【作者】 秦洁芳；

【导师】 贾晓平；

【作者基本信息】 上海海洋大学 ， 渔业资源， 2011， 硕士

【摘要】 邻苯二甲酸酯类化合物（PAEs）作为在工业中大量使用的人工合成有机物,其对水生生物的潜在毒性隐患已引起了人们的广泛关注,对其毒性毒理研究也成为目前环境科学研究中的一个热门问题。本研究以工业生产中使用量最大的两种PAEs（邻苯二甲酸二丁酯（DBP）和邻苯二甲酸二乙基己酯（DEHP））为研究对象,以南海区重要的经济养殖水产品（翡翠贻贝、红鳍笛鲷、紫红笛鲷）作为实验生物,利用生物体的抗氧化指标（超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）、谷胱甘肽-S-转移酶（GST）、丙二醛（MDA））,乙酰胆碱酯酶（AChE）,代谢酶解毒酶EROD以及功能基因CYP1A作为指标,并利用组织切片观察了DBP胁迫对鱼类组织的损伤情况,探讨了PAEs对海洋生物的毒性效应,为阐明PAEs对不同海洋生物的影响及作用机理提供参考数据。研究结果如下:1、DBP、DEHP对翡翠贻贝内脏团和外套膜中的SOD、CAT活性存在明显的胁迫效应。DBP胁迫下翡翠贻贝内脏团中的SOD、CAT活性主要表现被抑制;外套膜中的SOD活性主要表现为先抑制后诱导（P<0.05）,CAT活性变化波动较大,规律不明显。DEHP胁迫下翡翠贻贝内脏团SOD活性表现为先诱导升高后被抑制降低（P<0.05）,CAT活性则表现为先被抑制降低后诱导升高;DEHP胁迫初期外套膜中的SOD活性在低浓度组受到抑制,高浓度组被诱导,4d后SOD活性逐渐恢复对照组水平。MDA在DBP、DEHP胁迫过程中都有明显的增加,表明PAEs导致海洋贝类脂质过氧化损伤。翡翠贻贝在PAEs胁迫结束移入清洁海水后,其抗氧化酶和MDA含量的变化在DBP胁迫实验中能在较短的时间内恢复,恢复情况较DEHP好,表明DBP长期胁迫引起的毒性损伤较DEHP轻。2、急性毒性实验结果表明,DBP对红鳍笛鲷幼鱼的96hLC50为6.66 mg·L^-1,安全浓度为2.02 mg·L^-1;DEHP的96hLC50为10.73 mg·L^-1,安全浓度为3.01 mg·L^-1;从毒性上判断这两种PAEs化合物对红鳍笛鲷幼鱼都属于高毒物质,且在水体中DBP对生物的毒性高于DEHP,认为与这两种PAEs在水体中的溶解性有关。3、DBP对红鳍笛鲷肝脏和鳃组织中的SOD活性表现为先诱导后抑制,具有明显的时间-效应。DEHP胁迫下红鳍笛鲷肝脏中的SOD活性也被明显诱导（P<0.05）,鳃中则表现为先诱导后抑制。两种PAEs胁迫下红鳍笛鲷肝脏和鳃组织内的MDA都有显著的升高（P<0.05）,表明PAEs能在短时间内对生物体产生氧化胁迫效应,造成脂质过氧化损伤。另外,两种PAEs胁迫下红鳍笛鲷幼鱼脑组织AChE活性表现为一段时间后受到极为明显的诱导,表明PAEs对海水鱼类神经传导具有明显的刺激,AChE是一种对PAEs胁迫具有很强的指示性的指标。4、DBP胁迫下紫红笛鲷肝脏组织中的POD活性被显著诱导,鳃中则被抑制。肝脏和鳃组织中的GST活性在DBP胁迫下发现明显的变化,15d后0.5 mg·L^-1浓度组被显著诱导。5、紫红笛鲷肝脏中的代谢酶EROD活性在7d后相对于对照组活性升高,15d后又受抑制降低;而鳃中的EROD活性在3d、7d都表现为低于对照组,15d后才相对于对照组升高。EROD活性变化“时间-效应”明显。研究也表明肝脏组织中的EROD活性高于鳃,变化也更敏感。本研究从紫红笛鲷肝脏和鳃中克隆得出了CYP1A cDNA序列,序列分析表明了该基因为CYP1A亚家族成员。利用qRT-PCR定量检测DBP胁迫紫红笛鲷肝脏和鳃mRNACYP1A表达水平变化的结果表明,在DBP胁迫肝脏CYP1A mRNA表达先受到显著抑制其后被诱导最后又被抑制,鳃中则先抑制后诱导,这与EROD酶的结果基本一致,但与抗氧化酶间的关系不明显。6、DBP长期胁迫下紫红笛鲷肝脏和鳃组织结构发生了明显的改变,肝脏细胞出现肿大,细胞质中出现空泡,少数细胞核有变形的现象,脂肪粒沉积;鳃中鳃小片发生明显的肿胀、融合、卷曲变形、坏死脱落。7、DBP对翡翠贻贝造成的损伤较DEHP轻,而其对鱼类的毒性却高于DEHP;翡翠贻贝内脏团对PAEs胁迫指示性较外套膜高;鱼类肝脏组织内的抗氧化防御体系指示性较鳃高,其肝脏组织中的EROD酶和CYP1A基因表达变化较鳃组织敏感,脑组织中的AChE则是PAEs的指示性指标。更多还原

