YIC-IR  > 中国科学院烟台海岸带研究所知识产出
镉和砷对菲律宾蛤仔毒性的剂量-效应关系研究
Alternative TitleDose-effect relationship of toxicities induced by cadmium and arsenic to Ruditapes philippinarum
战君菲
Subtype博士
Thesis Advisor吴惠丰
2021-05-10
Training institution中国科学院烟台海岸带研究所
Degree Grantor中国科学院大学
Place of Conferral中国科学院烟台海岸带研究所
Degree Name理学博士
Degree Discipline海洋生物学
Keyword 菲律宾蛤仔 组学 剂量-效应关系 Cadmium Arsenic Ruditapes philippinarum Omics Dose-effect relationship
Abstract镉(Cd)和砷(As)是近海典型污染物,对海洋生态系统健康构成威胁。因此,深入研究Cd和As对海洋生物毒性的剂量-效应关系,并筛选敏感生物标志物指示近海Cd、As污染具有重要意义。本研究利用Meta分析探究海洋双壳贝类对Cd暴露的响应特征。基于Meta分析的结果选择菲律宾蛤仔整体组织为研究对象开展室内Cd、As暴露实验,利用转录组学、代谢组学等组学技术筛选响应指标,并结合组织病理损伤和生化等指标,利用基准剂量法(BMD)对响应指标的剂量-效应关系进行模型拟合,以期深入揭示Cd和As的毒性效应机制。同时,根据最优模型计算各生物指标的BMD值,筛选单调且灵敏响应的指标作为Cd和As毒性的生物标志物。主要研究结果如下: 1. Meta分析揭示了海洋双壳贝类对Cd暴露的响应特征 按照剔除和纳入标准,共纳入87篇相关文献,获得2042个数据。按照物种、组织、毒性效应指标以及暴露浓度进行Meta分组分析。结果显示,海洋双壳贝类对Cd暴露表现出显著的物种差异,其中蛤对Cd暴露响应程度最大,而各组织响应程度并未表现出显著差异;海洋双壳贝类体内各类生物指标的响应程度存在显著差异,其中解毒和基因毒性相关的指标响应程度最大;暴露浓度是导致Cd毒性差异的主要因素,随暴露浓度的升高,海洋双壳贝类生物指标响应程度显著增加;氧化应激和解毒相关的多个指标对Cd暴露浓度呈现多样化的剂量-效应关系,其中金属硫蛋白(MT)呈现先上升后基本不变的剂量-效应关系,谷胱甘肽过氧化物酶(GPx)和丙二醛(MDA)的剂量-效应曲线均呈倒U型,尤其GPx表现出典型的低剂量刺激、高剂量抑制的毒物兴奋效应,而超氧化物歧化酶和过氧化氢酶未表现出明显的剂量依赖效应。 2. Cd对菲律宾蛤仔毒性的剂量-效应关系 根据环境相关浓度及96 h半致死浓度设定Cd暴露梯度浓度(0、3、9、27、81和243 µg/L),实验室条件下暴露菲律宾蛤仔14 d。利用转录组学、代谢组学等技术对蛤仔整体组织响应基因、代谢物等指标进行高通量筛选,同时测定金属离子、酶活、组织病理损伤及细胞凋亡等毒理学指标。结果显示,随Cd暴露浓度升高,Cd在蛤仔体内富集量逐渐增加,必需金属含量也随之发生变化,且剂量效应曲线呈现多样性。结合转录组学分析,发现Cd暴露在基因调控水平对离子内稳态的影响非常有限,Cd主要通过竞争必需金属离子转运体和必需金属离子结合蛋白干扰离子内稳态。而必需金属含量的改变以及Cd对关键酶活性中心必需金属元素的竞争结合,导致相关基因(如漆酶laccase、谷氨酰胺转移酶TGs、金属还原酶STEAP、钙调蛋白calmodulin等)的异常表达。结合KEGG和GO富集分析发现,Cd暴露干扰细胞内氧化还原的平衡,引起氧化应激,激活细胞凋亡等信号通路,影响菲律宾蛤仔细胞粘附、肽交联、蛋白水解等正常生物学功能。此外,代谢组学、酶活力分析结果显示,Cd暴露以剂量依赖的方式影响了三羧酸(TCA)循环、糖酵解、氨基酸代谢以及氧化磷酸化等关键代谢途径。 利用BMD方法对多种响应指标的剂量-效应关系进行模型拟合,并计算BMD值。结果发现,TGs、MT、STEAP和laccase等DEGs呈单调变化,且BMD值较低,是理想的基因生物标志物。菲律宾蛤仔对Cd暴露最敏感的GO条目和KEGG通路分别为肽交联和细胞粘附通路。代谢组学和酶活力分析结果显示,丙氨酸、葡萄糖、AMP的含量变化和琥珀酸脱氢酶(SDH)的活力呈典型毒物兴奋效应;谷氨酰胺和葡萄糖-1-磷酸的含量变化以及己糖激酶(HK)和柠檬酸合酶(CS)的活力变化呈单调下调,其中CS活力是理想的酶类生物标志物。而谷胱甘肽(GSH)含量和谷胱甘肽巯基转移酶(GST)活力等传统生物标志物呈非单调的剂量-效应关系,不宜作为菲律宾蛤仔响应Cd暴露的生物标志物。 3. As(Ⅴ)对菲律宾蛤仔毒性的剂量-效应关系 根据环境相关浓度及96 h半致死浓度设定As(Ⅴ)暴露梯度浓度(0、0.2、0.5、1.25、2.5和5 mg/L),实验室条件下暴露菲律宾蛤仔14 d。利用高效液相色谱联用电感耦合等离子体质谱法测定菲律宾蛤仔体内砷形态及其含量,结果显示,随As(Ⅴ)暴露浓度的升高,有机砷含量基本不变,无机砷As(Ⅴ)和As(Ⅲ)含量增加,表明菲律宾蛤仔体内发生了As(Ⅴ)到As(Ⅲ)的转化。转录组学分析显示,在高浓度As(Ⅴ)暴露组硫氧还蛋白、GST和3-磷酸甘油醛脱氢酶(GAPDH)等参与As(Ⅴ)转化生成As(Ⅲ)过程的酶基因均显著上调,表明菲律宾蛤仔通过上调关键酶的转录表达促进体内累积的As(Ⅴ)转化生成As(Ⅲ)。 进一步整合转录组和代谢组结果,发现菲律宾蛤仔通过促进GSH的合成、增加对过氧化物等ROS的清除能力等方式抵抗无机砷富集引起的氧化应激。As(Ⅴ)暴露促进蛤仔体内糖酵解反应、TCA循环和氧化磷酸化等过程,为机体提供更多的能量,且关键差异代谢物和DEGs与As(Ⅴ)的暴露浓度呈现多样化的剂量-效应关系;鞘脂类去饱和酶(DEGS)、羟基类固醇脱氢酶(HSD17B6)、羟基丁酸脱氢酶(bdh)基因均显著上调表达,表明As(Ⅴ)暴露还促进脂肪酸氧化代谢等途径,为TCA循环提供更多的乙酰辅酶A。最高浓度As(Ⅴ)暴露组磷酸胆碱含量显著降低,也提示As(Ⅴ)暴露导致菲律宾蛤仔能量代谢紊乱。此外,As(Ⅴ)暴露还干扰了氨基酸代谢。 利用BMD方法对多种响应指标的剂量-效应关系进行模型拟合,并计算BMD值。GST、组织蛋白酶L(CTSL)、ATP结合盒转运蛋白(ABCC)、和maeB等多个DEGs的表达倍数均呈单调曲线,且BMD值较低,因此可作为As(Ⅴ)对菲律宾蛤仔毒性的生物标志物。通过对拟合到最佳模型的DEGs进行富集分析,发现对As(Ⅴ)暴露最敏感的GO条目和KEGG通路分别为半胱氨酸型肽酶活性和吞噬作用。 4. Cd和As(Ⅴ)对菲律宾蛤仔毒性作用机制比较 Cd和As暴露均干扰了菲律宾蛤仔TCA循环、糖酵解以及氧化磷酸化等关键代谢过程,且均表现出毒物兴奋效应;Cd和As(Ⅴ)均通过消耗GSH破坏细胞内氧化还原平衡,诱导氧化应激;菲律宾蛤仔鳃和消化腺的病理损伤指数均随Cd和As(Ⅴ)暴露剂量单调增加,菲律宾蛤仔消化腺组织对Cd和As(Ⅴ)暴露较鳃组织更敏感。同时,Cd和As(Ⅴ)对菲律宾蛤仔的毒性作用机制存在较大差异。Cd暴露导致葡萄糖、ATP和AMP等主要能量代谢物质的含量呈非单调变化,但As(Ⅴ)暴露后蛤仔体内葡萄糖、ATP和AMP的含量并未发生改变;对Cd和As(Ⅴ)暴露敏感的GO条目和KEGG通路完全不同;As(Ⅴ)还表现出对菲律宾蛤仔的生殖内分泌干扰效应
