- 學位論文
環境相關濃度鄰苯二甲酸二 (2-乙基己基) 酯對秀麗隱桿線蟲老化、肥胖、學習與記憶之毒性及調控機制探討
Toxicity of environmentally relevant concentrations of di(2-ethylhexyl)phthalate on aging, obesity, learning and memory and the underlying mechanisms in Caenorhabditis elegans
摘要
鄰苯二甲酸酯類 (phthalates) 常用於各種生活用品，造成環境與食品中的污染。由於鄰苯二甲酸二(2-乙基己基)酯 (DEHP) 常於食品、飲料、生物體及人體被檢測出，因此造成不容忽視的潛在的食品安全及健康危害。目前證據顯示暴露DEHP可能造成多種不良健康效應，包括過早衰老、肥胖以及認知能力受損等。然而目前對於DEHP透過何種生物調控機制造成上述毒性效應仍須進一步探討。 本研究的整體目標為探討暴露環境相關濃度DEHP對於老化、肥胖及記憶與學習所造成的影響及其生物調控機制，並利用模式生物秀麗隱桿線蟲 (C. elegans) 進行研究。本博士論文包含三個子目標：(1) 探討發育期間慢性暴露於DEHP對老化所造成的影響及其調控機制；(2) 探討慢性暴露DEHP誘導肥胖的效應及相關調控機制；及 (3) 探討發育期間慢性暴露於DEHP對C. elegans記憶與學習能力所造成的影響。 子目標 (1) 成果顯示，自L1時期慢性暴露於1.5 mg/L DEHP顯著造成整體健康狀態損傷，並縮短C. elegans之壽命。此外，DEHP也加劇C. elegans咽喉收縮與排泄行為隨老化過程之衰退。此外，DEHP亦造成老化相關指標如脂褐素 (lipofuscin)、脂質過氧化 (lipid peroxidation) 及氧化壓力累積增加。慢性暴露於DEHP也顯著抑制老化相關基因hsp-16.1、hsp-16.49及hsp-70之mRNA表達量。同時，本研究利用insulin/IGF-1 signaling (IIS) 相關基因 (daf-2、age-1、pdk-1、akt-1、akt-2及daf-16) 突變種C. elegans，發現IIS相關基因突變消除了DEHP所造成脂質過氧化累積增加效應，顯示IIS參與調控DEHP之毒性效應。另外，skn-1之突變則加劇DEHP所造成的脂質過氧化累積增加，顯示skn-1亦扮演關鍵角色。因此，發育期間慢性暴露於DEHP可能透過IIS及SKN-1加速C. elegans之老化，亦提供了可能的分子調控基礎。 本研究子目標 (2) 成果顯示早期生命暴露DEHP造成體內脂質及三酸甘油酯 (TG) 含量增加，且效應主要源自於DEHP而非其代謝物。此外，發育階段與暴露時機影響DEHP誘導TG累積增加之效應，且慢性暴露DEHP造成最顯著的效應。慢性暴露DEHP亦改變線蟲體內自由脂質與TG的脂肪酸組成，導致ω-6/ω-3脂肪酸比例增加。本研究發現，慢性暴露DEHP誘導TG增加的效應需脂質合成基因POD-2、FASN-1、FAT-6、FAT-7，以及SREBP同源轉錄因子SBP-1的參與。本研究亦發現慢性暴露環境相關濃度的DEHP誘導XBP-1所調控的內質網壓力，並驅動SBP-1表達增加。本研究成果顯示內質網壓力與SBP-1/SREBP調控的脂質合成可能與DEHP所誘導肥胖效應有關。 本研究子目標 (3) 成果顯示發育初期暴露DEHP抑制第0天成蟲的長期記憶能力。自L1幼蟲時期至第5天成蟲慢性暴露於DEHP，則導致長期記憶能力加速衰退。此外，本研究結果顯示DEHP對長期記憶隨老化衰退之效應與CREB同源轉錄因子CRH-1有關。將C. elegans IIS的唯一受體daf-2突變剔除後可逆轉DEHP對長期記憶能力的抑制，且此逆轉效應需daf-16的參與。同理，daf-2突變剔除後恢復原受到DEHP抑制的CRH-1表達量，且此效應需daf-16的參與。本子目標結果顯示發育初期及慢性暴露於DEHP透過CREB與IIS相關機制加速長期記憶隨老化的衰退。由於CREB與IIS皆在演化上具高度保守性，顯示DEHP亦可能對其他生物的長期記憶造成不良效應。 綜上所述，本博士論文成果顯示，於發育初期慢性暴露於環境相關濃度DEHP對線蟲造成老化相關生物指標表現下降、增加肥胖、以及惡化老化相關的學習與記憶衰退，且DEHP所造成的毒性效應與多種演化上保守的機制如IIS、SKN-1/NrF2、SBP-1/SREBP、內質網壓力及CRH-1/CREB有關。由於以上生物機制在演化上具高度保守性，表示DEHP也可能對其他物種造成相似毒性效應。此博士論文對於提供DEHP慢性毒性深入認知作出貢獻。
關鍵字
鄰苯二甲酸二 (2-乙基己基) 酯 ； 老化 ； 秀麗隱桿線蟲 ； insulin/IGF-1 signaling ； SKN-1/Nrf2 ； 脂質合成 ； SBP-1/SREBP ； 內質網壓力 ； 學習與記憶衰退 ； CRH-1/CREB並列摘要
Phthalates are widely used in various consumer products which lead to increasing occurrence of phthalates in the environment and food. Di(2-ethylhexyl)phthalate (DEHP) is one of the most commonly detected phthalates in food, beverages, biota, and human samples, raising food safety and health concerns. Recent evidence has suggested that exposure to DEHP may result in various adverse health conditions such as premature aging, obesity, and neurocognitive impairment. However, the underlying mechanisms of DEHP toxicity on aging, obesity, and neurocognitive impairment remain largely unknown. Herein the overall objective of this study is to investigate the toxicity of environmentally relevant concentrations of DEHP on aging, obesity, learning and memory and their underlying mechanisms in Caenorhabditis elegans. Three specific aims are included in this doctoral study: (1) to elucidate the toxicity of early-life DEHP exposure on age-related declines in C. elegans and its underlying mechanisms; (2) to investigate the obesogenic effects of DEHP following chronic exposure and the underlying mechanisms; and (3) to decipher the toxicity of DEHP in long-term associative learning and memory (LTAM) of C. elegans and the interaction with