Free Radical Biology and Medicine
Vol. 22, No. 4, pp. 669-678, 1997
©1997 Elsevier Science. Excerpt reprinted with permission.

QUERCETIN PROTECTS CUTANEOUS TISSUE-ASSOCIATED CELL TYPES INCLUDING SENSORY NEURONS FROM OXIDATIVE STRESS INDUCED BY GLUTATHIONE DEPLETION: COOPERATIVE EFFECTS OF ASCORBIC ACID

STEPHEN D. SKAPER, MICHELE FABRIS, VANNI FERRARI, MAURIZIO DALLE CARBONARE, and ALBERTA LEON

INTRODUCTION

Normal cell metabolism results in a continuous generation of reactive oxygen species, such as the superoxide radical or the nonradical hydrogen peroxide.¹ An imbalance between reactive oxygen species and the antioxidant defense mechanisms of a cell, leading to an excessive production of oxygen metabolites, creates a condition frequently termed "oxidative stress." Oxidative injury leads to lipid peroxidation, DNA breakage, and enzyme inactivation, including free radical scavenger enzymes. The molecular mechanisms responsible for this spectrum of biochemical damage are complex, but it has been established that superoxide radical and hydrogen peroxide are precursors of other reactive species, such as the hydroxyl radical.²

Lipid peroxides and reactive oxygen species are likely involved in numerous pathological events, including inflammation, radiation damage, metabolic disorders, cellular aging, and reperfusion damage.^2-4 Evidence for the potential role of oxidants in the pathogenesis of many diseases suggests that antioxidants may be of therapeutic use in these conditions. Flavonoids (plant phenolic pigment products) such as quercetin (3,5,7,3',4'-pentahydroxyflavone) may delay oxidant injury and cell death⁵ by: scavenging oxygen radicals;^6-9 protecting against lipid peroxidation^10,11 and thereby terminating the chain-radical reaction;¹² chelating metal ions,⁹ to form inert complexes that cannot take part in the conversion of superoxide radicals and hydrogen peroxide into hydroxyl radicals.

Cutaneous tissue sensitivity to oxidative damage is evident in disorders like insulin-dependent diabetes mellitus^13,14 and and in photoaging.¹⁵ In addition, oxygen radicals can arise in skin during inflammatory processes.¹⁶ For example, ultraviolet light induces a decrease in the cellular content of reduced glutathione (GSH), an increase in the level of oxidized glutathione (GSSG), a decrease in the level of all major lipophilic antioxidants, and peroxidation of lipids.^17,18 Also, cuItured human keratinocytes are described to be irreversibly damaged by prolonged oxidative stress caused by organic hydroperoxides.¹⁹ We have now examined the ability of several flavonoids to protect in vitro cell types characteristic of cutaneous tissue, from oxidative stress and death triggered by depletion of the critical antioxidant defense molecule GSH. A specific and essentially irreversible inhibitor of g-glutamyl-cysteine synthetase,²⁰ buthionine sulfoximine (BSO), was used to decrease the intracellular concentration of GSH and cause cytotoxicity without application of an external stress (e.g., increased oxygen, drugs, radiation). The results show that quercetin is especially effective in protecting cultured human skin fibroblasts, keratinocytes, and endothelial cells from a protracted oxidative injury, even following BSO withdrawal. Quercetin also reduced the BSO-dependent death of sensory neurons. Because ascorbic acid may have flavonoid-protective activity,^20,21 we asked if the antioxidative function of the tested flavonoids could be enhanced by ascorbate.

The beneficial effects of quercetin observed here presumably reflect the ability of this flavonoid to protect cells from the deleterious consequences of GSH deficiency. Glutathione represents a key cellular defense mechanism against oxidative injury, and a major consequence of GSH deficiency is mitochondrial damage. Hydrogen peroxide, produced by mitochondria, can cause extensive damage to this organelle when GSH levels are greatly decreased.^38-40 The cytotoxic effects of inhibiting GSH synthesis occurred without application of stress and were prevented by administration of GSH monoesters, as described by others.^34,38-40 Quercetin treatment did not lead to a recovery of cellular GSH, as verified by direct biochemical measurement. Further, using the probe 2,7-dichlorofluorescein diacetate, which becomes trapped in cells and fluoresces upon oxidation,⁴¹ quercetin was found to reduce intracellular peroxide accumulation in BSO-treated cultures (our unpublished observations). Because quercetin was active when first added after BSO removal, it is unlikely to interact with the inhibitor. The results thus show that quercetin is effective in preventing injury to dermal cells subjected to a long-lasting oxidative insult generated intracellularly.

Interactions between flavonoids and ascorbic acid have been documented.⁴² Ascorbate is reported to have flavonoid-protective^21,22 and flavonoid-enhancing^9,32-33 activities. Here, ascorbic acid was found to enhance the cytoprotective effects of quercetin and rutin against oxidative stress-induced death of human skin fibroblasts. Ascorbic acid both lowered the EC₅₀ and prolonged the time over which the flavonoid was active in rescuing cells from oxidative injury. The cooperative activities between quercetin or rutin and ascorbic acid may result from a reduction by ascorbate of oxidized quercetin (or rutin) and regeneration of the flavonoid. With quercetin and its 3-glycoside rutin, ascorbate regenerates the flavonoid from the respective aroxyl radical, although this remains to be established for more complex (whole cell) biological systems. Such an interaction could be evidenced as a synergistic effect, providing a constant resupply of the flavonoid acting as radical scavenger and to limit the amount of the aroxyl radical decaying by a second-order reaction.⁴² Alternatively, it may reflect a prooxidative effect of the flavonoids, if ascorbate is the more important cofactor.⁴³ The second possibility seems unlikely, given that ascorbic acid in the present system had no significant protective action, and actually became cytotoxic at the highest concentrations tested. Under some conditions, ascorbic acid may have prooxidant effects.^44,45 In any case, these data are the first to ascribe a synergistic action of flavonoids and ascorbic acid in rescuing cells from death caused by oxidative stress.

Cutaneous tissue injury involving dysfunction of the microvasculature can occur in disorders like insulin-dependent diabetes mellitus^13,14 and photoaging^15,62 where free radical oxidative stress may be an important factor. Endothelial cell oxidative injury^56,57 will likely increase capillary permeability,⁶³ leading to tissue entry of pro-oxidant species. Damage to intraneural capillaries can compromise the blood-nerve interface,⁶⁴ with subsequent breakdown in the homeostatic equilibrium of the intraneural microenvironment.^65,66 Antioxidant treatment can, in fact, effectively prevent the development of diabetic neuropathy.^67,68 Furthermore, quercetin and several related flavonoids reportedly reduce the increased cutaneous vascular permeability occurring in conditions of experimentally induced inflammation.⁶⁹ The present data propose that flavonoids like quercetin or rutin, alone or combined with ascorbic acid, may be effective in protecting neurovascular structures in skin and likely also those in other districts (e.g., mucosa and nerves) from oxidative stress and free radical-induced toxicity.

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