Reactive oxygen species (ROS) are products of normal metabolism and xenobiotic exposure, and depending on concentrations, ROS can be beneficial or harmful to cells and tissues. At physiological low levels, ROS function as "redox messengers" in intracellular signaling and regulation while excess ROS induce oxidative modification of cellular macromolecules, inhibit protein function and promote cell death. Additionally, various redox systems, such as the glutathione, thioredoxin, and pyridine nucleotide redox couples, participate in cell signaling and modulation of cell function, including apoptotic cell death. Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways, the death receptor-or mitochondria-mediated pathways. Various pathologies can result from oxidative stress induced apoptotic signaling that is consequent to ROS increases and/or antioxidant decreases, disruption of intracellular redox homeostasis, and irreversible oxidative modifications of lipid, protein or DNA. In the current review, we focused on several key aspects of ROS and redox mechanisms in apoptotic signaling, and highlighted the gaps in knowledge and potential avenues for further investigation. A full understanding of redox control of apoptotic initiation and execution could underpin the development of therapeutic interventions targeted at oxidative stress associated disorders. OVERVIEW OF REACTIVE OXYGEN SPECIES AND INTRACELLULAR SOURCESReactive oxygen species (ROS) is a collective term that broadly describes O 2 -derived free radicals such as superoxide anions (O 2 •− ), hydroxyl radicals (HO•), peroxyl (RO 2 •), alkoxyl (RO•), as well as O 2 -derived non-radical species such as hydrogen peroxide (H 2 O 2 ) [1]. The mitochondrion is a major intracellular source of ROS. Of total mitochondrial O 2 consumed, 1-2% is diverted to the formation of ROS, mainly at the level of complex I and complex III of the respiratory chain, and is believed to be tissue and species dependent [2,3]. Mitochondriaderived O 2 •− is dismutated to H 2 O 2 by manganese superoxide dismutase, and, in the presence of metal ions, highly reactive HO• is generated via Fenton and/or Haber-Weiss reactions, inflicting significant damage to cellular proteins, lipids, and DNA. To date ~10 potential mitochondrial ROS-generating systems have been identified [4]. Among these, Krebs cycle enzyme complexes, such as α-ketoglutarate dehydrogenase (α-KGDH) and pyruvate dehydrogenase have been implicated as significant mitochondrial O 2•− and H 2 O 2 sources [5].Address correspondence to: Tak Yee Aw, PhD, Department of Molecular & Cellular Physiology, LSU Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, Tel: 318-675-6032, Fax: 318-675-4217, taw@lsuhsc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the result...

Apoptosis or programmed cell death represents a physiologically conserved mechanism of cell death that is pivotal in normal development and tissue homeostasis in all organisms. As a key modulator of cell functions, the most abundant non-protein thiol, glutathione (GSH), has important roles in cellular defense against oxidant aggression, redox regulation of proteins thiols and maintaining redox homeostasis that is critical for proper function of cellular processes, including apoptosis. Thus, a shift in the cellular GSH-to-GSSG redox balance in favour of the oxidized species, GSSG, constitutes an important signal that could decide the fate of a cell. The current review will focus on three main areas: (1) general description of cellular apoptotic pathways, (2) cellular compartmentation of GSH and the contribution of mitochondrial GSH and redox proteins to apoptotic signalling and (3) role of redox mechanisms in the initiation and execution phases of apoptosis.

The objectives of this study were to (1) determine the time course of neutrophil adhesion to monolayers of human umbilical vein endothelial cells (HUVECs) that were exposed to 60 minutes of anoxia followed by 30 to 600 minutes of reoxygenation and (2) define the mechanisms responsible for both the early (minutes) and late (hours) hyperadhesivity of postanoxic HUVECs to human neutrophils. The results clearly demonstrate that anoxia/reoxygenation (A/R) leads to a biphasic increase in neutrophil adhesion to HUVECs, with peak responses occurring at 30 minutes (phase 1) and 240 minutes (phase 2) after reoxygenation. Oxypurinol and catalase inhibited phase-1 adhesion, suggesting a role for xanthine oxidase and H2O2. In comparison, platelet activating factor (PAF) contributed to both phases of neutrophil adhesion. Anti-intercellular adhesion molecule-1 (ICAM-1) and anti-P-selectin antibodies (monoclonal antibodies [mAbs]) attenuated phase-1 neutrophil adhesion, consistent with roles for constitutively expressed ICAM-1 and enhanced surface expression of preformed P-selectin. Phase-2 neutrophil adhesion was attenuated by an anti-E-selectin mAb, indicating a dominant role of this adhesion molecule in the late phase response. Pretreatment with actinomycin D and cycloheximide or with competing ds-oligonucleotides containing the nuclear factor-kappa B or activator protein-1 cognate DNA sequences significantly attenuated phase-2 response, suggesting a role for de novo macromolecule synthesis. Surface expression of ICAM-1, P-selectin, and E-selectin on HUVECs correlated with the phase-1 and -2 neutrophil adhesion responses. Collectively, these findings indicate that A/R elicits a two-phase neutrophil-endothelial cell adhesion response that involves transcription-independent and transcription-dependent surface expression of different endothelial cell adhesion molecules.

