Living in an oxygenated environment has required the evolution of effective cellular strategies to detect and detoxify metabolites of molecular oxygen known as reactive oxygen species. Here we review evidence that the appropriate and inappropriate production of oxidants, together with the ability of organisms to respond to oxidative stress, is intricately connected to ageing and life span.

Oxidants, oxidative stress and the biology of ageing.

Radicals are species containing one or more unpaired electrons. The oxygen radical superoxide (O 2 - ) and the non-radical oxidants hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) are produced during normal metabolism and perform several useful functions. Excessive production of O 2 - and H2O2 can result in tissue damage, which often involves generation of highly reactive hydroxy 1 radical (· OH) and other oxidants in the presence of “catalytic” iron or copper ions. A major form of antioxidant defence is the storage and transport of iron or copper ions in forms that will not catalyze formation of reactive radicals. Tissue injury, e. g., by ischaemia or trauma, can cause increased iron availability and accelerate free radical reactions. This may be especially important in the brain, since areas of this organ are rich in iron and cerebrospinal fluid cannot bind released iron ions. Oxidative stress upon nervous tissue can produce damage by several interacting mechanisms, including rises in intracellular free Ca2+ and, possibly, release of excitatory amino acids. Recent suggestions that free radical reactions are involved in the neurotoxicity of aluminium and in damage to the substantia nigra in Parkinson’s disease are reviewed. Finally, the nature of antioxidants is discussed, with a suggestion that antioxidant enzymes and chelators of iron ions may be more generally useful protective agents than chain-breaking antioxidants. Careful precautions must be taken in the design of antioxidants for therapeutic use.

Reactive Oxygen Species and the Central Nervous System

Insulin resistance is a cardinal feature of type 2 diabetes and is characteristic of a wide range of other clinical and experimental settings. Little is known about why insulin resistance occurs in so many contexts. Do the various insults that trigger insulin resistance act through a common mechanism? Or, as has been suggested, do they use distinct cellular pathways? Here we report a genomic analysis of two cellular models of insulin resistance, one induced by treatment with the cytokine tumour-necrosis factor-alpha and the other with the glucocorticoid dexamethasone. Gene expression analysis suggests that reactive oxygen species (ROS) levels are increased in both models, and we confirmed this through measures of cellular redox state. ROS have previously been proposed to be involved in insulin resistance, although evidence for a causal role has been scant. We tested this hypothesis in cell culture using six treatments designed to alter ROS levels, including two small molecules and four transgenes; all ameliorated insulin resistance to varying degrees. One of these treatments was tested in obese, insulin-resistant mice and was shown to improve insulin sensitivity and glucose homeostasis. Together, our findings suggest that increased ROS levels are an important trigger for insulin resistance in numerous settings.

Reactive oxygen species have a causal role in multiple forms of insulin resistance.

The redox state of the endoplasmic reticulum (ER) was measured with the peptide N-Acetyl-Asn-Tyr-Thr-Cys-NH2. The peptide diffused across cellular membranes; some became glycosylated and thus trapped within the secretory pathway, and its cysteine residue underwent reversible thiol-disulfide exchanges with the surrounding redox buffer. Glycosylated peptides from cells were disulfide-linked to glutathione, indicating that glutathione is the major redox buffer in the secretory pathway. The redox state of the secretory pathway was more oxidative than that of the cytosol; the ratio of reduced glutathione to the disulfide form (GSH/GSSG) within the secretory pathway ranged from 1:1 to 3:1, whereas the overall cellular GSH/GSSG ratio ranged from 30:1 to 100:1. Cytosolic glutathione was also transported into the lumen of microsomes in a cell-free system. Although how the ER maintains an oxidative environment is not known, these results suggest that the demonstrated preferential transport of GSSG compared to GSH into the ER lumen may contribute to this redox compartmentation.

https://science.sciencemag.org/content/sci/257/5076/1496.full.pdf

Oxidized redox state of glutathione in the endoplasmic reticulum

Glutathione and its role in cellular functions.

Glutathione deficiency induced in newborn rats by giving buthionine sulfoximine, a selective inhibitor of gamma-glutamylcysteine synthetase, led to markedly decreased cerebral cortex glutathione levels and striking enlargement and degeneration of the mitochondria. These effects were prevented by giving glutathione monoethyl ester, which relieved the glutathione deficiency, but such effects were not prevented by giving glutathione, indicating that glutathione is not appreciably taken up by the cerebral cortex. Some of the oxygen used by mitochondria is known to be converted to hydrogen peroxide. We suggest that in glutathione deficiency, hydrogen peroxide accumulates and damages mitochondria. Glutathione, thus, has an essential function in mitochondria under normal physiological conditions. Observations on turnover and utilization of brain glutathione in newborn, preweaning, and adult rats show that (i) some glutathione turns over rapidly (t 1/2, approximately 30 min in adults, approximately 8 min in newborns), (ii) several pools of glutathione probably exist, and (iii) brain utilizes plasma glutathione, probably by gamma-glutamyl transpeptidase-initiated pathways that account for some, but not all, of the turnover; thus, there is recovery or transport of cysteine moieties. These studies provide an animal model for the human diseases involving glutathione deficiency and are relevant to oxidative phenomena that occur in the newborn.

