N-acetylcysteine:not simply a glutathione precusor

The ischemic episodes to which transplanted organs are exposed present management problems extending from the immediate postoperative period. An early consequence−lung reperfusion injury after liver ischemia-reperfusion−is considered by Weinbroum and colleagues (1), who evaluated whether rat lung reduced glutathione (GSH) concentrations correlated with improvements in function using N-acetylcysteine (NAC) (in the effluent from ischemic livers with which the lungs were perfused). Ischemia is but an extreme of the physiological variability in oxygen concentrations within an organ (e.g., across the liver lobule). There are similar variations in oxygen consumption (e.g., during cell division) and in the release of cytotoxic products of oxidative metabolism, including the reactive oxygen species (ROS) superoxide (OJOURNAL/trans/04.03/00007890-200003150-00001/OV0254/v/2021-02-16T024319Z/r/image-png2) and peroxide (HOO−) and their reaction products (with Cl−, NO, and Fe2+): hypochlorous acid (HClO), peroxynitrite (ONOO−), and hydroxyl radical (OH·), respectively. A critical balance prevails between ROS production and their detoxication with an extensive intracellular armory comprising antioxidants/free-radical scavengers (e.g., GSH; vitamins A, C, and E; bilirubin, etc.), enzymes (e.g., superoxide dismutase, catalase, glutathione peroxidase, heme oxygenase, etc.), and nucleophilic proteins (e.g., metallothionein). Compartmentalizing and customizing cytoprotectants within the mitochondria, peroxisomes, and nucleus provides particular control over “local” events (e.g., mitochondrial uncoupling and transcription factor binding). Consequently, an increase in production of ROS (“oxidative stress”) that is transient and moderate can be accommodated within the normal range of cellular responses. Control over oxidative stress is also achieved via the multiple responses it induces to alleviate its adverse consequences (e.g., the induction of γ-glutamyl-cysteine synthase, the rate limiting step in GSH synthesis) (2, 3). The extent of control achieved can be judged from growing evidence for a regulatory role of ROS, including actions (especially for HOO−) as second messengers, at oxygen-sensitive response elements in gene promoters, and in modifying transcription factors binding to these promoters. Influences via the cell’s redox status (normally predominantly “reductive” as judged from reduced:oxidized glutathione ratios > 95:1) are frequently responsible. Such control is commensurate with the production of ROS as a unified response to many cytotoxins: e.g., radiation, inflammatory mediators, heat shock, and xenobiotics. To endow such systems with the rapid and reversible responses required, a chemical basis for regulation prevails and sulfhydryl groups within proteins act as primary targets for oxidation (by ROS) and can transform function via configurational changes, both within the proteins and at active sites. Reversal of these processes by simple chemical reduction of the same targets occurs endogenously (e.g., with GSH and cysteine) and with administered thiols and their precursors (e.g., β-mercaptoethanol, lipoic acid, and NAC, which has been widely used to combat tissue injury in clinical toxicology (4) and intensive care medicine). Reperfusion injury leads to extreme ROS production, either after cold ischemia (in organ transplantation) or warm ischemia (in tumor resection, aneurysm repair, myocardial infarction, or cerebral thrombosis). Of prolonged duration, such injuries may be homeostatically uncontrollable without intervention because they induce multiple responses which exacerbate ROS production by a self-perpetuating cascade of events described elsewhere in much greater detail (2, 3, 5) but including: (1) the activation of immediate-early genes, (2) the oxidation of the transactivating and DNA-binding domains of numerous transcription factors (e.g., AP-1, NF-κB, and the dissociation of IκB from NF-κB) and the activation of kinases and phosphatases—with differential effects on gene expression, and (3) induction of the mitochondrial membrane permeability transition (via oxidative changes at GSH and pyridine cofactor sites). The consequences include: (1) up-regulation of cell adhesion molecule expression leading to leukocyte adherence; (2) release of tumor necrosis factor-α, pro-inflammatory cytokines (interleukin [IL]-1, IL-6, and IL-8), vasoconstrictors (e.g., endothelin and thromboxane A2), and platelet activators; (3) mitochondrial uncoupling and dysfunction (promoting ATP depletion, DNA damage, apoptosis, and necrosis); (4) activation of endonucleases, phospholipases and proteases and increases in lipid peroxides, cytoplasmic Ca2+, Fe2+, and oxidized glutathione (GSSG); and (5) additional ROS production (from mitochondria and activated and sequestered cells). Perhaps rationalizing a common mediator of this response, Weinbroum and colleagues chose treatment with NAC alone. The dual perfusion circuit they describe is an ideal platform for studying the effects of soluble mediators of reperfusion injury because, as the authors stress, contributions from platelets, neutrophils, and complement do not apply. Xanthine oxidase was massively increased and suggested to be the likely major source of ROS, but tumor necrosis factor-α, another rapid inducer of OJOURNAL/trans/04.03/00007890-200003150-00001/OV0254/v/2021-02-16T024319Z/r/image-png2 (6) was not measured. Additional reservations can be raised on the length of liver ischemia (120 min), the lack of an oxygen carrier, and an excessively fast liver perfusion rate (4 ml/min/g). Perfusate NAC concentration (possibly at 0.7, 1.1, and 1.6 mM for the three doses) can only be approximated from the experimental details supplied. NAC was rapidly effective in limiting injury when present above the lowest concentrations used, implying that the variable responses to NAC described in previous publications may depend on the extent and duration of the oxidative stress treated. The disproportionality between efficacy and tissue GSH concentration may not be surprising given the actions of NAC as a chemical reductant of oxidized thiols, as a scavenger of ROS (especially OH·, HOCl, and ONOO− but not OJOURNAL/trans/04.03/00007890-200003150-00001/OV0254/v/2021-02-16T024319Z/r/image-png2 or HOO−) (7) and as a vasodilator (via nitrosothiols) as well as a GSH precursor. That its chemical properties dominate may be judged also by its rapid benefit in this study and by the similar potency of D- and L-NAC to reduce oxygen-induced lung edema (8) but with the former isomer unlikely to be incorporated into GSH. Based on its efficacy in reducing lung reperfusion injuries, can we conclude that NAC will protect the graft itself? The answer can only be equivocally affirmative. Several features of ischemia-reperfusion injury to grafts (e.g., endothelial cell shrinkage and detachment, the release of proteases from sinusoidal lining cells, etc.) may predate those events against which NAC appears most effective. In addition, multiple consequences of the “autonomic storm” and catecholamine release at brain death lead to organ damage in the donor (9) before NAC administration is feasible. Therefore, a more effective intervention may be to block the combined expression of these effects later in evolving cell injury—an approach tested successfully using an adenoviral vector for bcl-2 to limit apoptosis in cold-preserved mouse livers (10). Alternative approaches avoiding the unknown hazards of DNA transfection and its vectors may be simple organ “conditioning” with a brief ischemic episode, or the avoidance of severe ischemia with either rescue or continuous perfusion. Acknowledgment. The author is grateful to Wayel Jassem and Martin Phillips for helpful discussions during the preparation of this document.