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
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.
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
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