Original Contribution

QUERCETIN INHIBITS HYDROGEN PEROXIDE-INDUCED OXIDATION OF

THE RAT LENS

J

ULIE

S

ANDERSON

,* W. R

USSELL

M

C

L

AUCHLAN

,

†

and

G

ARY

W

ILLIAMSON

†

*School of Biological Sciences, University of East Anglia, Norwich, UK; and

†

Department of Biochemistry, Institute of Food

Research, Norwich, UK

(Received 8 July 1998; Accepted 14 August 1998)

Abstract—Cataract results from oxidative damage to the lens. The mechanism involves disruption of the redox system,
membrane damage, proteolysis, protein aggregation and a loss of lens transparency. Diet has a significant impact on
cataract development, but the individual dietary components responsible for this effect are not known. We show that low
micromolar concentrations of the naturally-occurring flavonoid, quercetin, inhibit cataractogenesis in a rat lens organ
cultured model exposed to the endogenous oxidant hydrogen peroxide. Other phenolic antioxidants, (

1)epicatechin and

chlorogenic acid, are much less effective. Quercetin was active both when incubated in the culture medium together with
hydrogen peroxide, and was also active when the lenses were pre-treated with quercetin prior to oxidative insult.
Quercetin protected the lens from calcium and sodium influx, which are early events leading to lens opacity, and this
implies that the non-selective cation channel is protected by this phenolic. It did not, however, protect against formation
of oxidized glutathione resulting from H

2

O

2

treatment. The results demonstrate that quercetin helps to maintain lens

transparency after an oxidative insult. The lens organ culture/hydrogen peroxide (LOCH) model is also suitable for
examining the effect of other dietary antioxidants.

© 1999 Elsevier Science Inc.

Keywords—Lens, Cataract, Antioxidant, Diet, Flavonoid, Quercetin, Calcium, Free radical

INTRODUCTION

In cataract, the normally clear and transparent lens in the
eye becomes opaque. Evidence from epidemiological
studies, model systems, and human lenses obtained after
cataract surgery, indicate a role for oxidation in this
opacification process [1,2]. Analysis of human cataracts
has demonstrated a loss of oxidized glutathione [3],
oxidation of membrane lipids [4] and oxidation of pro-
tein thiols, to form cysteic acid, methionine sulfoxide,
mixed disulphides and protein-protein disulphides [5,6].
In cortical cataracts, this is associated with an increase in
membrane permeability and intracellular calcium over-
load [7]. The increase in calcium concentration has been
directly linked to lens opacification by experimental
modelling of cataractogenesis in organ cultured lenses
[8]. Organ cultured lenses are an ideal model system to
examine the effect of antioxidants and other molecules
on cataractogenesis, and in this study we used the en-

dogenous oxidant H

2

O

2

to induce oxidative damage in

the rat lens, which we have termed the Lens Organ
Culture with Hydrogen peroxide (LOCH) assay. This
type of model of oxidative cataract has been well
characterized [1]. H

2

O

2

is present in the aqueous hu-

mor and so the lens is in contact with this oxidant in
vivo. The oxidative damage resulting from exposure to
H

2

O

2

parallels observed changes occurring to the hu-

man lens as a result of cataractogenesis, including loss
of GSH, increases in intracellular calcium and lens
opacification [1]. The model was used to investigate
the potential protective activity of the naturally-occur-
ring dietary flavonoid, quercetin. This has previously
been shown to inhibit the formation of diabetic cata-
ract [9,10].

MATERIALS AND METHODS

Lens Organ Culture with Hydrogen peroxide (LOCH)

Rat lenses used in this study were from 10 –15 wk old

Wistar rats. For each individual experiment, rats were

Address correspondence to: Julie Sanderson, School of Biological Sci-

ences, University of East Anglia, Norwich, NR4 7TJ, UK; Tel:

144 (0)

1603 592268; Fax:

144 (0) 1603 592250; E-Mail: [e-mail].

Free Radical Biology & Medicine, Vol. 26, Nos. 5/6, pp. 639 – 645, 1999

Copyright © 1999 Elsevier Science Inc.

