title: Copper-induced changes in intracellular thiols in two marine diatoms: Phaeodactylum tricornutum and Ceratoneis closterium.
abstract: Phytochelatins and glutathione (reduced (GSH) and oxidised (GSSG)) are important intracellular ligands involved in metal sequestration and detoxification in algae. Intracellular ratios of GSH:GSSG are sensitive indicators of metal stress in algae, and like phytochelatin production are influenced by metal speciation, concentration, exposure time and the biological species. This study investigated the effect of copper exposure on phytochelatin and glutathione content in two marine diatoms Phaeodactylum tricornutum and Ceratoneis closterium at various time intervals between 0.5 and 72h. Liberation of cellular glutathione and phytochelatins was optimised using freeze/thaw cycles and chemical extraction, respectively. Extracted phytochelatins were derivatised (by fluorescent tagging of thiol compounds), separated and quantified using HPLC with fluorescence detection. Glutathione ratios were determined using a commercially available kit, which uses the enzyme glutathione reductase to measure total and oxidised glutathione. Despite similarities in size and shape between the two diatoms, differences in internalised copper, phytochelatin production (both chain length and quantity) and reduced glutathione concentrations were observed. P. tricornutum maintained reduced glutathione at between 58 and 80% of total glutathione levels at all time points, which would indicate low cellular stress. In C. closterium reduced glutathione constituted <10% of total glutathione after 48h. P. tricornutum also produced more phytochelatins and phytochelatins of longer chain length than C. closterium despite the latter species internalising significantly more copper.
authors.affiliation: School of Chemistry, University of Wollongong, NSW 2522, Australia. Electronic address: cs713@uowmail.edu.au.
authors.forename: Cassandra L
authors.initials: CL
authors.lastname: Smith
authors.affiliation: School of Chemistry, University of Wollongong, NSW 2522, Australia.
authors.forename: Jessica E
authors.initials: JE
authors.lastname: Steele
authors.affiliation: CSIRO Land and Water, Locked Bag 2007, Kirrawee, NSW 2232, Australia.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Stauber
authors.affiliation: School of Chemistry, University of Wollongong, NSW 2522, Australia. Electronic address: djolley@uow.edu.au.
authors.forename: Dianne F
authors.initials: DF
authors.lastname: Jolley
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000633:toxicity*; D017377:Diatoms / Q000737:chemistry / Q000187:drug effects*; D005978:Glutathione / Q000032:analysis; D054811:Phytochelatins / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000032:analysis*; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D013438:Sulfhydryl Compounds; D014874:Water Pollutants, Chemical; D003300:Copper; D054811:Phytochelatins; D005978:Glutathione
keywords: Glutathione; Metallothionein; Phytochelatin; Redox; Reduced glutathione