Alterations in brain antioxidant enzymes and redox proteomic identification of oxidized brain proteins induced by the anti-cancer drug adriamycin: implications for oxidative stress-mediated chemobrain

Abstract

Adriamycin (ADR) is a chemotherapeutic for the treatment of solid tumors. This quinone-containing anthracycline is well known to produce large amounts of reactive oxygen species (ROS) in vivo. A common complaint of patients undergoing long-term treatment with ADR is somnolence, often referred to as "chemobrain." While ADR itself does not cross the blood brain barrier (BBB), we recently showed that ADR administration causes a peripheral increase in tumor necrosis factor alpha (TNF-alpha), which migrates across the BBB and leads to inflammation and oxidative stress in brain, most likely contributing to the observed decline in cognition. In the current study, we measured levels of the antioxidant glutathione (GSH) in brains of mice injected intraparitoneally (i.p.) with ADR, as well as the levels and activities of several enzymes involved in brain GSH metabolism. We observed significantly decreased GSH levels, as well as altered GSH/GSSG ratio in brains of ADR treated mice relative to saline-treated controls. Also observed in brains of ADR treated mice were increased levels of glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR). We also observed increased activity of GPx, but a significant reduction in GST and GR activity in mice brain, 72 h post i.p. injection of ADR (20 mg/kg body weight). Furthermore, we used redox proteomics to identify specific proteins that are oxidized and/or have differential levels in mice brains as a result of a single i.p. injection of ADR. Visinin like protein 1 (VLP1), peptidyl prolyl isomerase 1 (Pin1), and syntaxin 1 (SYNT1) showed differential levels in ADR treated mice relative to saline-treated controls. Triose phosphate isomerase (TPI), enolase, and peroxiredoxin 1 (PRX-1) showed significantly increased specific carbonylation in ADR treated mice brain. These results further support the notion ADR induces oxidative stress in brain despite not crossing the BBB, and that antioxidant intervention may prevent ADR-induced cognitive dysfunction.

Figure 1

Figure 1a and b. a) Reduced GSH levels in brain isolated from saline-injected and ADR-injected mice, 72 h post i.p. injections. A significant reduction in GSH level is seen in brain isolated from ADR-injected mice when compared to control. * p < 0.001. b.) Decreased GSH/GSSG ratio in brain isolated from saline injected mice and ADR mice. The data are presented as mean ± SEM expressed as percentage of control. (*p < 0.05, n=4 controls, n=5 ADR)

Figure 2

Figure 2a. Representative Western blot of brain GPx, GST, and GR 72 h post i.p. injection of saline or ADR in mice. GAPDH was used as a loading control. (n=4 controls, n=5 ADR) Figure 2b. Representative plot of GR levels in brain isolated from saline-injected and ADR-injected mice, 72 h post i.p. injections. A significant increase in GR levels is seen in ADR-injected mice brain when compared to control. * p < 0.05. The data are presented as mean ± SEM expressed as percentage of control. (n=4 controls, n=5 ADR) Figure 2c. Representative plot of GST levels in brain isolated from saline-injected and ADR-injected mice, 72 h post i.p. injections. A significant increase in GST level is seen in ADR-injected mice brain when compared to control. * p < 0.05. The data are presented as mean ± SEM expressed as percentage of control. (n=4 controls, n=5 ADR) Figure 2d. Representative plot of GPx levels in brain isolated from saline-injected and ADR-injected mice, 72 h post i.p. injections. A significant increase in GST level is seen in ADR-injected mice brain when compared to control. * p < 0.05. The data are presented as mean ± SEM expressed as percentage of control. (n=4 controls, n=5 ADR)

Figure 3

Representative 2D gels from brains of saline- and ADR-treated mice, respectively, showing geographical location of proteins on 2D gel identified by mass spectrometry that showed differences in expression or oxidation as a result of i.p ADR. Spots that showed a significant difference in expression or oxidation levels are boxed and labeled with the corresponding protein identity. (n=4 controls, n=5 ADR)

Figure 4

Magnified 2D gel map areas containing spots with significant expression differences in ADR and saline-treated mice brains. All proteins identified by arrows were significantly decreased in brain with ADR treatment.

Figure 5

Figure 5a & b. Magnified 2d gel and oxyblots containing significantly oxidized spots in ADR and saline-treated mice. All proteins identified showed an increase in specific oxidation in brain after i.p. administration of ADR.

Figure 5

Figure 5a & b. Magnified 2d gel and oxyblots containing significantly oxidized spots in ADR and saline-treated mice. All proteins identified showed an increase in specific oxidation in brain after i.p. administration of ADR.