Printed in the USA. All rights reserved

0891-5849/99/$–see front matter

PII S0891-5849(98)00262-7

639

born within 1 wk of each other. The lenses were dis-
sected using the posterior approach and incubated in
artificial aqueous humor (AAH): NaCl 130 mM; KCl
5 mM; CaCl

2

1 mM; MgCl

2

0.5 mM;

D

-glucose 2.5 mM;

NaHCO

3

5 mM; HEPES 20 mM; pH 7.3 for approxi-

mately 1 h at 35°C. The lenses were photographed using
back-scattered light by illumination from above from a
fiber-optic ring-light (1500 electronic, Schott), for as-
sessment of light scatter at t

5 0 h, and then placed

directly into AAH containing 1 mM H

2

O

2.

Quercetin in

dimethyl sulfoxide (DMSO) or DMSO alone was added
at the indicated concentration, and the final concentration
of DMSO was always less than 0.1 % v/v. Control
lenses remained in AAH for the duration of the ex-
periment. The lenses were photographed at 0, 4 and
8 h. After 12 h, the lenses were rolled on filter paper
to remove adhering tissue, frozen in liquid nitrogen
and stored at

270°C. The rate of detoxification of the

H

2

O

2

by enzymes in the lens was measured in freshly

dissected lenses over a 4 h period using a colorimetric
assay based on the formation of Fe

3

1

, which forms a

complex with Xylenol orange (PeroXOquant; Pierce
& Warriner Ltd, Chester, UK). The rate of metabolism
of H

2

O

2

by the lens was approximately 200 nmol/h,

equivalent to a loss of approximately 50% of the H

2

O

2

from the culture medium in 8 h.

For some experiments, lenses were treated with quer-

cetin in organ culture without H

2

O

2

for 24 h, the lenses

washed, and then treated with H

2

O

2

without quercetin as

follows: after dissection the lenses were transferred to
Eagle’s Minimal Essential Medium (EMEM) together
with 26 mM NaHCO

3

, 0.1% bovine serum albumin,

5

mg/ml transferrin and 5 ng/ml sodium selenite. In the

first hour, the EMEM was supplemented with 1000 U/ml
penicillin and 1 mg/ml streptomycin. Subsequently, the
penicillin/streptomycin concentration was reduced to
100 U/ml penicillin and 0.1 mg/ml streptomycin. Lenses
were incubated at 35°C in 5% CO

2

in 3 ml EMEM.

Following an overnight incubation the lenses were ex-
posed to quercetin for 24 h. They were then transferred to
AAH for 1 h prior to exposure to 1 mM H

2

O

2

in AAH.

The lenses were photographed at 0, 4 and 8 h. At 12 h the
lenses were frozen in liquid nitrogen and stored at
270°C.

For all lenses, the light scatter was measured by

scanning the photographic negatives in two dimensions
using a laser densitometer and assessed using Gelscan
XL 2.1 software (LKB-Pharmacia). Data are expressed
in arbitrary absorbance units per unit area (au/mm

2

). This

was computed from the optical density reading derived
from the photographic negatives of experimental lenses
divided by the total area occupied by the lens.

Glutathione assays

Preparation of rat lens extracts for glutathione assays.
Each rat lens was homogenized in 0.5 ml of 5-sulfosal-
icylic acid in a 0.5 ml centrifuge tube using a tissue
grinder. The homogenate was then centrifuged at
13,000

3 g for 5 min at ambient temperature. The

supernatant was then split 4:1 for the oxidized and total
glutathione assays respectively.

Quantification of total glutathione (GSH). The total glu-
tathione content of the rat lenses was determined using
the glutathione reductase recycling method of Tietze
[11]. Sample (maximum of 200

ml) was added to 700 ml

of 100 mM sodium phosphate buffer containing 1 mM
EDTA (ethylenediaminetetraacetic acid, tetra sodium
salt), 0.28 mM NADPH and 100

ml of 6 mM 5,59-dithio-

bis(2-nitrobenzoic acid) (DTNB). This mixture was in-
cubated at 30°C for 10 min and then 1 unit of glutathione
reductase (type III from Bakers Yeast) was added, and
the increase in absorbance at 412 nm over 6 min was
recorded on a Beckman DU 70 spectrophotometer. The
glutathione content of lens extracts was calculated from
a standard curve generated by plotting the rate of absor-
bance change at 412 nm against amount of glutathione
(0, 50, 250, 500, 750 and 1000 pmol).

