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 peroxidation10,11 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 mellitus13,14 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.

DISCUSSION

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-protective21,22 and flavonoid-enhancing9,32-33 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 mellitus13,14 and photoaging15,62 where free radical oxidative stress may be an important factor. Endothelial cell oxidative injury56,57 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.

REFERENCES

1. Bowling, A.C.; Beal, M. F. Bioenergetic and oxidative stress in neurodegeneative diseases. Life Sci. 56:1151-1171; 1995.
2. Wiseman, H.; Halliwell, B. Damage to DNA by reactive oxygen and nitrogen species: Role in inflammatory disease and progression to cancer. Biochem. J. 313:17-29; 1996.
3. Ames, B.N.; Shigenaga, M.K.; Hagen, T.M. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA 90:7915-7922; 1993.
4. Buttke, T.M.; Sandstrom, P.A. Oxidative stress as a mediator of apoptosis. Immunol. Today. 15:7-10; 1994.
6. Bors, W.; Heller, W.; Michel, C.; Saran, M. Flavonoids as antioxidants: Determination of radical-scavenging efficiencies. Methods Enzymol. 186:343-355; 1990.
7. Bors, W.; Michel, C.; Saran, M. Flavonoid antioxidants: Rate constants for reactions with oxygen radicals. Methods Enzymol. 234:420-429; 1994.
8. Jovanovic, S.V.; Steenken, S.; Tosic, M.; Majanovic, A.; Simic, M.G.Flavonoids as antioxidants. J. Am. Chem. Soc. 116:4846- 4851; 1994.
9. Afanas'ev, I.B.; Dorozhko, A.I.; Brodskii, A.V.; Kontyuk, V A.; Potapovitch, A.I. Chelating and free radical scavenging mechanism of inhibitory action of rutin and quercetin in lipid peroxidation. Biochem. Pharmacol. 38:1763-1768; 1989.
10. Laughton, M.J.; Evans, P.J.; Moroney, M.A.; Hoult, J.R.; Halliwell, B. Inhibition of mammalian S-lipoxygenase and cyclo-oxygenase by flavonoids and phenolic dietary additives. Relationship to antioxidant activity and to iron ion-reducing ability. Biochem Pharmacol. 42:3673-16813; 1991.
11. Dechameux, T.; Dubois, F.; Beauloye, C.; Wattiaux-DeConinck, S.; Waniaux, R. Effect of various flavonoids on lysosomes subjected to an oxidative stress. Biochem Pharmacol. 44:1243- 1248; 1992.
12. Torel, J.; Cillard, J.; Cillard, P. Antioxidant activity of flavonoids and reactivity with peroxy radical. Phytochemistry. 25:382-386; 1986.
13. Odetti, P.R.; Borgoglio, A.; DePascale, A.; Rolandi, R.; Adezad, L. Prevention of diabetes-increased aging effect on rat collagen-linked fluorescence by aminoguanidine and rutin. Diabetes. 39:796-801; 1990.
14. McCance, D.R.; Dyer, D.G.; Dunn, J.A.; Bailie, K.E.; Thorpe, S.R.; Baynes, J.W.; Lyons, T.J. Maillard reaction products and their relation to complications in insulin-dependent diabetes mellitus. J. Clin. Invest. 91:2470-2478; 1993.
15. Darr, D.; Fridovich. I. Free radicals in cutaneous biology. J. Invest. Dermatol. 102:671-675; 1994.
16. Darr, D.J. The biology of oxygen free radicals and their relevance to dermatology. In: Takase. Y.; Kligman, A.M. eds. Cutaneous aging. Tokyo: University of Tokyo Press; 1988:415- 423.
17. Wheeler, L.A.; Aswad, A.; Conner, M.J.; Lowe, N. Depletion of cutaneous glutathione and the induction of inflammation by 8-methoxypsoralen plus UVA radiation. J. Invest. Dermatol. 87:658-662; 1986.
18. Punnonen, K.; Puntala, A.; Jansen, C.T.; Ahotupa, M. UVB irradiation induces lipid peroxidation and reduces antioxidant enzyme activities in human keratinocytes in vitro. Acta. Dermatol. Veneral. 71:239-273; 1991.
19. Vessey, D.A.; Lee, K.H.; Blacker, K.L. Characterization of the oxidative stress initiated in cultured human keratinocytes by treatment with peroxides. J. Invest. Dermatol. 99:859-863; 1992.
20. Griffith, O.W.; Meister, A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J. Biol. Chem. 254:7558-7560; 1979.
21. Sorata, Y.; Takahama, U.; Kimura, M. Cooperation of quercetin with ascorbate in the protection of photosensitized lysis of human erythrocytes in the presence of hematoporphyrin. Photochem. Photobiol. 48:195-199; 1988.
