The responses of Ht22 cells to oxidative stress induced by buthionine sulfoximine (BSO)

Abstract

Background: glutathione (GSH) is the most abundant thiol antioxidant in mammalian cells. It directly reacts with reactive oxygen species (ROS), functions as a cofactor of antioxidant enzymes, and maintains thiol redox potential in cells. GSH depletion has been implicated in the pathogenesis of neurological diseases, particularly to Parkinson's disease (PD). The purpose of this study was to investigate the change of cellular antioxidant status and basic cell functions in the relatively early stages of GSH depletion.

Results: in this study, GSH was depleted by inhibition of glutamylcysteine synthetase using buthionine sulfoximine (BSO) treatment in Ht22, a neuronal cell line derived from mouse hippocampus. Treatment with BSO produced dose-dependent decreases in total GSH level, Fe3+-reducing ability (FRAP assay), Cu2+-reducing ability (Antioxidant Potential, AOP assay), and ABTS free radical scavenging ability (ABTS assay) of the cells, but the sensitivity of these indicators to dosage varied considerably. Most of the changes were completed during the first 8 hours of treatment. Cell viability was tested by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay, and cells at lower density in culture were found to be more sensitive to GSH depletion. The activity of antioxidant enzymes, such as glutathione peroxidase (GPx), glutathione reductase (GR), and copper/zinc superoxide dismutase (Cu/Zn-SOD) were affected by GSH depletion. A cDNA expression array assay of the effects of BSO treatment showed significantly decreased mRNA level for 3 genes, and significantly increased mRNA level for 10 genes, including the antioxidant enzymes Cu/Zn-SOD and thioredoxin peroxidase 2 (TPxII).

Conclusions: the study suggests that there are BSO-sensitive and BSO-resistant pools of GSH in Ht22 cells, and that different categories of antioxidant react differently to GSH depletion. Further, the effect of GSH status on cell viability is cell density dependent. Finally, the alterations in expression or activity of several antioxidant enzymes provide insight into the various cellular responses to GSH depletion.

Figure 1

Growth curve of Ht22 cells. Cell number counted by hemocytometer, three counts for each point.

Figure 2

Total intracellular GSH concentration in Ht22 cells. The GSH levels decrease with increasing cell density in culture.

Figure 3

Dose response of Ht22 cells to BSO treatment. The effects of 15-hour BSO treatment on GSH level and total antioxidant capacity in Ht22 cells were measured. Average value and SD are shown, N = 3. In comparison to 0.03 mM of BSO, increasing the concentration to 1 mM or higher caused significant decreases in GSH levels (P values ≤ 0.0399).

Figure 4

MTT assay for the viability of Ht22 cells treated with BSO for 15 hours. Average value and SD are shown (1e5 cells/ well, N = 9; 5e4 cells/ well, N = 5). At 3 and 10 mM BSO concentrations, the differences caused by cell density are significant (P values ≤ 0.0027).

Figure 5

Resazurin based bioreduction assay of Ht22 cells treated with BSO for 15 hours. Average value and SD are shown (1e5 cells/ well, N = 6; 5e4 cells/ well, N = 5). For 1e5 cells/well, treatment of 0.03 mM BSO caused significant increase in the bioreduction activity vs control (P = 0.0053); for 5e4 cells/well, the increases caused by treatments of 0.1 and 3 mM BSO were significant vs control (P ≤ 0.0175).

Figure 6

Time course of the responses of Ht22 cells to BSO treatment. The effects of total GSH level and total antioxidant capacities of Ht22 cells treated with 0.1 mM BSO for 4, 8, 12, and 15 hours were measured. Average value and SD are shown, N = 3. For total GSH, FRAP and ABTS, 4-hr treatment induced significant decreases vs control (P values ≤ 0.0001); from 4 hrs to 8 hrs, all of the assays showed significant decreases (P values ≤ 0.0326).

Figure 7

GPx activity of Ht22 cells treated with BSO for 15 hours. Average value and SD are shown, N = 3. Treatment with 1 mM BSO significantly increased the GPx activity vs control (P = 0.0401); and the decreases caused by 3 or 10 mM BSO are significant vs 1 mM BSO treatment (P values ≤ 0.0421).

Figure 8

GR activity of Ht22 cells treated with BSO for 15 hours. Average value and SD are shown, N = 3. At 10 mM BSO concentration, the decrease is significant (P = 0.045) vs control.

Figure 9

Cu/Zn-SOD activity of Ht22 cells treated with BSO for 15 hours. Average value and SD are shown, N = 3. Treatment of 10 mM BSO caused a trend of increase in SOD activity vs control (P = 0.0619).