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TP11 Metals in the Environment: Aquatic Biological Perspectives
(STO-1117-814258) Novel endpoints for measuring bioregulation of trace metals in phytoplankton.
Stoiber, T1, Hemming, J2, Karner, D2, Shafer, M1, 2, Armstrong, D1, 1 University of Wisconsin-Madison, Madison, WI, USA2 Wisconsin State Laboratory of Hygiene, Madison, WI, USA
ABSTRACT- Glutathione (GSH) is a tri-peptide and the most abundant thiol species in eukaryotic cells. GSH is important for many metabolic processes and functions to protect cells from environmental stressors such as reactive oxygen species, toxic metals, and harmful organic compounds. Glutathione is present in cells primarily in its reduced (GSH) form. The reduced species (GSH) is readily oxidized to form glutathione disulfide (GSSG). Reduction of GSSG to GSH is mediated by the enzyme glutathione reductase (GR). Intracellular ratios of GSH to GSSG may serve an indicator of algal cell health and the cells' ability to resist toxic stressors, and therefore could serve as a biomarker of exposure and effect. Similarly, the activity of GR may correlate with oxidative stress to the cell. 24-hour and 6-hour Cu exposure experiments were conducted with the green algae species Chlamydomonas reinhardtii. A colorimetric enzyme-recycling microplate assay was adapted and validated for measurements of GSH and GSSG in algal cells. This assay was applied in Cu dose-response experiments to determine the relationship of GSH/GSSG ratios to heavy metal stress. Glutathione reductase, the enzyme that catalyzes the reduction of GSSG to GSH, was also assayed using a similar colorimetric kinetic method. Conditions under which GSH, GSSG, and GR are efficiently extracted from algal cells, and speciation preserved, was also investigated. Trends of increasing levels of GSSG were apparent with increasing Cu levels. GSH/GSSG endpoints were sensitive biomarkers at 6-hour and 24-hour exposures.
Key words: algae, glutathione, copper
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