22. Jan, C.Y.; Takahama. U.; Kimura, M. Inhibition of photooxidation of a-tocopherol by quercetin in human blood cell membranes in the presence of hematoporphyrin as a photosensitizer. Biochim. Biophys. Acta. 1086:7-14; 1991.
32. Vrijsen, R.; Everaert, L.; Boeye, A. Antiviral activity of flavonols and potentiation by ascorbate. J. Gen. Virol. 69:l749-1751; 1988.
33. Kandaswami. C.; Perkins, E.; Soloniuk, D.S.; Dnewiecki, E.; Middleton, E., Jr. Ascorbic acid-enhanced antiproliferative effect of flavonoids on squamous cell carcinoma in vitro. Anti Cancer Drugs 4:91-96; 1993.
34. Jain, A.; Martensson, J.; Stole, E.; Auld, P.A. M.; Meister, A. Glutathione deficiency leads to mitochondrial damage in brain. Proc. Nat. Acad. Sci. USA. 88:1913-1917; 1991.
38. Martensson, J.; Jain, A.; Frayer, W.; Meister, A. Glutathione metabolism in the lung: Inhibition of its synthesis leads to lamellar body and mitochondrial defects. Proc. Natl. Acad. Sci. USA. 86:5296-5300; 1989.
39. Martensson, J.; Steinherz, R.; Jain, A.; Meister, A. Glutathione ester prevents buthionine sulfoximine-induced cataracts and lens epithelial cell damage. Proc. Nat. Acad. Sci. USA 86:8727-8731; 1989.
40. Martensson, J.; Jain, A.; Meister, A. Glutathione is required for intestinal function. Proc. Nat. Acad. Sci. USA 87:1715-1719; 1990.
41. Bass, D.A.; Farce, J.W.; Dechatelet, L.R.; Szejda, P.; Seeds, M.C.; Thomas, M. Flow cytometric studies of oxidative product formation by neutrophils: A graded response to membrane stimulation. J. Immunol. 130:1910-1917; 1983.
42. Bors, W.; Michel, C.; Schikora, S. Interaction of flavonoids with ascorbate and determination of their univalent redox potentials: A pulse radiolysis study. Free Radic. Biol. Med. 19:45-52; 1995.
43. Bentsath, A.; Ruaznyak, S.; Szent-Gyorgyi, A. Vitamin P. Nature 139:326-327; 1937.
44.Kanner, J.; Mendel, H. Prooxidant and antioxidant effects of ascorbic acid and metal salts in a b-carotene-linoleate model system. J. Food Sci. 42:60-64; 1977.
45. Wefers, H.; Sies, H.The protection by ascorbate and glutathione against microsomal lipid peroxidation is dependent on vitamin E. Eur. J. Biochem. 174:353-357; 1988.
50. Baynes, J.W. Role of oxidative stress in development of complications in diabetes. Diabetes 40:405-412; 1991.
56. Henricksen, T.; Evensen, S.A.; Carlander, B. Injury to human endothelial cells in culture induced by LDL. Scand. J. CIin. Lab. Invest. 39:361-368; 1979.
57. Kuzuya, M.; Naito, M.; Funaki, C.; Hayashi, T.; Asai, K.; Kuzuya, F. Protective role of intracellular glutathione against oxidized low density lipoprotein in cultured endothelial cells. Biochem. Biophys. Res. Commun. 163:1466-1472; 1989.
63. Alexander, J.J.; Graham, D.J.; Miguel, R. Oxygen radicals alter LDL permeability and uptake by an endothelial smooth muscle cell bilayer. J. Surg. Res. 51:361-367; 1991.
64. Weuasuriya, A.; Curmn, G.L.; Podulso, J.E. Blood-nerve transfer of albumin and its implications for endoneurial microenviromnent. Brain Res. 494:114-121; 1989.
65. Lundborg, G. Intraneural microcirculation. Orthoped. CIin. North Am. 19:1-12; 1988.
66. Olsson, Y. Microenvironment of the peripheral nervous system under normal and pathological conditions. Crit. Rev. Neurobiol. 5:265-311; 1990.
67. Bravenboer, B.; Kappelle, A.C.; Hamers, E.P.T.; van Buren, D.W.; Erkelens, D.W.; Gispen; W.H. Potential use of glutathione for the prevention and treatment of diabetic neuropathy in the streptozotocin-induced diabetic rat. Diabetologia 35:813-817; 1992.
68. Cameron, N.E.; Cotter, M.A.; Maxfield, E.K. Anti-oxidant trearment prevents the development of peripheal nerve dysfunction in streptozotocin-diabetic rats. Diabetologia 36:299-304; 1993.
69. Nakadate, T.; Yamamoto, S.; Aizu, E.; Kato, R. Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced increase in vascular permeability in mouse skin by lipoxygenase inhibitors. Jpn. J. Pharmacol. 38:161-168; 1985.