aging. The results from specific aim 1 showed that early-life (L1) exposure to 1.5 mg/L DEHP significantly impairs general health status and shortened the mean lifespan of the worms. DEHP exposure further adversely affected pharyngeal pumping rate and defecation cycle in aged worms. DEHP exposure also further enhanced accumulation of age-related biomarkers including lipofuscin, lipid peroxidation, and intracellular reactive oxygen species in aged worms. In addition, exposure to DEHP significantly suppressed gene expression of hsp-16.1, hsp-16.49, and hsp-70 in aged worms. Further evidences showed that mutation of genes involved in insulin/IGF-1 signaling (IIS) pathway (daf-2, age-1, pdk-1, akt-1, akt-2, and daf-16) restored lipid peroxidation accumulation upon DEHP exposure in aged worms, whereas skn-1 mutation resulted in enhanced lipid peroxidation accumulation. Therefore, IIS and SKN-1 may serve as an important molecular basis for DEHP-induced age-related declines in C. elegans. Results in specific aim 2 showed that early-life DEHP exposure resulted in an increased lipid and triglyceride (TG) accumulation mainly attributed to DEHP itself, not its metabolites. In addition, developmental stage and exposure timing influence DEHP-induced TG accumulation, and chronic DEHP exposure resulted in the most significant effect. Chronic DEHP exposure altered fatty acid composition of free fatty acids and TG, resulting in an increased ω-6/ω-3 ratio. The induced TG accumulation by chronic DEHP exposure required lipogenic genes POD-2, FASN-1, FAT-6, FAT-7, and SBP-1. Moreover, environmentally-relevant chronic DEHP exposure induced XBP-1-mediated endoplasmic reticulum (ER) stress and might lead to up-regulation of SBP-1. This study suggests the possible involvement of ER stress and SBP-1/SREBP-mediated lipogenesis in DEHP-induced obesogenic effects. In specific aim 3, the results showed that early-life exposure to DEHP reduced LTAM in wild-type worms at day-0 adulthood. Chronic exposure to DEHP from the L1 stage to day-5 adulthood worsens the age-dependent decline of LTAM. Moreover, the effect of DEHP on age-related LTAM requires CRH-1, a homologue of CREB. Mutations in daf-2, the sole receptor of C. elegans IIS, ameliorated the inhibition of LTAM by DEHP, and the effect depended on daf-16. In addition, daf-2 mutation restored the CRH-1 level in DEHP-exposed worms, and the effect required daf-16. Our study suggests that early-life chronic exposure to DEHP adversely affects age-related LTAM decline and the effect is associated with CRH-1 and IIS in C. elegans. The evolutionary conservation of IIS and CREB implies possible adverse effects by DEHP across species. Taken together, this doctoral dissertation shows that early-life and chronic exposure to environmentally relevant levels of DEHP results in declines of age-related biomarkers, increase of DEHP-induced obesogenic effects, and enhancement of age-dependent decline of LTAM in the nematode C. elegans. The adverse effects of DEHP involve several evolutionarily conserved molecular pathways including IIS, SKN-1/NrF2, SBP-1/SREBP, ER stress, and CRH-1/CREB. These findings also suggest that DEHP-induced toxicities might be conserved in other species due to evolutionary conservation of related pathways. This dissertation contributes to a better understanding for DEHP induced toxicity.