Gamma-glutamyl transpeptidase null mice fail to develop tolerance to coumarin-induced Clara cell toxicity.

Coumarin was used as a model Clara cell toxicant to test the hypothesis that tolerance to injury requires increased gamma-glutamyl transpeptidase (GGT) activity. Wildtype (GGT(+/+)) and GGT-deficient (GGT(-/-)) mice on a C57BL/6/129SvEv hybrid background were dosed orally with corn oil (vehicle) or coumarin (200 mg/kg). In vehicle-treated mice, Clara cell secretory protein (CC10) expression was distributed throughout the bronchiolar epithelium. After one dose of coumarin, CC10 expression was dramatically reduced and the bronchiolar epithelium was devoid of Clara cells in GGT(+/+) and GGT(-/-) mice. In wildtype mice, 9 doses of coumarin produced tolerance, characterized as a renewed bronchiolar epithelium with Clara cells expressing CC10 along with a 40% increase in total glutathione (GSH) and a 7-fold increase in GGT activity in the lung. In contrast, tolerance was not observed in GGT(-/-) mice. To assess whether changes in whole lung levels of GSH and GGT activity reflect Clara cell specific changes an enriched population of cells was isolated from female wildtype B6C3F1 mice made tolerant to coumarin. Compared to Clara cells from control mice, GSH and GGT activity increased 3- and 13-fold, respectively. Collectively, these data suggest Clara cell tolerance to coumarin toxicity requires increased GGT activity favoring enhanced GSH synthesis.

Mitochondrial glutathione and chemically induced stress including ethanol.

The mechanisms of alcohol toxicity as related to mitochondrial dysfunction and the glutathione-dependent protective systems are reviewed. The pathophysiology of ethanol-induced liver damage is defined in terms of an early phase and a late phase. CYP2E1 dependent toxicity appears closely related to oxidative stress injury with possible roles of peroxynitrite, TNFalpha, protein adducts, and enhanced protein expression. Modulation of mitochondrial glutathione affects mitochondrial function and cell survival with superoxide and hydrogen peroxide generation being crucial to mitochondrial membrane permeability transition and apoptosis.

Alkylation of protein disulfide isomerase by the episulfonium ion derived from the glutathione conjugate of 1,2-dichloroethane and mass spectrometric characterization of the adducts.

The reactivity of the episulfonium ion derived from S-(2-chloroethyl)glutathione (CEG), the glutathione conjugate of 1,2-dichloroethane, with the catalytic sites of protein disulfide isomerase (PDI) was investigated. The two cysteine residues of the two active sites of PDI are expected to be the major targets of alkylation. PDI was incubated with equimolar to 100-fold excess CEG. The activity of PDI was irreversibly inhibited with a concurrent loss of two thiols; however, PDI oxidative refolding activity was not completely inhibited. With mass spectrometry, sequencing PDI identified one alkylation event on each of the N-terminal cysteine residues in the two active site peptides. PDI appears robust and able to maintain some activity by steric constraint. We have established that the episulfonium ion of CEG can adduct PDI and may have important toxicologic significance for 1,2-dichloroethane toxicity.

Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry.

Hydrogen/deuterium (H/D) exchange in combination with electrospray ionization mass spectrometry and near-ultraviolet (UV) circular dichroism (CD) was used to study the conformational properties and thermal unfolding of Escherichia coli thioredoxin and its Cys32-alkylated derivatives in 1% acetic acid (pH 2.7). Thermal unfolding of oxidized (Oxi) and reduced (Red) -thioredoxin (TRX) and Cys-32-ethylglutathionyl (GS-ethyl-TRX) and Cys-32-ethylcysteinyl (Cys-ethyl-TRX), which are derivatives of Red-TRX, follow apparent EX1 kinetics as charge-state envelopes, H/D mass spectral exchange profiles, and near-UV CD appear to support a two-state folding/unfolding model. Minor mass peaks in the H/D exchange profiles and nonsuperimposable MS- and CD-derived melting curves, however, suggest the participation of unfolding intermediates leading to the conclusion that the two-state model is an oversimplification of the process. The relative stabilities as measured by melting temperatures by both CD and mass spectral charge states are, Oxi-TRX, GS-ethyl-TRX, Cys-ethyl-TRX, and Red-TRX. The introduction of the Cys-32-ethylglutathionyl group provides extra stabilization that results from additional hydrogen bonding interactions between the ethylglutathionyl group and the protein. Near-UV CD data show that the local environment near the active site is perturbed to almost an identical degree regardless of whether alkylation at Cys-32 is by the ethylglutathionyl group, or the smaller, nonhydrogen-bonding ethylcysteinyl group. Mass spectral data, however, indicate a tighter structure for GS-ethyl-TRX.

In vivo reversal of glutathione deficiency and susceptibility to in vivo dexamethasone-induced apoptosis by N-acetylcysteine and L-2-oxothiazolidine-4-carboxylic acid, but not ascorbic acid, in thymocytes from gamma-glutamyltranspeptidase-deficient knockout mice.

Cellular glutathione is released during apoptosis and may play a role in the regulation of the mitochondrial permeability transition pore. The question of whether only cytosolic glutathione is important in apoptosis, or whether mitochondrial glutathione also plays a role, was investigated using gamma-glutamyltranspeptidase-deficient knockout mice. Thymocytes from these mice were found to have both glutathione pools diminished and they were more susceptible to dexamethasone (DEX)-induced apoptosis. Supplementation with N-acetylcysteine (NAC) and L-2-oxothiazolidine-4-carboxylic acid replenished both glutathione pools and provided protection from apoptosis. Ascorbate supplementation was beneficial to the mitochondrial glutathione pool, but apoptosis was not prevented. NAC supplementation caused an increase in reactive oxygen species formation and cardiolipin oxidation, but had no adverse affect on the amount of apoptotic cells. Our results suggest that the glutathione status is an important factor in apoptosis and indirect evidence indicates that the cytosolic pool of glutathione may be important in DEX-induced apoptosis, with mitochondrial events being secondary, and may reflect the execution phase.