Other AbstractAs typical pollutants in coastal waters, cadmium (Cd) and arsenic (As) have posed serious threats on marine ecology and human health. Therefore, it is necessary to study the toxicological effects and mechanisms of Cd and As in marine organisms as well as to select sensitive biomarkers. In this work, meta-analysis was used to combine the response endpoints in marine bivalves treated with Cd, aiming to outline the response characteristics. The results showed that clams were the most sensitive bivalves to Cd exposure. In order to unravel the dose-effect relationships between pollutants and response endpoints, omics methods including transcriptomics and metabolomics as well as biochemical and histopathological analyses were integrated to identify the endpoints at multiple levels in clam Ruditapes philippinarum treated with Cd and As. Benchmark dose (BMD) analysis was conducted on the responsive endpoints to fit the best dose-response models. Meanwhile, BMD values were calculated based on the best-fitted models, and the endpoints presenting monotonic curves together with lower BMD values were regarded as potential biomarkers. The main results are as bellows:1. Characterization of responses in marine bivalves treated with Cd using meta-analysisAccording to the selection criteria, a total of 87 papers consisting of 2042 data were included for meta-analysis. The total data were grouped in terms of species, tissues, toxicological endpoints and exposure doses because of the high heterogeneity and inconsistency of data structure. The results showed that the responses of endpoints were species-dependent but tissue-dependent. Endpoints in clams were more sensitive to Cd than those in other bivalves. Detoxification and genotoxicity related endpoints presented responses with larger scales. Exposure dose was the primary factor contributing to the toxicity of Cd. The response extent was positive to exposure dose. Especially, endpoints associated with detoxification and genotoxicity presented multiple dose-effect relationships. For example, metallothionein increased in lower concentrations of Cd-treated groups, and maintained constant in higher concentrations of Cd-treated groups, and glutathione peroxidase (GPx) and malondialdehyde presented inverted U-shaped curves. In contrast, superoxide dismutase and catalase did not present significant dose-dependent responses. 2.The dose-effect relationships of Cd toxicity in R. philippinarum Clams R. philippinarum were exposed to six serial concentrations (0, 3, 9, 27, 81, 243 µg/L) of Cd for 14 d. The exposure concentrations were designed according to the environmentally relevant concentrations of Cd and 96 h LC50 of Cd in R. philippinarum. The altered genes and metabolites were identified by using high-throughput transcriptomic and metabolomic analysis, respectively. Conventional