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Advisor(s)
Abstract(s)
The green alga Pseudokirchneriella subcapitata
has been extensively used for the assessment of adverse
impacts of pollutants. Glutathione is involved in antioxidant
defence and drug detoxification. Intracellular reduced glutathione
(GSH) concentration can be used as an indicator of the
health of cells. This work describes a simple and fast fluorescent
cell-based assay for the evaluation of intracellular
GSH in the alga P. subcapitata, using monochlorobimane
(mBCl). Metabolically active algal cells incubated with
50 μmol L−1 mBCl form fluorescent bimane–glutathione
(B-SG) adducts that can be measured fluorometrically. The
distribution of GSH (B-SG adducts) in whole cells can be
observed by epifluorescence microscopy, in the form of blue
fluorescent spots. Depletion of cellular GSH with iodoacetamide,
inhibition of glutathione S-transferase with ethacrynic
acid or heat-induced death of the cells inhibited the
formation of fluorescent adducts in the presence of mBCl.
The fluorometric assay, using the 96-well microplate format,
was able to detect GSH depletion in algal cells. This cell-based
assay can be used to evaluate decreases in GSH content due to
exposure to toxicants. This assay is amenable to automation
and may be useful in high-throughput toxicity screening using
the alga P. subcapitata.
Description
Keywords
Fluorescence microscopy Glutathione Microplate assay Monochlorobimane Selenastrum capricornutum Toxicity assay
Citation
Publisher
Springer Verlag