title: Metallomics and NMR-based metabolomics of Chlorella sp. reveal the synergistic role of copper and cadmium in multi-metal toxicity and oxidative stress.
abstract: Industrial wastewaters often contain high levels of metal mixtures, in which metal mixtures may have synergistic or antagonistic effects on aquatic organisms. A combination of metallomics and nuclear magnetic resonance spectroscopy (NMR)-based metabolomics was employed to understand the consequences of multi-metal systems (Cu, Cd, Pb) on freshwater microalgae. Morphological characterization, cell viability and chlorophyll a determination of metal-spiked Chlorella sp. suggested synergistic effects of Cu and Cd on growth inhibition and toxicity. While Pb has no apparent effect on Chlorella sp. metabolome, a substantial decrease of sucrose, amino acid content and glycerophospholipid precursors in Cu-spiked microalgae revealed Cu-induced oxidative stress. Addition of Cd to Cu-spiked cultures induced more drastic metabolic perturbations, hence we confirmed that Cu and Cd synergistically influenced photosynthesis inhibition, oxidative stress and membrane degradation. Total elemental analysis revealed a significant decrease in K, and an increase in Na, Mg, Zn and Mn concentrations in Cu-spiked cultures. This indicated that Cu is more toxic to Chlorella sp. as compared to Cd or Pb, and the combination of Cu and Cd has a strong synergistic effect on Chlorella sp. oxidative stress induction. Oxidative stress is confirmed by liquid chromatography tandem mass spectrometry analysis, which demonstrated a drastic decrease in the GSH/GSSG ratio solely in Cu-spiked cultures. Interestingly, we observed Cu-facilitated Cd and Pb bioconcentration in Chlorella sp. The absence of phytochelatins and an increment of extracellular polymeric substances (EPS) yields in Cu-spiked cultures suggested that the mode of bioconcentration of Cd and Pb is through adsorption of free metals onto the algal EPS rather than intracellular chelation to phytochelatins.
authors.affiliation: Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore. chmlifys@nus.edu.sg.
authors.forename: Wenlin
authors.initials: W
authors.lastname: Zhang
authors.forename: Nicole G J
authors.initials: NG
authors.lastname: Tan
authors.forename: Baohui
authors.initials: B
authors.lastname: Fu
authors.forename: Sam F Y
authors.initials: SF
authors.lastname: Li
mesh_terms: D018533:Biomass; D001704:Biopolymers / Q000378:metabolism; D002104:Cadmium / Q000633:toxicity*; D002708:Chlorella / Q000166:cytology / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D002734:Chlorophyll / Q000378:metabolism; D000077194:Chlorophyll A; D003300:Copper / Q000633:toxicity*; D009682:Magnetic Resonance Spectroscopy; D055442:Metabolome / Q000187:drug effects; D055432:Metabolomics; D018384:Oxidative Stress / Q000187:drug effects*; D010455:Peptides / Q000378:metabolism; D025341:Principal Component Analysis; D066244:Proton Magnetic Resonance Spectroscopy; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013997:Time Factors
chemical_list: D001704:Biopolymers; D010455:Peptides; D013438:Sulfhydryl Compounds; D002104:Cadmium; D002734:Chlorophyll; D003300:Copper; D000077194:Chlorophyll A