endpoints such as metal concentrations, enzyme activities, histopathological indices and apoptosis in clam whole soft tissue were also measured. With the increase of Cd exposure concentration, both Cd and other essential metals were significantly changed and presented multiple dose-effect curves. Transcriptomic analysis revealed that gene regulation posed limited influences on ion homeostasis. Instead, the disruption of ion homeostasis was caused by Cd through competing for binding to active sites in transporter and enzymes, which subsequently induced the dysregulation of genes such as laccase, transglutaminases (TGs), metalloreductase (STEAP), calmodulin. KEGG and GO enrichment analysis found that differentially expressed genes (DEGs) induced by high concentrations of Cd were primarily enriched in focal adhesion, apoptosis, ubiquitin mediated proteolysis, and peptide cross-linking, suggesting that Cd exposure disrupted redox homeostasis and induced apoptosis, redox stress, and necrosis. Moreover, diverse metabolites and enzymes related to tricarboxylic acid (TCA) cycle, glycolysis, amino acid metabolism, and oxidative phosphorylation (OXPHOS) were also altered in dose-dependent manners.BMD analysis was performed on these endpoints to fit the best models, based on which the BMD values were calculated. TGs, MT, STEAP, and laccase were preferable biomarkers due to their monotonic curves and lower BMD values. Peptide cross-linking was the most sensitive GO term, and focal adhesion was the most sensitive KEGG pathway. Glucose, AMP contents as well as succinate dehydrogenase (SDH) activity showed hormetic effect induced by Cd in R. philippinarum. Glutamine and glucose-1-phosphate (G1P) as well as hexokinase (HK) and citrate synthase (CS) activity presented monotonic decrease trends, among which CS activity was regarded as a preferable biomarker because of its lower BMD value. Unexpectedly, glutathione (GSH) and glutathione S-transferase (GST) that were frequently used as biomarkers showed non-monotonic responses in this work, suggesting that GSH and GST in R. philippinarum were not suitable as biomarker of Cd stress. 3. The dose-effect relationships of As(Ⅴ) toxicity in R. philippinarumClams R. philippinarum were exposed to six serial concentrations (0, 0.2, 0.5, 1.25, 2.5 and 5 mg/L) of As(V) for 14 d. The exposure concentrations were designed according to the environmentally relevant concentrations of As(Ⅴ) and 96 h LC50 of As(Ⅴ) in clams R. philippinarum. High-performance liquid chromatography coupling of inductively coupled mass spectrometry (HPLC-ICP-MS) was used to measure As species in the whole soft tissues of clams. With the increase of As(Ⅴ) exposure dose, inorganic arsenics like As(Ⅴ) and As(Ⅲ) were significantly ascended, whereas organic arsenics showed no statistical changes, suggesting that the transformation of As(Ⅴ) into As(Ⅲ) occurred in As(Ⅴ)-treated clams and there was a threshold of As transformation. Transcriptomic analysis revealed that the DEGs involved in As