Xiaoli Liu 1,2 Linbao Zhang 1,2 Liping You 1,2 Junbao Yu 1 Ming Cong 1 Qing Wang 1 Fei Li 1 Lianzhen Li 1 Jianmin Zhao 1 Chenghua Li 1 Huifeng Wu 1 1 Key Laboratory of Coastal Zone Environment Processes, CAS; Shandong Provincial Key Laboratory of Coastal Zone Environment Processes, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, P. R. China 2 The Graduate School of Chinese Academy of Sciences, Beijing, P. R. China Research Article Assessment of Clam Ruditapes philippinarum as Heavy Metal Bioindicators Using NMR-Based Metabolomics There are mainly distributed three pedigrees (White, Liangdao Red, and Zebra) of Manila clam Ruditapes philippinarum in Yantai population along the Bohai marine and coast. However, the biological differences to environmental stressors have been ignored in toxicology studies, which could lead to the distortion of biological interpre- tations of toxicological effects induced by environmental contaminants. In this study, we applied a system biology approach, metabolomics to compare the metabolic profiles in digestive gland from three pedigrees of clam and characterize and compare the metabolic responses induced by mercury in clam digestive gland tissues to determine a sensitive pedigree of clam as a preferable bioindicator for metal pollution monitoring and toxicology research. The most abundant metabolites, respectively, included branched-chain amino acids, alanine, and arginine in White samples, glutamate, dimethylglycine, and glycine in Zebra clams and acetylcholine, betaine, glucose, and glycogen in Liangdao Red clams. After 48 h exposure of 20 mgL 1 Hg 2þ , the metabolic profiles from the three pedigrees of clams showed differentially significant changes in alanine, glutamate, succinate, taurine, hypotaurine, glycine, arginine, glucose, etc. Our findings indicate the toxicological effects of mercury exposure in Manila clams including the neurotoxicity, disturbances in energetic metabolisms and osmoregulation in the digestive glands and suggest that Liangdao Red pedigree of clam could be a preferable bioindicator for the metal pollution monitoring based on the more sensitive classes of metabolic changes from digestive glands compared with other two (White and Zebra) pedigrees of clams. Keywords: Bioindicator; Manila clam; Mercury; Metabolomics; NMR Received: September 24, 2010; revised: December 9, 2010; accepted: December 13, 2010 DOI: 10.1002/clen.201000410 1 Introduction Manila clam Ruditapes philippinarum is widely distributed and can been found at high densities in intertidal areas along the Chinese Bohai coast. This species is relatively tolerant to a wide range of temperature and salinity [1–3], and is readily sampled throughout the whole year and maintained in the laboratory. Due to the wide distribution, long life cycle, high tolerance to salinity and tempera- ture, ease of collection, and high bioaccumulation of heavy metals, Manila clam therefore meets most of the criteria that define a bioindicator for metal pollution monitoring and hence has been considered a good bioindicator in marine and coast ecotoxicology [4, 5]. Recent toxicological studies have elucidated distinct bio- chemical and genetic responses of R. philippinarum to heavy metal exposures and other toxic contaminants, which supports the prop- osition that R. philippinarum could be a useful biomonitor for marine and coastal pollutions [6–9]. However, there are dominantly distrib- uted three pedigrees (White, Liangdao Red, and White) of Manila clams in Yantai population along the Bohai coast. To our knowledge, there is a paucity of studies on the biological and biochemical differences between various pedigrees of Manila clams, which could induce distortion to the biological interpretations of toxicological effects. Thus it is extremely necessary to characterize the biological and/or biochemical differences and the sensitivity to the marine and coastal environmental contaminants to determine a preferable ped- igree of clam as bioindicator for metal pollution monitoring. Traditionally, toxicological approaches focus on the measure of specific responses, such as the lysosomal membrane stability to test for activation of catabolic processes or the glutathione peroxidase levels to test for oxidative stress [10]. Recent developments in genomics have greatly expanded the single biomarker approaches [11–13]. Proton NMR ( 1 H-NMR) spectroscopy-based metabolomics is a post-genomic approach that combines the high throughput meta- Correspondence: Professor H. Wu, Key Laboratory of Coastal Zone Environment Processes, CAS, Shandong Provincial Key Laboratory of Coastal Zone Environment Processes, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, P. R. China E-mail: hfwu@yic.ac.cn Abbreviations: AChE, acetylcholinesterase; ADP, adenosine diphosphate; ATP, adenosine triphosphate; BCAA, branched chain amino acids; LV, latent variable; PC, principal component; PCA, principal components analysis; PLS-DA, partial least squares-discriminant analysis; PR, pattern recognition Clean – Soil, Air, Water 2011, 39 (8), 759–766 759 ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com