice exhibited relatively restricted metabolic responses, mainly involving energy- and nucleotide-related metabolites. Female mice showed a wider range of metabolic changes at both exposure durations, with particularly prominent alterations following one month of exposure to 500 nm particles. These changes were associated mainly with glucose, amino acid, choline/phospholipid, and nucleotide metabolism. Taken together, the findings suggest that 50 nm particles were associated more prominently with intestinal metabolic alterations, whereas 500 nm particles produced broader hepatic metabolic responses, although these patterns varied according to sex and exposure duration. In conclusion, exposure to PS sub-MPs and NPs produced distinct metabolic responses across organs, particle sizes, sexes, and exposure durations rather than a uniform toxicological pattern. Despite relatively limited changes in body and organ weights, substantial metabolic alterations were observed, demonstrating that metabolomic profiling can reveal biological responses that may not be apparent from conventional physiological endpoints alone. The affected metabolic processes encompassed glucose and energy metabolism, amino acid metabolism, nucleotide metabolism, choline/phospholipid metabolism, and gut microbiota-related metabolism, with response patterns differing between the small intestine and liver. Collectively, these findings provide a basis for future studies integrating lipidomics, gut microbiome profiling, and other multi-omics approaches to further elucidate the mechanisms of PS-MNP toxicity and improve understanding of the potential long-term implications for human health.