Quantification of oxidized glutathione (GSSG). The ox-
idized glutathione content was determined by modifica-
tion of the glutathione reductase recycling method as
described previously [12]. Reduced glutathione is re-
moved from the assay by derivatization with 2-vinylpyri-
dine; 2

ml was added to rat lens extract (100 ml). Trieth-

anolamine (8

ml) was then added and the solution was

mixed for 30 s and then left at ambient temperature for
1 h. The samples were then assayed as described for the
total glutathione measurements except that glutathione
reductase (2 units) was added to start the reaction and the
standard curve was generated using GSSG. The reduced
glutathione content was calculated by subtracting the
amount of glutathione present as GSSG from the total
value. All data are presented as GSH equivalents in nmol
GSH/mg protein. One mol of GSSG is therefore ex-
pressed as 2 mol GSH equivalents.

Influx measurements

Influx experiments were carried out by incubating

lenses for 4 h in medium containing 40 kBq ml

21 45

Ca

2

1

and

22

Na

1

(Amersham International, Little Chalfont,

UK) at 35°C. At the end of the incubation period, the
lenses were washed in 5 ml non-radioactive medium for
1 min to remove excess external isotope. They were then
rolled on dry filter paper and weighed. Each lens was

640

J. S

ANDERSON

et al.

placed in a scintillation vial containing 2 ml AAH and
was left overnight to allow for efflux of the isotopes from
the lens into the external medium. After adding 10 ml
OptiPhase “SuperMix” scintillation fluid (Wallac Scin-
tillation Products, Milton Keynes, UK) to each vial, the
radioactivity was assayed using a Wallac 1409 liquid
scintillation counter. To obtain the specific activity of the
loading solution, 10

ml samples were removed from the

respective incubation media and added to 2 ml AAH and
10 ml scintillation fluid. The data were calculated on a
mol/lens wet weight basis.

Aqueous phase antioxidant activity

The Trolox equivalent antioxidant capacity (TEAC)

was measured by the method of Salah et al. [13]. The
assay is based on the relative ability of antioxidants to
scavenge the radical cation of 2,2

9-azinobis(3-ethyl-ben-

zothiazoline-6-sulphonate)

(ABTS).

The

extent

of

quenching of the ABTS radical is measured spectropho-
tometrically at 734 nm and compared to standard
amounts of Trolox. The values are expressed as Trolox
equivalents (mM).

RESULTS

Quercetin protects against H

2

O

2

-induced opacity in the

LOCH assay

Exposing lenses to 1 mM H

2

O

2

resulted in a loss of

lens transparency (Fig. 1). The increases in light scatter
occurred in the cortical regions of the lens. Within 1 h of
exposure to H

2

O

2

, most of the opacity was located in the

equatorial cortical region; there was also some opacity in
the central cortical region, which increased over the time
course of the experiment. Incubation with quercetin
(30

mM) almost completely abolished this increase in

opacity after 4 h (Fig. 1). We investigated this effect in
more detail. Quercetin protected against opacity in a
dose- and time-dependent manner (Fig. 2). Under these
conditions, 10

mM quercetin inhibited light scatter by

87% at 4 h and 41% at 8 h (p

, .05), and increasing the

concentration to 30

mM showed no further protection. As

a control, quercetin (50

mM) alone had no effect on light

scatter, indicating a lack of toxicity even at this higher
concentration.

The effect of quercetin is not due to direct free radical
scavenging

Quercetin is an effective antioxidant in most assay

systems, and so we compared the action of quercetin

Fig. 1. Photographs of typical results during the course of a rat lens organ culture with hydrogen peroxide (LOCH) assay. Rat lenses
were cultured in control AAH (c), 1 mM H

2

O

2

(h) or 1 mM H

2

O

2

together with 30

mM quercetin (hq). Photographs were taken after

4 h.

Fig. 2. Effect of quercetin on light scatter in the LOCH assay. Lenses
were incubated with H

2

O

2

together with 0

mM (F), 3 mM (Œ), 10 mM

(■) or 30

mM (‚) quercetin and compared to control lenses with no

H

2

O

2

(E). Light scatter is measured in arbitrary absorbance units per

mm

2

. Experimental points are the mean and standard deviation of 4

separate measurements.

