Analysis of Selenium Toxicity on Reduced Thiol Content

Abstract

Enterobacter sp. YSU a multimetal resistant strain, grew when exposed to 40 mM selenite by reducing it to elemental selenium and thus detoxifying it. Growth curves established in M-9 minimal medium showed that Enterobacter sp. YSU grew in the presence of 40 mM selenite only when supplemented with L-cysteine and died when exposed to selenite in the absence of L-cysteine. A previous study involving M-9 minimal medium suggested that when selenite concentration was higher than the sulfate concentration selenite could enter the Enterobacter sp. YSU cells through two pathways: a specific pathway and a non specific pathway (sulfate permease channel). The presence of L-cysteine prevents the entrance of selenite through non specific pathway. The actual toxicity mechanism of the selenium oxyanion, selenite (SeO32-), is unknown. To determine if the interaction of selenite with the reduced thiols is involved in the toxicity mechanism and the reason for oxidative stress, the RSH content of Enterobacter sp. YSU was assayed. This strain was grown under sensitive (no L-cysteine) and resistant (presence of L-cysteine) conditions. Selenite was added during the early log-phase. Cells were harvested before and after the exposure to selenite, lysed and reacted with RSH specific derivatizing agent 5, 5' -- dithiobis (2-nitrobenzoic acid) for thiol analysis. HPLC was performed for specific quantification of reduced glutathione. The derivative formed represented the level of oxidative stress. Cells were also harvested for Bradford assay to determine the cell protein concentration. It allowed for the RSH content to be normalized to total cell protein. Upon exposure of selenite, the RSH content of the cells grown under sensitive conditions decreased markedly. In the presence of L-cysteine, only a small fraction of RSH content became oxidized. Total protein concentration of the sensitive cells declined as well, as compared to the resistant cells. HPLC analysis showed that there was a decrease in reduced glutathione in sensitive cells. How