Poster Presentation Australian and New Zealand Metabolomics Conference 2018

Copper is widely used in industry and agriculture, and thus is a common chemical contaminant in aquatic environments. As filter feeders, bivalves are prone to accumulate certain heavy metals, such as copper. However, the toxicity mechanisms of copper in marine bivalves is not fully understood. In this study, we provide the first report on the combined approach of GC–MS-based metabolomics and novel flow cytometry techniques to characterize the toxic effects and elucidate the toxicity mechanism of copper on New Zealand Greenshell^TMmussel (Perna canaliculus) haemocytes. Mussel haemolymph samples were exposed to increasing concentrations of Cu²⁺ (0.0, 25.0, 62.5, 125.0 and 187.5 μM) for 24 hours. Then, flow cytometric assays (mortality, ROS production and apoptosis) and metabolomics analyses were conducted for all samples. Flow cytometric results showed significant increases in a dose dependent manner in cell mortality, production of reactive oxygen species (ROS) and apoptosis (via alteration of caspase 3/7 activation and mitochondrial membrane potential) of haemocytes exposed to increasing Cu²⁺concentrations. For metabolomics, we observed alterations of 25 metabolites within metabolites profile of Cu²⁺-exposed haemolymph (125 μM) compared to those of control samples. Changes in levels of these metabolites may be considered important signatures of oxidative stress (e.g., glutathione, cysteine) and apoptosis processes (e.g., alanine, glutamic acid) that may be involve in the transsulfuration pathway, glutathione metabolism and taurine metabolism. In this model, Cu²⁺exposure led to excessive ROS production that caused the decreases of GSH, methionine, cysteine then taurine. The decrease of taurine, in turn, would have led to a decrease in glutamic acid and accumulation of alanine. This study provides insights into the cellular and molecular mechanisms of oxidative stress and apoptosis in marine bivalves and highlights the applicability and reliability of metabolomics and flow cytometry techniques for immunotoxicological studies in marine organisms.