| 其他摘要 | As 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. |
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