Glutathione Depletion by DL-Buthionine-SR-Sulfoximine (BSO) Potentiates X-Ray-Induced Chromosome Lesions after Liquid Holding Recovery

Abstract

The impact of intracellular glutathione depletion on chromosome damage induced by X irradiation under aerobic conditions was investigated in two different cell lines, Ehrlich ascites tumor cells (EATC) and Chinese hamster ovary cells (CHO-K1). Thiol-depleted cell cultures in plateau phase were obtained by prolonged incubation in growth medium containing DL-buthionine-SR-sulfoximine (BSO), a specific inhibitor of gamma-glutamyl-cysteine synthetase. Cells were then assayed using the procedures of G. L. Ellmann (Arch. Biochem. Biophys. 82, 70-77 (1959)), F. Tietze (Anal. Biochem. 27, 502-522 (1969)), and J. Sedlack and R.H. Lindsay (Anal. Biochem. 25, 192-205 (1968)) for non-protein bound SH (NPSH), glutathione (GSH), and total SH (TSH). In both cell lines GSH was reduced to less than 10% of controls at higher BSO concentrations around 1 mM, whereas TSH and NPSH were affected to only 40-60%. In EATC pretreated with up to 1 mM BSO for 72 h, increased levels of spontaneously occurring micronuclei were found. At BSO concentrations above 200 microM, both cell lines showed a potentiation of chromosome lesions scored as micronuclei and induced under aerobic X irradiation when liquid holding recovery in the original nutrient-depleted medium was performed; the extent of chromosome damage eventually reached that which could be obtained by application of beta-arabinofuranosyladenine (beta-araA), known to inhibit DNA repair processes by blocking DNA polymerases. It is therefore suggested that GSH depletion causes impairment of repair of lesions leading to chromosome deletions and subsequently to micronuclei. In contrast to CHO cell cultures, EATC showed a reversion of the potentiation effect as indicated by a decrease in the micronucleus content during prolonged incubation in the presence of BSO in the millimolar range. This effect could not be correlated to the remaining GSH content of less than 10% but may be due to some accumulation of unknown NPSH components. Since addition of L-cysteine to EATC cultures pretreated with BSO decreased the micronucleus content, cysteine/cystine or a related thiol within the NPSH fraction may be involved in the reestablishment of repair. Thus at least in one cell line, a rather complex response to BSO administration indicated that not only GSH but also other thiols may determine the level of chromosome damage after liquid holding recovery.

Förster resonance energy transfer-based sensor targeting endoplasmic reticulum reveals highly oxidative environment

“…44 In spite of the 10-fold drop in GSH levels of CHO cells pretreated with 0.1 mmol/L BSO for 24 h, the FRET signal was unchanged at basal state (Figure 2b). Additional treatment with H 2 O 2 had no or little effect on both GSH level and the CY-RL7 response.…”

“…In addition to CDNB, intracellular GSH content was modulated with BSO, another commonly used glutathione depletor, which inhibits GSH synthesis with high specificity. 44 In spite of the 10-fold drop in GSH levels of CHO cells pretreated with 0.1 mmol/L BSO for 24 h, the FRET signal was unchanged at basal state (Figure 2b). Additional treatment with H 2 O 2 had no or little effect on both GSH level and the CY-RL7 response.…”

Featured Article: Inhibition of glutathione synthesis distinctly alters mitochondrial and cytosolic redox poise

“…46 GSH synthesis can be selectively decreased by BSO, which inhibits the gamma-glutamylcysteine synthetase that catalyzes the first step in GSH synthesis. 47 The effects of BSO on mammalian cell thiol content and ROS production are described elsewhere, and the relationship between GSH deficiency and subcellular oxidation depends on the dose and duration of BSO treatment. 47,48 Recent data obtained with genetically encoded redox-sensitive probes reveal an absence of cytosolic oxidation in rat hippocampal cells after overnight incubation with BSO.…”

“…47 The effects of BSO on mammalian cell thiol content and ROS production are described elsewhere, and the relationship between GSH deficiency and subcellular oxidation depends on the dose and duration of BSO treatment. 47,48 Recent data obtained with genetically encoded redox-sensitive probes reveal an absence of cytosolic oxidation in rat hippocampal cells after overnight incubation with BSO. 43 However, more extended treatment with BSO (72 h) significantly oxidized roGFP1 targeted to cytosolic and mitochondrion matrix compartments of human skin fibroblasts.…”