Analysis of glutathione endpoints for measuring copper stress in Chlamydomonas reinhardth

Abstract

Glutathione (GSH) is the most abundant nonprotein thiol in eukaryotic cells and it protects cells by functioning as an antioxidant and a metal-binding ligand. Because glutathione readily undergoes oxidation-reduction reactions to combat oxidative stress, intracellular ratios of the reduced (GSH) to the oxidized (GSSG) forms of glutathione may serve as an important biomarker of exposure and effect of trace metals in eukaryotic cells. We compared sensitivity of glutathione ratios in the freshwater alga Chlamydomonas reinhardtii to the traditional endpoints of cell growth rates and chlorophyll a following exposure to Cu for periods of 6 and 24 h. A response of the GSH:GSSG ratio to Cu concentration was observed at Cu levels of 40 and 80 nM after exposure for both 6 and 24 h. The concentration of total GSH at 24 h was roughly half the value at 6 h after exposure to either 40 or 80 nM Cu. A response for cell growth rate was observed only at 24 h, whereby the average specific growth rate decreased from about 1.1 to 0.4 d(-1). The total Cu concentrations eliciting a cell response of 50%, effect concentrations (EC50s), after 24 h of exposure were similar (49.2, 49.8, and 38.2 nM Cu) and not significantly different for GSH:GSSG ratio, GSH levels, and specific growth, respectively. Total cell-associated Cu concentrations after exposure for 24 h were calculated from the EC50 endpoints and ranged from 13.3 to 17.0 fg/cell. Overall, thiol ratios were indicative of toxicity resulting from exposure to Cu, but precision may be greater for the cell growth rate endpoints.

Copper Uptake, Intracellular Localization, and Speciation in Marine Microalgae Measured by Synchrotron Radiation X-ray Fluorescence and Absorption Microspectroscopy

“…for C. closterium). 11,55 This decrease in reduced GSH has significant impacts on cells, as most cellular processes utilise the reduced form. 56 The use of XANES in this study also highlighted differences in the Cu-binding ligand in Cu-exposed cells for the two diatoms C. closterium and P. tricornutum supporting the hypothesis that the two diatoms have different intracellular mechanisms for sequestering excess Cu.…”

Differential effects of copper and cadmium exposure on toxicity endpoints and gene expression in Chlamydomonas reinhardtii

“…Cadmium is generally considered to be nonessential and toxic at low concentrations. Whereas Cd is a known strong thiol inducer [9,10], Cu has been observed to deplete nonprotein thiols in cells, leading to toxic effects [11,12]. In addition, Cu promotes the oxidation of sulfhydral groups, resulting in the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) [13].…”

Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimane

“…This value is of same order of magnitude of the GSH concentration (384±89 amol cell −1 ) reported by Lavoie et al (2009) for P. subcapitata cells in stationary phase of growth. Nevertheless, Stoiber et al (2007) described a lower total GSH concentration (14-37 amol cell −1 ) for P. subcapitata in stationary phase of growth.…”

“…The depletion in GSH content in Scenedesmus bijugatus and Chlamydomonas reinhardtii algal cells exposed to copper has been described (Nagalakshmi and Prasad 2001;Stoiber et al 2007). Similarly, the GSH content of the green alga Scenedesmus acutus showed a downward trend with an increase in the Cr(VI) concentration in a sulphate-free buffer (Gorbi et al 2006).…”