Further ANNUAL REVIEWSAnnu. Rev. Pharmacol. Toxicol. 1985.25:715-744. Downloaded from www.annualreviews.org by University of Wisconsin -Madison on 10/04/12. For personal use only. HEPATOCYTE GSH REGULATION 717 SINUSOIDAL BLOOD Figure 1 The regulation of hepatic glutathione. Reaction 1: 'Y-glutamylcysteine synthetase; reaction 2: GSH synthetase; reaction 3: GSH peroxidase; reaction 4: GSSG reductace; reaction 5: thioltransferase; reaction 6: GSH S-transferase. This model does not show the mitochondrial pool, which seems to have the same enzymes and distinct GSH regulation. [Reprinted with permission (2).]ing (GSH S-transferase) reactions, and the rate of GSH export from hepato cytes ( Figure I) . Enzymes of GSH BiosynthesisThe enzymes of GSH synthesis were first described and characterized over 30 years ago by Bloch and coworkers (18,19). The synthesis of GSH from its constituent amino acids, L-glutamate, L-cysteine, and L-glycine, involves two ATP-requiring enzymatic steps. The first , which is rate-limiting in GSH synthesis, is the formation of 'Y-glutamylcysteine from L-glutamate and L cysteine. 'Y-Glutamylcysteine synthetase, the enzyme that catalyzes this reac tion, is specific to the 'Y-glutamyl moiety and is regulated by (a) feedback competitive inhibition of the 'Y-glutamate binding site by GSH (Ki = 2.3 mM) (20, 21) and (b) the availability of its precursor, cysteine (22-24) . The Km of 'Y-glutamylcysteine synthetase fo r its two substrates, cysteine and glutamate, are 0.35 mM and 2 mM (21). Recent studies of this enzyme have concentrated on the purification, identification, and characterization of the nature of its active site (25 , 26) . Purification of the enzyme from rat liver has not been reported, but the purified 'Y-glutamylcysteine synthetase from rat kidney has a Mr of 100,000 daltons, with heavy (Mr = 74,000) and light (Mr = 24,000) Annu. Rev. Pharmacol. Toxicol. 1985.25:715-744. Downloaded from www.annualreviews.org by University of Wisconsin -Madison on 10/04/12. For personal use only. Annu. Rev. Pharmacol. Toxicol. 1985.25:715-744. Downloaded from www.annualreviews.org by University of Wisconsin -Madison on 10/04/12. For personal use only. of Glutathione: Biochemical. Physiolog ical, Toxicological, and Clinical As pects. New York: Raven. 393 pp. 8. Smith, M. T., Loveridge, N., Wills, E. Annu. Rev. Pharmacol. Toxicol. 1985.25:715-744. Downloaded from www.annualreviews.org by University of Wisconsin -Madison on 10/04/12. For personal use only.

The intestinal epithelium sits at the interface between an organism and its luminal environment, and as such is prone to oxidative damage induced by luminal oxidants. Mucosal integrity is maintained by the luminal redox status of the glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) couples which also support luminal nutrient absorption, mucus fluidity, and a diverse microbiota. The epithelial layer is uniquely organized for rapid self-renewal that is achieved by the well-regulated processes of crypt stem cell proliferation and crypt-to-villus cell differentiation. The GSH/GSSG and Cys/CySS redox couples, known to modulate intestinal cell transition through proliferation, differentiation or apoptosis, could govern the regenerative potential of the mucosa. These two couples, together with that of the thioredoxin/thioredoxin disulfide (Trx/TrxSS) couple are the major intracellular redox systems, and it is proposed that they each function as distinctive redox control nodes or circuitry in the control of metabolic processes and networks of enzymatic reactions. Specificity of redox signaling is accomplished in part by subcellular compartmentation of the individual redox systems within the mitochondria, nucleus, endoplasmic reticulum, and cytosol wherein each defined redox environment is suited to the specific metabolic function within that compartment. Mucosal oxidative stress would result from the disruption of these unique redox control nodes, and the subsequent alteration in redox signaling can contribute to the development of degenerative pathologies of the intestine, such as inflammation and cancer.