641

Dietary antioxidants and cataract

to that of other phenolic free radical scavengers, chlo-
rogenic acid and (

1)epicatechin. The structures of

these compounds are shown in Fig. 3. The ability
of the compounds to scavenge the radical cation of
ABTS compared to a 1 mM standard solution of the
water soluble vitamin E analog, Trolox, was mea-
sured. Total antioxidant activities (expressed as
Trolox equivalents) are: quercetin, 4.43

6 0.03 mM;

(

1)epicatechin, 2.23 6 0.3 mM; chlorogenic acid,

1.24

6 0.01 mM (mean and standard deviation of 3

determinations).

These phenolics were compared to quercetin in the

LOCH assay. No significant protection of transpar-
ency was observed at up to 1 mM chlorogenic acid, but
(

1)epicatechin (50

mM) showed a weak and non-

significant (p

5 .1) inhibition of opacification by 38%

after 4 h. The latter effect is much weaker than that
observed for quercetin. If activity in the LOCH assay
was dependent exclusively on free radical scavenging,
then chlorogenic acid would be expected to exhibit
some protection and (

1)epicatechin would be more

effective. We can therefore conclude that the ability to
scavenge free radicals is not sufficient for activity in
the LOCH assay.

Pre-treatment of lenses with quercetin also protects
against lens opacification in the LOCH assay

It is possible that quercetin reacts with compounds

formed by H

2

O

2

in the culture medium, and so pre-

vents radicals reaching the lens. To test for this, lenses
were treated with quercetin (30

mM) for 24 h. Lenses

were then washed with culture medium to remove the
quercetin, and then introduced into the LOCH assay.
Control lenses without quercetin were treated in the
same way. Quercetin protected against lens opacifica-
tion by 46% after 8 h (p

5 .05) in this assay (Fig. 4).

Fig. 3. Structures of (a) quercetin, (b) (

1)-epicatechin and (c ) chlo-

rogenic acid.

Fig. 4. Effect of pre-treatment of lenses with quercetin on light
scatter in the LOCH assay. Control lenses were cultured in AAH for
24 h, washed with AAH, and then incubated in AAH with (■) and
without (

h) H

2

O

2

. Lenses were also treated with quercetin (30

mM)

in AAH for 24 h, washed with AAH alone and then incubated in
AAH (with no quercetin) for 8 h with (Œ) and without (

D) H

2

O

2

.

Light scatter is measured in arbitrary absorbance units per mm

2

.

Experimental points are the mean and standard deviation of 4
separate measurements.

642

J. S

ANDERSON

et al.

To demonstrate that the quality of the lenses had not
changed during the pre-incubation procedure, we mea-
sured levels of oxidized and reduced glutathione.
Freshly dissected lenses contained 26.1

6 5.7 nmol

GSH/mg protein and 0.22

6 0.02 nmol GSSG/mg

protein (n

5 4) with a GSH/GSSG ratio of 119. After

48 h culture, the GSH content was not significantly
different (24.0

6 6.8 nmol GSH/mg protein) but

GSSG was slightly increased (0.38

6 0.06 nmol

GSSG/mg protein); GSH/GSSG

5 63. Both of these

markers remained unchanged after incubation with
quercetin relative to controls. Further, after 4 h of
exposure to H

2

O

2

, the levels of GSH decreased by

45% and GSSG increased by 12.5-fold. Pre-treatment
with quercetin did not affect these changes.

The protective effects of pre-incubation with querce-

tin on transparency shows that quercetin is either taken
up by the lens, or elicits some change(s) in the lens, that
protect in the LOCH assay. The mechanism of action is
not dependent on removing or inactivating H

2

O

2

from

the culture medium, and pre-incubation with quercetin
does not affect GSH/GSSG ratios.

Quercetin inhibits

45

Ca

2

1

and

22

Na

1

flux into the lens

Modification of lens membrane channel proteins by

diamide or mercury-containing compounds, which oxi-
dize a sulphydryl group on the surface of the channel,
leads to an influx of Ca

2

1

and Na

1

, a loss of ionic

homeostasis and loss of lens transparency [14,15]. We
measured transmembrane ionic fluxes using

45

Ca

2

1

and

22

Na

1

tracer techniques to further elucidate the mecha-

nism of action of quercetin in the LOCH model. H

2

O

2

(1

mM) induced a 235% increase in Na

1

influx and a 58%

increase in

45

Ca

2

1

influx (Fig. 5). Quercetin significantly

inhibited the H

2

O

2

-induced Ca

2

1

and Na

1

influx by 80%

and 45% respectively after 4 h, and this effect is coupled
with a protection in the LOCH assay, as described above.
On the other hand, epicatechin exhibited a much smaller
effect on Ca

