秀麗隱桿線蟲砷的毒理機制

Abstract

砷為一自然環境中存在的物質，因此人類有很高的機率暴露到。本研究目的為探討砷在Caenorhabditis elegans (C. elegans)中的毒理機制，包括以下3個研究目標：（1）探討砷是否造成神經毒性並探討其相關作用機制;（2）探討砷是否加速老化並探討其相關作用機制;（3）探討砷造成的生殖毒性是否會傳遞至子代並探討其相關作用機制。 研究目標1，探討在C. elegans中氧化壓力於砷所造成的神經毒性中扮演的角色。研究結果顯示暴露砷(100 µM)造成C. elegans運動行為（body bends, head thrashes及reversals的頻率）的降低。此外砷(100 µM)也造成熱感應行為的改變，並造成AFD感覺神經元的結構特性損壞。此外砷(100 µM)造成C. elegans體內氧化壓力的增加。預處理抗氧化劑薑黃素(curcumin) (100 μM)則能改善其不利效應。研究結果顯示氧化壓力在砷所造成的神經毒性中扮演關鍵的角色。 研究目標2，探討慢性暴露砷是否造成C. elegans老化加速及其相關機制。結果顯示長期暴露砷(100、500、1000 μM)造成C. elegans壽命顯著的降低。此外砷暴露(100 μM)也造成C. elegans與老化相關的生物指標顯著的變化，包括減少排便次數，腸脂褐質和脂質過氧化的積累過多的現象。進一步的證據顯示，暴露砷造成老化的C. elegans體內氧化壓力顯著增加。此外暴露砷(100 μM)亦造成與老化相關的熱擊蛋白 (HSP-16.1，HSP-16.49，和HSP-70)之mRNA表達量顯著增加。最後，daf-16轉錄因子突變株中對於砷(100 μM)所造成加速老化效應更為顯著，並且在砷暴露(100 μM)下無法誘導超氧化物歧化酶(SOD-3)之mRNA的表達。研究顯示慢性砷暴露造成C. elegans老化加速。此外體內氧化壓力的增加以及轉錄因子DAF-16/ FOXO在砷暴露所造成加速老化作用中扮演關鍵角色。 在研究目標3中，以C. elegans探討母代(F0)暴露砷所造成的繁殖毒性是否會傳遞到子代(F1至F4)。結果顯示母代(F0)暴露砷(1 mM)造成C. elegans產卵數顯著降低，並且觀察到在後代世代（F1至F4）即使沒有暴露砷也造成產卵數顯著減少。此外在母代(F0)幼蟲時期(L4)於1 mM砷暴露24小時後，在F0和F1都發現體內砷的累積。母代暴露砷(1 mM)後，spr-5 (H3K4me2 demethylase LSD/KDM1)之mRNA表達量亦顯著降低。同樣結果亦可以在F1至F3子代中發現，母代暴露造成F1至F3子代spr-5的mRNA之表達量顯著降低。此外在F0至F3之C. elegans體內H3K4me2之含量亦顯著提高。此結果顯示母代暴露砷後，經由影響組蛋白H3K4甲基化作用，因而造成的表觀遺傳的改變可能是砷造成繁殖毒性傳遞的因素。

Arsenic (As) permeates the environment. As a result, humans are continually exposed to it. This study investigates the toxicity regulation of arsenic in Caenorhabditis elegans. The specific aims of the research in this study are: (1) to determine whether or not arsenic exerts neurotoxic effects and what factors and mechanisms are involved; (2) to determine whether or not arsenic accelerates aging process and what factors and mechanisms are involve; and (3) to determine whether or not arsenic exerts transgenerational reproductive toxicity and what factors and mechanisms are involved. For specific aim 1, we investigated the possible roles of oxidative stress in arsenite (As(III))-induced neurotoxicity in C. elegans The results showed that exposure to As(III) (100μM) caused a decrease in locomotor behaviors (frequencies of body bends, head thrashes, and reversals) of C. elegans. In addition, As (III) (100 μM) exposure decreased thermotactic behaviors, and induced severe deficits in the structural properties of the amphid finger neuron (AFD). Exposure to As(III) (100 μM) also caused an elevated production of reactive oxygen species (ROS). Pretreatment with the antioxidant curcumin (100 µM) ameliorated the decrease in locomotor and thermotactic behavior, and the formation of deficits in the structural properties of AFD sensory neurons in As(III)-exposed nematodes. Our study suggests that oxidative stress plays a crucial role in the As(III)-induced neurotoxic effects on locomotor behavior and the structures and function of AFD sensory neurons in As(III)-exposed nematodes. For specific aim 2, we investigated the effects and the underlying mechanisms of chronic arsenite exposure on the aging process in C. elegans. The results showed that prolonged arsenite (100, 500, and 1000 μM) exposure caused significantly decreased lifespan compared to non-exposed ones. In addition, arsenite exposure (100 μM) caused significant changes of age-dependent biomarkers, including a decrease of defecation frequency, accumulations of intestinal lipofuscin and lipid peroxidation in age-dependent manner in C. elegans. Further evidence revealed that intracellular reactive oxygen species (ROS) level was significantly increased in age-dependent manner upon arsenite exposure (100 μM). Moreover, the mRNA levels of transcriptional makers of aging (hsp-16.1, hsp-16.49, and hsp-70) were increased in aged worms under arsenite exposure (100 μM). Finally, we showed that daf-16 mutant worms were more sensitive to arsenite exposure on lifespan and failed to induce the expression of its target gene sod-3 in aged daf-16 mutant under arsenite exposure (100 μM). Our study demonstrated that chronic arsenite exposure resulted in accelerated aging in C. elegans. The overproduction of intracellular ROS and the transcription factor DAF-16/FOXO play key roles in mediating the accelerated aging process by arsenite exposure in C. elegans. This study implicates a potential exoctoxicological and health risk of arsenic in the environment. For specific aim 3, the transgenerational reproduction defects of arsenite on C. elegans were investigated by examining the parental generation (F0) to the fourth offspring generation (F4). The results showed that the total brood size of the C. elegans was significantly reduced (33%) in the F0 by arsenite exposure (1 mM), and the reduction of the brood size was also observed in the offspring generations (F1 to F4), after the arsenite exposure was removed. In addition, both adult worms from F0 and F1 generation accumulated arsenite and arsenate when L4 larvae of F0 were exposed to 1mM arsenite for 24h. Moreover, the mRNA level of the H3K4me2 demethylase LSD/KDM1, spr-5, was significantly reduced in F0 by arsenic exposure (1 mM). Likewise, the mRNA level of spr-5 was also significantly reduced in F1 to F3 generations. In addition, di-methylation of global H3K4 was increased in F0 to F3 generations. Our study suggests that prenatal arsenic exposure causes transgenerational reproduction defects in C. elegans which might be attributed to epigenetic change via Histone H3K4 di-methylation.