Effect of glutathione depletion on formation of paramagnetic chromium in Chinese hamster V‐79 cells

Abstract

Incubation of Chinese hamster V-79 cells with either buthionine sulfoximine @SO) or diethylmaleate @EM) prior to exposure to Na&rQ resulted in a depletion of GSH. ESR study shows that the depletion of GSH by BSO caused an increase in the cellular level of Cr(V) intermediate without affecting the level of Cr(III) complex, whereas the levels of Cr(V) and (III) were both suppressed by the depletion of GSH by DEM. GSH depletion by DEM decreased cellular uptake of chromate more than that by BSO. Glutathione reductase activity in cells, which is capable of reducing Cr(VI), was una.tkcted by BSO, however a strong inhibition was observed in cells treated with DEM, indicating that DEM affects not only GSH levels but also Cr(VI)-reducing enxyme activity. Thus, the depletion of GSH in cells increases Cr(V) formation, based upon the effect of BSO. The role of intracellular GSH in Cr(V1) reduction is discussed. Key work Glutathione;Chromium; ESR; Chinese hamster V-79 cell IntroduetlonCr(V1) compounds are well known to be human carcinogens [l]. They have been shown to produce DNA single strand breaks and DNA-protein crosslinks and to inhibit the activity of enzymes such as glutathione reductase (GR) in mammalian cells [24]. In contrast to Cr(III), Cr(V1) actively enters cells by the sulfate anion transport system and is then believed to be reduced through reactive intermediates such as Cr(V) and (IV) to more stable Cr(II1) by cellular reductants including glutathione (GSH), ascorbic acid, riboflavin and flavoenzymes including GR [24]. Thus, the generation of paramagnetic chromium may play an important role in the cellular damage caused by Cr(V1). For instance, Cr(V) complexes have been reported to induce DNA breakage and %hydroxy-2'-deoxyguanosine in vitro as well as mutation in bacterial systems, and recent in vitro studies indicated that not only Cr(VI)/(V) but also the Cr(III)/ (II) redox couple serve as cyclical electron donors, in a Fenton-like reaction, to produce active oxygen species [5-81. Among the biological reductants, GSH has been reported to be present normally in millimolar concentrations in intact cells, and the modification of cellular GSH in cultured cells has been shown to alter the levels of DNA damage caused by Cr(VI) [9]. Furthermore, ESR studies have shown that Cr(II1) and Cr(V) complexes are formed during reduction of Cr(V1) by GSH in vitro, and *Corresponding author. Fax: (81) (942) 31 4377. the formation of Cr(V) in rat thymocytes and human red blood cells treated with Cr(VI) has been reported to be suppressed by thiol-reactive agents such as diethylmaleate (DEM) and N-ethylmaleimide, respectively [lO,l 11. However, none of these thiol depletors are specific for GSH alone, and the influence of GSH depletion by an agent such as buthionine sulfoximine (BSO) which can deplete the cellular store of GSH via competitive inhibition of y glutamylcysteine synthetase [12], on the formation of paramagnetic chromium in intact cells has not been investigated.In the present study, the effect of GSH depleti...

Reductive activation of hexavalent chromium by human lung epithelial cells: Generation of Cr(V) and Cr(V)-thiol species

“…6), thiols are not necessary for the generation of this signal. This is consistent with observations in other studies, including: (a) thiols such as GSH may not contribute significantly to Cr(V) formation in Chinese hamster V-79 cells, [69]; (b) thiol depletion of chick embryos did not affect the level of hepatic Cr(V) after 70 min [68]; and (c) GSH depletion increased the Cr(V) signal in the kidney (but not the liver) of rats injected with Cr(VI) [70].…”

Electron Spin Resonance Study of Chromium(V) Formation and Decomposition by Basalt-Inhabiting Bacteria

“…The formation of various Cr(III) complexes by different cellular reductants is one of the possible mechanisms. The complexes of various types due to Cr(III) ions are well determined in different medium ( − ).…”

A comparison of hepatocyte cytotoxic mechanisms for chromate and arsenite

“…Incubating isolated rat hepatocytes with 1 mM Cr (VI) inactivated more than 40% GSH reductase in an hour (Ueno et al, 1989), and oxidized more than 85% GSH in just 5 min (Pourahmad and O'Brien, 2001). Interestingly, the rapid sequestration and reduction of intracellular Cr (V1) by GSH may increase the entry of Cr (VI) into the cells as GSH depleted cells accumulated less Cr (VI) (Sugiyama and Tsuzuki, 1994).…”

“…There are few cellular studies in the literature about the molecular cytotoxic mechanisms of chromium (Ueno et al, 1989;Sugiyama and Tsuzuki, 1994;Blankenship et al, 1994;Susa et al, 1997) and arsenic (Hei et al, 1998;Larochette et al, 1999). In the following, we have compared the cytotoxic mechanisms of chromate (Cr (VI)) and arsenite (As (III)), and have shown that there are significant differences in molecular aspects (e.g.…”