https://www.pnas.org/content/pnas/88/5/1913.full.pdf

Glutathione deficiency leads to mitochondrial damage in brain

Glutathione deficiency in newborn rats, produced by administration of L-buthionine-(S,R)-sulfoximine, a transition-state inactivator of gamma-glutamylcysteine synthetase, decreases ascorbate levels of kidney, liver, brain, and lung. These tissues, especially their mitochondria, undergo severe damage and the animals die within a few days. When glutathione levels are markedly decreased, ascorbate levels decrease leading to formation of dehydroascorbate, which is degraded. Ascorbate has high antioxidant activity, but it (and other antioxidants such as alpha-tocopherol) must be maintained in reduced forms. These studies show in vivo that an important function of glutathione is to maintain tissue ascorbate. Administration of large doses of ascorbate (but not of dehydroascorbate) to buthionine sulfoximine-treated newborn rats decreases mortality, leads to normal levels of ascorbate, and spares glutathione. Newborn rats given lower doses of buthionine sulfoximine develop cataracts that, as shown previously, can be prevented by giving glutathione monoester; as found here, such cataracts can be partially prevented by administration of high doses of ascorbate or dehydroascorbate. Ascorbate spares glutathione indicating that these compounds have similar antioxidant actions. Ascorbate may have reductive functions that are not efficiently performed by glutathione. Although glutathione normally functions to maintain ascorbate, alpha-tocopherol, and other cellular components in reduced states, ascorbate can serve as an essential antioxidant in the presence of severe glutathione deficiency.

Glutathione deficiency decreases tissue ascorbate levels in newborn rats: ascorbate spares glutathione and protects

Glutathione (GSH) deficiency produced in mice by giving buthionine sulfoximine leads to severe degeneration of the epithelial cells of the jejunum and colon. This is prevented by giving GSH monoester (orally or i.p.) and also by giving GSH (orally, but not i.p.). The i.p. administration leads to high plasma levels of GSH but does not appreciably increase GSH levels in intestinal mucosa or pancreas. These and previous studies on lens, lung, lymphocytes, liver, heart, and skeletal muscle indicate that there is very little, if any, transport of intact GSH from plasma to these tissues. Cells can use extracellular GSH by a pathway involving its cleavage, uptake of products and intracellular GSH synthesis. Epithelial cells of the gastrointestinal tract may use this pathway and can also take up lumenal GSH (which arises partly from the bile) by a mechanism(s) that may involve transport of dipeptides or of GSH. It is suggested that biliary GSH normally functions in the protection of intestinal mucosa. Administration of GSH may be protective of the gastrointestinal epithelium and may also serve as a good source of cysteine moieties for intracellular GSH synthesis in the gastrointestinal tract and in other tissues. Administration of GSH delivery agents such as GSH esters is more effective than administration of GSH in increasing cellular and mitochondrial levels of GSH.

/pdf/glutathione-is-required-for-intestinal-function-2zcconldfa.pdf

Glutathione is required for intestinal function

Glutathione, an essential cellular antioxidant required for mitochondrial function, is not synthesized by mitochondria but is imported from the cytosol. Rat liver mitochondria have a multicomponent system that underlies the remarkable ability of mitochondria to take up and retain glutathione. At external glutathione levels of less than 1 mM, glutathione is transported into the mitochondrial matrix by a high-affinity component (Km, approximately 60 microM; V max, approximately 0.5 nmol/min per mg of protein), which is saturated at levels of 1-2 mM and stimulated by ATP. Another component has lower affinity (Km, approximately 5.4 mM; Vmax, approximately 5.9 nmol/min per mg of protein) and is stimulated by ATP and ADP. Both components are inhibited by carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), glutamate, and ophthalmic acid. Increase of extramitochondrial glutathione promotes uptake and exchange; the intermembranous space seems to function as a recovery zone that promotes efficient recycling of matrix glutathione. The findings are in accord with in vivo data showing that (i) rapid exchange occurs between mitochondrial and cytosolic glutathione, (ii) lowering of cytosolic glutathione levels (produced by administration of buthionine sulfoximine) decreases export of glutathione from mitochondria to cytosol, and (iii) administration of glutathione esters increases glutathione levels in mitochondria more than those in the cytosol.

https://www.pnas.org/content/pnas/87/18/7185.full.pdf

High-affinity transport of glutathione is part of a multicomponent system essential for mitochondrial function.

Skeletal muscle degeneration associated with mitochondrial damage was found after marked depletion of glutathione produced by administration to mice of buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase. No mitochondrial damage was found in heart. These studies show that in the absence of applied stress (such as ischemia, drug toxicity), very marked depletion (to approximately 3% of the controls) of glutathione must occur before skeletal muscle mitochondria are affected and thus suggest that muscle has a large excess of glutathione. Depletion of glutathione followed a biphasic pattern in skeletal muscle and heart, probably reflecting, in the slow phase, loss of glutathione from mitochondria. Skeletal muscle degeneration did not occur when glutathione monoisopropyl ester was given together with buthionine sulfoximine; it did occur, however, when glutathione was given together with buthionine sulfoximine. Administration of the glutathione monoester (but not of glutathione) prevented the marked decline of mitochondrial glutathione produced by buthionine sulfoximine in skeletal muscle and increased the level of glutathione in heart mitochondria to values higher than the controls. The findings suggest that glutathione monoesters may be useful agents for protection of heart and skeletal muscle against toxicity.

Johannes Martensson

Papers

Glutathione deficiency leads to mitochondrial damage in brain

Glutathione deficiency decreases tissue ascorbate levels in newborn rats: ascorbate spares glutathione and protects

Glutathione is required for intestinal function

High-affinity transport of glutathione is part of a multicomponent system essential for mitochondrial function.

Mitochondrial damage in muscle occurs after marked depletion of glutathione and is prevented by giving glutathione monoester.