Digilio Giuseppe, Sforzini Susanna, Cassino Claudio, Robotti Elisa, Oliveri Caterina, Marengo Emilio, Musso Davide, Osella Domenico, Viarengo Aldo
Department of Sciences and Technological Innovation (DiSIT), University of Piemonte Orientale "Amedeo Avogadro", Viale T. Michel 11, 15121 Alessandria, Italy.
Department of Sciences and Technological Innovation (DiSIT), University of Piemonte Orientale "Amedeo Avogadro", Viale T. Michel 11, 15121 Alessandria, Italy.
Comp Biochem Physiol C Toxicol Pharmacol. 2016 May-Jun;183-184:61-71. doi: 10.1016/j.cbpc.2016.02.003. Epub 2016 Feb 17.
Numerous studies on molluscs have been carried out to clarify the physiological roles of haemolymph serum proteins and haemocytes. However, little is known about the presence and functional role of the serum metabolites. In this study, Nuclear Magnetic Resonance (NMR) was used to assess whether changes of the metabolic profile of Mytilus galloprovincialis haemolymph may reflect alterations of the physiological status of the organisms due to environmental stressors, namely copper and temperature. Mussel haemolymph was taken from the posterior adductor muscle after a 4-day exposure to ambient (16 °C) or high temperature (24 °C) and in the absence or presence (5 μg/L, 20 μg/L, or 40 μg/L) of sublethal copper (Cu(2+)). The total glutathione (GSH) concentration in the haemolymph of both control and treated mussels was minimal, indicating the absence of significant contaminations by muscle intracellular metabolites due to the sampling procedure. In the (1)H-NMR spectrum of haemolymph, 27 metabolites were identified unambiguously. The separate and combined effects of exposure to copper and temperature on the haemolymph metabolic profile were assessed by Principal Component Analysis (PCA) and Ranking-PCA multivariate analysis. Changes of the metabolomic profile due to copper exposure at 16 °C became detectable at a dose of 20 μg/L copper. Alanine, lysine, serine, glutamine, glycogen, glucose and protein aliphatics played a major role in the classification of the metabolic changes according to the level of copper exposition. High temperature (24 °C) and high copper levels caused a coherent increase of a common set of metabolites (mostly glucose, serine, and lysine), indicating that the metabolic impairment due to high temperature is enforced by the presence of copper. Overall, the results demonstrate that, as for human blood plasma, the analysis of haemolymph metabolites represents a promising tool for the diagnosis of pollutant-induced stress syndrome in marine mussels.
为阐明血淋巴血清蛋白和血细胞的生理作用，已对软体动物开展了大量研究。然而，关于血清代谢物的存在及其功能作用却知之甚少。在本研究中，利用核磁共振（NMR）评估地中海贻贝血淋巴的代谢谱变化是否可能反映由于环境应激源（即铜和温度）导致的生物体生理状态改变。在贻贝暴露于环境温度（16℃）或高温（24℃）4天后，从后闭壳肌采集血淋巴，且分别在不存在或存在（5μg/L、20μg/L或40μg/L）亚致死铜（Cu(2+)）的情况下进行采集。对照贻贝和处理过的贻贝血淋巴中的总谷胱甘肽（GSH）浓度均极低，这表明采样过程未导致肌肉细胞内代谢物的显著污染。在血淋巴的氢核磁共振（(1)H-NMR）谱中，明确鉴定出了27种代谢物。通过主成分分析（PCA）和排序主成分分析（Ranking-PCA）多变量分析评估了铜暴露和温度对血淋巴代谢谱的单独及联合影响。在16℃下，当铜暴露剂量达到20μg/L时，血淋巴代谢谱的变化就变得可检测到。根据铜暴露水平，丙氨酸、赖氨酸、丝氨酸、谷氨酰胺、糖原、葡萄糖和蛋白质脂肪族在代谢变化分类中起主要作用。高温（24℃）和高铜水平导致一组常见代谢物（主要是葡萄糖、丝氨酸和赖氨酸）一致增加，这表明高温导致的代谢损伤因铜的存在而加剧。总体而言，结果表明，与人类血浆一样，血淋巴代谢物分析是诊断海洋贻贝中污染物诱导应激综合征的一种有前景的工具。