【Abstract】 The phthalic acid esters （PAEs） is one of the most extensively used synthetic organic chemicals. More and more attention was paid to its toxicity to the aquatic organism. Recently, the study on the toxicity of PAEs is highlighted. The present study aimed to elucidate the toxic effect of dibutyl phthalate （DBP） and di-（2-ethylhexyl） phthalate （DEHP） on Green mussel （Perna viridis）, Crimson snapper （Lutjanus erythropterus） and River snapper （Lutianus argentimaculatus）. The super oxide dismutase （SOD）, catalase （CAT）, peroxidase （POD）, glutathione Stransferase （GST）, malondialdehyde （MDA） contents and acetylcholinesterase （AChE） in brain, EROD activity and CYP1A gene expressions were examined. In addition, the morphology of liver and gills of river snapper were analysized using routin histological technique and light microscope analysis.1、The SOD and CAT activities of green mussel were significantly influenced by the stresses of DBP and DEHP. When exposed to DBP, the activities of SOD and CAT of the visceral mass were significantly restrained. Whereas,the activity of SOD of the mantle was firstly significantly increased and then decreased. There was an obvious fluctuation inCAT activity and no regular changing pattern existed. When exposed to DEHP, the activity of SOD of the visceral mass was generally followed the same pattern as that of DBP. But CAT was adverse. In the beginning, the SOD activity of the mantle was decreased under a low dose of DEHP and increased under a high dose of DEHP. However, four days later, the mussel in all treatment groups recovered. The MDA content increased with increasing DBP, indicated that PAEs resulted to a lipid peroxidation （LPO） damage on shellfish in the ocean. During the period of purification, the time for the activities recovery of SOD, CAT and the content of MDA lowered to the controlled level was shorter than under DBP stress than that of DEHP.2、The 96h LC50 of DBP was 6.66 mg·L^-1, and the safe concentration （SC） in our study was 2.02 mg·L^-1 on crimson snapper; 96h LC50 of DEHP was10.73 mg·L^-1, the SC was 3.01 mg·L^-1. Thus, the two poisonous chemicals were highly toxic materials for crimson snapper. Furthermore, our data indicated that DBP was even worse than DEHP, due to its higher dissolvability in seawater.3、The SOD activity of the liver and gills of crimson snapper firstly increased and then inhibited by DBP. Under DEHP stress, SOD activity was increased in liver, but decreased in gills. With the, MDA activity significantly increased with increasing PAEs concentration, indicated oxidation stress to fish in a short time and leading to LPO. Moreover, it was demonstrated that the PAEs induced the activity of AChE as one of effective index, implying that it significantly stimulated the nerve conduction of marine fish.4、Under DBP stress, POD activity was increased in liver, but decreased in gills of river snapper; GST significantly fluctuated in these two tissues and dose was induced after 15d exposed 0.5 mg·L^-1.5、For crimson snapper, the EROD in the liver under DBP stress was higher than the controlled level after 7d and then decreased on the 15d. While, in the gills the EROD were lower than the control group on either 3d or 7d, but higher on the 15d. The results suggested that the activity of EROD in the liver was higher than in the gills and more sensitive. The CYP1A cDNA cloned from the tissues was one of the subfamily members. The quantitative examination by qRT-PCR showed that the change of the expression level of CYP1A mRNA was generally similar to the EROD activity.6、The results from the morphology of the two tissues of river snapper revealed that cells enlarged, vacuoles occurred in the cytoplasm, and the karyomorphism was changed in liver. Also, branch leaf was significantly swelled, blended, fused, necrotic and scaled in gill.7、For the green mussel, the toxic effects of DBP was less than that of DEHP. However, the toxic effects of DBP were higher than that of DEHP. Under the exposure of PAEs, the activity of enmity in the visceral mass was more sensitive than mantle forgreen mussel, and liver was better than gills. The antioxidase, EROD and CYP1A expressions in fish liver were more sensitive than in gills. AChE in brain was an early-warning index to PAEs exposure.更多还原