Correlation of transcriptomic responses and metal bioaccumulation in Mytilus edulis L. reveals early indicators of stress

Marine biomonitoring programs in the U.S. and Europe have historically relied on monitoring tissue concentrations of bivalves to monitor contaminant levels and ecosystem health. By integrating ‘omic methods with these tissue residue approaches we can uncover mechanistic insight to help link tissue concentrations to toxic effects. In an effort to identify novel biomarkers and better understand the molecular toxicology of metal bioaccumulation in bivalves, we exposed the blue mussel, Mytilus edulis L., to sub-lethal concentrations (0.54 M) of cadmium, lead, and a Cd+Pb mixture. Metal concentrations were measured in gill tissues at 1, 2, and 4 weeks, and increased linearly over the 4 week duration. In addition, there was evidence that Pb interfered with Cd uptake in the mixture treatment. Using a 3025 sequence microarray for M. edulis, we performed transcriptomic analysis, identifying 57 differentially expressed sequences. Hierarchical clustering of these sequences successfully distinguished the different treatment groups demonstrating that the expression profiles were reproducible among the treatments. Enrichment analysis of gene ontology terms identified several biological processes that were perturbed by the treatments, including nucleoside phosphate biosynthetic processes, mRNA metabolic processes, and response to stress. To identify transcripts whose expression level correlated with metal bioaccumulation, we performed Pearson correlation analysis. Several transcripts correlated with gill metal concentrations including mt10, mt20, and contig 48, an unknown transcript containing a wsc domain. In addition, three transcripts directly involved in the Unfolded protein response (UPR) were induced in the metal treatments at 2 weeks and were further up-regulated at 4 weeks. Overall, correlation of tissue concentrations and gene expression responses indicates that as mussels accumulate higher concentrations of metals, initial stress responses are mobilized to protect tissues. However, given the role of UPR in apoptosis, it serves as an early indicator of stress, which once overwhelmed will result in adverse physiological effects. A total of 52 samples including initial samples and 3 time-points following exposure to cadmium, lead, or a mixture. There were four replicate samples for each treatment and time-point. An additional three replicate samples were used for RT-qPCR.

美国及欧洲的海洋生物监测项目历来依托双壳类动物的组织污染物浓度监测，以评估污染物水平与生态系统健康状况。通过将组学(omic)方法与传统组织残留检测手段相结合，本研究可揭示机制性见解，助力阐明组织浓度与毒性效应之间的关联。

为识别新型生物标志物并更深入理解双壳类动物体内金属生物积累的分子毒理学机制，我们将紫贻贝（Mytilus edulis L.）暴露于亚致死浓度（0.54 μM）的镉、铅以及镉-铅混合溶液中。分别在暴露1、2、4周后对鳃组织中的金属浓度进行测定，结果显示其在4周暴露周期内呈线性上升趋势。此外，实验结果表明混合处理组中的铅会干扰镉的吸收过程。

本研究使用针对紫贻贝的3025序列微阵列(microarray)开展转录组学分析，共鉴定出57个差异表达序列。对上述序列进行层级聚类分析后，成功区分了不同处理组，证明各处理组的基因表达谱具有良好的可重复性。

对基因本体(Gene Ontology, GO)术语进行富集分析发现，多个生物过程经金属处理后发生扰动，包括核苷磷酸生物合成过程、mRNA代谢过程以及应激响应过程。

为筛选表达水平与金属生物积累量相关的转录本，我们开展了皮尔逊(Pearson)相关分析。最终鉴定出多个与鳃组织金属浓度显著相关的转录本，包括mt10、mt20以及contig 48——一种含有wsc结构域的未知功能转录本。此外，在暴露2周的金属处理组中，3种直接参与未折叠蛋白响应(Unfolded Protein Response, UPR)的转录本被诱导表达，并在第4周进一步上调。

总体而言，组织金属浓度与基因表达响应的相关性表明，随着贻贝体内金属积累量升高，机体将启动初始应激响应以保护组织。然而，鉴于未折叠蛋白响应在细胞凋亡中的作用，该响应可作为应激的早期预警指标，一旦应激过载则会引发不利的生理效应。

本研究共包含52份样本，涵盖初始样本以及暴露于镉、铅或混合溶液后的3个时间点样本。每个处理组与时间点均设置4份生物学重复样本。另有3份重复样本用于实时定量聚合酶链反应(Reverse Transcription Quantitative Polymerase Chain Reaction, RT-qPCR)验证。