transformation, including thioredoxin, glutaredoxin, glutathione synthetase (GS), GST, and glyceraldehyde-3-phosphate dehydrogenase, were significantly up-regulated, which might contribute to the transformation of As(Ⅴ) into As(Ⅲ).The up-regulation of glutamylcysteine synthetase, GST, GPx and GS transcripts in As(Ⅴ)-treated groups suggested that GSH biosynthesis and elimination of reactive oxygen species were enhanced to resist the oxidative stress induced by As(Ⅴ) and As(Ⅲ). Furthermore, the up-regulation of phosphoenolpyruvate carboxylase, malate dehydrogenase (maeB), ATP synthase, and NADH dehydrogenase transcripts, together with the increases in oxaloacetate, citrate, fumarate, and pyruvate, suggested that As(Ⅴ) promoted glycolysis, TCA cycle, OXPHOS, aiming to provide more energy. As(Ⅴ) promoted pathways such as fatty acid oxidation and hydroxybutyrate metabolism to provide more acetyl-CoA for TCA cycle. The decrease in phosphorylcholine in the highest concentration of As(Ⅴ)-treated group was consistent with the dysregulation of energy metabolism induced by As(Ⅴ). Furthermore, As(Ⅴ) disrupted amino acid metabolism.BMD analysis was performed on the endpoints of transcriptome and HCIs to fit the best models, based on which the BMD values were calculated. GST, cathepsin L, ATP-binding cassette, carbonyl reductase, and maeB in R. philippinarum were preferable biomarkers of As(Ⅴ) toxicity. Phagocytosis was the most sensitive KEGG pathway, and cysteine-type peptidase activity was the most sensitive GO term. 4. The comparison of toxicological mechanisms of Cd and As(Ⅴ) in R. philippinarumBoth Cd and As(Ⅴ) disrupted TCA cycle, glycolysis, OXPHOS in dose-dependent manners, and the responses of these processes presented hormesis. They also induced oxidative stress because of GSH consumption and subsequently disrupting redox homeostasis. The HCIs in gills and digestive glands of R. philippinarum from Cd- and As(Ⅴ)-treated groups showed similar monotonic increase trends, and digestive gland was more sensitive to gill in both Cd- and As(Ⅴ)-treated R. philippinarum. However, there were still several differences between the toxicological mechanisms of Cd and As(Ⅴ) in R. philippinarum. For example, glucose, ATP, and AMP presented U-shaped or inverted U-shaped in Cd-treated R. philippinarum, whereas no significant alterations of these energy related metabolites occurred in As(Ⅴ)-treated R. philippinarum. Peptide cross-linking and focal adhesion were more sensitive in Cd-treated R. philippinarum, while phagocytosis and cysteine-type peptidase activity were more sensitive in As(Ⅴ)-treated R. philippinarum. What is more, As(Ⅴ) induced reproductive endocrine-disrupting effects in R. philippinarum.
Pages207
Language中文
Document Type学位论文
Identifierhttp://ir.yic.ac.cn/handle/133337/30826
Collection中国科学院烟台海岸带研究所知识产出
Recommended Citation
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战君菲. 镉和砷对菲律宾蛤仔毒性的剂量-效应关系研究[D]. 中国科学院烟台海岸带研究所. 中国科学院大学,2021.
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