塑化劑對酵母菌基因穩定性之影響

Abstract

塑化劑是種廣泛用於工業用途上的化合物，常被使用添加於塑膠製品中，而使用最普遍的則是一群稱為鄰苯二甲酸酯類的化合物。目前已知鄰苯二甲酸酯類是種環境賀爾蒙，會造成類似雌激素的作用，由動物實驗已知會造成生殖毒性、DNA損傷等，且在雄性子代中會造成隱睪症、尿道下裂症、不孕症和睪丸異常等生殖相關疾病。其他研究也指出在小鼠實驗發現，會造成其肝臟損傷以及過氧化體增生，造成致癌，且也發現會誘發氧化壓力產生及粒線體的功能缺失。然而，塑化劑對於動物體甚至於人體之基因穩定性及基因突變率的影響仍是未知的。因此，我們以酵母菌做為模式生物，探討塑化劑對細胞基因穩定性之影響。我們首先利用不同劑量的塑化劑dibutyl phthalate (DBP)和dimethyl phthalate (DMP)，將其溶解在液態培養液中與正常及錯誤配對基因剔除 (msh2Δ, msh3Δ, msh6Δ)的酵母菌共培養，觀察其不同天數的生長曲線和細胞存活率。而對於基因穩定性之影響則是藉由基因突變分析法 (CANr, Hom+, Lys+)觀察其突變率。結果顯示，塑化劑DBP及DMP對於正常及突變的酵母菌細胞的生長曲線皆有抑制效果，其中以最高濃度的DMP (5mM)最為顯著。而突變率分析試驗則是發現塑化劑DMP會造成msh6Δ突變株的酵母菌細胞之突變率顯著增加。而在ROS產生量方面，正常及突變細胞在加入塑化劑後，其過氧化氫及超氧陰離子的含量與控制組相比並無差異，但是對於正常細胞在加入DMP後，其過氧化氫量隨天數有顯著增加，此點與Lys+的結果相同。根據我們的研究，塑化劑對於正常細胞及突變細胞都會造成影響，且塑化劑會造成錯誤配對修復缺陷菌株之突變率增加，尤其以DMP會造成msh6Δ細胞株突變率有最明顯的上升，不過對於ROS的產生量，除了加入DMP的正常細胞外，其他組別並沒有顯著的影響。

Plasticizers are abundant compounds used in the manufacturing industry. Some are known as endocrine disruptors, such as xenoestrogens. Others induce reproductive toxicity and damage to DNA. It was shown to cause liver damage, peroxisome proliferation, and induce carcinogenesis in mouse. Animal studise also showed that DEHP caused oxidative stress, mitochondrial dysfunction and contributed phthalate-induced disruption of spermatogenesis. However, it is not known whether DEHP and other derivatives can cause genome instability, which is a hallmark of cancer. Thus, the objective of this study is to study the effect of DBP and DMP on genome stability using S. cerevisiae as a model. We treated yeast cells with various concentrations (0.05mM, 0.5mM, 5mM) of DBP and DMP determined cell growth and viabilities. Normal cells and DNA repair defective cells (msh2Δ, msh3Δ, msh6Δ) were cultured with DBP and DMP or without. Genome stability can be evaluated by measuring rates of gene mutation, including CANr, Hom+ and Lys+ mutation assays. Results showed that plasticizers inhibited cells growth and cell viabilities. Particularly, five milimole of DMP showed the most effective inhibition. We observed that DMP induced the highest mutation rate in msh6Δ cells compared to WT, msh2Δ and msh3Δ cells. There was no significant difference on ROS levels in cells treated with or without DBP and DMP. Our results suggest that DBP and DMP may have adverse effect on genome stability in mismatch repair defective cells.