In vivo administration of D609 leads to protection of subsequently isolated gerbil brain mitochondria subjected to in vitro oxidative stress induced by amyloid beta-peptide and other oxidative stressors: relevance to Alzheimer's disease and other oxidative stress-related neurodegenerative disorders

Abstract

Tricyclodecan-9-yl-xanthogenate (D609) has in vivo and in vitro antioxidant properties. D609 mimics glutathione (GSH) and has a free thiol group, which upon oxidation forms a disulfide. The resulting dixanthate is a substrate for glutathione reductase, regenerating D609. Recent studies have also shown that D609 protects brain in vivo and neuronal cultures in vitro against the potential Alzheimer's disease (AD) causative factor, Abeta(1-42)-induced oxidative stress and cytotoxicity. Mitochondria are important organelles with both pro- and antiapoptotic factor proteins. The present study was undertaken to test the hypothesis that intraperitoneal injection of D609 would provide neuroprotection against free radical-induced, mitochondria-mediated apoptosis in vitro. Brain mitochondria were isolated from gerbils 1 h post injection intraperitoneally (ip) with D609 and subsequently treated in vitro with the oxidants Fe(2+)/H(2)O(2) (hydroxyl free radicals), 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH, alkoxyl and peroxyl free radicals), and AD-relevant amyloid beta-peptide 1-42 [Abeta(1-42)]. Brain mitochondria isolated from the gerbils previously injected ip with D609 and subjected to these oxidative stress inducers, in vitro, showed significant reduction in levels of protein carbonyls, protein-bound hydroxynonenal [a lipid peroxidation product], 3-nitrotyrosine, and cytochrome c release compared to oxidant-treated brain mitochondria isolated from saline-injected gerbils. D609 treatment significantly maintains the GSH/GSSG ratio in oxidant-treated mitochondria. Increased activity of glutathione S-transferase, glutathione peroxidase, and glutathione reductase in brain isolated from D609-injected gerbils is consistent with the notion that D609 acts like GSH. These antiapoptotic findings are discussed with reference to the potential use of this brain-accessible glutathione mimetic in the treatment of oxidative stress-related neurodegenerative disorders, including AD.

Figure 1

Shows the increment in protein carbonyl formation in brain mitochondria isolated from saline-injected gerbils and treated with various oxidants [AAPH, Fe²⁺/H₂O₂ or Aβ (1-42)] compared to control. The protective effects of D609 against protein carbonyl formation in brain mitochondria isolated from gerbils injected i.p. 1h before sacrifice with D609 and treated with AAPH, Fe²⁺/H₂O₂ and Aβ (1-42) also are shown. ^*p<0.01 and ^**p<0.001 compared to control and # p<0.01 compared to oxidant treatment. The data are presented as mean ± SEM expressed as percentage of control (n=6).

Figure 2

Shows the increment in 3-NT levels in brain mitochondria isolated from saline-injected gerbils and subsequently treated with AAPH, Fe²⁺/H₂O₂ or Aβ (1-42) compared to 3-NT levels in brain mitochondria isolated from saline-injected gerbil that received no treatment of any oxidant, ^*p<0.01 and ^**p<0.001. This figure also shows decreased 3-NT levels shows in brain mitochondria isolated from gerbils previously injected i.p. with D609 1 h before sacrifice and treated with AAPH, Fe²⁺/H₂O₂ or Aβ (1-42) compared to the oxidant treatment but no prior injection of D609, # p<0.01. The data are presented as mean ± SEM expressed as percentage of control (n=6).

Figure 3

Shows the significantly elevated protein-bound HNE content in brain mitochondria isolated from saline-injected gerbils and treated with different oxidants [AAPH, Fe²⁺/H₂O₂ or Aβ (1-42)]. The protective effects of D609 against HNE formation of protein-bound HNE in brain mitochondria isolated from gerbil injected i.p. with D609 1 h before sacrifice and treated with AAPH, Fe²⁺/H₂O₂ or Aβ (1-42) also are shown. ^*p<0.01 and ^**p<0.001 compared to control, # p<0.01 and ## p<0.001 compared to oxidant treatment. The data are presented as mean ± SEM expressed as percentage of control (n=6).

Figure 4A

Shows a significant decrement in GSH levels in brain mitochondria isolated from saline-injected gerbils and subsequently treated with AAPH, Fe²⁺/H₂O₂ or Aβ (1-42) compared to GSH levels in brain mitochondria isolated from saline-injected gerbils not subjected to treatment of any oxidant. Also shown is the protection of GSH levels in brain mitochondria isolated from gerbils previously injected i.p. with D609 1 h before sacrifice and treated with AAPH, Fe²⁺/H₂O₂ or Aβ (1-42) compared to GSH levels in brain mitochondria isolated from saline-treated gerbils and then treated with oxidants. ^*p<0.01 and ^**p<0.001 compared to control, # p<0.01 and ## p<0.01 compared to oxidant treatment. The data are presented as mean ± SEM expressed as percentage of control (n=6).

Figure 4B

Shows the increased level of GSSG in brain mitochondria isolated from saline-injected gerbils and subsequently treated with AAPH, Fe²⁺/H₂O₂ or Aβ (1-42) as compared to GSSG levels in brain mitochondria isolated from saline-injected gerbils but not subjected to treatment of any oxidant. The reduction in GSSG level shows in brain mitochondria isolated from gerbils previously injected i.p. with D609 1 h before sacrifice and treated with AAPH, Fe²⁺/H₂O₂ or Aβ (1-42) compared to GSSG levels in brain mitochondria isolated from saline-treated gerbil and then treated with oxidants. ^*p<0.01 and ^**p<0.001 compared to control, # p<0.01 and ## p<0.01 compared to oxidant treatment. The data are presented as mean ± SEM expressed as percentage of control (n=6).

Figure 4C

Shows the ratio of GSH/GSSG, decreased in brain mitochondria isolated from saline-injected gerbils and subsequently treated with AAPH, Fe²⁺/H₂O₂ or Aβ (1-42), compared to the GSH/GSSG ratio in brain mitochondria isolated from saline-injected gerbils but not subjected to treatment of any oxidant. The increment in the ratio of GSH/GSSG in brain mitochondria isolated from gerbils previously injected i.p. with D609 1 h before sacrifice and then treated with AAPH, Fe²⁺/H₂O₂ or Aβ (1-42) compared to this ratio determined in brain from mice treated with oxidant but no pre-injection of gerbils with D609 is also shown. ^*p<0.01 and ^**p<0.001 compared to control, # p<0.01 and ## p<0.01 compared to oxidant treatment. The data are presented as mean ± SEM expressed as percentage of control (n=6).

Figure 5

Shows the increased level of cytochrome-c released from brain mitochondria isolated from saline-injected gerbils and treated with various oxidants (AAPH, Fe²⁺/H₂O₂ or Aβ (1-42) as compared to cytochrome-c released from brain mitochondria isolated from saline-injected gerbils but not subjected to treatment of any oxidant. Also shown is the decrement of cytochrome-c release from brain mitochondria isolated from gerbils previously injected i.p. with D609 1 h before sacrifice and treated with AAPH, Fe²⁺/H₂O₂ or Aβ (1-42) compared to that released from brain mitochindria isolated from gerbils subjected to oxidant treatment. The D609 only treatment shows significantly less cytochrome-c release compared control. ^*p<0.01 as compared to control, # p<0.01 compared to oxidant treatment. The data are presented as mean ± SEM expressed as percentage of control (n=5).