Figure 5.

Selective Depletion of Mitochondrial GSH and Inactivation of TrxR2 by MitoCDNB within Tissues In Vivo

Mice were injected with MitoCDNB (5 mg/kg) and tissues harvested for analysis of total and mitochondrial GSH content, and for TrxR activity.

(A) Liver levels of MitoCDNB and MitoGSDNB were quantified by LC-MS/MS.

(B) Blood samples were analyzed for MitoCDNB, MitoGSDNB, and MitoCysDNB by LC-MS/MS.

(C) Spot urines 24 hr after MitoCDNB injection were quantified for MitoCDNB, MitoGSDNB, and MitoCysDNB by LC-MS/MS.

(D) Liver mitochondrial levels of MitoCDNB and MitoGSDNB were quantified by LC-MS/MS.

(E) GSH depletion by MitoCDNB in vivo. Mitochondrial and whole tissue fractions from liver, heart, and kidney at various times after MitoCDNB injection. Data are a percentage of control mice culled at the same time. The levels of GSH in the whole tissue and mitochondria, respectively, were: liver = 30.56 ± 3.7, 3.71 ± 0.6; kidney = 1.71 ± 0.3, 2.14 ± 0.52; heart = 4.15 ± 0.54, 2.36 ± 0.1 (nmol GSH/mg protein).

(F and G) Expression of γ-glutamylcysteine ligase (γGCL) and glutathione synthetase (GS). Mice were injected with MitoCDNB and 24 hr later liver levels of γGCL and GS were analyzed by western blot (F). Each lane is a separate mouse, and quantified relative to GAPDH (G).

(H) Liver and mitochondrial fractions at indicated times after MitoCDNB injection were assessed for TrxR activity.

Data are means ± SEM, N = 4 or 5 (E). *p < 0.05, **p < 0.01, ***p < 0.001 relative to control, time zero (H), or time-matched controls (E). See also Figure S6.