2

1

influx and no effect on Na

1

influx, and

was much less active in protecting against opacification
in the LOCH assay. Chlorogenic acid showed no effect
on protection against ion flux and was also inactive in the
LOCH assay. These data provide good evidence that the
protective effect against H

2

O

2

-induced opacification by

Fig. 5. Effect of quercetin (30

mM), (1)epicatechin (60 mM) and chlorogenic acid (120 mM) on H

2

O

2

-induced (a) calcium and (b)

sodium influx (n

5 4). Each compound is at a comparable concentration based on the ability to scavenge the ABTS radical.

643

Dietary antioxidants and cataract

phenolics involves protection of lens membrane channel
proteins.

DISCUSSION

Several epidemiological studies have indicated that a

lowered risk of cataract (all types) is correlated to higher
plasma concentrations of antioxidant vitamins and/or
intake of antioxidants [16,17]. However, the protective
effects of individual dietary components against catarac-
togenesis are much more difficult to ascertain. The ma-
jority of laboratory experiments on cataract have focused
on the antioxidant vitamins C and E. Both have been
shown to have protective effects in some in vivo and in
vitro models of cataract [18 –21] although it is notewor-
thy that in vitro experiments with vitamin C tend to
report a protection against oxidative damage rather than
protection of transparency. In the experiments described
in this paper, transparency was considered to be the
parameter of primary importance.

In the LOCH assay, quercetin is an inhibitor of lens

opacification and improves transparency even at low
concentrations (3

mM). Other phenolic free radical scav-

engers are either much less effective or are ineffective.
Quercetin does not protect against loss of glutathione,
and most important, the major function is not to prevent
hydrogen peroxide or derived compounds in the culture
medium from reaching the lens. Quercetin shows a pro-
nounced inhibition of Ca

2

1

and Na

1

influx, which either

leads to a protection or is a consequence of action of
quercetin at another site. A probable target for protection
is the non-selective cation channel that is activated by
oxidative stress to the lens [14,15], as well as other
tissues [22,23]. It is likely that prevention of Ca

2

1

influx

is an important mechanism by which quercetin acts in the
LOCH assay, since increases in intracellular Ca

2

1

are

linked with lens opacification and activation of calpain (a
calcium dependent protease) [8,24]. When lenses were
treated with quercetin before the LOCH assay, quercetin
could possibly bind to the non-selective cation channel
and remain at this site after washing of the lens and
removal of quercetin from the culture medium.

Flavonoids and related polyphenols are potent inhib-

itors of aldose reductase, that is significant in the devel-
opment of diabetic cataract [9]. Quercetin either applied
topically to the lens, or in the diet, protected against lens
opacification in two in vivo models of diabetic cataract
[10]. It has since become apparent that diabetic cataract
also involves oxidative mechanisms [25], and so quer-
cetin may also exhibit some protection against diabetic
cataract. Further, a decreased risk of cataract is associ-
ated with a daily tea consumption of greater than 500 ml
[26]: tea is a major source of quercetin, and flavonoid
intake was found to be highly correlated with consump-

tion of tea [27]. These data, together with the mechanis-
tic data presented in this paper using the LOCH assay,
suggest that dietary quercetin may reduce the risk of
certain types of cataract. Cataract is the major cause of
blindness worldwide [28] and cataractous lens removal is
the most common surgical procedures performed in the
elderly [29]. Optimization of dietary intake of protective
nutrients could be an effective approach towards reduc-
ing the incidence of this disease.

Acknowledgements — We would like to thank Geoff Plumb for TEAC
measurements, Gre´goire Thomas for help with graphics, and George
Duncan for helpful discussions and critical reading of the manuscript.
Funded by the Ministry of Agriculture, Fisheries and Food, UK.

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ABBREVIATIONS

AAH—artificial aqueous humor
BHT— butylated hydroxytoluene
DMSO— dimethyl sulfoxide
EMEM—Eagles minimal essential medium
LOCH—lens organ culture with hydrogen peroxide
TAA—total antioxidant activity
TEAC—trolox equivalent antioxidant capacity

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Dietary antioxidants and cataract